PPU2014-40 PhD PROPOSAL FOR THE PASTEUR-PARIS UNIVERSITY INTERNATIONAL PROGRAM

 

Deadline for full application: December 15th, 2013

Interviews: March, 2014

Start of the Ph.D.: October 1st, 2014

 

 

 

Department: Developmental & Stem Cell Biology

Title of the PhD project: Role of the chromatin in the regulation of the alternative splicing

Name of the lab: Unit of Epigenetic Regulation

Head of the lab: Christian MUCHARDT

PhD advisor: Eric BATSCHE

Email address:  batsche@pasteur.fr ; muchardt@pasteur.fr

Web site address of the lab:

http://www.pasteur.fr/ip/easysite/pasteur/en/research/scientific-departments/developmental-e-stem-cells-biology/units-and-groups/epigenetic-regulation/index

Doctoral school affiliation and University: iViV (ED387) Paris 6/7

 

 

Presentation of the laboratory and its research topics:

 

Histones organize the cellular DNA into chromatin. These histones are subject to numerous post-translational modifications and function as a storage medium for epigenetic information. The writing of this information involves enzymes guided to their target sites by transcription factors, but also RNAs and RNA-binding proteins, often as a response to activated signal transduction pathways. The resulting patterns of histone modifications in turn affects both the machineries involved in transcription and those involved in maturation of the transcripts. Thus, chromatin defines the cellular transcriptome in a broad sense. Our objective is to explore the crosstalk between the chromatin factors, the transcription machineries and the RNAs that they produce to gain a better understanding of transcriptional regulation in the context of chromatin.

 

Chromatin factors and the transcriptional regulation of inducible genes during development and in diseases

The advent of protocols allowing generation of induced pluripotent stem cells has revealed that genes turned off during cell differentiation are stably but not definitively repressed, and that the “silencing” machineries involved in this repression share mechanisms and properties with the more transient repression detected at inducible promoters.

H3K9 trimethylation (H3K9me3) are among the histone marks that function in both stable “silencing” of repeat rich regions and transient repression of euchromatic genes. Consistent with this, HP1 proteins that bind this histone modification are enriched at pericentromeric and interspersed repeats, while they also function as very general regulators of inducible genes involved in development, cell differentiation, cell cycle, and immune response.

Consistent with the role of HP1 proteins in the transcriptional control of both inducible genes and repeated DNA sequences, we have now shown that in patients with Multiple Sclerosis a defected in HP1-mediated silencing causes reactivation of both pro-inflammatory cytokines and human endogenous retroviruses (HERVs). In a subset of the patients, this reactivation is correlated with increased activity of the peptidyl arginine deiminase PADI4 that interferes with the binding of HP1 proteins to H3K9me3 by converting the neighbouring arginine 8 into a citrulline.

Currently, we are exploring additional mechanisms of interaction of HP1 proteins with chromatin. In this context, we find that HP1 proteins contact not only the histone H3 tails, but also regions inside the nucleosomal barrel and that this interaction is regulated by several histone modifications.

Finally, we are screening for small molecules allowing for a chemical control of HP1-mediated silencing, with a special interest for molecules able to facilitate induction of pluripotent stem cells.

 

Chromatin factors and the regulation of alternative splicing

Alternative splicing is a major source of diversity for the proteome. A few years ago, our laboratory provided pioneering data showing that chromatin and chromatin factors play a role the regulation of this alternative splicing.

Along this track, we have now shown that alternative splicing is affected by a novel machinery combining nuclear proteins involved in RNAi and splicing factor.

Within this machinery, Argonaute 1 and 2 (AGO1 and AGO2) participate in the recruitment to intragenic chromatin of both chromatin-structuring factors (HP1 and histone methylases) and components of the spliceosome in response to activation of the MAP kinase pathway.

Altogether, our data suggests that this machinery facilitates inclusion of alternative exons by locally assisting the rapid assembly of a functional spliceosome and by generating chromatin structures interfering with the elongation rate of the RNA polymerase II (RNAPII).

Currently, we are exploring the mechanism allowing for the targeted recruitment of the machinery inside the coding region of actively transcribed genes with a special interest for the nature and the origin of the small RNAs associated with AGO1 and AGO2 in the nucleus.

In parallel, we have elaborated an in vitro transcription-splicing system on chromatinized templates that allows us to biochemically explore the connections between chromatin and splicing.

 

 

Description of the project:

 

Nearly 90% of genes are alternatively spliced (AS) and disruptions of this process are the cause of many diseases. Pre-messenger RNAs (pre-mRNAs) maturation is initiated cotranscriptionally and chromatin-borne information participates in the regulation of alternative splicing. Our team was first to show the involvement of chromatin regulators in the AS regulation (Batsché et al., 2006) and pioneered the field linking chromatin to splicing. The regulation of AS by the chromatin may be mediated by modulation of the speed of the elongating RNAPII, and/or by the recruitment of splicing factors. Any obstacle on the path of the RNAPII could contribute to a change in the expression of splice variants. We have found recently that elevated levels of trimethylation of histone H3 on Lys9 (H3K9me3) and the chromodomain protein HP1γ are a characteristic of the alternative exons of several genes including CD44 (Saint-André et al. 2011). On this gene, we have shown that the endogenous RNA interference pathway is involved in the H3K9me3 targeting on the variant exons through the chromatin recruitment of the nuclear argonaute proteins (AGO1 or AGO2) (Ameyar-Zazoua et al. 2012). These results suggest that long intragenic non-coding RNAs (LincRNA) are involved in the AS regulation through the recruitment of the AGO-dependent silencing machinery.

 

In this project, we propose to characterize the effects of cryptic intragenic transcription on AS of protein-coding genes. A first task will involve exploration of the mechanisms that promote changes in AS mediated by the expression of lincRNAs. This task will combine large scale genomic and transcriptomic approaches to identify cryptic transcripts associated with specific splicing events. A second phase will be dedicated to the exploration of the role lincRNAs in aberrant splicing during carcinogenesis. Indeed, splicing defects can contribute to several aspects of tumor progression, including the control of cell proliferation, apoptosis, and differentiation. While levels of splicing factors are affected in most types of cancer, some breast cancers seem devoid of changes in these factors. Our working model is here that alterations in the structure of chromatin and expression of lincRNAs could explain the splicing defects observed in for example breast carcinomas. This paradigm shall be challenged using a well-described cellular model of breast tumor progression. Functional analysis of the genes identified as affected by alternative spliced in these cells will further allow determining what kind of regulatory chromatin complexes controls the expression of cryptic transcripts as wel as their influence on AS regulation.

Defects of LincRNA expression and of their potential regulatory complexes could open new perspectives to understand disregulations occurring in cancer cells without involving DNA mutations.

 

 

References:

 

Ameyar-Zazoua M, Rachez C, Souidi M, Robin P, Fritsch L, Young R, Morozova N, Fenouil R, Descostes N, Andrau JC, Mathieu J, Hamiche A, Ait-Si-Ali S, Muchardt C*, Batsché E* Harel-Bellan A*, (2012) Nat. Struc. Mol. Biol 19:998-1004. Argonaute proteins couple chromatin silencing to alternative splicing.

 

Saint-André V., Batsché E., Rachez C. and Muchardt C. (2011) Nat. Struc. Mol. Biol   18:337-44.

Histone H3 lysine 9 tri-methylation and HP1γ favor inclusion of alternative exons. Recommended by F1000 Biology

 

Batsché E, Yaniv M and Muchardt C (2006) Nat. Struc. Mol. Biol   13:22-9.

The human SWI/SNF subunit Brm is a regulator of alternative splicing.

 

 

Expected profile of the candidate (optional):

Skills in Bioinformatic will be an advantage

 

 

Contact:

 

Christian Muchardt

Director, Unit of Epigenetic Regulation

Deputy Director, Dpt of Developmental and Stem Cell Biology

URA2578 CNRS - Equipe FRM

Jacques Monod Bldg.

Institut Pasteur

25 rue du Dr-Roux

75724 PARIS CEDEX 15

FRANCE

Tel: 33 1 45 68 85 25 / 33 1 45 68 86 25

Cellphone: 33 6 76 09 84 37

Fax: 33 1 45 68 89 76

Mail: muchardt@pasteur.fr

Mis à jour le 16/09/2013