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DNA methylation basics Acetylation Acetyltransferases/Deacetylases

Transcription Regulation And Gene Expression in Eukaryotes

Cycle G2 (lecture 13709) FS 2014 P Matthias & RG Clerc

P. Matthias, 16 April 2014

TFIIB

RNAPII

TFIIA TFIID

TFIIH TFIIF TFIIE

The Players in Transcription Regulation

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

Gene

Basal Machinery

DNA-binding TFs

Chromatin regulators Mediator

Co-activator

Wikipedia

DNA methylation basics

DNA methylation is usually associated with gene inactivity

Methyl-C, the 5th base

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

DNA methylation and demethylation

TET: Ten eleven translocation

7th and 8th base

5-formylcytosine 5-carboxylcytosine

HDAC

HAT

Sparmann A et al., 2006

Chromatin structure and histone modifications in eukaryotes

PresenterPresentation NotesAs all of you know, in eukaryotic cells DNA is packaged into a nucleosome structure which is composed of DNA and histone octarmer complexes. In a past decade it has been shown these histone molecules are able to be modified by several kinds of post translational modification such as acetylation, phos, met and many others. These combinations of histone modifications alter chromatin structure and binding capacity to DNA and protein, as a result they act in diverse biological processes such as gene regulation, DNA repair, DNA replication and more. Acetylation on histone lysine residue is the one of the well characterized modification among them. Generally speaking histone hyper acetylation makes chromatin structure opened and facilitate transcription factors which lead to the gene activation. On the other hand histone hypoacetylation makes chromatin structure tighten and prevent binding of them as a result gene expression is silenced. Histone acetylation is catalysed by two opposing activity enzymes. One is called HAT and the other called HDAC. HDAC remove acetyl group from histone and then shut down gene expression. In this sense HDAC is working as a gene repressor and maintaining histone as a hypo acetyl state.

23. April 2014

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/Vorinostat Zolinza®

TSA

Romidepsin Istodax®

HDAC inhibitors: TSA, SAHA, VPA, LBH …

HDAC inhibitors as drugs Cancer,

Leukemia,

PolyQ disorders (neurodegeneration),

Autoimmune and inflammatory diseases,

…

23. April 2014

Deacetylation of histones and various kinds of transcriptional regulators

Cell cycle arrest

Apoptosis

Differentiation,…

>300 clinical trials (completed or planned)

HDACs

PresenterPresentation NotesWhat we know about HDACs are mainly caming from numbers of studies about HDACis because HDACis have been known to be good candidates for cancer therapy. Importantly one HDAC inhibitor was approved for a rare type of T cell leukemia (cutaneous T cell lymphoma ) and many others are being clinical trial. Recently several publication have shown HDACis are not only working for cancer therapy but also apply different sorts of diseases such as polyQ, autoimmune, inflamatory disease…In this aspect, to study HDAC in vivo is important. How HDACi nhibitor function? They inhibit several HDACs in cells since they don’t have clear specificity for HDACs and as a outcome cell stop dividing, induce apoptosis and differentiation. These previous inhibitor work suggesting that HDACs are important for them.

(H)DAC inhibitors approved as drugs

Zolinza® (Vorinostat): FDA-approved on 6 Oct. 2006 for cutaneous T cell leukemia (CTCL) MOA: “The mechanism of the antineoplastic effect of Vorinostat has not been fully characterized” Istodax® (Romidepsin): FDA-approved on 6 Nov. 2009 for cutaneous T cell leukemia (CTCL) MOA: “The mechanism of the antineoplastic effect of Romidepsin observed in nonclinical and clinical studies has not been fully characterized”

23. April 2014

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

Widespread lysine acetylation

HDACis in the food: broccoli sprouts Dietary compounds: Na-butyrate, diallyl disulphide, sulphoraphane Found at high levels in cruciferous (Brassicaceae) vegetables

Human healthy volunteers

Human colon cells

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

Modiefied from: Knoepfler and Eisenman (1999). Sin meets NuRD and other tails of repression. Cell 99, 447-450.

Gene-specific Transcription Factors

Other Gene-specific Transcription Factors

HDAC recruitment: core complex

HDAC1 and 2 complexes

PresenterPresentation NotesHDAC1 and HDAC2 are known to be components of 3 well characterized multisubunit complexes called, sin3, co-resr and nurd. All complexes seem to contain HDAC1 and 2 as a heterodimer. According to this distribution of HDAC1 and 2 in protein complexes, they might have a redundant function in vivo.Few years ago HDAC1 straight KO mouse was reported and it have shown that KO mouse was embryonic leathal. Importantly HDAC2 expression was detected during the time period for leathality. This fact indicates that HDAC1 has specific functions.Since KO mouse is leathal, HDAC1 function in different tissue, adult mouse remain unclear. Therefore we started to analyse function of HDAC1 in tissue specificaly using Cre-loxP conditional KO system Later on to compare both HDAc1 and 2 functions in vivo, we started to generate HDAC2 conditional mouse.

• The PAH domains are suggested to be organized into two α-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 independent associations 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

PresenterPresentation NotesComprehensive HDAC interaction network. Cytoscape interaction network representing 180 SAINT‐filtered putative HDAC–prey interactions. HDAC–prey interactions were visualized by network edges. Preys were manually classified and color coded by biological processes. If known, preys were also grouped by subcomplex or function. Diamond and circle nodes indicate previously identified and uncharacterized HDAC interactions, respectively. Edge thickness indicates log2‐transformed prey spectral counts.

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

Sin3 schematic structure

ClassII HDACs as “signal transducers”

ClassII HDACs as “cytoplasmic regulator”: HDAC6

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