Chromatin Structure and its Effects on

Transcription

Epigenetics 2014 by Nigel AtkinsonThe University of Texas at Austin

From Weaver 4th editionand Armstrong 1st edition

What is the point?• DNA is not naked. Histones and other proteins

organized it into a fantastically compact unit.

• The presence of nucleosomes can interfere with the binding of TFs to enhancers and with the preinitiation complex to the promoter. = Repression

• When other proteins (simple TFs) are bound to the DNA they can prevent the histones from binding. This is competitive inhibition of histone binding. Called de-repression or antirepression in the book by Weaver.

Histone size amino acids Molecular weight

H2A 150 14,000

H2B 150 13,770

H3 150 15,400

H4 100 11,340

H1 (H5) 200 21,500

Eukaryotes five different Histone Classes

Eukaryotes contain many copies of each histone gene.10-20 in mice, 100X in Drosophila

human chromosome about 10 cm long. nucleus 10 umeter2m DNA in a human cell.

A) General information about the histones 1) Basic Histones rich in K and R which gives them a basic charge at neutral pH. Positive charge at neutral pH means that they are resistance to acid extraction (this is unusual) and migrate in the opposite direction during electrophoresis than most proteins. Eukaryotes contain many copies of each histone gene. 10-20 in mice, 100X in Drosophila Positive charge helps them interact with the negatively charged DNA. 2) Histones are evolutionarily ancient and strongly conserved molecules Histones evolved very early during the history of life on our planet. In fact, histone H3 and H4 are the most conserved proteins known. In most organisms, their amino acid sequence is identical or only slightly changed. For example, histone H4 from cows and from Mendel's pea plant are identical in all but 2 amino acids. Furthermore, these two changes are conservative amino acid changes. This indicates that the sequence of histone H4 has not significantly changed in the 109 years since plants and animals diverged (Freifelder and Malacinski, Essentials of Molecular Biology 2nd ed). 3) Compaction A chromosome is much more compact that the DNA molecule it contains. For example, if stretched out the DNA in just a single human chromosome would be about 10 cm long. A human cell contains about 2m of DNA. But a eukaryotic cell is only about 3 - 10 um in diameter and the nucleus is smaller than this. 4) Interaction with DNA DNA wraps around histone core 1.65 timesThe amount of DNA that interacts with the core is 147 bpHistones H-bind to the DNAHistones organize DNA into a nucleosome structureLinker joins the nucleosomes to each other - core + linker = ~200 bp (length of the linker is species dependent)

Details

5) Nucelosome (fig. from Tsankova et al 2007)

6) Compaction Naked -> Beads on a string -> 30 nm fiber -> etc 7) Histone H1 8) 30 nm fiber and the Tetranucleosome

9) Loops ! as regulatory units It is only recently that we have started to think about chromosome shapes and organization as a regulatory mechanism. Manipulation of chromosome conformation may be the most important regulatory mechanism. Subnuclear localization is also an important manipulation (for instance - heterochromatin is often associated with the nuclear envelope).

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

Details

Nucleosome

Images from Watson et al Molecalr Biology of the Gene

6th edition

1.75

Compaction

Doublehelix

BeadsOnAstring

zig-zag SnakeTheSolenoidPR 25

700 nm fiber

Probably involve SARS

Figure from http://upload.wikimedia.org/wikipedia/commons/4/4b/Chromatin_Structures.png

Two competing models

Images from Watson et al Molecular Biology of the Gene

6th edition

tetrasome model

Histone H1

Images from Watson et al Molecalr Biology of the Gene

6th edition

Tetranucleosome Fig 13.7

Tetranucleosomehttp://www.nature.com/nature/journal/v436/n7047/suppinfo/nature03686.html

35-80 kb loopsEach loop is very independent

(e.g. independently supercoiled).

Histones repress transcription

• Activators are responsible for much of the variation in levels of transcription of different genes.

• Why? See above!

Histones represses transcription

✤ Natural break on runaway transcription

✤ Naked DNA can be transcribed. Histones repress transcription.

✤ A difference between prokaryotes and eukaryotes?

✤ prok require active repression

✤ Histones act as basal repressors.Euk require activation to remove basal repression Figure 18.13

Weaver 4th edition

Moving them can be an act of activation!!! - Remodeling means to move them.

Modifying them can make them moveable.

The cell does this

Example given:swi/snf proteins

Histone acetylation

• amino groups of lysine side chains

• unacetylated histones tend to repress transcription

• acetylated histones tend to activate transcription

• Histone acetyl transferase (HAT)

• Histone deacetylase (HDAC)

HAT type A• Nuclear, involved in regulating gene expression

• Have bromodomain (HAT B’s do not)

• Binds aceylated lysines. So HAT A’s can recognize partially acetylated histone tails.

• Think of it like this: the work of one HAT can attract other HATs.

• Examples p55, Gcn5p CBP/p300, TAFII250

• What mechanism does this suggest? In the context of TAFII250 and core promoter recognition?

HAT type B

• cytoplasmic, acetylate newly synthesized histones H3 and H4 so that they can be assembled into a nucleosome.

• These acetylations will later be removed in the nucleus.

• no bromodomain

• NOT thought to be used in the regulation of gene expression.

Acetylation continued

• Acetylation of histone tails neutralizes some of the positive charge, causing them to relax their grip on the DNA.

• Reduces nucleosome cross-linking. That is; the interaction between histones in neighboring nucleosome. eg. basic N-terminal tail of H4 in one nucleosome and an acidic pocket in H2A-H2B dimer in the next nucleosome

Acetylation continued

• Also some TFs recognize acetylated histones. eg. TAFII250 has a double bromodomain and recognizes low level acetylated histones. Once bound it is a HAT and increases acetylation.

• low level acetylation of histones occurs in inactive chromatin.

Deacetylation

• Most eukaryotic repressors recruit corepressors

• Most corepressors complexes include histone deacetylases

• Example, NCoR/SMRT in mammals

Controlling DNA accessibility

• Histones are repressors - by regulating how tightly histones bind the DNA we regulated access to transcriptional control regions

• Acetylation helps to make DNA more accessible but by itself it is not enough. Also need additional chromatin remodeling.

• Acetylation is a form of chromatin modification that can make remodeling easier.

Chromatin remodeling

• Clearest definition

• ATP-dependent mobilization movement of nucleosomes so that DNA is accessible.

Weaver pg 382Armstrong pg 30

Chromatin remodeling

• List of protein complex familiesSWI/SNF - SwitchSniffISWI - iimitation switchCHD - Chromodomain and helicase-like domain ATPases (NuRD)INO80

• Produce nucleosome free regions around enhancers & promoters

pg 382 in Weaver 4th editionArmstrong Chapter 4

Constituitive heterochromatin,

Facultative heterochromatin,

Euchromatin

Additional levels of organization that are regulatory in nature

45-80 kb loops

InsulatorsProvides a barrier through which activation or repression cannot pass.Some also block the encroachment of condensed chromatin.Some do one, some the other.What we know is based on very few examples.Probably many different types exist.There are probably many different mechanisms.

One way that they are thought to work

Haini Cai&

Ping Shen

45-80 kb loops