Lecture 8: Histone Acetylation

Explain what histone acetyltransferases do chemically, acetylation of the ε-amino group of lysine with acetyl-CoA as the donor, say what this does to the net positive charge of the residue, and note that acetylation also occurs non-enzymatically

• Histone acetylation is done by enzymes called histone acetyltransferases (HATs)

• Acetylate the ε-amino group (epsilon group) of lysine residues

• ε-amino group provides positive charge to lysine

• Acetylation decreases net positive charge of lysine

• Acetyl-CoA acts as the acetyl donor, linking acetylation to the metabolic state of the cell

• Acetylation also occurs non-enzymatically

• Molecular basis of substrate specificity is not well understood

Name the three major HAT families, GNAT, MYST and p300/CBP, give one founding member of each, state that HATs also carry a KAT designation, give the histone specificity of at least one enzyme from each family, and say which genomic features H3K9ac and H3K27ac mark

• Three major families

• Each HAT has a KAT number and target residues (table below)

• H3K9ac marks active promoters; H3K27ac marks active enhancers

• MYST family: Founded by MOZ (human monocytic leukemia zinc-finger HAT), yeast YBF2, SAS2, and TIP60. All members contain a conserved region called the MYST domain

• GNAT family: Gcn5-related N-acetyltransferase

• p300 family

Explain the two modes of regulating acetyltransferase activity, assembly into multiprotein complexes, which sets substrate specificity and supplies the partner subunits a HAT needs to act on nucleosomes, and autoacetylation, and name the complexes formed by the Gcn5/pCAF and MYST families with the histones each acetylates.

Two major modes of regulation:

1. Assembly into multiprotein complexes:

• Assembly regulates activity and sets substrate specificity, which residue of which protein is targeted (see the HAT table)

• Assembly also supplies the partner subunits a HAT needs: Gcn5 alone acetylates free histones but requires Ada2 and Ada3 to acetylate nucleosomes

• Gcn5/pCAF form SAGA and SLIK in yeast, TFTC and STAGA in humans, and acetylate H2B, H3 and H4

• MYST form NuA3 and NuA4, and acetylate H3 and H4

2. Autoacetylation of the enzyme itself

• Certain HATS can autoregulate by autoacetylation

Explain autoacetylation of p300/CBP, including the activation loop, the hyperacetylated and hypoacetylated states, and the deacetylase that reverses it

• p300/CBP family is well known example:

1. Contains 40 lysine residues in its activation loop

2. Protein is active when hyperacetylated

3. Protein is inactive when hypoacetylated

4. SIRT2 removes these marks and inactivates p300

Explain how HATs promote transcription by delivering TBP to the core promoter, and contrast the TFIID and SAGA routes

• HATs are part of complexes that deliver TATA-binding protein (TBP) to core promoter:

1. As part of TFIID complex

2. TAF145 has HAT activity

• SAGA (SPT-ADA-GCN5 acetyltransferase)

• TFIID and SAGA are two routes to the same end

Define a bromodomain, explain why bromodomains occur in tandem arrays, and describe what the six bromodomains of PB1 do

• Proteins with bromodomain recognize and bind to acetylated lysine

• Bromodomain exists in multiples:

1. Number varies by organism: six in PB1, one or two in yeast Rsc

2. These may provide specific binding to different acetylated histone lysines

• PB1 (Polybromo 1) can have six bromodomains:

1. Four domains bind H2A, H2B, H3, H4, respectively

2. The other two may provide stability to the complex by binding non-specifically to any acetylated lysine once the complex is formed by the first four bromodomains

Describe how bromodomain-containing proteins couple acetylation to nucleosome remodeling through Swi/Snf, and state what remains unknown about their sequence specificity

• The bromodomain containing proteins form complex with other proteins. Alter chromatin

• Swi/Snf complexes have bromodomain containing proteins that bind to acetylated lysine. Remodel nucleosomes

• Knowledge about sequence dependent recognition by bromodomain containing proteins is still limited

Name the two HDAC families and the four classes, say which cofactor each family requires, and say where in the cell different HDACs are found

• Two families:

1. Histone deacetylase family

2. Sir2 regulator family

• Four classes

• Class I, II, and IV belong to histone deacetylase family. These use zinc as a cofactor for deacetylation

• Class III belong to Sir2 regulator family. These use Nicotinamide Adenine Dinucleotide (NAD+) as a cofactor for deacetylation

Explain why HDAC1, HDAC2 and HDAC3 have little activity alone, name the four complexes that activate them, give the four-protein core shared by Sin3 and NuRD, and explain how these complexes couple deacetylation to DNA methylation and ATP-dependent remodeling, from methylated CpG through MeCP2 and Sin3A to transcriptional silencing

• Classes I, IIa, IIb and IV are the classical HDACs and use zinc

• Class III are the sirtuins and use NAD+

• Localization varies: nuclear, cytoplasmic, mitochondrial or nucleolar

• Regulation is achieved by interaction with other proteins in a complex

• HDAC1, 2 and 3 have very low enzymatic activity individually

• These HDACs must be present in complexes with other proteins

• HDAC1, 2 and 3 are found in four complexes: Sin3, NuRD, CoREST and NCoR/SMRT

• Sin3 and NuRD share a common core of four proteins: HDAC1, HDAC2, RbAp48 and RbAp46

• Involved in ATP-dependent remodeling – via Mi2 subunit

• Binds to methylated DNA via MBD3

• Presence of MBD allows deacetylation of histones of the nucleosomes bound to methylated DNA

• NURD complex also binds to transcriptional repressors for specific genes

• NURD is an example of how histone deacetylation, DNA methylation, and chromatin remodeling work together to suppress gene activity

• Acts as a scaffold protein for HDAC complexes

• Recruited by protein-protein interactions with sequence-specific DNA-binding proteins

• Methyl-CpG binding proteins (e.g., MeCP2) and ATP dependent chromatin remodeling proteins also bind to Sin3 complex

• MeCP2 at methylated CpG recruits Sin3A, HDACs and the BRM remodeler, silencing the gene

Explain how phosphorylation regulates HDACs, including why it activates HDAC1, HDAC2 and HDAC8 but inactivates HDAC4 by driving 14-3-3 binding and export to the cytoplasm

• Phosphorylation activates HDAC1,

• HDAC2 and HDAC8, but inactivates HDAC4

• Dephosphorylation reverses each of these effects

• Phosphorylation can affect localization of HDAC in the cell

1. Ex: By affecting its interaction with 14-3-3 protein

2. This affects the activity of HDAC