Gene Products

What is polydactyly?

It is a mutation that results in extra digits due to the overexpression of limb genes. This mutation occurs due to a single nucleotide change (G → A) in a cis-regulatory element about a million bases away from the gene body, which allows the gene to turn on in a place where it shouldn’t turn on

What are the requirements for the production of translatable mRNA from specific genes?

1. accessibility of gene to transcription factors (TFs)

2. interactions of TFs with cis-regulatory elements

3. initiation, elongation, and termination of RNA transcript

4. processing (capping and splicing) of newly transcribed RNA

5. transport of processed mRNA to cytoplasm

What does the interaction of TFs with cis-regulatory elements allow for?

It allows for the basal transcriptional apparatus (mainly RNA Polymerase II) to assemble and begin transcription

What binds to cis-regulatory elements?

Transcription factors (proteins)

What state does chromatin need to be in in order for DNA to be transcribed?

The chromatin needs to be more open so it is more easily accessible by TFs and basal transcriptional apparatus

What is the structure of chromosomes in cells?

Each chromosome is a single long molecule of double-stranded DNA and it exists as chromatin, which is about 1/3 DNA, 1/3 histones, and 1/3 other proteins; DNA does not exist on its own in cells, always associated with protein

Are histones present in every species?

Yes, histones and the sequence of individual histones are highly conserved across all species, which points to a very ancient and very important function

How are histones arranged in chromatin?

They exist in the form of nucleosomes, which consists of two copies of each H2A, H2B, H3, and H4 core histones which the DNA wraps arounds. There is also a linker histone, H1 that is outside the core part but still closely associated to the DNA

How do histones change the length of DNA?

They reduce the length of DNA 7-fold

What is the difference between euchromatin and heterochromatin?

• Euchromatin → more open and accessible to RNA polymerase and TFs; more likely to be transcriptionally active

• Heterochromatin → more compact less accessible to RNA polymerase and TFs; less likely to be transcriptionally active; often gene-poor and repeat-rich, and localized to the periphery of the nucleus

Where would TEs and SSRs/microsatellites be likely to be found in chromatin?

They would be more likely to be found in heterochromatin

Can regions of chromatin switch between being euchromatic and heterochromatic?

Yes, most regions can depending on the cell type and activity, but some regions are always going to be heterochromatic or always euchromatic

What does the state of chromatin depend on?

Histone modification → Histone tails are exposed beyond the nucleosome, and they can be post-translationally modified by acetylation, methylation, phosphorylation, and ubiquination

What are the two broad classes of ways histones can be modified?

1. Changing the charge on histones (acetylation) → alters DNA accessibility

2. Changing the interaction of histones with other proteins (methylation) → alters recognition by cofactors

These modifications are also called histone marks

What modifications change the charge on histones?

This depends on the acetylation of lysine (K) in specific spots on the histone tails. The side chain of lysine is typically positively charged, and this allows for interaction with the negatively charged backbone of DNA → strong association between histone and DNA, hard for TFs to access DNA. This interaction can be modified by HAT (histone acetyl transferase) which acetylates those lysines, neutralizing the positive charge and reducing the interaction of the histone with the DNA → easier to access by TFs; this can be reversed by HDAC (histone deacetylase) which deacetylates histones, making the interaction with DNA stronger → less accessible DNA

What would HAT do if added to DNA?

It would acetylate the lysines on the histones around which the DNA is wrapped. This would neutralize the previously positive lysines, reducing the interaction of the histone with the negatively charged DNA, making the DNA more accessible for transcription

What would HDAC do if added to DNA?

HDAC would deacetylate any acetylated lysines on the histones, restoring the positive charge to the lysines, increasing the interaction of the histone with the DNA, making it less accessible for transcription

What residues on a histone can be acetylated?

Lysines (K)

What residues on a histone can be methylated?

Lysines (K) and Arginines (R)

How does the methylation of arginine work and what is the outcome?

Arginine gets methylated by protein arginine methyltransferase (PRMT); PRMT can add multiple methyl groups or just one; depending on the histone type and the specific residue number, methylation of arginine can enhance or repress transcription

How does the methylation of lysine work and what is the outcome?

Lysine gets methylated by histone methyltransferase (HMT); HMTs can add multiple methyl groups or just one; depending on the histone type and the specific residue number, methylation of lysine can enhance or repress transcription

Will highly acetylated nucleosomes by condensed or uncondensed?

They will be uncondensed

Will sparsely acetylated nucleosomes by condensed or uncondensed?

They will be condensed

How is H3K9me read?

Histone 3, lysine 9 (from the N-terminus), methylation

What is the outcome if H3K27 is methylated?

This will strongly repress transcription; Polycomb Complex proteins (PCs) can bind to H3K27me, and this enforces transcriptional silencing

What is the outcome if H3K27 is acetylated?

This is strongly promoting of transcription

What is the outcome if H3K9 is methylated?

This will strongly repress transcription; H3K9me is recognized by Heterochromatin Protein-1 (HP-1) and HP-1 binds to it, and this enforces transcriptional silencing

What are ATP-dependent remodelers?

They are a class of proteins that recognize modifications in histones and require ATP to do their job; they can move around, add, or eject nucleosomes or alter the composition of histones, dramatically changing the state of chromatin; the ATPase activity helps overcome the nucleosome-DNA interactions; a remodler may come in and add nucleosomes to a region of chromatin, making it more compact; a remodeler may come in and eject or reposition nucleosomes to make DNA more accessible; a remodeler may come in and alter the composition of histones (change the dimers and variant present) to change DNA accessibility; ex. SWI/SNF proteins, chromodomain proteins (CHD), ISWI proteins

What is are some ways to determine the level of compaction of a region of DNA?

• You can look for specific histone alterations; ex. if you want to know which regions of DNA are condensed, look for H3K27me because that alteration is associated with transcriptional repression

• You can look for copies of specific TFs or of RNA polymerase, because if there are many copies of these present at a site, it is likely that it is transcriptionally active

What is ChIP-Sequencing and how does it work?

Chromatin immunoprecipitation with sequencing; the goal is to capture portions of the genome that are associated with whatever it is you want to understand (ex. H3K27me, RNA polymerase, etc.); to do this, must use an antibody for the protein of interest:

1. isolate chromatin from whole genome (not just DNA)

2. fragment chromatin into smaller pieces

3. incubate chromatin fragments with antibody for protein of interest (incluidng modification, ex. H3K27me) and precipitate out the antibody-chromatin complexes

4. Discard rest of chromatin

5. Then isolate DNA from precipitated material and sequence it via Illumina

6. compare the sequence from the region of activity against reference genome → the more reads there are of a particular site in the genome, the more likely it was that the protein of interest was bound to that site in the gene

This is a very good way to understand if certain modifications or TFs are good predictors of active transcription

How can cis-regulatory elements be determined in the genome?

Using ChIP-Seq, if you have an antibody to a TF that might be binding to that cis-regulatory element, you can use ChIP-Seq to determine if that TF is actually binding to a particular site of interest

What are some downsides to using ChIP-Seq?

You need a lot of DNA to do it and you need really good, specific antibodies

Do we understand the outcome of every histone modification?

No

Is each histone encoded by one gene?

No, there are multiple genes that encode different versions of the same histone, and they can be substituted out for one another at different times to serve different functions

What are the core canonical vs. variants for H3?

• The core canonical histone is H3

• One variant is H3.3, which looks a lot like H3, but H3.3 is specifically associated with maintaining transcriptional activation by keeping chromatin open; H3.3 is less methylated than H3

• Another variant is CENPA, which is a centromere associated protein that is inserted during mitosis; it helps kinetochores attach to centromeres

What is a core canonical histone?

The main structural histone protein (ex. H2A, H2B, H3, and H4)

What are histone variants?

different versions of core canonical histones that can be inserted at different points to serve different functions

Are H3 and H3.3 encoded by the same gene?

No, they are different proteins, just similar versions of each other, so they are encoded by different genes

What are the core canonical vs. variants for H2?

• the core canonical histone is H2

• One variant is H2AX which is substituted for H2A when the cell is trying to repair DNA damage

Can core canonical histones be present at the same time as their variants?

Yes, sometimes they can be present together or they can be substituted for one another

What traits do cancer cells evolve that make them different from other cells?

• sustained proliferative signaling

• evade growth suppressors

• activate genes involved in metastasis

• resistant to immune system

• disregulated metabolism

• replicative immortality

• induce angiogenesis

• resist cell death

What type of mutation is common in pediatric cancers (specifically glioma)?

H3 mutations, specifically H3K27M, which changes lysine 27 to a methionine → affects the ability of H3K27 to get methylated (which is usually strongly suppressing of transcription)

Would you expect cancer cell DNA to be highly or sparsely acetylated?

Highly acetylated