Histone code hypothesis

From Chapter 13 WeaverChromatin Structure and its Effects on Transcription continued

Epigenetics 2012 by Nigel AtkinsonThe University of Texas at Austin

Idea of the histone code

• The combination of histone modifications on a given nucleosome affects efficiency of transcription of the gene.

How many characters are there in this code?• More than 100 modifications have been

detected by NMR.

• Many scientists find this to be disquieting because you can make a huge number of unique patterns (code words) with a 100 character alphabet.

• Modifications in one histone can affect (allow or prevent) a modification in an another histone.

human interferon-beta gene (IFN-ß)

• Dimitris Thanos

• human interferon-beta gene (IFN-ß)

• viral infection activates it.

• TF activators bind nucleosome free areas near promoter generating an enhanceosome.

enhanceosome

enhanceosome recruits GCN5

The GCN5 HAT acetylatesH4 K8 & K3 K9

Histone code is complete & now proteins use it. Acetylated H4K8 is recognized by SWI/SNF.SWI/SNF remodels nucleosome.

Kinase binds enhanceosome& phosphoryaltes H3 S10. NOW GCN 5 can acetylate H3 K14.

TFIID is attracted by acetylated H3 K9 and H3 K14.Nucleosome now lets TFIID have access to the TATA box. which it binds.Then it bends the DNA and the nucleosome moves.

Histone code hypothesis/model

The ChIP assaychromatin immunoprecipitation

• Allows one to determine which proteins and molecules are present on a transcriptional control region.

• Allows one to detect changes in abundance of proteins and molecules on a transcriptional control region.

Decoding the Histone Acetylation Code383

Figure 1. Virus Infection Induces a Distinct Pattern of Histone Acetylation at the IFN-! Promoter In Vivo

(A) HeLa cells were either mock- or virus-infected with Sendai virus for the indicated amounts of time. Cross-linked chromatin was immunopre-cipitated with the indicated antibodies and the IFN-! promoter was detected by PCR in these samples using promoter-specific primers. Thebottom part of the figure shows the abundance of the IFN-! mRNA as detected by RT-PCR.(B) Cross-linked chromatin precipitated with the histone H4 (K5, K8, K12, and K16), H4 (K5), and H4 (K16) acetylation-specific antibodies (lane1), or beads (lane 2) was separated by SDS PAGE along with input chromatin (lane 3) followed by Western blot using the same antibodies asimmunoprobes as indicated in the figure.(C) The IFN-! enhanceosome was assembled on a biotinylated promoter fragment ("143 to #183) bearing the nucleosome ("15 to #132)followed by incubation with HeLa nuclear extracts in the absence (lanes 1 and 2) or the presence of Acetyl-CoA. The proteins were analyzedby SDS PAGE and specifically acetylated histone lysine residues were detected by Western blotting using the antibodies shown on the leftpart of the figure.(D) Same as in (C) except the templates were incubated either with complete nuclear extracts (lanes 1 and 2), CBP/p300 depleted extracts(lanes 3 and 4), GCN5/PCAF depleted extracts (lanes 5 and 6), or BRG1/BRM depleted extracts (lanes 7 and 8) followed by Western blotanalysis using an antibody that recognizes specifically acetylated H4K8.(E) Shown is a Western blot using the indicated antibodies (shown on the left) and either complete (lanes 1, 3, and 5), or CBP/p300-depleted(lane 2), or GCN5/PCAF-depleted (lane 4) or SWI/SNF-depleted-HeLa nuclear extracts (lane 6).

to occur (Agalioti et al., 2000). The acetylated lysine of CBP/p300 did not affect histone acetylation (lanes 3and 4). As a control, we showed that depletion of theresidues in histones H3 and H4 were detected by West-

ern blot analysis using specific antibodies. In agreement SWI/SNF complex did not affect the pattern of histoneacetylation (lanes 7 and 8). The Western blot of Figurewith the in vivo results (Figure 1A), the enhanceosome

recruits HAT proteins in vitro, which acetylate H4 and 1E shows that removal of GCN5/PCAF from the extractsdid not lead to codepletion of CBP and that depletionH3 at K8 and K14 (and K9, data not shown), respectively.

Thus, both in vivo and in vitro, the enhanceosome in- of CBP did not lead to depletion of GCN5/PCAF. Theseexperiments suggest that GCN5 is the primary histoneduces acetylation of histones H3 and H4 at the same

residues. To determine which of the HAT proteins re- acetylase that functions in the site-specific acetylationof the nucleosome at the IFN-! promoter. This conclu-cruited by the enhanceosome causes site-specific acet-

ylation, we repeated the above experiment using ex- sion is consistent with the observation that histone acet-ylation at the IFN-! promoter in vivo correlates bettertracts depleted for GCN5/PCAF or CBP/p300. Figure 1D

shows that depletion of GCN5/PCAF abolishes H4 K8 with the recruitment of GCN5 than with that of CBPduring the time course of virus infection (Agalioti et al.,(and H3 K14, data not shown) acetylation in vitro (com-

pare lanes 1 and 2 with 5 and 6). Surprisingly, depletion 2000, Lomvardas and Thanos, 2002).

Agalioti et al 2002Laboratory of Dimitris Thanos

Time course of Sendai virusinfection of HeLa cells.

Weaver Fig 13.30 4th ed.

• Repressed

• Stimulated

Early evidence that the histone modifications can be read in as combinations of letters.

Histone methylation in Drosophila

H2Nlys4

methyl

lys9 H3

H2Nlys4

methyl methyl

lys9 H3methyl

H4H2Nlys20

H4H2Nlys20

Now can be bound by the activator Brahmabut not by the repressors HP1 and polycomb.

Interpreted to mean that the proteins are interpreting a code.Methylated K9 has a different meaning when it is in the presence of methylated K4 and methylated K20.

Repressors HP1 & polycomb can bind.

pg 392 Weaver 4th ed.

End

MM

MM

M

a

b

c

Histone tail

DNA

Histone

P

P

P

P

Histones Histonetail

DNA

Co-Act

Basal transcription complex

Active

Permissive

Repressed

Inactive

P P

RepRep

Rep

RepRepRep

?

Histonetail

H3 K4 K9 S10 K14 K18

K23 K27 S28 K36 K79

A

A

AA A

A

AA A

A

A

A AA A

A

M M M

MMM

M

MMM

M

M

M

M M

M M

M

M

MM

MM

Deacetylation

HAT HDAC

Acetylation(activating)

PK PP

Phosphorylation(activating)

Dephosphorylation

Demethylation

Methylation(activating)

Methylation(repressing)

Demethylation

HMT HMTHDM HDM

Acetylation

Methylation

Phosphorylation

H2B

H2A

H3

H4

Transcription factor+

Overview of epigenetic mechanismsChromatin is the complex of DNA, histones and non-histone proteins in the cell nucleus. Remodelling of chromatin is a dynamic process that modulates gene expression. The fundamental unit of chromatin is the nucleosome, which consists of ~147 base pairs of DNA wrapped around a core histone octamer (~1.65 turns). Each octamer contains two copies each of the histones H2A, H2B, H3 and H4 (FIG. 1a). The nucleosomal struc-ture of chromatin allows DNA to be tightly packaged into the nucleus by organized folding5. Intricate chroma-tin remodelling mechanisms ensure that DNA remains accessible to the transcriptional machinery. These epigenetic mechanisms alter gene activity by modulat-ing DNA–protein interactions without changing the genetic code.

In simplified terms, chromatin exists in an inac-tivated, condensed state, heterochromatin, which does not allow transcription of genes, and in an activated, open state, euchromatin, which allows individual genes to be transcribed (FIG. 1b). The opening of chroma-tin is associated with acetylation of nearby histones, although it remains unclear whether acetylation mediates or reflects chromatin decondensation. In reality, chromatin can exist in many states in between

Figure 1 | General scheme of chromatin remodelling. a | Picture of a nucleosome showing a DNA strand wrapped around a histone octamer composed of two copies each of the histones H2A, H2B, H3 and H4. The amino (N) termini of the histones face outward from the nucleosome complex. b | Chromatin can be conceptualized as existing in two primary structural states: as active, or open, euchromatin (top left) in which histone acetylation (A) is associated with opening the nucleosome to allow binding of the basal transcriptional complex and other activators of transcription; or as inactive, or condensed, heterochromatin where all gene activity is permanently silenced (bottom left). In reality, chromatin exists in a continuum of several functional states (active; permissive (top right); repressed (bottom right); and inactive). Enrichment of histone modifications such as acetylation and methylation (M) at histone N-terminal tails and related binding of transcription factors and co-activators (Co-Act) or repressors (Rep) to chromatin modulates the transcriptional state of the nucleosome. Recent evidence suggests that inactivated chromatin may in some cases be subject to reactivation in adult nerve cells, although this remains uncertain. c | Summary of common covalent modifications of H3, which include acetylation, methylation and phosphorylation (P) at several amino acid residues. H3 phosphoacetylation commonly involves phosphorylation of S10 and acetylation of K14. Acetylation is catalysed by histone acetyltransferases (HATs) and reversed by histone deacetylases (HDACs); lysine methylation (which can be either activating or repressing) is catalysed by histone methyltransferases (HMTs) and reversed by histone demethylases (HDMs); and phosphorylation is catalysed by protein kinases (PK) and reversed by protein phosphatases (PP), which have not yet been identified with certainty. K, lysine residue; S, serine residue. Panels a,c modified, with permission, from Nature Rev. Neurosci. REF. 62 ! (2005) Macmillan Publishers Ltd.

REVIEWS

356 | MAY 2007 | VOLUME 8 www.nature.com/reviews/neuro

B) It's memory! Maintenance of the modification Acetylation Propagation of the modification Methylation and X-chromosome inactivation

Tsankova, N, Renthal, W, Kumar, A, Nestler, EJ (2007) Epigenetic regulation in psychiatric disorders. Nat Rev Neurosci, 8:355–367.

Extra for later

• H3K9 recruits HP1

• HP1 has chromodom and showdow chromodomain (binds nucleosomes together)

• binds Su(var)3Y9 histone methyl transferases (H3K9methylation) which triggers spreading.