Molecular Biology Final

Role of HAT and HDAC in chromatin remodeling

- Acetylation of Lys neutralizes positive charges on histones - Coactivator complexes - loss of histone H1 modifications (such as acetylation, phosphorylation and methylation) → euchromatin - Co repressor complexes - Histone deacetylation, dephosphorylation and demethylation → heterochromatin

Role of HAT and HDAC in chromatin remodeling

- Acetylation of Lys neutralizes positive charges on histones - Transcription regulation by HAT and HDAC - Change in environmental signaling

Coactivators and Corepressors:

- Act indirectly by binding other proteins (transcription factors) rather than DNA, sometimes exhibit activities of nucleosome modification. - Are required for essential communication between the DNA-binding trans-activators and the complex composed of Pol II and the general transcription factors. - Sometimes, a variety of repressor proteins can interfere with communication between Pol II and the DNA-binding trans-activators, resulting in repression of transcription. Some proteins act as an activator or coactivator at one promoter and a repressor at another promoter.

Mediator Complexes

- Are known to consist of more than 20 subunits and to play key roles in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. - Mediate both the opening of chromatin structure and transcriptional activation - Mediates the interaction between Pol II CTD and other DNA-bound activators - Helps initiation by regulating the CTD kinase in TFIIH

Mechanism of activation and repression of eukaryotic gene expression

- CAP activates the lac genes by directly interacting with RNA Pol - (a)Transcription activators and coactivators bound to distant regulatory sites recruit components of Pol II transcriptional machinery to the promoter. - (b)Repression is mediated by proteins that disrupt or prevent essential contacts between Pol II and activators or coactivators.

Example: Role of coactivators (CBP and PCAF) in regulation of the inflammatory gene

- Coactivators simultaneously interact with transcriptional activators for its activation, in many cases, by modifying nucleosomes. - NF-kB (p50/p65): Gene-specific transcription factor for inflammatory genes - PCAF: co-activator - CBP/HAT: histone tail modification for gene transcription - NcoR/HDAC: histone tail modification for gene suppression - Positive regulation: inflammatory stimuli - Negative regulation: corticosteroids

Epigenetics in Cancer

- Deregulation of gene expression is a hallmark of cancer. - Although genetic lesions have been the focus of cancer research for many years, it has become increasingly recognized that aberrant epigenetic modifications also play major roles in the tumorigenic process. - These modifications are imposed on chromatin, do not change the nucleotide sequence of DNA, and are manifested by specific patterns of gene expression that are heritable through many cell divisions - DNA methylation, histone modification, nucleosome remodeling, and RNA-mediated targeting regulate many biological processes that are fundamental to the genesis of cancer. - This information, along with the promising clinical and preclinical results seen with epigenetic drugs against chromatin regulators, signifies that it is time to embrace the central role of epigenetics in cancer.

What is epigenetics?

- Epigenetics, literally "on" genes, refers to all modifications to genes other than changes in the DNA sequence itself. - Epigenetic modifications include addition of molecules, like methyl groups, to the DNA backbone. Adding these groups changes the appearance and structure of DNA, altering how a gene can interact with important interpreting (transcribing) molecules in the cell's nucleus. - Epigenetic modifications may condense or relax nucleosomes

1. Eukaryotic genes have nucleosomes and their modifiers

- Eukaryotes are wrapped in histones to form nucleosomes. - The transcription machinery is presented with a partially concealed substrate, which reduces the expression of many genes in the absence of regulatory proteins - Several enzymes that rearrange or chemically modify histone alter nucleosomes in ways that transcriptional machinery and DNA-binding proteins can bind and operate.

How do epigenetic modifications affect genes?

- Every cell in the body has the same genetic information; what makes cells, tissues and organs different is that different sets of genes are turned on or expressed. - Because they change how genes can interact with the cell's transcribing machinery, epigenetic modifications, or "marks," generally turn genes on or off, allowing or preventing the gene from being used to make a protein. On the other hand, mutations and bigger changes in the DNA sequence (like insertions or deletions) change not only the sequence of the DNA and RNA, but may affect the sequence of the protein as well. - There are different kinds of epigenetic "marks," chemical additions to the genetic sequence. The addition of methyl groups to the DNA backbone is used on some genes to distinguish the gene copy inherited from the father and that inherited from the mother.

Characteristic of Gene-Specific Transcription Factors in Gene Regulation

- Gene-specific Transcription factors (TFs) are regulatory proteins whose function is to activate (or to inhibit) transcription of DNA by binding to specific DNA sequences. - TFs have defined DNA-binding domains with up to 10^6-fold higher affinity for their target sequences than for the remainder of the DNA strand. - Transcription factors can also be classified by their three-dimensional protein structure, including basic helix-turn-helix, helix-loop-helix, and zinc finger proteins. - These different structural motifs result in transcription factor specificity for the consensus sequences to which they bind. - In eukaryotes, regulation of gene expression by transcription factors is said to be combinatorial, in that it requires the coordinated interactions of multiple proteins. - Compare the number of gene-specific TFs (~3,000 factors) with number of genes (~25,000 genes) in eukaryotic cells

DNA methylation and gene expression

- Genomic regions rich in CpG dinucleotides; tend to be near by promoters - CpG island becomes methylated - Recruit HDAC - HDAC attaches via MeCPs (methylated-CpG binding proteins) - → packages chromatin and decrease gene transcription

Epigenetic Therapy in Cancer: HDAC (histone deacetylase) inhibitors

- HDAC inhibitors for the treatment of cutaneous T-cell lymphomas -- Vorinostat (trade name Zolinza) The first FDA-approved HDAC inhibitor for cutaneous T cell lymphoma in 2006. -- Vorinostat is an inhibitor of histone deacetylase (HDAC). -- ⬆ acetylation = ⬆gene expression = ⬆ anti-tumor activity of T cells

2. Eukaryotic genes have more regulators and more extensive regulatory sequences

- Increasing complexity of regulatory sequences from a simple bacterial gene to a human gene controlled by multiple activators and repressors - Individual regulator binds short sequence in bacteria, but in eukaryotes, these binding sites are often more numerous and positioned further from the start site of transcription - More extensive signal integration

DNA methylation

- Methyl group (an epigenetic factor found in some dietary sources) can tag DNA and activate or repress genes

Epigenetic Therapy in Cancer: DNMT (DNA methyltransferase) inhibitors

- Nucleoside analogues - Most DNA methylation inhibitors (DNMT inhibitors) that have been clinically tested belong to the nucleoside analog family.

Histone modification

- The binding of epigenetic factors to histone "tails" alters the extent to which DNA is wrapped around histones and the availability of genes in the DNA to be activated - Histones are proteins around which DNA can be wind for compaction and gene regulation

Types of Epigenetic Modifications

1. Covalent modifications of histone tails that alter chromatin state (acetylation, phosphorylation...) 2. Covalent modification of DNA (methylation of CpG islands near promoter region)

Transcriptional Regulation in Eukaryotes

1. Eukaryotic genes have nucleosomes and their modifiers 2. Eukaryotic genes have more regulators and more extensive regulatory sequences

Transcriptional Regulation Overview

1. Transcription is initiated by interactions between activators, coactivators and RNA polymerase. 2. Repressors displace the activators, and a corepressor inhibits RNA polymerase. 3. A histone deacetylase associates with the corepressor, and RNA polymerase dissociates from DNA. 4. Histone deacetylation leads to chromatin condensation, further repressing transcription of the gene.

Epigenetic Modification Involving DNA Methylation and Cancer Connection

A previously unmethylated Tumor Suppressor (TS) gene such as p53 becomes methylated. Transcription factor(s) (TF) can no longer bind the promoter region, the gene is not expressed, and damaged cells are allowed to proliferate and become cancerous. A proto-oncogene is demethylated, allowing TFs to initiate transcription and express the protein product. As in (A), uncontrolled cell growth ensues and leads to cancer.

Having more extensive regulatory sequences means that some regulators bind sites far from the genes they control, hundreds kilobases or more. How does this raise problems?

Activation at a distance raises problems since there may be several genes within range of an activator.

Upstream Activator Sequence (UAS)

An enhancer-like sequence located just upstream of the genes they regulate.

What triggers epigenetic changes?

Epigenetic mechanisms are affected by these factors and processes: - Development (in utero, childhood) - Environmental chemicals - Drugs/pharmaceuticals - Aging - Diet Health endpoints: - Cancer - Autoimmune disease - Mental disorders - Diabetes

High Mobility Group (HMG)

Facilitates a DNA looping

Transcriptional activators and coactivators help assembles _______ ____________ _______

General Transcription Factors

Histone tail modification by ___ and ____

HAT: Histone Acetyltransferase HDAC: Histone Deacetylase

Nucleosome modifications for transcriptional activation come in two types:

Histone acetyltransferase (HATs): add acetyl group Nucleosome modifier: ATP-dependent activity of SWI/SNF: displace the nucleosome

1. Covalent modifications of histone tails that alter chromatin state (acetylation, phosphorylation...)

Histone tail modification by HAT and HDAC HAT: Histone Acetyltransferase Lysine in histone tail + acetyl CoA -HAT→ CoA-SH + H+ HDAC: Histone Deacetylase CoA-SH + H+ -HDAC→ Lysine in histone tail + acetyl CoA

__________ block activation of the promoter by activators bound at the enhancer.

Insulators

Additional factors required for efficient transcription initiation in vivo

Mediator complexes

2. Covalent modification of DNA (methylation of CpG islands near promoter region)

Modification of DNA (methylation and demethylation) in CpG site of promoter can also affect gene expression - CpG: Cytosine-phosphate-Guanine - Human promoters have a high CpG content. - 5'-CpG-3' - CpG: regions of DNA where cytosine occurs next to a guanine - Epigenetic marks include a variety of gene regulatory events, such as chromatin structure remodeling, histone modifications, DNA methylation, and small noncoding RNAs, that do not entail changes in the DNA sequence. Gene expression is directly associated with RNA polymerase and transcription factors that bind to regulatory sequence elements, such as promoters and enhancers. Epigenetic events regulate gene expression at both transcription (histone modification and DNA methylation) and translation (small noncoding RNA) levels. Specifically, epigenetic regulation is involved in genomic imprinting, X chromosome inactivation, and gene silencing. It is thus closely correlated with disease mechanisms. In 1975, two independent research groups suggested that the methylation of cytosine might play a pivotal role as an epigenetic mark in animals. DNA methylation was found to occur predominantly on cytosines followed by guanine residues (CpG). This type of methylation is referred to as CpG methylation, and cytosine methylated at the fifth carbon of the pyrimidine ring is called 5-methylcytosine (5mC). The major function of DNA methylation is the suppression of gene expression. DNMT: DNA methyltransferase - Cytosine -DNMT→ 5-methylcytosine -- Methylated DNA -- Gene silence - 5-methylcytosine -Demethylase→ cytosine -- Unmethylated DNA -- Gene activation

Epigenetic patterns in tumor suppressor gene in normal and cancer cells: Chromatin and Histone Modification

Normal: Nucleosomes with H3K4 methyl mark and acetylation mark Cancer: repressive complex (e.g. PcG) on nucleosomes, methylated DNA binding protein and H3K9/K27 methyl mark

Epigenetic patterns in tumor suppressor gene in normal and cancer cells: DNA Methylation

Normal: unmethylated cytosine before/on exon and methylated going forward Cancer: methylated cytosine before/on exon and unmethylated going forward

GTF's and Rpol II (forms transcription initiation complex) bind to specific sequence elements in the ________. Activators (enhancer) and repressors (silencer) bind to regulatory sites on DNA nearby promoter regions. ____________ and ___________ are transcriptional coregulators that bind to activators or repressors and either increase or decrease the rate of transcription.

Promoter Co-activators and Co-repressors Different genes have distinct gene regulatory proteins and their binding locations

Summary: Transcriptional regulation in Eukaryotes

Transcription factors work together with co-activators (or co-repressors) and nucleosome modifiers for the regulation of gene transcription.

Assembly of the pre-initiation complex in the DNA template in vivo

Transcriptional regulator proteins called activators help recruit polymerase to the promoter, stabilizing its binding there. This recruitment is mediated through interactions between DNA-bound activators, chromatin modifying and remodeling factors, and parts of the transcription machinery. Mediator complex is associated with the transcription machinery by touching the CTD tail of the polymerase subunit while presenting other surfaces for interaction with DNA-bound activators. In addition, mediators help initiation by regulating the CTD kinase in TFIIH. This may explain the need for Mediator to achieve significant transcription in vivo.

Epigenetic Therapy in Cancer

Various compounds that alter DNA methylation and histone modification patterns are currently being examined as single agents or in combination with other drugs in clinical settings.

Successful binding of Pol II holoenzyme at one of its promoters usually requires the action of three types of regulatory proteins:

general transcription factors, DNA-binding transcription activators, and coactivators.

Recruitment of __________ _________ can activate a gene packaged within chromatin

nucleosome modifiers

Epigenetic modifications can lead to...

stable, heritable alterations in phenotypes without alterations in DNA sequences