www.fmi.ch/training/teaching

DNA methylation basics

Acetylation

Acetyltransferases/Deacetylases

Transcription Regulation And Gene Expression in Eukaryotes

Cycle G2 (lecture 13709), P. Matthias, April 20th, 2016

Epigenetics & Chromatin

TFIIB

RNAPII

TFIIATFIID

TFIIH TFIIFTFIIE

The Players in Transcription Regulation

• DNA-binding transcription factors (upstream factors)• Chromatin regulators• Coactivators and corepressors: Mediator, etc..• Basal Machinery: RNA PolII, GTFs

Gene

BasalMachinery

DNA-binding TFs

Chromatin regulatorsMediator

Co-activator

Wikipedia

DNA methylation basics

DNA methylation is usually associated with gene inactivity

Methyl-C, the 5th base

B. Lewin, Genes VIII

B. Lewin, Genes VIII

Maintenance of DNA methylation

Distribution of methyl groups in the genome: CpG islands

Distribution of methyl groups in the genome: CpG islands

CpG islands are not methylated

Genes & Dev, 2: 1127 (1988)

Genes & Dev, 3: 612 (1989)

24/31 DECEMBER 2015 | VOL 528 | NATURE | 575

Cell, Vol. 64, 1123-1134, March 22, 1991,

DNA methylation and demethylation

TET: Ten eleven translocation

7th and 8th base

5-formylcytosine5-carboxylcytosine

3. Mai 2016

HDAC

HAT

Sparmann A et al., 2006

Chromatin structure and histone modifications in eukaryotes

NaButyrate-treated

Control

H3, H4 mononucleosomes can be acetylated

NaButyrate-treated

+DNAseI

NaButyrate-treated

H3, H4 mononucleosomes

Ratio H4-3/H4-0 = 0.9

Ratio H4-3/H4-0 = 1.9

TJ Richmond, Nature 1997 vol. 389 (6648) pp. 251-60

Reversible Lysine acetylation

The effects of Histone lysine acetylation

P. Jenö, Lecture 4610

In-gel assay

Histones BSA

Tetrahymena crude Macronuclear activity

Some of the chromatin-binding domains present in HAT complexes in yeast

HATs: Complexes and single proteins

More HATs: CIITA, TFIIB, CLOCK,…

Histone Acetyltransferases (HATs): GNAT family

Histone Acetyltransferases (HATs): MYST family

Some HAT inhibitors: anacardic acid, curcumin, garcinol…

Elp3: HAT of GNAT family

P. Jenö, Lecture 4610

Some HDAC inhibitors

SAHA/VorinostatZolinza®

TSA

RomidepsinIstodax®

HDAC inhibitors:

TSA, SAHA, VPA, LBH …

HDAC inhibitors as drugs

Cancer,

Leukemia,

PolyQ disorders (neurodegeneration),

Autoimmune and inflammatory diseases,

…

3. Mai 2016

Acetylation of histones, transcriptional regulators and other

targets

Cell cycle, Apoptosis, Autophagy,

M tilit

Hundreds of clinical trials

HDACs

MOA: “The mechanism of the antineoplastic effect of Vorinostat/ Romidepsinhas not been fully characterized”

“We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275.”

Widespread lysine acetylation

HDACis in the food: broccoli sproutsDietary compounds: Na-butyrate, diallyl disulphide, sulphoraphaneFound at high levels in cruciferous (Brassicaceae) vegetables

Human healthy volunteers

Human colon cells

Seminars in Cancer Biology 17 (2007) 363-369

Classical Zn-dependent HDACs

NAD-dependent HDACs: Sirtuins

Rajundran et al., JBB 2011, doi:10.1155/2011/368276

Ahringer (2000). NuRD and SIN3. Trends Genet. 16, 351-356.

NuRD and Sin3 Complexes

NuRD Sin3

HDAC1 and 2 complexes

• The PAH domains are suggested to be organized into two a-helices separated by a flexible spacer region

• They are among the most evolutionarily conserved regions of the large SIN3 proteins (100–170 kDa)

• These domains are important for Sin3 function through their independentassociations with various repressors and other associated proteins

• PAH2 is both necessary and sufficient for interaction with the Mad proteins

HDAC Interaction Domain

Paired Amphipathic Helix (PAH)Domains

Sin3 schematic structure

Sin3 schematic structure

Comprehensive HDAC interaction network.

Joshi P et al. Mol Syst Biol 2013;9:672

©2013 by European Molecular Biology Organization

Phenotypes of class I HDACs mouse knockouts

Phenotypes of class I HDACs mouse knockouts

Reichert, Choukrallah and Matthias, CMLS, 2012 DOI 10.1007/s00018-012-0921-9

ClassII HDACs as “signal transducers”

ClassII HDACs as “cytoplasmic regulator”: HDAC6

Cre transgenic mouse Cre

Cell type specific expression

HDAC1

HDAC2

482 aa

489 aa

Exon 6HDAC domain

HDAC1HDAC2

KYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYYFPGTGDLRKYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYYFPGTGDLR

Hdac2 floxed mouseExon 14Exon 1

Hdac1 floxed mouseExon 6 Exon 15Exon 1

Essential for catalytic activity** *

Conditional inactivation of HDAC1 and HDAC2 in specific lineages

Residue specificity for HDAC1 and 2

p21 and p57 are upregulated in HDAC1 and HDAC2 deficient cells

HDAC1HDAC2

days0 0.5 1 2 3 4 5

HDAC2 Kd cells HDAC1/2 KO/Kd cells

p21

Days afterTamoxifen:

Actin

0

1

2

3

4

5

6

7

8

9

10

0 1 1 2 3 4 5-0,1

0,1

0,3

0,5

0,7

0,9

1,1

1,3

1,5

HDAC-1p21

Upregulation of p21 and p57 in MEFs lacking HDAC1 and 2

Yamaguchi et al., G&D 24: 455-469

Direct regulation of the p21 and p57 genes

Yamaguchi et al., G&D 24: 455-469

3. Mai 2016Orkin & Zon, Cell 132, 2008

Mb1-Cre

HDAC inactivation in the B cell lineage

B cells are almost lacking in vivo after deletion of HDAC1 and 2

Early B cell development

Bone Marrow Spleen, periphery

Plasma cell

V(D)J recombination

Pre-BCR BCR

VJ recombination

mb1-Cre

CD4

CD

8

T cells

T cells

Spleen cellsSpleen cells

WT

B220

IgM

Mature Bcells

HDAC1/2 DKO

Mature Bcells

Differentiation

Bone Marrow cells

B220

IgM

Pro-pre B cells

B cells

B cells

Pro-pre Bcells

HDAC1/2 dKO mice have strongly reduced B cell populations

B cells are almost lacking in vivo after deletion of HDAC1 and 2

Block at preB cell stage in absence of HDAC1 and 2

mb1-Cre

4x

Pre BI

100x

Pre BI Pre BIIPro B

Pre BI Pre BII

Cell cycle block at preB cell stage in absence of HDAC1 and 2

7-AAD

Brd

U

3. Mai 2016

Pre BII

WT

dKO

Pre BI

Yamaguchi et al., G&D 24: 455-469

Apoptosis at preB cell stage in absence of HDAC1 and 2

HDAC1/2 are associated with p21 and p57 promotersp21 and p57 are directly regulated by HDAC1 and 2

December 13, 2010

Normal mature resting B cell numbers in absence of HDAC1 and 2

cd23-Cre

Normal mature resting B cell numbers in absence of HDAC1 and 2

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