Exam 1 Study Sheet - Epigenetics of Neuro

Explain how CpG methylation can be used as a form of cellular memory.

Creates heritable changes in gene expression without altering DNA sequence

Allows cells to remember past states (developmental decisions, environmental exposures)

Methylation → repression

After DNA replication, CpG sites hemimethylated → DNA methyltransferase restores methylation → copies methylation pattern to daughter → gene expression state maintained through cell division

Methylation patterns influenced by environmental conditions, DNA damage, chemical agents, aging, etc

Some immortalized cell lines do not express a number of commonly expressed genes. It was thought that these cell lines carried mutations in these genes. However, treatment of the cells with 5-azacytidine was shown to reactivate these genes (cause them to be expressed). Tell me why the original cells did not express the genes and everything that you can about how and why 5-azacytidine restored expression.

Genes epigenetically silenced by DNA methylation rather than DNA sequence mutations

5-azaC restores expression by disrupting DNA methylation maintenance → silenced genes demethylated

5-azaC incorporated into DNA as cytosine analog and inhibits DNA methyltransferases by covalently trapping

After replication → hemimethylated CpG sites not restored → methylation lost subsequently

Loss of CpG methylation removes transcriptional repression

TFs can bind and RNA polymerase initiates trnascription

The mutation rate in cultured rodent and human cells are about the same. But the transformation of rodent cells is about 1.9x10-6 and humans is 10-9. This is not likely to be due to differences in the rate of mutation. Here, transformation means a transition to a cancer cell-like state that includes loss of growth control and perhaps immortality. What does the Robin Holliday paper suggest (albeit stated rather torturously) might be the cause of the higher transformation rate in the rodent cells.

Rodent cells experience more epigenetic changes more frequently than human cells

DNA damage and replication errors → loss of CpG methylation → altered gene regulation

Transformation in two-steps: epigenetic defect → mutation or additional epigenetic change

First step more common in rodents so transformation frequency much higher

In animals, the TFIID complex is subject to substantial variation. There are a number of paralogs that encode TAF variants. This means that there is great diversity in the types of TFIID complexes that can be produced. What purpose might this diversity serve for the cell?

Allows fine tuning of transcription rather than uniform gene activation

Different TAF paralogs → different TFIID complexes with distinct promoter specificities → selective regulation of specific gene sets

Cell type/developmental stage specific gene expression

Increasing regulatory flexibility and robustness

Describe for me what the Mediator complexes do and what the types of complexes they interact with.

Bridge between gene specific transcription factors and RNA polymerase II

Integrates regulatory signals from activators and repressors bound at enhancers and promoters

Facilitates recruitment, positioning, and activation of RNA polymerase II at promoters

Helps regulate tracscription initiation and transition to elongation

Interact with sequence specific transcription factors, RNAPII and other transcription machinery, chromatin modifying and remodelling complexes, other regulatory complexes

With respect to gene expression what is meant by the term melting? What purpose does it serve?

Local unwinding of DNA double helix at the promoter to create an open complex

Exposes template strand so RNA polymerase II can initiate RNA synthesis and begin transcription

__________ plays a central role in holding enhancers near to the core promoter they regulate.

Cohesion

Capping of the mRNA is thought to be important for release from promoter pausing (transcription pausing).

True

Which protein or protein complex recognizes core promoters by itself?

Transcription factors

What is the minimum function that a DNA sequence must have in order to be considered a core promoter? What does it not have to do?

Specifies where to start and which direction for transcription

Does not specify if it will occur

What is the minimum function that a DNA sequence must have in order to be considered an enhancer sequence? What does it not have to do?

Specifies how much and when for transcription

Does not specify start or direction

During the process of core promoter activation, which of the following is most likely the first protein or protein complex to recognize (bind) a core promoter element?

TFIID

A DNA enhancer sequence is 85 kb from a core promoter (that's pretty far). Could this enhancer regulate the core promoter or is it too far away?

Could do it

A DNA enhancer is inside of the transcribed region of a gene. Could this enhancer regulate the core promoter or is this not likely because it is inside the transcribed region?

Could do it

Core promoter elements are simple degenerate sequences that appear often in the genome. Is this statement true?

Yes, it is true. They occur frequently which makes it all the more amazing that transcription factors can rapidly find them.

Define the term "transcription factor". Also name two groups of DNA- associated proteins that are not transcription factors.

DNA-binding proteins that regulate transcription

Recognize specific DNA sequences

Influence recruitment, assembly, or activity of RNA polymerase II

Not included: RNA polymerase II, histones

Which component of TFIID has histone acetylase activity?

TAF 1

It is uncommon for a mammalian gene to have two different core promoters.

False

Which class of enzyme can directly produce nucleosome-free regions at core promoters and enhancers?

Remodeling enzymes

What does a bromodomain do? In what sense can it help to promote transcription?

Protein domains that recognize and bind acetylated lysine residues of histone tails

Recruit transcription factors, co-activators, and chromatin-remodeling complexes to acetylated chromatin

Stabilizes transcriptionally active reions

Promotes assembly of the transcriptional machinery

Helps maintain open chromatin state that favors transcription

Adjacent nucleosomes can interact with each other to condense the chromosome. Specifically, how do they interact with each other?

Stack together via electrostatic histone tail interactions and H1 linker histones

Is the highly acetylated state or the deacetylated state associated with

condensation?

Deacetylated

Describe in great detail how the acetylation of histones promotes an increased level of gene expression.

DNA not naked in eukaryotes → wrapped around histone octamers to form nucleosomes

Packaging essential for genome stability → physical and functional barrier to transcription

Regulatory DNA sequences obscured by nucleosomes → TFs and RNAPII cannot bind

Histones = repressors → change in chemistry = regulate gene expression

Histone tails rich in lysine → positive charge

DNA backbone → negative charge

Positive lysine stabilizes negative DNA

Acetylation neutralizes positive lysine → reduced affinity for DNA

Doesn’t activate transcription alone but makes more permissive for chromatin remodeling enzymes

Chromatin folds into compact fibers that strongly repress transcription

Unacetylated chromatin: basic H4 tail from one nucleosome inserts tail into acidic pocket from H2A-H2B dimer of neighboring chromosome → promote nucleosome-nucleosome contact → condensed 30nm chromatin fiber

Acetylated histone H4: positive charge neutralized → no tail insertion → no 30nm fiber → open conformation → regulatory DNA elements more accessible → transcription strongly enhanced

Acetylation actively recruits transcription-promoting factors

Bromodomain containing proteins (mainly TFs and histone acetyltransferases) recognized acetylated lysines

Recruit: components of pre-initiation complex, co-activators to stabilize TF binding, other HATs to reinforce acetylation (positive feedback)

Linker H1: binds DNA between nucleosomes, promotes chromatosome formation (favors chromatin compaction and repression), inhibits HAT in nearby histone. recruits HDACs (remove activating acetyl marks)

Acetylation counters by reducing H1-mediated condensation (favors open chromatin) and recruiting TFIID (part of pre-initiation complex)

Positive deacetylated histone partially neutralize negative charges on opposing DNA strands

Neutral acetylated histones → negative charges unmasked → increased repulsion between strands

Tell me what the chromatin immunoprecipitation assay is used for and explain in detail how the assay works. Also explain to me why measuring the input DNA is important and how one could use this information to normalize the data.

Used to determine which proteins or histone modifications are associated with specific DNA regions in living cells and how those associations change over time and conditions

1. Living cells + formaldehyde → covalent crosslinks between nearby proteins and DNA → protein-DNA interactions frozen

2. Cells lysed to release chromatin

3. Chromatin sheared using sonication or micrococcal nuclease

4. Protein or histone-specific antibody added

5. Beads washed multiple times in increasingly stringent conditions → remove DNA fragments and non-target protein complexes

6. Protein-DNA cross links reversed and proteins degrades by protease treatment → DNA purified

7. Recovered DNA → qPCR, microarrays, and/or highthroughput sequencing → amount of DNA in region reflects strength and frequency of target protein associated

Must measure input DNA to control for chromatin accessibility, copy number, PCR and sequencing bias, distinguish true enrichment from background

Done using percent input method (ChIP signal/input signal x 100)

Synapsins are a family of neuron-specific phosphoproteins common to all neurons. You harvest two samples of chromatin. Sample 1 is from the liver and sample 2 is from the brain. In one of the chromatin samples the synapsin I core promoters are extremely sensitive to digestion with DNase I (enzyme that non-specifically cuts DNA) whereas in the other sample this exact same region is resistant to digestion with DNase I. Explain what has happened to make it extremely sensitive?

In sample 2 the synapsin I core promoter is extremely sensitive to DNase I digestion.

Synapsin I gene expressed in neurons (not liver) → packaged in ope chromatin

Nucleosome at promoter displaced or destabilized by chromatin-remodeling complexes, histones highly acetylated, histone-DNA interactions weakened → site is hypersensitive to DNase I → DNA exposed and easily cut

Synapsin I not expressed in liver → promoter remains in compact chromatin → protected from DNase I digestion

Describe the composition of a core nucleosome, include the names of the proteins, and all of the details about each of the proteins you can remember. Include details about its interaction with DNA.

Core nucleosome = histone octamer + ~147 base pairs of DNA

Octamer = 2(H2A) + 2(H2B) + 2(H3) + 2(H4)

Central tetramer = 2(H3-H4 heterodimers)

Opposite sides of tetramer = 2(H2A-H2B heterodimers)

Stable protein scaffold around which DNA wraps

Each histone has globular histone fold domain (mediates histone-histone interactions) → octamer core and N-terminal tail

Tails unstructured and flexible, extend beyond DNA, major sites of post-transcriptional modification, regulate chromatin structure and gene expression by affected nucleosome interactions and protein recruitment

DNA interacts with histone electrostatically (positive histone contacts negative DNA at regular intervals)

Describe the autocatalytic chain reaction that forms S. cerevisiae heterochromatin. Be sure that you convey the idea of how it moves down the chromosome. Finally, be sure to tell me what stops the heterochromatin from propagating down the entire length of the chromosome.

Sir2 deacetylates H3 and H4 tails → bind Sir3 → recruits Sir4 → Sir3-Sir4 complex recruits additional Sir2 → acetylates neighboring nucleosomes → cycle repeats propagating heterochromatin down chromosome

Propagation blocked by boundary/insulator elements (regions with actively transcribed genes, specific DNA-binding proteins, histone modifications preventing Sir protein binding)

This work was done in S. cerevisiae. In Panel A we see that the URA3+ gene has been inserted at the edge of a telomere. All of the yeast are of this genotype. Panel B shows the outcome when drops of two cultures are grown on rich non-selective media (YPD). Panel C shows the outcome when drops of the two cultures are grown on media containing 5-fluoro-orotic acid (5-FOA). WT - wild type for the sir2 gene sir2delta - mutant at the sir2 gene Why does the sir2delta mutant grow more poorly on the 5-FOA media?

URA3⁺ inserted adjacent to telomere (normally forms heterochromatin in S. cerevisiae)

WT: Sir2 deacetylated histones → promotes telomeric heterochromatin spreading → silences URA3 → 5-FOA only toxic when URA3 expressed → WT survive on 5-FOA

Sir2Δ mutant: Sir2 absent → heterochromatin not formed or propagated → URA3 expressed → converts 5-FOA to toxic product → kills cells/inhibits growth

What are three fundamental principles of heterochromatin spreading that were observed studying Saccharomyces cerevisiae?

Nucleation

Propagation

Epigenetic inheritance

Name three activating histone marks, one inactivating histone mark, and one inactivating DNA mark. By activating and inactivating I mean that they are most often associated with this transcriptional state. The term mark refers to a chemical modification of the histone.

Activating histone: H3K4me3, H3K9ac, H3K27ac

Inactivating histone: H3K9me3

Inactivating DNA: 5mC

With regards to this paper, write a single sentence that tells me what the paper demonstrates.

DNA methylation changed at conserved CpG sites can build epigenetic clocks that measure biological age across mammalian species and tissues

Aging follows evolutionarily conserved programs linked to development

How would the authors demonstrate that their algorithm could predict the age of lions?

Train the algorithm on a dataset that contains all available mammals except for lions

In this paper, in Figure 4a, what do the most positive peaks on a Manhattan plot represent?

Genomic regions in which the level of CpG methylation is positively statistically correlated with age.

In this paper, what is represented on the X axis of the Manhattan plot?

All of the chromosomes of a species, in order from left to right.

Explain LOSO training as used in the development of the algorithm.

Leave-One-Species-Out: cross-validation strategy, epigenetic clocks trained on DNA methylation data from all but one mammalian species then tested to predict left-out species, process repeated for every species

Demonstrates that algorithm can accurately predict age for a species it has never seen

Provides evidence that DNA methylation aging signal is universal and conserved across mammals

Explain in detail how the bisulfite reaction is used to determine whether a cytosine is methylated.

Genomic DNA treated with sodium bisulfite → selectively reacts with unmethylated cytosines → deamination of unmethylated cytosines → converted to uracil → treated DNA amplified by PCR or sequencing → DNA polymerase reads uracil as thymine → original unmethylated C detected as T → compare sequence before and after to determine methylation status of each cytosine