Lectures 1-4 Epigenetics

What are the three regions on every chromosome?

Telomeres, Replication Origins, Centromeres

What is the function of telomeres?

Located at the end of chromosomes, they contain repetitive sequences to protect chromosome ends from degradation and allow replication.

What is the function of replication origins?

Sequences where DNA replication begins.

What is the function of centromeres?

Specialized, highly conserved sequences in the center where spindle fibers attach during division (mitosis/meiosis) for segregation.

How does genome organization in the nucleus ensure efficiency and accuracy?

Organization allows the massive genome (2 meters) to fit into a small nucleus (5-8µm) while remaining accessible and regulated.

What are chromosome territories?

Chromosomes occupy distinct non-random spaces in the nucleus to prevent tangling.

What factors determine chromosome location?

Gene density, chromosome size, interactions with nuclear proteins.

Why is a non-random arrangement of chromosomes beneficial?

Helps regulate gene expression (e.g., clustering active genes) and maintains genome stability.

What is the composition of a chromosome?

Chromatin: long linear double-stranded DNA associated with proteins (histones and non-histones).

What is the smallest fundamental unit of the chromosome?

Nucleosome, which consists of 147 base pairs of DNA wrapped around a histone octamer core.

What are the different histones that form the core of the nucleosome?

H2A, H2B, H3, H4.

What type of interaction occurs between histones and DNA in the nucleosome?

Histones are positively charged and interact with the negatively charged phosphate backbone of DNA.

What led to the discovery of the nucleosome structure?

Ada and Don Olins published electron microscope images in 1974 showing a beads-on-a-string structure.

What are histone variants?

Histones with slightly different amino acid sequences that replace standard histones to alter function.

How is the nucleosome structure dynamic?

Nucleosomes can alter their structure and position to expose different parts of DNA for cellular processes.

What drives the plasticity of the nucleosome structure?

Nucleosomes are not static; they can slide, partially disassemble, replace histone parts with variants, or undergo chemical modifications.

What is nucleosome occupancy?

Where nucleosomes are placed on DNA and whether they are present or absent at a specific location.

How is nucleosome occupancy related to being dynamic?

Nucleosomes are dynamic, meaning their occupancy is not permanent and can change to control access to DNA.

How is nucleosome occupancy connected to the NFR in the gene promoter region?

Dynamic movement creates a nucleosome-free region (NFR) that allows transcription factors to bind and start gene expression.

What are fuzzy nucleosomes?

In active genes, nucleosomes constantly slide back and forth to let enzymes pass, resulting in shifting occupancy.

What is the benefit of having a dynamic nucleosome structure?

Allows the genome to open or close as needed for processes like transcription, replication, and repair.

How may histone variants alter nucleosome function?

Histone variant H3.3 is incorporated in active genes and is critical for heart development.

What are chaperone proteins?

Proteins that assist in folding or assembling other proteins (like histones) and prevent inappropriate clustering.

What is the role of histone chaperones in regulating transcription?

Histone chaperones help load H3.3 into active genes, keeping chromatin open for transcription.

What are the main differences between genetics and epigenetics?

Genetics involves static DNA code, while epigenetics involves reversible changes to chromatin structure that regulate gene expression.

What is epigenetics?

Changes in gene expression that occur without changing the DNA sequence itself.

What was the Waddington thesis regarding epigenetics?

Described development as a landscape where a cell rolls down valleys representing decisions that direct the cell into a specific identity based on environmental influence.

What is the epigenome?

A complex set of chemical modifications on the DNA and histone proteins across the entire genome.

How are interphase chromosomes packed?

Heterochromatin is condensed/closed (silent genes), while euchromatin is extended/open (active genes).

What are ATP-dependent chromatin remodeling complexes?

Multi-subunit protein machines that use energy from ATP hydrolysis to physically move or alter nucleosomes.

What is the function of the catalytic subunit in chromatin remodeling complexes?

It contains an ATPase domain that performs ATP hydrolysis to generate mechanical force for disrupting contacts between DNA and histones.

What are accessory subunits in chromatin remodeling complexes?

Various non-catalytic proteins that attach to the ATPase subunit and assist in the remodeling process.

What are the four proposed mechanisms of chromatin remodeling by the remodeling complexes?

1. Binding: ATPase subunit anchors to nucleosomal DNA. 2. Energy Conservation: Hydrolyzes ATP to release energy. 3. DNA Translocation: Uses energy to pump or twist the DNA double helix. 4. Outcome: Mechanical movement breaks histone-DNA interaction, resulting in sliding, ejection, unwrapping, or exchange.

How do the SWI/SNF complexes activate transcription?

By sliding and ejecting nucleosomes, recruited to specific DNA sites by binding to transcription factors or specific histone markers.

What are the examples of silencing complexes?

Polycomb Repressive Complexes (PRC1 and PRC2) that mediate chromatin remodeling and transcription.

What does PRC2 do?

Adds methyl groups to Histone H3 at Lysine 27, creating the H3K27me3 marker.

What does PRC1 do?

Attaches ubiquitin to Histone H2A at Lysine 119, creating the H2AK119ub1 marker.

What is the effect of histone acetylation on transcription?

It adds an acetyl group to lysine tails, neutralizing the positive charge and weakening the histone-DNA attraction, allowing transcription machinery easier access to DNA.

What are the different modifications of histones?

1. Acetylation, 2. Methylation, 3. Ubiquitination, 4. Phosphorylation.

How does histone methylation influence transcription?

Creates binding sites for specific proteins that either activate or silence transcription based on the location of the methyl tag.

What is the role of writers in histone modifications?

Enzymes that add chemical marks, such as methyltransferases.

What is the role of erasers in histone modifications?

Enzymes that remove chemical marks, such as demethylases.

What is the role of readers in histone modifications?

Proteins that recognize and bind to specific marks to execute a function.

What are the consequences of not having HATs and HDACs?

Chromatin remains permanently closed or relaxed, leading to uncontrolled gene expression or gene silencing.

What is the effect of histone phosphorylation?

It adds a phosphate group to histones, often associated with gene activation.

Where and when are histones modified?

Post-translationally on specific amino acids (typically lysine) on the tails.

What are the general features of histones fundamental to their function?

Unstructured N-terminal tails that extend out of the nucleosome and are primary sites for chemical modifications.

How do histone modifications maintain a balance between stability and reversibility?

They must be reversible to allow cells to switch genes on/off rapidly in response to signals.

What are nucleosome-free regions (NFRs) and their significance?

Regions created by chromatin remodeling mechanisms that allow transcription factors to bind and activate genes.

What is the effect of histone ubiquitination on transcription?

It can signal either active or silent chromatin depending on the specific ubiquitination pattern.

What is the role of bromodomains in SWI/SNF complexes?

They recognize acetylated histones, which are active markers.

What happens during the sliding mechanism of chromatin remodeling?

The DNA moves along the histone, exposing previously hidden sequences.

What happens during the ejection mechanism of chromatin remodeling?

The histone octamer is physically pushed off the DNA entirely.

What happens during the unwrapping mechanism of chromatin remodeling?

The DNA is loosened from the edge of the nucleosome.

What happens during the exchange mechanism of chromatin remodeling?

The complex facilitates the removal of a standard histone and the insertion of a variant.

What is the significance of the unstructured N-terminal tails of histones?

They are primary sites for chemical modifications that regulate DNA accessibility.

What is the effect of histone methylation on chromatin structure?

It can signal either activation or repression of transcription based on the specific lysine modified.

Why is histone acetylation important for gene expression?

It opens up chromatin structure, allowing transcription machinery to access DNA.

What are the two enzymes that catalyze histone modification with the acetyl group?

HATs (Histone Acetyltransferases) add acetyl groups; HDACs (Histone Deacetylases) remove them.

What is the role of specific histone marks in gene regulation?

They act as signals to recruit proteins that either activate or silence transcription.

How do transcription factors target specific genes?

By binding to specific gene sequences and recruiting the SWI/SNF complex.

What is the turnover rate in methylation?

It refers to how fast a chemical mark (methylation) is added and removed.

What does high turnover in methylation indicate?

It indicates that methylation marks are added and removed very quickly, allowing for immediate reactions.

What does low turnover in methylation indicate?

It ensures that cells maintain their identity and do not change accidentally, while still being reversible.

What are the roles of methyltransferase and demethylase enzymes?

Methyltransferase are 'writers' that add methyl groups, while demethylase are 'erasers' that remove them.

How are methyltransferase and demethylase recruited to their genomic destinations?

They are guided by DNA sequence, non-coding RNA, and existing marks (crosstalk).

What does it mean that the effects of methylation are context-dependent?

The outcome of adding a methyl group depends on its location, affecting whether genes are turned on or off.

What are the consequences of dysregulation of histone methylation?

It can lead to cancer and intellectual disabilities due to improper cell growth and function.

Which nucleotide is modified by the methyl group in DNA methylation?

Cytosine, specifically at the 5th carbon in the ring.

What are the functions of DNA methylation?

It serves as a silencing marker, maintains cell identity, and is involved in mitotic heritability.

What are the writers of DNA methylation?

The DNMT family, including DNMT3A, DNMT3B (de novo methyltransferases) and DNMT1 (maintenance methyltransferase).

What happens if there is a mutation or no expression of DNMT enzymes?

Cells cannot establish new instructions or maintain their identity, leading to inappropriate gene expression.

What is the general methylation pattern of the epigenome?

It is mostly hypermethylated, with 70% of all CpG sites having a methyl group attached.

What is epigenetic memory?

The ability of cells to 'remember' their identity through stable propagation of epigenetic marks.

What is the significance of epigenetic reprogramming after fertilization?

It resets cells to a totipotent state, allowing them to become any cell type.

What is Embryonic Genome Activation (EGA)?

It is when the embryo starts transcribing its own genome, coinciding with major chromatin remodeling.

What is the difference in reprogramming of epigenetic marks in paternal and maternal pronuclei?

Paternal reprogramming is extensive and fast, while maternal reprogramming is slow and gradual.

Why is slow maternal reprogramming significant?

It ensures the embryo retains critical instructions for survival during the early days of development.

What happens to repetitive DNA in terms of methylation?

Repetitive DNA is heavily methylated (hypermethylated) to keep it quiet and stable.

What is the role of non-coding RNA in methylation?

Non-coding RNA acts as a GPS system to guide enzymes to specific DNA sequences.

What are the consequences of having no DNMT1 expression?

Cells forget their identity, leading to dilution of methylation marks and inappropriate gene activation.

What is the role of DNMT1?

It is a maintenance methyltransferase that copies methylation patterns from parent to daughter strands during replication.

What does passive demethylation refer to?

It refers to the dilution of methylation marks during cell division, leading to their eventual loss.

What is the function of hypermethylated regions in the genome?

They keep dangerous or useless regions permanently silenced, maintaining genomic stability.

What is the significance of hypomethylated regions?

They are located at CpG islands near gene promoters, allowing transcription factors to bind and express genes.

What is the role of DNMT3 enzymes?

They establish new methylation patterns from scratch during early development.

What are the effects of active methylation marks?

They are associated with open promoters and gene activation.

What are repressive methylation marks associated with?

They are associated with gene silencing.

What is the process of passive demethylation?

It allows methylation marks to dilute out during cell division.

What is the function of methylation in gene promoters?

Methylation in promoters is typically absent (hypomethylated) to allow gene activity.

What is the role of existing marks in crosstalk?

They act as landing pads for enzymes to bind and add their own marks.

What is the significance of the methyl group in DNA methylation?

It plays a crucial role in gene regulation and maintaining cell identity.

What is the impact of environmental signals on gene methylation?

They can influence the addition or removal of methylation marks in response to stress or other factors.

X-inactivation

A process in female mammals (XX) where one X chromosome is randomly silenced to equalize gene dosage with males (XY).

X-inactivation center (XIC)

The region on the X chromosome that controls the X-inactivation process.

Xist

A long non-coding RNA (lncRNA) transcribed from the XIC that coats the X chromosome that produced it and recruits silencing factors to shut it down.

Mutation of Xist

If Xist is deleted/mutated on one X, that X cannot be inactivated, leading to non-random inactivation.

Mechanism of X-inactivation

Xist binding leads to exclusion of RNA Polymerase II, loss of active histone marks, gain of repressive marks, and DNA methylation.

Mosaicism

The random choice of which X to silence results in adult females having patches of cells expressing the maternal X and patches expressing the paternal X.

X-Chromosome Reprogramming in Germ Cells

Primordial Germ Cells (PGCs) must reactivate the silenced X chromosome so that every egg produced has an active X chromosome.