11.29.11 - LCBG Department Journal Club

Journal ClubJournal Club

November 29, 2011

Farhoud Faraji Kent Hunter

Gene Expression "Programs"

• Def: A gene network whose coordinated expression affects the overall phenotype of a cell.– Proliferation, differentiation, cellular maturation

• Regulated by a concerted interplay between transcription factors, epigenetic modulation, and signaling mediators

Objective

• To gain insight into the mechanisms of "epigenomic control" of regulated programs of gene expression

• Model: Serum-induced proliferation program

E2F coordinates transcription of genes required for entry into S-phase

Regulation:

Also:• Post-translational modification• Regulation of intracellular localization

Suv39h1

• H3K9 methyltransferase– Recruits Polycomb proteins -> Heterochromatin

spreads -> Genes silenced

• Known to associate with Rb/E2F complex

• Suv39h1 transgenic mice– Overexpression: homeostatic cell proliferation– h1/h2 dbl null: viability at embryonic stage

growth as adults

Hypothesis• Suv39h1 may function as a transcriptional

repressor of E2F1

• Support:– Transgenic mouse phenotypes– Complexes with Rb/E2F– Interacts with Cbx4(Pc2) of PRC1• Cbx proteins may play a critical role in cell-cycle

progression

Subnuclear Structures

• Polycomb group protein bodies (PcG bodies)– Polycomb complex-associated heterochromatin– Transcriptionally repressive environment– Markers: Ring1A, Bmi1 (components of PRC1)

• Interchromatin granules (ICGs)– Transcriptionally active environment– Thought to couple transcription and pre-mRNA

splicing– Marker: SC35, NEAT2

Saitoh et al. Mol Bio Cell. 2004Saurin et al. J Cell Biol. 1998

Margueron & Reinberg. Nature. 2011

PRC1 compacts chromatin and catalyses the monoubiquitylation of histone H2A

Margueron & Reinberg. Nature. 2011

PRC1 compacts chromatin and catalyses the monoubiquitylation of histone H2A

Pc2

LncRNAs associate with polycomb group proteins to modify chromatin state

Aguilo et al. Cancer Res 2011Gupta et al Nature 2011

ANRIL HOTAIR

Wilusz et al. Genes Dev. 2009

Known lncRNA functions

Key Hypotheses

• Lysine methylation by histone methyl-transferases can regulate the fate of non-histone proteins

• Crosstalk between subnuclear architectural features and ncRNA could be a possible mechanism for controlling mitogenic gene expression programs

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

Pc2 is a substrate for Suv39h1 in vitro

Strategy:1. Coexpress FLAG-Pc2 and wt Suv39h1 or

Suv39h1 H324L1. anti-FLAG IP2. Trypsin Digest3. Mass-spectrometry

Which residue(s) of Pc2 are methylated?

Suv39h1 dimethylates Pc2 at K191

Pc2 K191R mutant abolishes methylation

Suv39h1 specifically methylates Pc2 in vivo

Can histone demethylases demethylate non-histone substrates such as Pc2?

KDM4C demethylates Pc2K191me2 in vitro

Is Pc2K191me2 regulated under conditions of E2F1 activation?

Pc2 methylation negatively correlates with serum stimulation

Serum-induced demethylation of Pc2 on growth-control gene promoters

Pc2K191me2

Pc2K191

Does KDM4C occupy growth control gene promoters?

Does KDM4C demethylate Histone H3K9 in response to serum stimulation?

No, KDM4C does not demethylate H3K9me2 on growth-control gene promoters in response to serum stimulation

KDM4C is recruited to growth control gene promoters and demethylates Pc2K191me2 in response to serum stimulation.

But does recruitment of KDM4C affect the expression of these genes?

KDM4C is required for serum-induced growth-control gene expression

What about Pc2?

Pc2 is essential for serum-induced growth control gene expression

Pc2 expression and KDM4C recruitment to promoters are required for expression of growth control genes.

But does the change in expression of these genes actually impact cell proliferation?

Pc2 knockdown diminishes serum-induced entry into S-phase of Hela cells ...

... and primary human fibroblasts

K191 methylation is a key regulator for cell proliferation

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

Pc2K191me2 but not unmethylated Pc2 colocalizes with Bmi1 and Ring1A

Bmi1 and Ring1A are markers of PcG bodies, a transcriptionally

repressive chromatin environment

Unmethylated Pc2 but not Pc2K191me2 colocalizes with SC35

SC35 is a marker of ICGs, a transcriptionally active

chromatin environment

Do Pc2-associated E2F1 targets relocate from PcG bodies to ICGs

upon serum stimulation?

Serum-induced relocation of growth-control gene loci between PcG bodies and ICGs

Similar analysis conducted with PCNA and MSH2

Pc2K191me2 but not unmethylated Pc2 interacts with components of PRC1

The effect of Pc2, Bmi1, and PHC1 KD on relocation of MCM3 locus between PcG

bodies and ICGs

Suggests Pc2 is required for relocation of MCM3 to ICGs

KDM4C is required for serum-induced dissociation from PcG bodies

Demethylated Pc2 is required for relocation of MCM3 locus

Recap• Pc2 is essential for serum-induced growth-

control gene expression and cell proliferation• Suv39h1 dimethylates Pc2 at K191• Methylation of Pc2K191 is a key regulator for

the expression of E2F1-dependent growth-control genes and cell proliferation

• In the absence of serum, Pc2K191me2 occupies growth control genes and relocates these transcription units to PcG body markers in a methyl-Pc2 dependent manner

Recap (cont'd)• KDM4C demethylates Pc2• In the presence of serum, KDM4C is recruited

to promoters of growth-control genes and is required for their expression

• In the presence of serum, Pc2K191 occupies growth control genes and relocates these transcription units to ICGs in a KDM4C dependent manner

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

Might Pc2 bind ncRNAs?

• Evidence suggests that ncRNA may contribute to maintaining integrity of subnuclear bodies

• Chromodomains reported to bind RNA

Pc2K191me2 and unmethylated Pc2 bind to RNAs

TUG1 – previously shown to repress cell-cycle genes

NEAT2 – exclusive signature ncRNA for ICGs

Does Pc2 binding to ncRNAs affect its histone code reading ability?

• Histone array with TUG1 or NEAT2 alone showed no interaction with histone marks

• ncRNA guide chromodomain recognition of histone marks• switch preferential binding from repressive to

activating histone marks

TUG1 colocalizes with PcG bodies

TUG1 KD: growth-control gene expression and proliferation(similar to Pc2 K191R mutation)

NEAT2 KD: growth-control gene expression and proliferation

Identifying TUG1 and NEAT2 interactors

Proteins involved in transcriptional repression

Proteins involved in transcriptional activation and splicing

ncRNAs may impose a chromatin-remodeling environment by selectively interacting with chromatin modifier proteins

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

Upshifted E2F1 band induced by serum stimulation is due to post-translational modification

E2F1 SUMOylated in vitro and in vivo at K266

SUMOylation is required for E2F1 activation of growth-control genes

Is E2F1 SUMOylation RNA-dependent?

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

Outline

1. The effects of Pc2 methylation/demethylation on growth

2. The effects of Pc2 methylation/demethylation on its subnuclear localization

3. The role of Pc2-ncRNA interaction in subnuclear localization

4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation

5. The effects of E2F1-SUMOylation on chromatin architecture

MS analysis of proteins exclusively copurified with wt E2F1 but not K266R identifies CDCA7L

CDCA7L is recruited to growth-control gene promoters and is required for growth control

gene activation in the presence of serum

CDCA7L is required for serum-induced histone H2B ubiquitination on growth-control gene promoters

CDCA7L links E2F1 SUMOylation and H2B ubiquitination in growth-control gene activation

Summary

Pc2 methylation/demethylation is a molecular switch for the inactivation/activation of the E2F1-mediated mitogenic gene expression program.

In absence of mitogenic stimuli (i.e. serum)• Non-histone methylation of Pc2 by Suv39h1

induces Pc2 to bind growth gene promoters• TUG1 sequesters Pc2K191me2 to PcG bodies

thereby repressing transcriptional activation of growth control genes

Summary

Summary (cont'd)In the presence of mitogenic stimuli:• KDM4C demethylates Pc2K191me2

• Unmethylated Pc2 associates with NEAT2 and localizes to ICGs

• Pc2 results in the Ubc9 and NEAT2-dependent SUMOylation of E2F1

• SUMO1-E2F1 binds CDCA7L which ubiquitinates H2B at promoters of growth-control genes

• Transcription of growth control genes is activated and cells enter S-phase

Thank you for your attention

Questions?

Comments/criticisms to farajif@mail.nih.gov