Genetics II: Baniahamd Part

The cell, nucleus, nucleolus:

The cell nucleus is inside the cell and has a double layer membrane. Inside the cell’s nucleus has a dark
area called “Nucleolus”. The nucleolus is a site of inside nucleus that ribosome subunits are composed.
So, regarding the structure of nucleolus, reflection in the micrographs is darker.
The nuclear membrane is also darker than the cell nucleus due to the similar situation of nucleolus.
Also, inside the nucleus, there are other darker areas called “Heterochromatin” which means very
tight compacted chromatin. The heterochromatin mostly is located on the inner side of bilayer nuclear
membrane. The “Chromatin” defines as a complex of DNA and protein. In the other words, proteins
associated with DNA, such as histones and other proteins and many other factors.
Each nucleus contains:
(1) Nucleolus is located inside the nucleus where the ribosome subunits are produced.
(2) DNA Strands are moving into the nucleolus that encode “Ribosomal RNA (rRNA)”
(3) Spackles or Spliceosomes where the mature mRNAs from pre-mRNA are produced, then
transported to the cytoplasm.
(4) Heterochromatins are darker areas in the electron microscopic figures which contains the
inactive chromatin.
(5) Euchromatin which indicates more open chromatin.
(6) Nuclear envelope which contains two-layer lipid membrane. The Endoplasmic Reticulum can
make nuclear envelope.
(7) Nuclear Pores with the Nuclear Pore Complexes are located on the bilayer membrane that
allow transport various molecules between the cytoplasm and the nucleus and vice versa e.g.,
factors, nucleotides, RNAs, proteins, etc.
(8) Nuclear Lamina Layer which is located underneath the nuclear envelope where
heterochromatin attached to it.
Nuclear Envelope:
The nuclear envelope is composed by bilayer lipid membrane and Nuclear Pore Complexes (NPCs).
However, on the Inner Nuclear Membrane (INM), the specific receptor for lamina is located which
called “Lamin Binding Receptor (LBR)”. The LBR can bind to the chromatin by “Heterochromatin
Protein 1 (HP1)” factor. Usually, NPCs are crossed on both membranes i.e., INM and ONM, and interact
with actin filaments.
Nuclear Pore Complex (NPC):
The nuclear pore complex is composed from different components, and it has a very “complex” and
“conserved structure” among eukaryotes that means the structure between yeasts, mammalians,
plants and insects are same. The complex contains:
(1) nuclear basket which is connected to the central pore complex by the nuclear ring.
(2) cytoplasmic filaments which are connected to the central
pore complex by the cytoplasmic ring.
(3) central pore complex which interacts to the nuclear envelope.
The Nuclear Pore Complex Proteins (Nup) are highly conserved during
evolution. Research on the various eukaryotes showed the similarity
between Nups in yeast (C. cerevisiae) and other vertebrata. The
nuclear pore complexes in their structure have a quite similarity and
this homology caused by the function. It means the sequences are
different, but the functions are same. The functions of Nups mostly
are done by signaling proteins such as NLSs (Nuclear Localization Signals) that leads by importin for
import or exportin proteins for export.
Nuclear pore complex has diverse functions due to the conserved structure. The functions are
included:
(1) involving in the SUMOylation controlling (SUMOylation is a kind of molecular modification
such as ubiquitination that makes a covalent linking between peptides. SUMOylation can lead
to the protein stability, protein localization, and controlling of DNA repair)
(2) involving in the ubiquitination (ubiquitination is a kind of molecular modification such as
SUMOylation that adds a ubiquitin functional group to the molecule. More ubiquitination of
proteins mostly leads to the degradation by proteasomes. The proteasome activity is very
important to controlling many factors.) Usually, ubiquitination via the nuclear pore complex is
done for (a) Replication damage/ Persistent Double Strand Break (DSB) or (b) Repair and
replication restart.
(3) involving and association with telomere activity. (Telomeres are very important parts of
chromosomes which located in their ends. Telomeres are DNA sequences which play roles of
chromosome stability. If telomeres are not extended after each cell division by telomerase
enzymes, this will lead to cell death. In the human stem cells and germ cells, telomere activity
has been seen).
(4) involving in the repairing of subtelomeric DSBs by Non-Homologous End Joining (NHEJ)
activity.
(5) involving into the mitosis when the microtubules are associated with kinetochore.
(Kinetochores are protein complexes that can attach to the chromatins’ centromeres. In this
case, numerous Nups are involving between microtubules and kinetochores. On the side, Nups
can control cell division’s spindle. The spindle duty is segregation chromatids to the cellular
poles.) involving controlling the assembly of kinetochores and spindle via microtubules.
(6) involvement in the early prophase via centromere interaction (in the beginning of the
prophase, chromosomes must be condensed then before this phase i.e., in the early prophase,
one of the factors can be involved in the centromere attaching to the nuclear envelope).
Summary of Nup functions:
(1) DNA repair: translocation of damaged DNA to the NPC and NHEJ repair.
 (2) Trafficking between nucleus and cytosol
(3) Telomere length control
(4) Chromosome segregation and kinetochore assembly
(5) Posttranslational modifications (SUMOlation, ubiquitination)
(6) Chromatin organization
Important Note: during interphase whole structure of nuclear envelope is stable and fixed. However,
when prophase is started, the nuclear pore envelope disappeared and therefore Nups also segregated
with other parts of nuclear envelope. During anaphase the Nups are re-assembled and finally at the
telophase the nuclear envelope is re-assembled and Nups are active again. It means Nups are
connected to bilayer nuclear envelope and there is not a degradation during cell division.
Nuclear Laminas:
Underneath inner membrane of bilayer nuclear envelope, a tiny layer of intermediate filament
proteins is set which called “Nuclear Lamina” which its proteins called Lamin A, Lamin B and Lamin C.
The lamina proteins have a connection to cytoskeleton by different factors. Also, lamina interacts with
chromatin and other factors on the inner membrane. The nuclear pore complex is associated with
lamina, too. Lamin B protein can interact to the chromatin. Lamina has different functions that are
included:
(1) provide the nucleus shape.
(2) interact with chromatin.
(3) interact with transcription factor that are bound to the lamina.
(4) fast response and immediately activation to the extracellular signals by allowing the
transcription factors activation if they received the signals from outside of nucleus.
(5) store different transcription factors and interact with chromatin (eu-, heterochromatin) due
to the releasing and binding fast to the DNA sites.
Laminophaty:
There are several diseases that based on lamin genes which means pathological disorders of lamin
genes. For example, Lipodystrophy (genes 1A and 2B), Cardiomyopathy and Myopathy (genes 1A, 2A,
1B, 2B), Muscle Dystrophy and Myopathy (genes 1A, 2A, 1B, 2B), Progeria syndrome (genes 1A, 1B).
Hutchinson – Gilford syndrome is an example of laminopathy. In this case a mutant in the lamin A gene
caused an autosomal – dominant inherited disease that called “Progeria” or “early unset aging”. This
syndrome is a part of progeria. It is important to know, the nuclear shape in this disease have been
changed dramatically that caused by malfunctioning of Lamin A protein. Patients in their first years
develop normally but very rapidly loose hair, and then show rapid aging phenotypes such as
osteoporosis.
The model of chromatin organization:
The lamina can control chromatin organization. There are “Lamin Associated-chromatin Domains
(LADs)” which means the DNA domains can interact with lamina. The chromatin strains are located on
the lamina and hanged into the nucleoplasm (looping out) with the “Transcription Machineries (TMs)”
which means there are huge complexes of transcription factors that can transcribe the genes. In the
words, it can be possible that two different chromatin strain can be shared on a same TM. The looping
of these chromatin strains can be controlled by a specific factor called “CCCTC-binding Factor (CTCF)”.
The CTCFs can recognize the CTCF-rich sequence and then bind to them. This binding allows to the
chromatin strains that be stabilized looping and looping is essential for transcription.
Lamin Associated-chromatin Domains (LADs):
LADs are often repressed from their interaction with laminas. However, they can also associate with
transcription factories. Some LADs can interact with other repressive environments like
pericentromeric heterochromatin or the nucleolus.
By using techniques such as Hi-C method, it could be seen and had experiment that LAD’s location
inside the nucleus. The Hi-C method is used for analyzing DNA looping. Therefore, this technique can
be applicable for LADs. The LADs can be captured by heterochromatins and between them in this
interaction region which called “inter-LADs”.
The LADs are not stable, and experiments showed their dynamics. When chromatin becomes active,
the positions of the LADs can be changed and it will be more flexible, and if chromatin will change to
the heterochromatin and becomes inactive, it will move and attach to the lamina. This dynamic
behavior is very important due to the chromatin competition.
Heterochromatin changes and its role in the cell fate:
The heterochromatization caused by different stimuli in different patterns in young and old cells. It
would be possible to have different chromatin compactization and interactions with lamina. in this
case for senecios cells, different heterochromatin foci could be seen. these foci are very famous in
human cells. the cell nucleus is the highly structured organelle, and everything is not located randomly.
feature LADs Inter-LADs
gene density low (more stable) high
gene expression low high
Hi-C compartments B A
replication timing late early
sequence A/T content high low
histone marks H3K9me2 (di) H3K4me1
H3K9me3 (tri) H3K27ac
H3K27me3 (tri) H3K4me3
[for heterochromatin] [for active chromatin]
retroelements LINE SINE
Lamin Associated-chromatin Domains (LADs) identification by Hi-C method:
the associated domains such as LADs and TADs (Topologically Associated Domains) can be analyzed by
Hi-C (Conformation Chromosome Capture Assay) that analyzing looping by DNA sequences through
protein with others. the results showed by colors when colors became darker it means more looped
and interacted strongly.
regarding the knowledge about the proteins that are known as cross linked chromatin, then by using
restricted enzyme the fragments of chromatin are ready for analysis. then, the restricted fragments
and ligated and by purification and PCR analysis or sequencing of ligation products, the main materials
are ready.
there are different types of analysis if it is "3C" one-to-one and analysis one gene. "4C" one-to-all and
analysis one gene to whole genome. "5C" means many genes to many genes of genome, and "Hi-C"
means all-to-all that is analysis of whole genes with whole genome.
the experiment that is shown chromosomal organization in the cell nucleus:
There were two hypotheses about chromosomal organization in the cell nucleus: (1) chromosome
territory model, (2) random organization model. for both hypotheses, there were two different stories
if a laser damage in a part of the nucleus was happened: (I) for chromosomal territory model: some
chromosomes will show laser damage. (II) for random organizations model: all chromosomes will show
the laser damage. for illustration, scientists used fluorescent nucleotides in the media, after laser
damage, for repairing machinery. In the end, just "some" chromosomes showed laser damage, via
fluorescence dyes.
By labeling Fluorescence In Situ Hybridization (FISH) and having chromosome specific labeling via PCR
(fluorescence dyes). in the metaphase can be denatured DNA and allow hybridization of fluorescent
generating PCR to see where is in the nucleus. Also, if specific areas or sequences in the chromosomes
are labeled, it could be shown as different segregated chromosomes in the micrographs. It could be
done by the whole human chromosomes and their labeling. this labeling could be done by FISH-probes
(specific probes) that can hybridize the nuclear DNA.

Methods is mentioned in nuclear compartments detection:

• Transcriptional centers: overlaps in fluorouracil/anti ribosome immunofluorescence:
➢ Alternative: RNA FISH; immune-RNA/DNA FISH
• Splice complexes (speckles): immune-RNA FISH
• Repair complexes: ssDNA probes with anti H2AX (histone variant) antibodies
Chromosome conformation capture techniques refer to a plethora of analysis techniques that analyze
the spatial organization of chromatin in the cell. There are multiple variations of this technique, but
the common thread between all of them is as follows:
1. Chromatin is crosslinked with formaldehyde, so that any inter/intrachromosomal interactions
may be preserved.
2. Crosslinked chromatin is digested and ligated, so that each interaction locus is reduced to a
single molecule.
3. Heat is introduced, reversing crosslinking, and having only the ligate left.
The analysis methods used differ by their scope:
o 3C (one vs. one) quantifies interactions between a single pair of genomic loci. For example, an
enhancer-promoter interaction can be tested, provided all the interacting regions are already
known. The ligated fragment is enhanced using qPCR with a known primer.
o 4C (one vs. many; CCC on chip) involves a second ligation step, where the DNA fragments are
circularized (inverse PCR). Only one interacting sequence needs to be known in this method,
and primers specific to this sequence can be used to amplify the unknown sequence ligated to
it. Analysis can be performed via a microarray or by sequencing.
o 5C (many vs many; CCC carbon copy) ligates universal primers to all fragments, allowing for
amplification of all possible interactions and analysis via sequencing. However, this requires
millions of 5C primers if genome-wide complex interactions are to be used.
o Hi-C (all vs all) involves a combination of 3C and sequencing techniques, where the relative
abundance of ligation products is correlated to the probability of the respective fragments
interacting in 3D space.
 Before ligation, all fragmented chromatins are marked with a biotinylated nucleotide, allowing for
purification by streptavidin-coated magnetic beads. PCR amplifies the entire library, which allows
for analysis via high throughput sequencing.
o Another alternative that determines chromatin interactions is ChIA-PET (Chromatin
Interaction Analysis by Paired-End Tag Sequencing). It is considered a hybrid of CCC and ChIP
methods.
The 3D genome organization:
The double membrane nucleus has nuclear pores, nuclear lamina, and its inside nuclear speckles that
are large protein complexes, composed from different proteins. Also, beside the nuclear speckles, the
nucleolus is a site for ribosome subunit organization.
One of the features of organized nucleus is the hierarchal organization of the 3D genome. Inside the
interphase nucleus, the individual chromosomes can be seen that occupied separated territories. In
the nucleus, the nuclear compartization where are areas with compacted or opened chromatin or
more controlled can be seen. These are different compartments inside the nucleus which are never
seen by electron microscopy. Also, there are a cluster of DNA-looping specially into these active or
inactive regions. Even chromosome can loop in the same area and share some compartments.
The inactive regions are heterochromatin, differentially by the nuclear manner, in the nuclear lamina
and nucleolus in the similar manner. The euchromatins are looping to the open areas due to the being
more active, more transcribed in the same area. If the compartment A and B are magnified, there are
many lopping areas of different organization in the chromatin. Locally, genomic domains show strong
self-interactions and are insulated from nearly regions, forming Topologically Associated Domains
(TADs). Also, the CCCTC-binding factor (CTCF)-binding sites on chromatin are preferentially found at
TAD boundaries.
Inside of each TAD, there are a couple of different factors which can control these domains. The
cohesion is very important factor that involves in the stability of looping beside CTCFs. On the other
hand, near the boundaries of TADs, these are several factors such as NIPBL (nipped B like protein), and
MAU2. The main function of these proteins is looping.
The different moods of interactions among cis-regulatory elements in development:
There is a different possibilities and options for interactions among the cis-regulatory elements.
Chromosome structure has a flexibility and dynamic structure, also different generate with different
enhancers then these moods can be classified into these types:
(I) A transcribed gene associates with different enhancers
(II) An enhancer regulates several stages – specific genes.
(III) Stable and gained promoter – enhancer interaction to induce some gene expression.
Chromatin architecture dynamics:
By using Hi-C method it can be possible to study on chromosome structure and genome width. For
example, during the spermatogenesis, mitosis, meiosis (I) and meiosis (II) and also for the haploid
periods. Then by this method it can be illustrated where the homologous chromosomes looping out
and how reprogramming occurs. This time – dependence chromosome architecture is called "4-D
chromosome" or "4-D chromatin architecture".
For example: during mammalian spermatogenesis, TADs and loops are only at diplotene stages of
meiosis (I). it illustrates that these specific TADs and loops only for this development time.
For example: some diseases in human caused by TADs' mis-regulation. In the BW syndrome, there is a
change in the expression (abnormal expression) of the IGF2 gene that not correctly controlled by the
enhancers. In the normal situation there are two TADs for regulation, however in the cancerous gene,
there are three TADs (de novo TAD) because of the super-enhancer duplicated and IGF2 gene locus
and this duplication allows to super-enhancers to active strongly and then gene expression. IGF2 is an
Insulin Growth Factor that leads to tumor growth. Therefore, the de novo TAD leads to activates the
super-enhancer. However, in the normal situation TADs inhibit the gene expression.
Structure variation in the 3D genome:
In our genome, we have different types of structural variants or SVs. The SVs can be classified into two
variations:
a. Sequence Variation: SNPs, insertion, Deletion.
b. Structural Variation: Insertion, Deletion, Inversion, Duplication, Translocation.
How these variations can be detected?
For small changes, the Sanger and NGS are useful in deletion, however these methods cannot be
applied for the repetitive sequences. For large changes, the microarray, FISH are useful (megabases).
For cytogenetic > 5 – 10 Mb; molecular cytogenetic > 50 kb; Array-CGH > 50 kb.
Inversion: changing the order of loci.
Frequency and sizes of SVs:
The SVs can be occurred in the different sizes such as megabase (Mb), kilobase (kb). For example, 200
bp to 10 kb for insertion, 1 bp to 1 Mb for deletion. It's unclear between frequency and transcription
rated and the 3D architecture.
Interpretation of SVs:
The interpretation of SVs also is very difficult especially about "de novo SVs" and "so reduced
penetrance" and variable expression is happened in terms of phenotype correlation. Also, many SVs
are within non-coding DNA. The non-coding DNA means they are not involving in the gene expression.
However, the non-coding DNA can effect on the genome architecture, therefore, there is a hypothesis
about SVs can change genome architecture.
The main component of the human genome:
➢ Only 1.5% of genome just involves in the protein coding.
➢ The DNA transposons (3%) are originally from viruses. These elements are the coding DNA but
not for protein coding (retrotransposons).
➢ SINEs (Small Interspersed Nuclear Element): 3%
➢ LINEs (Long Interspersed Nuclear Element): 20%
Both SINEs and LINEs are derived from the viruses.
About 25% of human genome regarding the evolutionary evidence are from viruses.
➢ Miscellaneous parts (20%)
• Miscellaneous heterochromatin (8%)
• Miscellaneous unique sequences (12%)
➢ Introns (26%)
The gene structure:
Each gene contains:
• Promoter: the transcribe start site (TSS)
• UTR: short form of “Un-Translation Region”. Mostly causes transcription stop at 3’ UTR.
• The transcription elements: enhancers, insulators, promoters, and silencers. These elements
can control gene expression that could be located on downstream or upstream of genes, even
on the introns.
• The difference in the variations of our genome can be affected by the gene position and can
effect on gene expression. Therefore, these different positions can regulate the gene
expression. Although the gene itself, the coding region (includes within 5’UTR and 3’UTR that
has exons and introns) has been not mutated, the surrounding area, the 3D structure can
manipulate the gene expression.
Cohesin and Condensin:
Cohesin and Condensin are involved in the 3D structure (architecture) of DNA. These molecules
provide the genome looping. Also, these molecules are composed from different subunits. Condensin
leads to intra-molecular looping event and TAT. However, there are chromosome interactions such as
meiosis that homologous pairs are paired and cohesin is responsible about it. Cohesin is involved in
the inter-molecular events and DNA pair repairing. In the codensin interaction, codensin has a looping
structure inside it and protein structures and DNA loops inside its loop. Then the two DNA stands have
enough space to interact together.
NOTE: condensin activity ↑ = DNA mobility↓
The looping models of cohesin and condensin:
Two models are proposed regarding the functions of cohesin and condensin which the last one is
accepted regarding the biophysical studies. The new models describe how two DNA strains can go
inside the loop of condensin and cohesin. Regarding the models, these molecules can be charged by
different subunits. By looping these molecules can generate different loops.
Functions in the interphase:
The condensin is known for its functions during the interphase. It can be analyzed by 3C, and Hi-C (for
condensin interactions) in the model organisms (e.g., Drosophila melanogaster, C elegans,
Saccharomyces cerevisiae, Arabidopsis thaliana, mouse, cyanobacteria, microalgae). For eukaryotes,
condensin detected as a key molecule at interphase of polytene chromosome of salivary glands in
fruit fly. The molecule can parallelize DNA-sane molecules and chromosomes for creating giant
chromosomes or polytene chromosomes. Also, they are some hypotheses that condensin plays roles
during transfection, physical distortion, dosage compotation (paternal and maternal genes),
stabilizing ribosomal DNA (rDNA), the culturized tRNAs within one locus (leads co-expression) and
allowing the looping.
The nuclear bodies:
Inside the cell nucleus, in addition to the spliceosomes, heterochromatin, laminas, euchromatin, there
are many other bodies inside the nucleus. Most of them were detected by antibodies that labeled
fluorescent, for example: PcG body (Polycom Group) for gene expression, Cajal bodies, Cleavage
bodies, Nuclear speckles, Transcription factories, and PML bodies for cell senescence.

The transcription:
By using fluorescent dyes which has uridine as a couple system. The uridine can send from DNA to RNA
then the labeled uridines’ pulses only can be seen in the short time, after washing and analyzing how
many dots were observed. In sum up:
➢ By fluorescence uracil (Fl-U): transcription foci (5000 ~ 10000) = polymerase spackles.
➢ By bromo-domain uracil (Br-dU): replication foci (1000) = in the S-phase, in the spackles.
➢ By γ-H2AX: repair (radiation and) = 20 ~ 50.
PML bodies:
The PML bodies are a few sides, inside the nucleus. The name is derived from Pro Myelocytic Leukemia
bodies. These bodies were isolated, tagged by fluorescent. The analyzed data show the PMLs are
responsible for:
• Antiviruses
• Apoptosis
• DNA damage
• Cellular senescence
• Proteolysis
• Gene regulation (as much as suppression)
The mechanism for activation or suppression is not clear but regarding the structure, it’s known the
PML bodies within the situation, they can release or recruit a mechanism that affect by its sides.
Transcription factories (hubs, nuclear factories):
The nuclear factories are highly active transcription sites and inside the nucleus which can involve into
the many genes’ transcription that need be co-transcribed. The RNA Pol II can conglomerate with
different factors and proteins. Usually, transcription factories can cause chromatin loop formation. The
DNA is looping into these spackles and transcription factories. For example, Hbb (hemoglobin) and Klf1
and Hba genes are co-transcribed at the same time, and these genes are from different chromosomes.
In the transcription factories, the RNA Pol II has a carboxyl terminal domain can be phosphorylated
and for antibody experiments are useful. The specific antibodies can bind in the active form during
phosphorylation. Then the results show:
➢ RNA Pol II antibody staining: locations of RNA Pol II active sites.
➢ BrUTP incorporation: BrUTP antibody staining
Structure of a transcription factory:
The RNA Pol II in the transcription factory, DNA loops into that and RNA will be synthesized. Due to the
transcription factories are unlikely to move, because of looping into that, probability they are linking
systems, DNA moves into and out of the transcription factory to produce RNA transcribes. The
stationary RNA Pol II molecules could reel the transcribed DNA through the factory. Also, beside the
transcription factories, there are some organizer factories where they have transcription occurring,
looping, and enhancer interaction with the promoter. The intergenic transcription that probably is
much more complicated. Then, the organizer factories that allow transcription with the interaction of
enhancers, silencers, and promoters. But also, CTCFs and boundary terminators are not to providing.
Colocalization:
Basically, it suggests that gene colocalization is in the nucleus. This colocalization is for gene expression
and co-regulation. There is a chromosome territory and looping sites of active gene transcription at
the active compartment.
Summary: organization of eukaryotic genome:
The DNA is a double helix but not naked. It is associated with core histones. Then each nucleosome
(core histones + DNA) forms chromatin fibers. Then chromatin fibers can be magnified to the
chromatin loops (megabases. Kilobases). Then regarding the looping, they will become chromatin
domains. Then, chromatin compartments and finally chromatin territories will be formed. Inside the
nucleus, there are chromosomes, active and inactive compartments, lamin associated domains (TADs)
and nucleolus.
The principle of genome organization is the interplay several fundamental principles that grown on
genome organization.
1. Dynamic chromatin: chromatin is highly dynamic, and it is highly flexible system and not fixed.
2. Phase separation: heterochromatin, euchromatin (active and inactive chromatin separated).
3. Architecture elements: such as nuclear lamina, nucleolus, etc.
4. Heterogeneity and stochasticity.
5. Polymer – polymer interactions. DNA, RNA, and proteins are polymers, and they have
interactions to each other’s.
The transcription mechanism:
Activation by p300/CBA activator and co-activator molecules. They don’t bind to DNA directly, but they
can interact with transcription factors.
- Bridge between the TFs and basal machinery that RNA Pol II can bind to this bridge and can
induce a looping.
- p300 and CBA molecules can also interact with chromosome modifying enzymes like HAT
(Histone Acetyl Transferase), NAP (Nuclear Assembly Protein) that can assemble nucleosome.
This function is known as the scaffold.
- Histone modifications: adding or removing chemical radical(s) such as methylation,
acetylation, etc. Histone acetylation can activate gene expression locally.
- DNA binding TFs can be classified as: “Transcription activator” and “Transcription repressor”.
As a transcription activator, Jun and Fos that are heterodimer and also known as “proto-
oncogenes”. Usually, both proteins are overexpressed in the cancers. They have interaction
sites for DNA composed with the basic area of amino acids (basic amino acids can interact with
acidic backbones of DNA, phosphatic parts). In addition, they have zipper that such as a real
zipper can stabilize heterodimer in leucin as hydrophobic parts. Therefore, leucin – leucin
zipper together led to stabilize heterodimer complex DNA.
- TATA-Box Binding Protein (TBP) is another important factor for transcription. This monomeric
factor can bind to DNA and will recruit a signal for other TBP interacting factors, and RNA Pol
II will bind to the site and start transcription. Also, TBP alters genes are turned on or expressed.
The nuclear hormone receptors:
The glucocorticoid receptors are classified as the nuclear hormone receptors with the zinc finger for
DNA binding domains. The zinc atoms at the Zinc-Finger DNA binding domains, can stabilize the alpha
helix which is recognizing DNA sequence on the DNA. The glucocorticoid receptor binds to
glucocorticoid hormones in the hormone suppression, such as cortisol. The binding mechanism is
known as “homodimer”.
- Zinc-Finger is a homodimer and glucocorticoid receptor. It leads to the DNA transcription via
the factor – DNA interaction.
- Jun and Fos (=AP1) are heterodimer and known as basic leucin zipper.
- TBP is working for both groups.
The nuclear organization:
For gene regulation, it is not enough to have enhancers, promoters, silencers, and TFs. It needs
organization of TADs, looping, CTCFs and insulators.
 - Insulators such as CTCFs have cohesin and condensin that can stabilize DNA looping and have
poly-com proteins (PCs) and TFs interactions.
- CTCFs insulator and TFIIIC insulator are known in mammalians. However, fruit flies have more
insulators such as dCDCF (Drosophila CTC- binding factor) insulators, GAGA insulators, etc. The
reason for being more insulators in fruit flies is the genome size that is smaller. Therefore, the
genes are located closely to each other. To promote the correct expression in development or
the tissue specific expression, or the time-depending expression, it is important to insulate
genes, then regarding the insulators and tightly genes’ localization, they have more insulators.
- The nuclear organization is related to the gene expression. A looping can have a couple of
genes and CTCFs can allow to express gene or not. The CTCF can by insulating cause expression
or suppression. However, both mechanisms caused by histone marks.
- Today’s technology let us detect close location, cross, and analyzing insulators, CTCFs also,
close-related promoter and enhancer to the gene.
- Taking count of looping that some enhancers are very far away still cannot loop into close
promoter destiny or proximity. Also, too many loops are colocalized and having coexpression
system that interact either with CTCF bodies, polyCom bodies, and looping outside and inside
of coexpression. The chromosome takes compact structure or linear with or without loops. It
can have linker sequence to chromatin.
Superenhancers:
Beside of normal enhancers, other class that called “superenhancers”. The difference is the size,
normal enhancers are between 200 bp to 1 kb, and many have binding factors. However,
superenhancers are bigger (between 10 to 15 times larger) than the average and limited by genome.
It seems that superenhancers are very important for developmental aspects and differentiation
processes. Genes that are regulated by superenhancers could be found in stem cells for developmental
aspects. However, regular enhancers can just regulate one or few genes.
Due to the superenhacner’s size, they have more binding sites rather than normal one. It seems to
different cellular system, they involve into the different processes and side effects, e.g., stem-cellness
or stemness is proofed by Oct4, Sox2, Nanog, KII4 and Errb pathways that can bind to the same
superenhancer can control stemness and genes’ expression.
Nucleosome positioning:
It seems that nucleosome free sites are enhancers, around transcription start sites, also termination
sites. The high nucleosome positioning alters the first nucleosome of starting transcriptional site. For
example, in the one steady on Saccharomyces cerevisiae the nucleosome free positioning could be
found around the (before) termination site.
Controlling of gene expression:
The controlling can be possible via these ways:
- Transcription rate controlling of how many per time is produced: mRNA, tRNA, rRNA, long non-
coding RNA, micro-RNA.
- Promoting, inhibition of transcription initiation complex.
- At the level of DNA methylation and epigenetic silencing.
- Recruiting of remodeling and mediator complexes.
- Binding of transcriptional activators/ repressors to enhance/ silence the elements.
- Opening/ repression of chromatin accessibility (nucleosome assembly, remodeling, histone
exchange, histone modifications).
 - Localization within the nuclear structures.
- Allowing DNA looping.
Transcriptional Regulation of RNA Pol II promoter:
There are three of RNA Polymerase (RNAP) in the eukaryotes:
1. RNA Polymerase I (RNAPI)
2. RNA Polymerase II (RNAPII)
3. RNA Polymerase III (RNAPIII)
The RNAPI is a polymerase that can transcribe the coding of protein genes. Although the amount of
coding proteins in the genome is very low, many experiments have been shown towards analysis this
RNAP promoters. Since proteins are major parts of regulatory pathways and phenotypes.
The structure of RNAPII promoters contains: (a) transcription start site which defines as “+1”, (b) the
upstream of this start site in about 40 ~ 45% of all genes is “TATA-Box” (not all genes!), then (c)
different promoter elements that are upstream of TATA-Box.
For initiation of transcription is opening chromatin, then allowing the first factor binds to the TATA-
Box is “TBP” (TATA-Box Binding Protein). The TBP can bind too much to the DNA, also TBP can bind
with “TFIID” complex. The TF IID has several subunits. After TF IID binding, other TFII molecules will be
recruited such as “TFIIA”, “TFIIB”. then, RNA Pol II can be bound via “TFIIE”, “TFIIF” and “TFIIH” to the
chromatin. (Chromatin (TATA-box) + TBP > + TFIID > + TFIIA, + TFIIB> +TFIIE, +TFIIF, +TFIIH > RNAPII
binding)
RNAPII never binds directly to DNA; it binds to the factors that are bound to the promoter. This is
different rather than prokaryotes. Then, RNAPII needs phosphorylation for activation of RNA
polymerization.
The Promoter and RNAPIIs:
Each promoter has different parts such as “regulatory promoter” and “core promoter”. The core
promoter contains:
- The TATA-box is located around “-25 bp”.
- The TFIIB recognition element site around “-35 bp”.
- Initiator element site at “+1 bp”.
- Downstream Core Promoter element around “+30 bp”.
Some genes do not have TATA-box then the inhibition element that is around the transcription start
site (TSS), sometimes exists at downstream of core promoter. Each elements plays an important role
e.g., TBP at the initiation level that TFIIA binds to it and recruits other TFII elements. In the end, there
are a core promoter which can initiate RNA polymerization as a protein complex that includes TFIIB,
TFIID, TFIIE, TFIIF, TFIIH and RNAPII called RNAPII holoenzyme. The key factors are TBP and TATA-box
complex that can bind directly or indirectly via TFIIA.
The recruitment rate is regulated by initiation activity of RNAP. Also, the regulation is happened by
other DNA elements like silencers, enhancers, that can be at up-/downstream of promoter. The
enhancers can bind to the transcription activators. The silencers act as transcription repressors. These
elements can affect the promoter via looping that could be a further coregulator.
Coregulator can make mediation at the initiation level between transcription factors that bind to
regulatory elements, and the DNA up- and downstream of promoter and basic transcriptional
machinery.
A looping between transcription activators and distal sites of the promoter leave loop into the density
and proximity of promoter region. Promoter region is composed of TBP, subunits of TFIIB, RNAPII,
TFIIF, TFIIE, and TFIIH.
The transcription activator in Drosophila melanogaster is Hunch Back (HB) and Bicoids (BCD) that are
able to connect directly to TFIID subunits. By recruiting these TFIID subunits, the recruitment of TFIID
complex is much faster because the high affinity of promoter, therefore the transcription rate is faster.
Transcriptional regulation of RNAPII promoters:
The regulation has a reputation from the elements, and different stepwise recruitments of the big
protein complex. TBP is one of the big factors that associated with the TFIID which is recruited the
TATA-box or initiator elements, around the transcription start site (TSS) to TFIIA. This initiation complex
recruits other factors like TFII protein complexes (TFIIA, TFIIB, RNA Pol II, TFIIH, TFIIF, TFIIE, and
mediator). This protein complex is now built up at around the transcription start site. Then, other
factors such as silencers can loop into it to get closer to the promoter and then regulate the
transcription activity of RNAPII. These transcription factors can either be linking there, interactions
through coregulators, coactivators, corepressors; or directly interaction with mediators or even basic
transcription machinery.
The RNAPIII promoters:
RNAPIII transcribes special RNAs that are small. They have ribo-small RNAs or 5S-rRNAs which is
generated by RNAPIII, also tRNAs, some U6-RNAs and other splicing RNAs. Promoters and other
regulatory elements play main roles for RNAPIII identification. For example, on tRNA genes, only
RNAPIII with other initiation factors can transcribe, because the promoters and up-/downstream of
gene(s) are the main identifying elements for RNAPIII activation. Similarly, for 5S-rRNA genes,
promoters lay in the gene. Then, can be identified by sequence comparison of different elements,
called “A” and “B” (and sometimes “C” also) that are recognized by proteins. Others do have TATA-
box, such as U6-RNA and another element. It is very surprising that RNAPIII doesn’t need a specific
region or sequences and it’s highly flexible.
Sum up for two main concerning of RNA Pol III: (a) concerning the location of promoters, (b) which
types of elements exist.
The factors that can binding to the promoters can be identified by three factors: (a) TFIIIB, (b) TFIIIC,
and (c) TFIIIA. The genes have internal promoter, first recruit TFIIIC, then binds to the conserve
sequencing of A and B-box, then TFIIIB recruited and finally, both recruit RNAPIII (+TFIIIC A&B-box>
+TFIIIB >> RNAPIII). Those have internal promoters but different boxes, first TFIIIC then TFIIIB and both
recruit RNAPIII (+TFIIIC > +TFIIIB >> RNAPIII). Basically, there are three major subunits of protein
complex. Those genes have external promoter of transcribing genes, some of them have TATA-box
(e.g., IIIB-like protein complex) and this recruits RNAPIII. Another scenario is TFIIIB only recruits RNAPIII
(+TFIIIB >> RNAPIII). This is a flexibility for RNAPIII to recognize and bind the promoters.
Assembly of RNAPIII and related TFs:
The recognition of TFIIIA, TFIIIB, and TFIIIC is done by many fraction assays. The 5S-rRNA gene, first by
TFIIIA is bound (a protein with many Zinc-fingers), then TFIIIC is bound and finally TFIIIB with some
subunits such as TBP (for TATA-box). Then, the RNAPIII will start transcription.
Comparing 5S-rRNA gene vs. tRNA gene:
tRNA gene contains Box A and Box B; first TFIIIC, then TFIIIB and finally RNAPIII.
5S-rRNA: gene contains Box A and Box C; first TFIIIA, then TFIIIC, third TFIIIB and finally RNAPIII.
Overview on composition of RNA Pol I, II, and III in the Pre-Initiation Complex (PIC):
- RNAPI: TFIs (SL-1) > TBP (lateral part)
- RNAPII: TFIIs > TBP (central part)
- RNAPIII (different types of promoters): Internal (or) External gene: TFIIIs > TBP (central part
and key role)
RNAPII recruitment and elongation:
The RNAPII needs to recruit and then start polymerization. The rate of recruitment controls the
expression rate. Furthermore, Pre-initiation complex (PIC) recruitment, the transcription rate also
controlled by elongation rate. Elongation rate means how fast the entire gene can be transcribed. It's
important to notice that elongation is related to the recruitment.
The RNAPII has CTD (Carboxy-Terminal Domain) that is a long tail of heptide repeats. The heptide
repeat means seven amino acids are repeated. This heptide in mammalian's CTD of RNAPII is repeated
52 times which is consistent with Tyrosine – Serine – Proline – Threonine – Serine – Proline – Serine.
If you look at this structure via biochemistry, that tyrosine, serine, and threonine have hydroxyl groups
that are very important for PIC regulation due to the final phosphorylation.
When RNAPII is recruited for PIC, then CTD is phosphorylated. The phosphorylation is happened when
RNAPII is bound to initiation complex. Also, only Ser-2 and Ser-5 are phosphorylated. Then, RNAPII
starts transcription and elongation. When, the transcription is terminated, the phosphorylated amino
acids will have dephosphorylation. The Ser-2 and Ser-5 of CTD phosphorylation are important because
they are a part of the regulatory system for controlling elongation via the affection the elongation rate.
CTD phosphorylation of RNAPII:
Regarding the experiments, if only RNAPII is recruited, there is no transcription, because the Ser-5 or
Ser-2 were not phosphorylated. If both Ser-5 and Ser-2 are phosphorylated, then the transcription
will be started. The Ser-2 and Ser-5 phosphorylation is highly limited and compressed. However, this
phosphorylation is not sufficient for elongation rate. Some of the peptides has glycosylation in their
combination. This combination allows for elongation rate; for instance, in mammals 52 repeats, in
yeasts 26 repeats are reported for heptides and it seems mammals have more sites for controlling the
phosphorylation level that which amino acids in which level is phosphorylated and then glycosylation
rate. There are many sides here to regulate the elongation rate.
Inhibition of RNAPII and RNAPIII:
• Alpha-Amanitin (α-Amanitin) is very specific inhibitor of RNAPII and RNAPIII. It is a cyclic
peptide that contains 8 amino acids. α-Amanitin is derived from a toxic fungus called Amanita
phalloides that its toxicity is caused by presenting of α-Amanitin. α-Amanitin is a lethal
compound.
• Actinomycin D is another inhibitor that prevents elongation of RNA chain by RNAPII. This
molecule is an antibiotic with three major cycles. It's important to note that Actinomycin D is
more specific rather than α-Amanitin.
Active sites of transcription:
In the nucleus there are some active sites of transcription. One uses antibody against Ser-5 or Ser-2
phosphorylation of RNAPII. These sites are loci that active transcription parts of nucleus and show as
where and the nuclear architecture of active gene transcription.
The analysis with antibodies against phospho-Ser2 will show specific loci like dots in the nucleus.
Therefore, regarding this method if Actinomycin D or α-Amanitin is used, the results will be something
else with different rates. In the α-Amanitin treatment, it's illustrated that less dots which means more
power for reducing and then major effects on RNAP inhibition.
However, Actinomycin D can inhibit less than α-Amanitin, i.e., more dots than α-Amanitin treatments.
It's also suggested that experiments showed the phosphorylation event of Ser-2 and Ser-5 in the
presence of Actinomycin D, still not sufficient to explain a very good elongation rate. But it suggests
that some sites are active, and their elongation rate will not be possible due to the additional steps
that will need to be phosphorylation.
Regulation of gene expression:
At the RNA level:
- mRNA stability/ degradation
- splicing
- Editing
- Export / Import to the cytoplasm/ nucleus
- Binding to ribosome/ efficacy of translation (miRNAs)
Splicing:
The term of splicing basically means to remove “introns” of pre-mature mRNA that was transcribed
form gene(s) and then fusing the “exons” together to generate a normal mRNA. The mRNA will not be
matured after splicing, it needs to add 5’-end capping and 3’-end tailing (Poly-adenine tail: ~ 100 – 300
adenines) to obtain the mature mRNA. Then it's transported to the cytoplasm for protein synthesis.
The splicing is mediated by spliceosome that is very similar to ribosome and consists of a huge RNA –
Protein complex. The U6RNA is a part of the spliceosome. The spliceosome stabilizes the enzymatic
reactions that the RNAs cut between the intron-exon boundaries and generates the “Lariat Structure”
or “intron Lariat structure”. Also, the spliceosome can catalyze the second reaction to fuse two exons
together. The introns usually will be discarded but still some introns would be microRNA source or
loan-work RNA source. After splicing, two processes will be done: 5’-end capping and 3’-end tailing
(poly-A tail).
Alternative splicing:
By intron-exon structure, it can be possible to fuse different exons together or combine them to
generate different proteins from one gene. The splice variants (different proteins) come from one
gene, the alternative splicing can overcome by using the alternative promoter that leads to different
splicing or different splice sites. The splice site can be in exon, and then fuse to other exon, or one can
be removed or added exon(s), one can have alternative poly-A site to have pre-mature stop codon,
and mutually exclusive exons. The alternative splicing has a high flexibility for protein synthesis.
RNA editing:
Beside DNA editing by CRISPR-CAS9 method, there is an edition for RNA was discovered. It means that
RNA sequence will be modified after transcription. For example, apo-B gene that has two different
RNA transcriptions in liver and intestine cells. In liver, no editing, apo-B mRNA is generated with
capping and poly-A tail and UAA stop codon (a 100 kD protein). However, in intestine, a specific
modification on one cytosine (C) which is modified into the uracil (u), and this leads a stop codon
mutation (CCA → UAA: stop codon). Then, it's a pre-mature stop codon compared to the liver and has
48 kD protein.
How is it controlled (RNA editing)?
The amino acid sequence is encoded by DNA and has a transcription which a complimentary sequence,
except thymine (T) that is exchanged to uracil (U) in the mRNA, then the exist of guide RNA that can
bind to RNA. However, the guide RNA has differences. These differences sometimes generate gaps,
then by edition can add the additional nucleotides. Finally, it can show the different amino acids. This
is very famous for transposons. By this evidence, it is not possible to guess what the genetic
𝑡𝑟𝑎𝑛𝑠𝑐𝑟𝑖𝑝𝑡𝑖𝑜𝑛
information was because due to the RNA editing, everything has been changed (DNA → Pre-
𝑏𝑖𝑛𝑑𝑖𝑛𝑔
→ 𝑒𝑑𝑖𝑡𝑖𝑛𝑔 𝑡𝑟𝑎𝑛𝑠𝑙𝑎𝑡𝑖𝑜𝑛
RNA (Pre-edited RNA) ← guide RNA → edited RNA→ protein).
𝑎𝑙𝑖𝑔𝑚𝑒𝑛𝑡

Regulation of gene expression:

At protein level:
- Protein stabilizing / degradation
- Protein activation (cofactors, proteolytic processing, pro-hormone into the hormone, pro-
enzymes into the enzyme)
- Protein modifications (glycosylation, methylation, acetylation, alkylation, phosphorylation,
SUMOylation, proline isomerization, etc.)
- Protein translocation – specific site of action
Chromatin and histone codes:
An overview of the nucleosome structure and histone modification:
The nucleosomes are composed of DNA and wrapped around the eight histones which are dimers of
each four: H3, H4, H2A and H2B. The H1 is a linker histone that can be attached two nucleosomes and
make more compacted. Important thig is the “arms” of histones. These arms are flexible and have
enrichment of basic amino acids such as lysine (Lys), arginine (Arg), serine (Ser) and threonine (Thr).
Usually
- Ser and Thr can be phosphorylated,
- Lys can be methylated, acetylated,
- Arg can be methylated.
These modifications can control the chromatin structure and then gene expression.
Histone modification:
Some marks are available around transcription sites of active chromatin. The markers distribution is
different, some of them at upstream of promoter, some within the gene (perhaps for elongation),
some at the end of gene (perhaps for termination). These codes, the posttranscriptional codes are
very important. For example, H3.3 is a histone variant that cooperated in the nucleosomes. The normal
H3 is replaced by H3.3. The H3.3 is a mark for active chromatin (for the genes that are expressed
highly).
NOTES:
• H3K9me3 and H4K20me3 are together enriched at imprinted genes.
• H3K9me3 and H4K20me3 do not show a correlation with developmental regulation.
• H3K27me3 and H3K4me3, however, are more often associated with developmental
regulation.
• A specific tri-mark signature comprising H3K4me3, H3K9me3 and H4K20me3 has been
identified at all imprinting control regions.

DNA, nucleosome, and chromatin regulation:

DNA and chromatin can be compacted a lot. At the non-metaphasic state the chromatin can be
compacted regionally. This compaction forms from interphase to prophase, and during metaphase can
also be for gene regulation. The naked DNA is about 2 m which assembles to the nucleosomes by
histone core then be structured as the beads-one-string which by additional histone H1 that can be
compacted to 30 nm fibers. This structure has fewer active genes because of more compacted
structure. By additional scaffold proteins, this compaction can be more. The scaffold proteins can
allow larger loops (compacted loops) and additional more scaffold proteins allow chromosome to the
metaphasic state.
Also, the compaction will not be happened just at the meiosis or mitosis, it is happened in one of the
female’s X-chromosomes called “X-chromosome inactivation” that known as “Bar body”. Bar body as
a different modification of histone and DNA is compared to other X-chromosome of females.
Also, changing 30 nm fiber to open chromatin, is used for the cell fate, different stages, and gene
expression level. Furthermore, the active chromosome during interphase that has a large looping, can
again loop out and allows regulation.
So far, the regulation of chromatin at above the 30 nm fiber level have been discussed (looping, TADs,
transcriptional foci, etc.) However, there is also epigenetic regulation at the nucleosome (beads on a
string) level, depending on which the chromatin becomes heterochromatin or euchromatin.
Nucleosome: this an octamer of histones with DNA that is wrapped around 114 bp. The core histones
are H2B, H2A, H3 and H4. Each of these histones has “arms” or “tails” which are highly flexible for
modifications. The histone H1 or “Linker” is responsible for changing between heterochromatin and
euchromatin. H1 is not part of the histone core complex, but it’s added for compaction.
Histone tails:
Histone tails have enriched amino acids that consist of the basic, hydroxyl groups. DNA is wrapped
around octamer and histone tails are sides of them that can have extensively posttranslational
modifications, are the basis of epigenetic regulation. There are many modifications can be possible for
histone tails, like acetylation, methylation, SUMOylation, ubiquitination, phosphorylation, etc.
For example, H3 modifications related to the active and inactive chromatin (one amino acid can be
modified by different factors such as methylation, acetylation, etc.):
 - Lys (4)/ H3K4: acetylation / methylation
- Lys (9)/ H3K9: acetylation / methylation
- Ser (10)/ H3S10: phosphorylation
- Thr (11)/ H3T11: phosphorylation
Usually, acetylation on lysine leads to gene activation, however, methylation cannot be predictable. It
depends on which amino acids are involved during the modification. For example, methylation in H3
(Lys4: H3K4me) leads to activation, but in H3 (Lys9: H3K9me) leads to repression. (Lys4 + HATs: gene
induction; Lys9 + TET: gene repression) [HAT: Histone Acetyltransferase]
Generally:
𝑙𝑜𝑠𝑠 𝑜𝑓 𝐻1 𝑣𝑖𝑎 ℎ𝑖𝑠𝑡𝑜𝑛𝑒 𝑚𝑖𝑑𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 (𝑒.𝑔.𝑚𝑒𝑡ℎ𝑦𝑙𝑎𝑡𝑖𝑜𝑛)
𝐻𝑒𝑡𝑒𝑟𝑜𝑐ℎ𝑟𝑜𝑚𝑎𝑡𝑖𝑛 → 𝐸𝑢𝑐ℎ𝑟𝑜𝑚𝑎𝑡𝑖𝑛
(𝑐𝑜𝑚𝑝𝑎𝑐𝑡𝑒𝑑) ← (𝑚𝑜𝑟𝑒 𝑜𝑝𝑒𝑛)
𝑎𝑑𝑑𝑖𝑡𝑖𝑜𝑛 𝑜𝑓 𝐻1 𝑣𝑖𝑎 ℎ𝑖𝑠𝑡𝑜𝑛𝑒 𝑚𝑖𝑑𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 (𝑒.𝑔.𝑎𝑐𝑒𝑡𝑦𝑙𝑎𝑡𝑖𝑜𝑛)

Bromo-, Chromodomain binding proteins and Heterochromatin Protein 1 (HP1):

The heterochromatin protein 1 (HP1) is one of the important factors for “gene repression”. The HP1 is
a small protein that binds to the methylated histones, specifically to the trimethylated Lys9 (K9me3)
(like H3K9me3: trimethylated Lys9 in H3). Then, HP1 recognizes K9me3 and it’s a little bit compacted
because it can be multimerized. The multimerization of HP1 then can move to the nucleosome to close
together and then leads to gene repression (i.e., making the wrapped DNA less accessible). Before of
the HP1 binding, the nucleosome must be deacetylated and usually DNA would be methylated by
DNMT (DNA methyltransferase). Lys9 can become trimethylated (K9me3) by DNMT and recognizable
for HP1.
For gene activation (reverse of HP1 function), the HP1 be removed. The Histone Acetyltransferase
(HAT) that allows more open construction, then HDMT (Histone de-methyltransferase) will
demethylate Lys4, then, Lys9 will be demethylated and there are TET factor. TET factors can
demethylate DNA. Finally, it allows the more open construction and to gene-associated expression
complex. The trimethylated Lys-A is bound by chromo-/bromodomain proteins that stabilize the open
chromatin and allows the basic recruitments of basic transcription machinery to allow gene expression.
Bromo-/chromodomain binding proteins can read acetylated histone marks, which leads to
recruitment of other coactivators, increasing transcriptional accessibility.
NOTE: when heterochromatin is condensed, TATA-box cannot be expressed.

Distinct sites on histones and DNA are modified by specific enzymes:

There is a switch between euchromatin and heterochromatin that is mediated by DNA methylation.
Heterochromatin with Lys9, is trimethylated (K9me3) by many histone modifying enzymes. Histone
demethylase (HDM) that can demethylate the Lys4. Euchromatin is associated with Lys4 and
unmethylated DNA.
CpG (Guanine on the side of Cytosine that called C-phospho-G island) can be methylated and
unmethylated that can cause gene repression. CpG islands can be directly methylated by de novo
methyl transferases (DNMT’s) to silence expression of the genes they encode. This process is reversible
by the TET (ten-eleven translocation) DNA demethylase. The methylation of DNA causes
heterochromatinization and the unmethylated DNA leads to the more open chromatin. The acetylation
causes by HAT (Histone Acetyltransferase) like TIP60 that is an open marker. The contra-actor is a
Histone Deacetylase (HDAC) that removes acetyl groups from the acetylated histone. Chromodomains
(CHDs) can stabilize structures. (HP1 + K9me3 → multimerizing HP1 → condensing molecules)

Modifications on Lysine:
- Acetylation: lysine has an amino group, then after acetylation by HAT, it has been protonated
(charged possibility) at pH=7 that can interact strongly with DNA phosphoryl backbone (due to
the negative charge). This strong interaction between ionic positive and negative charges
between lysine and phosphoryl group of DNAs is very tight and can be elevated by acetylation,
because acetylation removes the positive charge. Then, lysine is not positively charged, and
the interaction is reduced. [Normally: Lys+ ↔ DNA- (interaction, because of amino and
phosphoryl groups); Acetylated: Lys0 ×DNA- (no interaction, because of reducing positive
charge of amino group)]
- Methylation: the methylation on the lysine is the opposite of acetylation and it’s not changing
the charge of lysine, i.e., methylated lysine still has positive charge, because methyl groups are
very small and cannot change the charge too much. However, trimethylated lysine 4 (K9me3)
is associated with gene expression. Because Bromo-/chromodomain proteins can bind and
recognize this histone mark and then it leads to binding other factors and mediates gene
activation in expression. Lysine can be mono-, di-, and trimethylated by KMT (Lysine
Methyltransferase). Also, it can be possible to demethylate by an enzyme called KDM (Lysine
Demethylase). The lysine methylation is not specific for histones, and other factors like
transcription factors (TFs) also can be modified posttranslationally by methylation.

Modifications on Arginine:
Arginine has a positively charged amino acid, and methylation doesn’t have changing on its positive
charge. However, arginine can be mono-, dimethylated. The arginine methylation can be symmetric or
asymmetric. Symmetric dimethylation means each amin groups has methylation, however,
asymmetric methylation is caused on an imine group and an amin group. In this case, the methylation
is done by “PRMT” (Protein Arginine Methyltransferase). The symmetrical methylation is done by
“PRMT type II” and asymmetrical by “PRMT type I”. The monomethylation is catalyzed by “PRMT type
III”.
The PRMT5 and PRMT9 are for symmetrical dimethylation and knock off the genes have severe effects
on the development. It seems that there is no irrelevant process and dimethylation of arginine
symmetrical or asymmetrical have different effects on biochemical pathways.

Histone code:
DNA methylation on CpG islands → repression [mostly caused by environmental effects then leads to
modifications on genes and finally an epigenetically changes]
The histone posttranslational modification (PTM):
➢ Acetylation on lysine (Kac): activation
➢ Phosphorylation on seine (Tph) or threonine (Thph): activation
➢ Methylation on arginine (Rme): activation [different between symmetric and asymmetric]
➢ Methylation on lysine of H3 (H3K4me/ H3K36me/ H3K79me): activation
➢ Methylation on lysine of H3 (H3K9me/ H3K27me) or H4 (H4K20me): repression
 ➢ Ubiquitination on lysine (Kub): activation / repression
➢ SUMOylation on lysine (Ksu): repression
➢ Isomerized proline (Pisom): activation / repression [it’s not clear].

Interference among histone modifications:

There is interference of histone modifications to neighboring modifications, i.e., direct effects, it can
be the case where one histone modification interferes with the downstream effects of another, e.g.:
- H3: methylation will block the acetylation of Lys9 (H3K9me ⊣ H3K9ac)
- H3: phosphorylation of serin 10 will promote the acetylation of lysine (H3S10ph → H3Kac)
- H3: methylation Lys4 will activate the transcription (H3K4me → transcription)
- H3: methylation Lys9 will represent the transcription (H3K9me ⊣ transcription)

Readers, Writers, and Erasers:

It can be possible to classify histone markers to modify specific amino acid or within the modifications.
This is a contra-acting enzymatic activity that is very important for development. For instance:
• Histone Acetyltransferase (HAT): writer
• Histone Deacetylase (HDA): eraser
• Lysine Methyltransferase (KMT): writer
• Lysine Demethylase (KDM): eraser
The “readers” of the histone code can make the chromatin more / less accessible. MECP2, a reader of
direct DNA modifications (methylated cytosine), recruit histone deacetylases and directly block
transcriptional accessibility of the methylated DNA.
Beyond the biochemical basis of accessibility, a wide variety of “readers,” “editors,” “erasers,”
corepressors and coactivators play a role in regulating the accessibility of the chromatin. It can be the
case where a single histone modification leads to an entire complex of activation or repression.
DNA methylation regulates gene expression:
DNA can be methylated at the cytosine and only for that cytosine which is close to the Guanine (CpG
islands). Only “C” of CpG islands can be methylated by DNMT (DNA methyltransferase) enzymes (i.e.,
DNMT1, DNMT3A, DNMT3B). these enzymes can add “S-Adenosyl-Methionine” (SAM) as a methyl
donor. The vitamin B12, cobalamin, is a reason of “SAM”. The vitamin B12 can act depression for the
mental health. The open chromatin is associated with unmethylated cytosine genes can be switched
on condense (close) chromatin that is mediated by methylation of many these CPG islands leading to
switching off gene expression.
Epigenetic regulation: interaction between DNA methylation and the histone modification:
𝐷𝑁𝑀𝑇1 𝐻𝐷𝐴𝐶 (𝐻𝑖𝑠𝑡𝑜𝑛𝑒 𝐷𝑒𝑎𝑐𝑒𝑡𝑦𝑙𝑎𝑠𝑒)
𝐶𝑝𝐺 → 𝐶𝑝𝐺 − 𝑚𝑒 → 𝐷𝑁𝑀𝑇1 → 𝐻𝑖𝑠𝑡𝑜𝑛𝑒 𝐷𝑒𝑎𝑐𝑒𝑡𝑦𝑙𝑎𝑡𝑖𝑜𝑛
𝑚𝑒3 𝑚𝑎𝑟𝑘 𝑓𝑜𝑟 𝑆𝑖𝑙𝑒𝑛𝑡
𝐷𝑁𝑀𝑇1 + 𝐻𝑀𝑇 (𝐻𝑖𝑠𝑡𝑜𝑛𝑒 𝑀𝑒𝑡ℎ𝑦𝑙𝑡𝑟𝑎𝑛𝑠𝑓𝑒𝑟𝑎𝑠𝑒) → 𝐾9𝑚𝑒 → →
ℎ𝑒𝑡𝑒𝑟𝑜𝑐ℎ𝑟𝑜𝑚𝑎𝑡𝑖𝑛 𝐶ℎ𝑟𝑜𝑚𝑎𝑡𝑖𝑛
• Lysine: activation of transcription
𝐻𝐴𝑇 𝐻𝐴𝑇
𝑙𝑦𝑠 → 𝐿𝑦𝑠 − 𝐴𝑐 → 𝑃𝑜𝑙 𝐼𝐼 → 𝑡𝑟𝑎𝑛𝑠𝑐𝑟𝑖𝑝𝑡𝑖𝑜𝑛 → 𝑎𝑐𝑡𝑖𝑣𝑒 𝑐ℎ𝑟𝑜𝑚𝑎𝑡𝑖𝑛
Control of cytosine methylation:
To analysis the epigenome of DNA methylation of 5-metylcytosine (5-mC), it needs a very specific
antibody against 5-meC. During the demethylation process, it is not just removing the methyl group.
The "TET" enzyme first hydroxylates the methyl group that is bound to 5-hydroxymethylcytosine (5-
hmC). The same enzyme changes that to aldehyde group called 5-formylcytosine (5-fC) then to the
acetylated cytosine called 5-acetylcytosine (5-caC) and then by DNA repairing system the base
exchanging repair, this nucleotide will remove, and normal cytosine will be replaced.
𝐷𝑁𝑀𝑇 𝑇𝐸𝑇 𝑇𝐸𝑇 𝑇𝐸𝑇 𝑇𝐷𝐺+𝐵𝐸𝑅
𝑐𝑦𝑡𝑜𝑠𝑖𝑛𝑒 → 5 − 𝑚𝐶 → 5 − ℎ𝑚 → 5 − 𝑓𝐶 → 5 − 𝑐𝑎𝐶 → 𝑐𝑦𝑡𝑜𝑠𝑖𝑛𝑒
BER: Base Exchange Repair

Methylation and demethylation of DNA:

There are three classes of enzymes for their functions regarding the methyl groups:
➢ Writers: put the methyl group to the CpG island (DNMT3A, DNMT3B, DNMT3L)
➢ Editors: demethylation by oxidation of 5-mC to 5-hmC (TET1, TET2, TET3)
➢ Readers: bind to the mC, recognizes that, and then leads to repression (MECP2, MBD)
MBD: Methyl-Binding Domains
With different levels that control methylation, and demethylation of DNA, MBD and MECP2 can
recognize just the methylation modification (not for others like 5-hydroxymethylcytosine (5-hmC)).
Because of MDB and MECP2 binding to this modification and recruit histone deacetylase (HDA) activity.

Acetylation and deacetylation of histones:

There are three classes of enzymes for their functions regarding the acetyl groups:
➢ Writers: put the acetyl group (HAT: histone acetyltransferase)
➢ Editors: deacetylation (HDAC: histone deacetylase)
➢ Readers: bind to the acetyl (bromodomain proteins)

Phosphorylation and dephosphorylation of histones:

There are three classes of enzymes for their functions regarding the phosphoryl groups:
➢ Writers: put the phosphoryl group (kinases; RPS6KA5, RPS6KA4, BAZ1B)
➢ Editors: dephosphorylation (PPP: histone deacetylase)
➢ Readers: bind to the phosphoryl (BRCTS)

The stepwise of changing and modifications of histone (an example):

Androgens induce phosphorylation of threonine 11 of Histone H3 (H3Thr11ph) to stimulate
transcription (highly controlled):
- Androgen Receptor (AR) is a transcription factor that is in the cytoplasm; by ligating,
transferring to the nucleus that can bind to DNA. When androgen receptor interacts with
 kinase PRK1, leads to threonine phosphorylation of H3 (H3T11ph) that leads demethylation of
lysine 9 (H3K9) and due to the recruiting and gene is still off, the acetylation locally will be
occurred. However, gene is still off. Only when Pol II comes cand PRK1 phosphorylates serine
5, then gene will be activated.
- Sum up: (i) ligand – binding stimulates the association of AR with PRK1, (ii) AR – PRK1
interaction causes phosphorylation of H3T11, (iii) demethylation of H3K9 by a lysine
demethylase (KDM)) is enhanced, (iv) phosphorylation of RNA Pol II on serine 5 activate
transcription.

Remodeling:
Chromatin remodeling complexes (CRCs) are versatile proteins that is also used for gene expression/
suppression. CRC changes the nucleosome positions, modifies histone markers, and adjusts/removes
histones from the DNA, or replaces one histone with a histone variant. It needs ATPs and usually by
moving the DNA around the nucleosomes, works. DNA is wrapped around the nucleosome and a short
push for looping can distorted loop and then loop walks around the nucleosome by using ATPs and
then DNA will shift a little bit around the nucleosome (position shifting). CRC’s can also remove H1
histones from the nucleosome, which are key components of heterochromatin. Depending on the
needs of the cell, certain histone variants can be recruited by CRC’s after adding/erasing the
appropriate histone markers.
One form of remodeling is adjusting the looping of the DNA so one part of the loop encoding the TATA
box is looser and more exposed, increasing transcriptional accessibility.
For chromatin and transcription this remodeling is done by "SWI-SNF Complex". The SWI-SNF complex
uses ATPs and moves far away a long DNA. Then the histone acetyltransferase (HAT) complex can
acetylate the histones, then chromatin will be more open. The SWI-SNF complex recruits histone
deacetylase (HDAC) to allow for moving across the DNA to gene repression. This complex is known for
RNAPII recruitment and opening a site for TBP binding. This also needs ATPs. Another remodeling is
removing one nucleosome from three nucleosomes by SWI-SNF complex and then allows the
transcription.

Histone variants:
One of the levels of regulation is “histone variants”. The typical histones are histone core (H2A, H2B,
H3, and H4). However, there are different histone variants:
- H3.3: for highly expressed genes (associated with transcriptional activation, a marker of active
chromatin)
- CENPA: for kinetochore assembly (microtubule association with centromeres), just in
centromere can be found and important in mitosis.
- H2AX: for DNA repairing, recombination while H2AZ plays roles in gene
expression/chromosome segregation.
- macroH2A: for X-chromosome inactivation in human females (mostly) and transcriptional
repression.
NOTE 1: CENPA is localized in the kinetochores. Research with antibodies’ staining showed that at
metaphase CENPA is associated with kinetochore complexes.
NOTE 2: Entire complexes of activation/ repression can be ubiquitylated and degraded by the
proteasome, depending on the extent of ubiquitination.
NOTE 3: histone variant H3.3 is detected as marker in active chromatin (puffing)
Histone exchange requires chromatin remodeling:
The histone variant needs to exchange, it means in cooperation of variants like H3.3 or H2AZ into the
nucleosome. It means, normally H3 or H2A must be removed, then isoforms must be incorporated.
This also needs Histone Remodeling Complexes (HRCs) that can open the nucleosomes then
incorporate a dimer (H3.3 + H4 = dimer), to the nucleosome by using ATPs.
Three main mechanisms to change chromatin structure:
➢ Chromatin remodeling (needs ATPs): changing nucleosome positions or sliding.
➢ Posttranslational modifications of histones (methylation, acetylation, etc.)
➢ Histone variant exchange (H2AZ → H2A)
NOTE: Bar body just in humans, in fruit flies is double expression.

Histone modification and heterochromatic gene silencing:

Coactivators and Corepressors:

The transcription factors bind to the enhancers and silencers. After chromatin remodeling has been
occurred that can bind to DNA. Then, they recruit various complexes could be histone acetyltransferase
(HAT), etc. then, interact locally with histones, the loop and modified a nucleosome. Where close after
the looping, also loop close intensity to the promoter site where the TBP is bound to the TAFs and then
allows basically the initiation of transcription (allows of the pre-initiation complex or “Pic”.)
Gene activation and repression by chromatin remodeling and modifying the enzymes:
Coactivators has the “HAT (=histone acetyltransferase) activation function”. They could be like
CBP/p300, the platform protein to allows looping. These nuclear hormone receptors are very good
module system that analyze gene expression. Basically, one can manipulate just by ligand and it’s
rapidly by tissues and cells. One can transcription very fast in experiment.

Highly dynamic chromatin recruitment:

By inducing hormones and using α-Amanitin for controlling, the activity of different factors can be seen
such as Pol II, SWI-SNF, etc. within 20 minutes, others.

Cancer pathways: Mechanisms of p53 tumor suppressor and myc protooncogene function, lncRNAs
as regulators of gene expression
The p53 tumor suppressor
One of the most well-known and studied tumor suppressor genes is p53. P53 is normally inhibited by
Mdm2, a ubiquitin ligase. However, certain post translational modifications like phosphorylation lead
to Mdm2 inhibition and tetramerization of p53 at p53 promoter elements, leading to the upregulation
of genes related to cell cycle arrest, apoptosis, DNA repair, senescence, autophagy, and
downregulation of metabolism, angiogenesis, migration, and metastasis.
Although a tumor suppressor gene, p53 is actually an example of haploinsufficiency, where a single
recessive mutation leads to a recessive phenotype. This is because four functional p53 molecules are
needed for tetramerization to occur, the odds of that happening being 20 percent if one dysfunctional
allele is present. Treatment of haploinsufficiency are therapeutic blockers that interfere with Mdm2
binding with p53.
p53 has seven domains of activity.
1. N terminus activation domain (AD1), which activates transcription factors.
2. A second activation domain AD2, important for apoptotic activity.
3. Proline rich domain important for apoptotic activity of p53 by nuclear exportation
4. Central DNA binding domain, which contains one zinc atom and several arginine amino acids.
5. A nuclear localization signaling domain.
6. Homo oligomerization domain
7. A C terminus involved in downregulating the DNA binding of the central domain.
Out of the seven, Mdm2 can interact with serines of the AD1, AD2 and C terminus domains.
To fulfill a particular function, p53 needs to first be phosphorylated at its self-associating DNA binding
domain, as well as interact with other factors. For example, Tip60, an acetyl transferase, often
associates with p53 in different contexts.
When Tip60 acetylates histone H4 at the p21 promoter in association with p53, the cell cycle is
arrested. On the other hand, if Tip60 is acetylated at K120, binding to apoptotic gene promoters is
selectively increased. If MOZ, instead of Tip160, acetylates K120 of p53, p21 induced senescence,
instead of cell death occurs.
Phosphorylation of p53 occurs under a variety of contexts, such as detection of oncogenes (ARF),
double/single strand breaks (ATM/ATR), as well as hypoxia (HIF1alpha). Thus, the outcome of
regulation depends on the concentration of active p53. If a moderate amount of p53 is active, the cell
cycle will be arrested, and repair will occur. However, if a high amount is active, apoptosis will occur.
The Myc oncogene
The oncogene Myc has two domains: a leucine zipper for heterodimerizing and a basic helix loop helix
region for binding to DNA (primarily F-box regions.). Myc has two primary association partners: Mad
and Max. When Myc heterodimerizes with Max, the heterodimer serves as a transcription factor by
binding to enhancer box sequences and at promoters of mitogenic genes. When Myc binds with Mad,
however, a corepressor complex is recruited, which contains a histone deacetylase. Being an
oncogene, Myc is regulated by miRNAs, ubiquitin ligases (such as Fbw7), kinases (like ERK or Src), and
phosphatases (like PP2A). In other words, multiple regulators from different classes modulate the
activity of Myc.
In addition to serving as a transcription factor, Myc also regulates the pause-release of RNA-Pol II by
upregulating phosphorylation of the C-terminal domain. In c-Myc, a member of the Myc family, P-TEFb
(positive transcription elongation factor b), a cyclin dependent kinase is recruited.

Long non-coding RNAs and circRNAs

Long non-coding RNAs (lncRNAs), to be distinguished from short non-coding RNAs like microRNAs, play
a key regulatory role. Just like normal coding proteins, they can:
Be regulated by transcription factors
Be transcribed by RNA pol II
Be spliced and alternatively spliced
lncRNAs can have five points of origin.
From intergenic regions
Intronic regions
Antisense
Bi-directional
Or exist as a transcript from an enhancer region.
Another type of lncRNA, circRNA, has coding and regulatory capacity. Being circularized, it is resistant
to exonuclease activity and is therefore more stable than normal messenger mRNA. It is also
abnormally expressed in certain cancers.
There are four ways to make circular RNA:
1. Exon splicing
2. Intron pairing-driven circularization
3. Exon skipping
4. Derivation from an intron lariat
LncRNAs can regulate several levels of gene expression via:
- Histone modifications
- Transcription factor recruitment
- Alternative splicing
- mRNA stability
- miRNA interaction
- As a circRNA sponge for miRNA
- Production of miRNA
- Being degraded by miRNA
- Compete with miRNA for target mRNA
- Polysome recruitment
- Packaging into extracellular vesicles to modulate gene expression in neighboring cells
- Furthermore, lncRNA can also play a time-dependent role, where its regulation would be
appropriate in embryogenesis but oncogenic in adult tissue.

Nuclear hormone receptors (HNRs) as a good model system to analyze gene expression:
The hormone receptors are known as a model system to analyze the concepts of gene expression.
There are two types of hormone receptors: (1) membrane – bind receptors, (2) nuclear receptors.
The hormone receptors have four domains: a highly variable domain, a highly conserved DNA binding
domain (zinc finger), a hinge region, and a moderately conserved hormone binding region.
Membrane – bind receptors, or protein hormone receptors are for peptide hormones that cannot cross
and penetrate the cell membrane. This kind of receptors are outside the cell (on cell membrane) and
after peptide binding, they release the signals inside the cell. Usually, they have transmembrane
domain, extracellular domain, and intracellular domains, such as tyrosine-kinase receptor where
growth hormone can bind there. After binding, tyrosine is phosphorylated then cascade cellularly
happened with different mechanisms. Usually there are phosphorylation events for activation of other
kinases such as RAF, RAS, MAPK, ERK, etc. These factors transmit signals into the nucleus. Then,
transcription factors will be activated for gene expression. There are also 7-transmembrane proteins,
there are g-couple receptors that activate cAMP signaling activation protein kinase A, then leading the
signaling into the nucleus.
Nuclear hormone receptors or lipophilic hormone receptors are for lipophilic hormones that can
diffuse through the cell membrane and into the nucleus, binding to transcription factors or activator
proteins there to stimulate transcription. These are classically seen as the “slow” acting hormones.
Because they can enter the membrane, and act inside the cell. The lipophilic hormones and ligase have
nuclear hormone receptors, such as glucocorticoids, vitamins, androgens, estrogen, etc. These
receptors can bind directly to DNA. They have DNA binding domains that are activated by binding to
the ligands.
The nuclear hormone family:
This family can bind to (a) steroids (androgens, estrogens, progestins, glucocorticoids, and
mineralocorticoids), and (b) non-steroids (thyroid hormone, retinoic acid, vitamin D3, prostaglandins).
There are five classes of steroids:
- Androgens (like testosterone) play a role in muscle hypertrophy and the maturation of the
secondary male sex organs, like the maturation of the Wollfian duct into the epididymis and
the vas deferens, as well as the maturation of the genital tubercle and the urogenital sinus into
the secondary male sex organs. There are three peaks of androgen levels: during the second
trimester, the first year of birth, and during/after puberty. Mutations in the androgen receptor
can lead to a female phenotype, even though the genotype is XY.
- Estrogens, in addition to playing a role in female sex determination (such as maturation of the
follicles), also increases the rate of adiposity and calcium absorption. Mutations in the receptor
can lead to forms of breast cancer, which is later discussed.
- Progesterone plays a role in pregnancy maintenance. The endometrium is thickened,
increasing the likelihood that the zygote will attach to the uterine wall. It also helps with bone
growth and serves as a diuretic, maintaining salt and body fluid levels. Progesterone belongs
to a bigger class of hormones known as progestins, some of which serve as a contraceptive by
building a mucal barrier in the cervix.
- Glucocorticoids increase the rate of gluconeogenesis and downregulates the immune system
by upregulating the transcription of inflammatory proteins as well as antagonizing leukocyte
migration. Mutations in the glucocorticoid receptor can lead to Cushing’s syndrome, which is
characterized by abdominal obesity (but not in the extremities), high blood pressure, and poor
healing skin.
- Mineralocorticoids, like aldosterone are responsible for the reuptake of fluids and sodium, as
well as the secretion of potassium. Mutations in the mineralocorticoid receptor, depending on
the nature of the mutation, can lead to either hyperaldosteronism, which is characterized by
hypertension and edema, or hypoaldosteronism, which can lead to hypotension and
hyponatremia, which can be deadly if left unchecked.
Four non-steroids:
- Thyroid hormones are involved in the myelination of neurons, heat generation via signaling to
the hypothalamus, and metabolism via increased sodium pump signaling. Problems in the
pathway can lead to retardation and weight gain.
- Retinoic acids are instrumental in limb and eye development, but also in the maintenance of
white blood cells. Translocations involving this receptor are discussed later.
- Vitamin D3 is responsible for calcium and phosphate absorption, as well as the upregulation
for the innate immune system and the dampening of the adaptive immune system. Defects in
this receptor include rickets and osteoporosis.
 - Different prostaglandins of the same family can have antagonizing roles, but prostaglandins
are involved in vasodilation and inflammation, as well as the inhibition of blood platelet
aggregation.
Schematic receptor structure:
A C-terminal binding domain to where these receptors can bind to the indicate the ligand. They have
amino terminus which is flexible and allows gene regulation.
Mechanisms of nuclear hormone receptors (NHRs):
Generally, nuclear hormone receptors are inactive in the cytosol by heat shock proteins. If a hormone
(agonist or antagonist) binds to the protein via its hormone binding domain, the conformation of the
protein changes and heat shock factors disassociate from the protein, allowing it to serve as an
activator or a repressor of gene transcription via histone modifications and association with the RNAPII
holoenzyme, along with the transcription initiation complex. Other hormone receptors are already in
the nucleus.
A modification of this model is the helix-12 domain. When not bound to a hormone, a nuclear
hormone receptor can have a conformation where the helix 12 motif has an affinity for corepressors,
thus allowing the molecule to have repressive activity in the nucleus. On the other hand, when the
ligand binds, the helix 12 motif changes conformation and the receptor increase its affinity for
coactivators. Thus, the same receptor can actively repress and activate the same genes, based on the
conformation of this helix 12 motif.
The RAR-PML fusion protein is the result of a translocation between the 15th and 17th chromosomes.
The tumor suppressor protein PML (promyelocytic leukemia) is fused to the retinoic acid receptor, and
as a result, its activity, being dependent on the retinoic acid receptor, greatly decreases, and a greater
susceptibility to leukemia results. Treatments of all trans retinoic acid in high quantities maintain the
activity of this protein, and only high quantities are sufficient to replace the corepressors with
coactivators.
Other translocations of the RAR protein are not as treatable. For example, the PLZF-RAR translocation
between the 11th and 17th chromosome produces a fusion of the retinoic acid receptor and
promyelocytic leukemia zinc finger tumor suppressor protein. Treatment of high amounts of all trans
retinoic acid leads to only partial coactivator recruitment and corepressor removal, which is
insufficient for the PLZF tumor suppressor to function.
There are deviations from the classical model of hormone receptors, where applying hormone either
represses its activity or stimulates the recruitment of HDACs. Furthermore, even though corepressors
are bound to an NHR when bound to the DNA in the nucleus, those corepressors can have coactivator
function, which depends on the posttranslational modifications that they have.
Hormone agonists activate the hormone receptor by changing the receptor conformation, while
hormone antagonists (artificial in nature) change the receptor conformation into a repressive state.
This can be done by blocking nuclear translocation or allowing the binding of corepressors.
Mamma carcinoma, a type of breast cancer, can be caused by hyperactivation of the estrogen
receptor. A common estrogen antagonist, tamoxifen, is used, and the estrogen receptor enters the
nucleus and represses gene transcription with the help of corepressors via RNAPII holoenzyme
blocking and chromatin modification. However, over time, mitogenic signaling from receptor tyrosine
kinase signaling (like the MAP kinase) pathways override this repressive activity by phosphorylating
corepressors, making the estrogen receptor change to an activator conformation.
Mechanism of “direct” gene regulation by nuclear hormone receptors
There are two distinct models: (1) receptors can bind to HSP factors in cytoplasm, (2) receptors can
bind to DNA without any ligands.
For first model, such as androgen receptor, the ligand binding to hormone leads conformation change
and dissociated by HSP factors, from the receptors. Receptors can be dimerized, and ligand receptor
can bind directly to DNA and allow gene expression.
For second model, such as thyroid hormone receptor, hormone can bind directly to the DNA without
any ligand. There is a possible to repress gene activity, then thyroid hormone is leading the
conformation change and then gene activation. This receptor tends to be heterodimerized by RXR.
Sum up: Model (1): hormone x > receptor (conformation change) > gene (target, e.g., androgen).
Model (2): hormone x > gene1 > gene2 (target, e.g., thyroid hormone)
Crosstalk of nuclear receptors with other transcription factors (no “direct” DNA binding)
The nuclear hormones can inhibit receptors (indirectly downregulate receptor pathways e.g., CREB,
NFκB, Jun/Fos or upregulating other pathways e.g., STAT), such as tyrosine kinase (RTK) activity at level
of Jun/Fos transcription factor which mentioned before as a leucine zipper heterodimer. The nuclear
hormone receptors can repress NFκB pathway or cAMP- protein kinase A (PKA) pathway that is induced
by transmembrane protein. Also, they can activate STAT signaling that is activated by JAK-STAT. all
these pathways are without directly binding to the DNA. Both peptide and lipophilic hormones also
have rapid effects of activation, activating pathways like PLC-PKC, Src-ERK/MAPK, PI3K-PKB/AKT, or
the PKA pathways. The hormone response element (HRE) assay characterizes the nature of a protein
(often a nuclear hormone receptor)’s DNA binding domain. There are non-genomic effects that
premeditatedly by two pathways:
1. High concentration of ligands to activate G-coupled receptors.
2. Nuclear hormone receptors that still acting in the cytoplasm. Small protein remains in the
cytoplasm. For example, lipophilic hormones can interfere with the Src, AKT, cAMP pathways.
Homologies of nuclear hormone receptors
The nuclear hormone receptors are a big family that are highly conserved. The most conserved
domains that is shared by nuclear hormone receptors, is the DNA binding domain (DBD) and second
important structure is conserved, the ligand binding domain (LBD). The variation can be seen only at
the amino parts. For steroids, they have three main conserved parts: ligand binding domain (LBD),
DNA binding domain (DBD), and N-terminal domain (NTD).
Steroidal hormones in insects:
➢ Ecdysone:
This is an important hormone between insects. This hormone is responsible for
metamorphosis (timing) in the insects. Ecdysone is a steroid hormone that can bind to the
nuclear hormone receptor which called it “ecdysone receptor”. Ecdysone interacts with
polytene chromosomes in insects, which develop from the chromosomes of diploid nuclei by
endopolyploidy, or the successive duplication of each chromatid without their segregation.
Drosophila and Diptera insects develop from egg, through different larva stages, then pupa,
and finally provide the adult. For these different steps of development, the ecdysone hormone
where have different triggers that can control different stages in the development from larva
status to pre-pupa and then to the pupa and an adult insect.
Polytene chromosome: interphase chromosomes generated by endopolyploidy in salivary glands of
Diptera.
Polytene chromosomes that are generated by endopolyploidy, can have bands. The interbands of
these polytene / giant chromosomes usually are mentioned as “euchromatins” and bands as
“heterochromatins”.
Puffing
In salivary glands of Diptera larvae, such as Drosophila, there are giant chromosomes called polytene,
that can transcribe many different digestive enzyme mRNAs. Because larva must produce a lot of
enzymes for digestion from its salivary glands (fast eating is the main aim). Therefore, the growth and
cell division are caused by polytene chromosomes. In these glands for fast eating and digestion, all
chromosomes are amplified and aligned exactly on top of each other’s that called “endopolyploidy”
(multimerization of genome about 1000-folds). Each polytene chromosome is seen a 1000-fold copies
and arranged as a similar manner. These giant chromosomes can be seen via the light microscopes.
The interphasic structures of polytene chromosomes have puffing that caused by ecdysone hormone.
The ecdysone binds to the ecdysone receptors that is a transcription factor, then leading to gene
activation. Also, ecdysone is a major regulator of DNA looping out (puffing generation) in the polytene
chromosomes. Depending on the developmental stage, ecdysone can induce puffs in different areas,
but because it doesn’t bring about a constant pattern of puffing in the genome, it is most likely not the
master epigenetic regulator. The puffing means loops of DNA, which are regions of higher transcription
that lack histones and too many transcription factors, chromosome remodeling complexes, etc., that
can be seen via the light microscopes. The puffing is an active transcription site whereas shown very
early by incorporation of UTB of these transcriptional sites.
Polytene chromosomes in plants: Phaseolus vulgaris
Phaseolus vulgaris has large polytene chromosomes were found in the large cells of embryo suspensor
of the legume. These chromosomes are for feeding the future embryo, there is a DNA amplification to
allow very rapid the production of large amounts of proteins to be able for growth supporting of
embryo.
Lamp brush chromosomes: a model for chromosomes looping (DNA looping)
These chromosomes are also visible by light microscopes, there aren’t amplified or endopolyploidy.
They are packed with so many proteins. These interphasic chromosomes can be found in frogs,
salamanders, certain birds, and fishes’ oocytes. These have in the meiosis, very interesting structure
that can be seen a looping out of DNA.
Each of these looping out is an active looping with controlling of CTCFs, cohesin and condensin
proteins. The homologous chromosomes are attached to each other in the central parts and chiasmata
with large lateral loops can be seen. Each loop consists of many genes.
Sum up: the lamp brush chromosomes are generated from the homologous chromosomes, and
chiasmatic regions. They have large lateral DNAs (looped out), and each loop consists of many gene.

Setup: introduction of a cDNA (coding for the nuclear receptor) and HRE/luciferase reporter plasmid
(containing the DNA binding domain) into the target cell genome.
The cDNA plasmid must contain:
- SV40 viral promoter/enhancer/ori (very strong viral-derived promoter, ensuring cDNA
expression
- The expressed cDNA, coding for the nuclear hormone receptor, flanked by a polyadenylation
sequence.
- An ampicillin resistance gene to be selected after cell transfection.
- The reporter plasmid must contain:
- An origin of replication
- An ampicillin resistance gene
- A luciferase reporter gene with a response element-specific enhancer (site of molecule binding
- Procedure: introduce both plasmids into the nucleus of the cell. cDNA will be expressed, and
the receptor will be expressed on the outer membrane of the cell (or in cytosol, depending on
the nature of the receptor). If a hormone is introduced and the receptor binds to the HRE in
 the nucleus, luciferase will be expressed, indicating a hormone response element compatible
with the hormone receptor.

Further characterization of the hormone response element can be done via introduction of point
mutations, deletions, and insertions to find out what the DNA binding domain recognizes (palindromic
repeats? Parallel repeats?)

At the end of lecture, Baniahmad proposed an exam question along the lines of the following:
How is gene activation mediated?
(This is an exhaustive answer. It’s meant to give a profile of what both repressed and activated states
would look like, just in case we were asked the opposite question on the exam- how is gene repression
mediated?)
Name the different levels that mediate gene activation.
Before genes are activated, they must be in a repressed state. This repression exists at both the 90 nm
fiber (euchromatin vs heterochromatin) level and at the nucleosome level.
At the nucleosome level, both the CPG islands of DNA and K9 of the H3 histone tail are methylated (by
maintenance and de novo methyl transferases for the DNA and histone methyl transferases for the
histones). Repressive methylations are read by heterochromatic protein 1, which polymerizes with
other copies of itself upon binding, thus making the promoter inaccessible. Histones are also
deacetylated. The combination of proteins responsible for repressive methylation, deacetylation, and
blocking/inhibiting transcription at the TATA box is collectively known as the corepressor complex.
At the 90 nm fiber level, the facultative heterochromatin is held together tightly by condensins as well
as H1 histones, which are responsible for bridging organization between the nucleosome and the 90
nm fiber levels. It is also repressed by nuclear lamins, which generally sequester facultative
heterochromatin near the nuclear membrane region in a transcriptionally inactive state. The chromatin
is isolated into topologically associated domains (TADs), which prevent inappropriate interactions with
extra-TAD chromatin. CTCF insulators mark the boundary of these sites.
Name the factor/family of factors involved in gene activation and give the order of gene activation.
Generally, the first steps of activation are mediated by either a chromatin remodeling complex or by a
histone writer/eraser (although CRC’s can also perform this function).
Chromatin remodeling complexes (like CRC1 or SWI/SNF) can remove H1 histones from the desired area
of activation, making the nucleosomes more accessible to transcriptional machinery. They can also
rotate the DNA looped around the histones, so the TATA box is on the looser end of the double loop.
CRCs also can erase and write histone modifications, which function like histone acetylases (lysine),
histone demethylases (demethylating H3K9me3), histone methyltransferases (methylating H3K4me3
with S-adenosyl methionine as a methyl donor) histone kinases, etc. This not only reduces the overall
attraction interactions between the histones and the DNA but also forces the disassociation of the
multimerized heterochromatic protein readers. In their place, bromo-chromodomain binding proteins
are recruited, which read H3K4me3 and promote transcription of the respective region.
Finally, CRCs can recruit histone variants, which are recruited based on what function the transcribed
gene will provide (CENPA for mitotic spindle assembly, H2AX for DNA repair, macroH2A1 for X
chromosome inactivation, etc.) Chromatin modifications (methylation, for example) are required for
variant exchange to occur.
Some other downstream effects:
Directly methylated cytosines of DNA in CPG islands are demethylated via a TET (ten-eleven
translocation) enzyme, decreasing transcriptional repression.
Increased chromatin accessibility leads to binding of activators to enhancer regions, which interact with
the promoter region and further stabilizes RNA pol II binding, enhancing the rate of transcription. This
can be mediated by either the random interactions within a TAD, or by similar
dimerization/oligomerization sequences at both the promoter and the enhancer.
Increased activation decreases the affinity of silencers for enhancer regions or downregulates them
outright.
Many parts of the pre-existing corepressor complex can be degraded by the proteasome to make way
for components of the coactivator complex.
It is at this point that the RNA polymerase II holoenzyme can bind. However, deviations from classical
models of transcription suggest that RNA pol II don’t go to activated genes but rather the other way
around. Loops of chromatin that are transcriptionally active are brought to transcription factories,
which are RNA pol II- rich sites of transcriptional activity at non-repressive domains of the nucleus.
The order of RNA pol II binding to the promoter is: 1. TATA binding protein to the TATA box with TFIID,
2. TFIIA+B, TFIIE+F+H, 3. mediator protein and RNA pol II, 4. Transcriptional activators at enhancers.
This order of binding mediates transcriptional initiation.
To bridge from initiation to elongation, RNA pol II’s heptapeptide repeats on its serine-rich C terminal
tail domain must be sufficiently phosphorylated so that attenuated pausing is overridden. One common
protein that mediates this is Myc, which after dimerizing with Mad, indirectly phosphorylates the C-
terminal tail.
How would you analyze that Myc inhibits pausing effects of transcription?
Use a coimmunoprecipitation assay, where an antibody against the C-terminal domain of RNA
polymerase II is applied in a Western blot.
You have a new transcription factor, and you don’t know how it works. What would you do to
analyze its activity?
Use ChIP to analyze all sequences it binds to.
Genetics II: Von Eyss Part
Introduction:
• Epigenetics is study of heritable changes in genome function that occur without a change in
DNA sequence. For example, methylation on histones or CpG islands of DNA. A proper
epigenome is responsible for maintaining/establishing a correct cell dedifferentiation state.
The absence of this can lead to cell dedifferentiation, loss of tissue homeostasis and disease.
• Epigenetics is important: because of focusing on (1) transcription regulation (which genes are
“on” or “off”), (2) cell identity, (3) adaption and evolution.
• DNA is not just a sequence of bases; DNA can be modified and covered with proteins which
can be modified, these modifications change DNA accessibility, epigenetic modifications will
determine which genomic regions are transcribed into RNA.
• During the embryotic development, DNA methylation is reset after zygote formation and the
re-established gradually during cell differentiation. When 5mC level in both paternal and
maternal nuclei are analyzed, after unification (zygote formation), the 5mC level in paternal is
reduced rapidly, meanwhile maternal very slowly. They will have very low 5mC level until the
blastocyst level when implantation is happening.
• A proper epigenome is necessary for maintaining / establishing a correct cell differentiation
state. The absence of these can lead to cell de-differentiation, loose of tissue homeostasis and
disease development. ↑DNA methylation: ↓de-differentiation potential: ↑cell specification.
• Adult stem cells are different from embryotic stem cells. During cell division of stem cells, the
differentiation will not be happened. The Division is symmetric and daughter cells are like
mother; that is why one of them can generate another stem cell line again (via “self-renewal”
pathway). Although the newly synthesized DNA strands are unmethylated, DNA
Methyltransferase1 (DNMT1) precisely maintains the DNA methylation status.
• Epigenetics can play important roles in other organisms, for example queen bee can be queen
by effect from environment (royal jelly feeding) that leads silencing the expression of DNA
methyltransferase3 (DNMT3) therefore, it leads to differentiation of a bee into a queen bee.
• Nucleosomes are made of DNA and histone proteins that important for DNA packaging. Due
to the long size of DNA (2 m) that must be organized at the very small nucleus (10 μm), it needs
to be packing in different levels.
• Eukaryotic DNA has different features. Metaphasic chromosomes have a protein scaffold to
which the loops of supercoiled DNA are attached. Chromosome scaffold is a proteinaceous
structure in the shape of a sister chromatid pair, generated when chromosomes are depleted
of histones. DNA of interphase chromatin is negatively supercoiled into independent domains
of ~ 85 kb. Nuclear matrix a network of fibers surrounding and penetrating the nucleus. (naked
DNA > DNA + histones = nucleosomes: “beads-on-a string structure” > nucleosomes + H1
histones: “30 nm fiber structure” > fibers + protein scaffolds: “loops” that extended scaffolds
> “condensed scaffolds” > metaphasic chromosome.
• Chromosomes occupy distinct territories within the nucleus even during the interphase:
There are two ways of chromatin compacting: euchromatin, heterochromatin. This
classification is based on electron microscopy that heterochromatin lies against the nuclear
envelope in patches and is broken up at the site of the nuclear pore. This compaction (more
or less) causes heterochromatin and the euchromatin. The factors which lead these
 compactions are histones and histone modifications lead also. Euchromatin means DNA is
accessible and heterochromatin means “NOT”. When a gene wants to be expressed, gene will
change its position in the nucleus. When cell recruited the signals its gene location will be
changed regarding the expression or silencing. The silencing neighborhood is near the nuclear
envelope and expression neighborhood on the specific speckles. There are heterochromatic
(periphery, pericentric chromosomes, nucleoli) and euchromatic regions (nuclear foci like
transcription factories). It is thought that LLPS is responsible for the affinity of the chromatin
to these particular sections in the nucleus, depending on the proteins that bind.
Changes in a gene’s subnuclear location correlate with changes in the gene expression. This
can proof that due to the low DNA methylation, the size of stem cell nucleus is bigger than
differentiated nucleus cells. In the other words, progressive differentiation, and loss of
euchromatin mobility leads to changes in nuclear architecture during differentiation.
• Chromatin fibers can be seen in two levels: “11 nm fibers” or “beads” and “30 nm fibers”. The
beads highly acetylated histones core (H3 and H4) that leads to reduce level of histone H1,
gene transcription possible. The 30 nm fibers are charged positively at N-termini (binding DNA
on neighboring nucleosomes) that leads high level of histone H1 and therefore “no” gene
transcription.
• The structure of nucleosomes is composed from histone proteins (histone core + H1) and
wrapped DNA. Histone structure is an octamer that has twofold symmetry (twice H3, H4, H2A,
H2B). The N-termini of histones (core histones) are important for modifications. They are
happening out around the histone cores. Only Archaea and eukaryotes have histone proteins
in their cells. Histone structures illustrate that they will have completed each other by the
“Handshake” motif. This handshake motif caused by two histones; each histone has a “Histone
folding domain”.
Histone variants: Histone H3 (H3, H3.3 [a transcriptional activity mark], CenpA [a kinetochore
assembly mark]), Histone H2A (H2A, H2AZ [a chromosomal segregation and gene expression
mark], H2AX [DNA repair recombination mark in the yeasts], macroH2A [X-chromosome
transcriptional repression mark]).
All core histones have the structural motif of histone folding that N-terminal tails extend out
of the nucleosome. DNA and histone tails can be modified.
• DNA methylation consists in methyl addition to 5’ position of cytosine at CpG islands (in
mammalians, DNA methylation occurs at CpG dinucleotide, and it is highly symmetric). This
modification correlates with compacted DNA and transcriptional repression. Also, it’s
regulated by intrinsic and external factors. One of famous enzymes for methylation is “DNA
methyltransferase” or DNMT and by S-Adenosylmethionine (SAM).
DNA methylation can act in three ways: (1) maintenance (by DNMT1), (2) de novo (by
DNMT3A, DNMT3B), (3) inactive (by DNMT3)
Due to its symmetry, CpG methylation can be stably propagated during DNA replication by the
maintenance methyltransferase DNMT1. Once established by the de novo methyltransferases
DNMT3A and DNMT3B, the process of DNA methylation maintenance allows inheritance
during cell division.
De novo methylase DNMT3s add a methyl group to an unmethylated target sequence on DNA.
Fully methylated site is a palindromic sequence that is methylated on both strands of DNA and
maintained by DNMT1. TET enzymes oxidize the methyl group to demethylate [TET proteins
 use the same reaction mechanism as Jumanji demethylases,
𝑇𝐸𝑇1,2,3+𝐹𝑒(𝐼𝐼)
5𝑚𝐶 → 5ℎ𝑚𝐶].
2 − 𝑂𝐺 + 𝑂2 → 𝑠𝑢𝑐𝑐𝑖𝑛𝑎𝑡𝑒 + 𝐶𝑂2
Unmethylated DNA → activity. Methylated DNA (+ HP1, + MeCP2) → silence.
• The act of demethylating DNA has both a passive and an active basis. In the passive pathway,
TET enzymes hydroxylate a methylated cytosine so that it cannot properly interact with
DNMT1. Furthermore, there is also an active pathway. In active demethylation, several
oxidation steps are undergone from 5 hydroxymethyl cytosine to 5-formyl cytosine to 5-
carboxy cytosine. In all these cases, O2 from alpha ketoglutarate is the donor and the rest is
given to succinate.
Afterwards, thymine-DNA glycosylase creates an abasic site, and base excision repair is
undergone via apurinic endonuclease and DNA ligase.
A potential problem to transcribing the gene is the possibility of recognizing promoter-like
sequences downstream of the promoter proper. This is referred to as spurious transcription.
The epigenome has been fashioned in such a way where the gene bodies downstream are
methylated to prevent this.
One of the most common mutations in DNA is due to base deamination. When a methylated
cytosine is demethylated, the intermediate is thymidine, which creates a mismatch against
the guanidine nucleotide. Base excision repair could potentially create a mutation at this
base pair.
NOTE: Housekeeping genes, which themselves have CpG islands, are barely methylated on
the DNA and therefore do not have this problem.
NOTE: DNA methylation can regulate transcription initiation, the chromatin structure itself,
alternative splicing, and transcription elongation.
• Histones regarding their modifications, have three kinds of enzymes. Writers are enzymes that
can add a specific modification on histone tails. Erases are enzymes that can remove a specific
modification on histone tails. Readers are enzymes that can bind the modification and recruit
other steps of pathway (main function).
• Chromosome remodeling complex (CRC) is responsible for different possibilities of chromatin
remodeling that can see: (1) nucleosome sliding, access the gene, (2) histone exchange,
variance, (3) nucleosome eviction, removing histone. Remodeling factor needs energy (ATPs).
Chromosome remodeling factors probably embrace the nucleosome and pull DNA into the
nucleosome.
• Hypersensitive sites are a short region of chromatin detected by its extreme sensitivity to
cleavage by DNase I nucleosome are excluded.
• Histone modifications: acetylation, methylation, ubiquitination, SUMOylation,
phosphorylation.
Three models for effects of posttranslational modifications; model 1: chromatin structural
change, model 2: inhibit binding of negative – acting factor, model 3: recruit positive – acting
factor.
• Histone acetylation is done by histone acetyltransferase (HAT) can recruit transcription factors
for gene activation. Normally it needs cofactors and HAT + Acetyl-CoA can change the amino
acid charge.
 Tetrahymena is a unicellular organism with two nuclei, macronucleus (active) and
micronucleus (inactive). For illustrate activity or inactivity of nuclei, people produce
radioactive dNTPs then exposure the microorganism. Then via fluorescence and radioactivity,
they could show MAC has active transcription. They used [3H] sodium acetate for labeling.
Then nuclei were isolated, and histones extracted, and then 2D-SDS-PAGE applied (by
Coomassie and autoradiogram dyes). As results, MAC had banding on both dyes, but MIC only
on Coomassie. It means, only radioactive nucleus had active transcription.
• Histone acetylation by HATs has different functions: (1) loosening of chromatin, (2)
recruitment of additional factors. Acetylated histones can be read by “bromo-domains” of
proteins.
Eukaryotes mRNAs are transcribed by big protein complexes called “RNAPII”. RNAPII needs to
bind to promoter then recruit, then gene transcribing. However, RNAPII cannot bind
everywhere, its regulation is not easily possible. Therefore, first recruitment, then joining
other transcription factors such as TFIID as basal transcription factor.
SWI/SNF or Switch/ Sucrose non-fermenting
(Brg1) is a complex that identified as CRC. This
complex protein was detected a mutation on
digestion of sucrose in yeast. Normally yeast can
grow on glucose and sucrose media, but mutants
can grow on glucose media. SWI/SNF can bind on
two binding sites by bromodomains, then (1)
SAGA via T+A and acetylation, (2) remodeler via
bromodomain and T+A factors can be recruited by
acetylated histones, (3) DNA becomes less
compacted and ready for gene transcription.
In yeast genetic experiments, histone deacetylation was shown to be a mode of epigenetic
repression. In yeast, Sir2 is a histone deacetylase, while Sir3/4 are histone binding proteins
that bind to unacetylated histones to mediate condensation, as well as telomere association,
which is associated with heteronuclear foci.
The reagent 5-fluororotic acid, if processed by URA3 competent yeast, produces 5 fluorouracil,
an agent of DNA damage. As a control, knockouts of yku70, a NHEJ protein, show lower survival
rates in dilution titers in comparison to wild type. Furthermore, knockouts of sir2, a histone
deacetylase, produce a similar phenotype. Sir2 therefore prevents 5-FU from associating with
the DNA due to repression.
• Histone deacetylase (HDACs) can convert acetyl-lysine (acK) to lysine by using H2O and H+.
Histone acetylation/ deacetylation in gene activation and repression:
Acetylation: DNA binding domain (DBD) and other TFs can bind to recruited HATs. Then this
modification leads to switch on and then needs to be switched off by deacetylation.
Question: how to analyze changes in histone acetylation in vivo?
Answer: by using the Chromatin – immunoprecipitation (ChIP) technique: (1) cross – linked
chromatin, (2) isolate and shear chromatin mechanically, (3) add antibody specific for
acetylated N-terminal histone tail, (4) immunoprecipitation, (5) release and purified
immunoprecipitated DNA and assay by PCR.
To obtain the antibodies needed, use a peptide with 10-15 amino acids where the amino acid
of interest is acetylated. Introduce into bloodstream of a rabbit with a carrier (BSA, for
 example) that helps efficiently induce an immune response. Additionally, add CFA (crushed
bacteria). For quality check, use an affinity column (1. Enrichment column 2. Depletion
column).
1- Attach peptide antigen to spin column and allow serum to flow so antibody binds. Wash
and elute.
2- In the depletion column, use an affinity column with peptides without acetylation to
ensure purification. Antibody of interest is in flow-through.
To control for change in expression of acetylation, dot blot different histone modifications (K9
vs K27, for example).
• Histone methyltransferase (HMT) causes methylation by using SAM (S-adenosylmethionine)
as methyl donor. Sometimes, same lysine residue can be methylated multiple times:
monomethyl-lysine, dimethyl-lysine, trimethyl-lysine.
Question: how to analyze changes in histone methylation in vivo?
Answer: by using the Chromatin – immunoprecipitation (ChIP) technique: (1) cross – linked
chromatin, (2) isolate and shear chromatin mechanically, (3) add antibody specific for
methylated N-terminal histone tail, (4) immunoprecipitation, (5) release and purified
immunoprecipitated DNA and assay by PCR and finally by using deep-sequencing.
Answer: 35S-labelled HP1 + biotinylated peptide → streptavidin pulldown → SDS-PAGE
NOTE: HATs → acetylation (loosening of chromatin; recruitment of additional factors) =
activation. HMTs → methylation (recruitment of additional factors) = activation or repression.
NOTE: specific domains for binding to methylated lysine (Kme): (1) chromodomains (HP1 >
H3K9me3), (2) Tudor doamains (PHF1 > H3K36me3), (3) plant homeodomains (PHD) (BPTF>
H3K4me3).
NOTE: metagene graphs are used for ChIP-Seq experiments. Metagene is a summarized gene
expression correlates with active promoters based on average. Each pick shows high possibility
of expressing.
• H3K4me3 recruits proteins that facilitate transcription: (1) CHD1 (chromatin remodeling
protein), (2) TAF3 (subunit of TFIID).
• Lysine methylation is reversible by specific enzymes:
(1) FAD-containing oxidases (LSD1/2) → mono-/ dimethylated lysine. For example:
demethylation of H3K4me1/ H3K4me2 by LSD1/2 enzymes.
(2) Jumanji protein (dioxygenase) → mono-/di-/trimethylated lysine. For example:
demethylation of H3K4me3 by Jumonji enzymes (hydroxylases).
•