Investigating a role for the cohesin complex in chromatin looping, gene regulation and development

Lead Research Organisation: University College London
Department Name: Cancer Biology

Abstract

Cell division poses one of the central questions of biology, namely, how does a single cell manage to replicate and separate its entire genome ensuring that both new cells receive an exact copy of each chromosome. To do this, a specialized group of proteins, known as the cohesins, play a central role ensuring that newly replicated DNA molecules do not prematurely separate, resulting in abnormal chromosome distribution and ultimately cell death. However, recent studies have shown that cohesin proteins do more then simply hold DNA strands together. Evidence from human diseases and animal models have suggested cohesins also control gene expression in the cell. My research will focus on how cohesin proteins, with their unique ability to hold DNA molecules together, influence gene expression during cellular development and if this can be explained by their role in establishing the correct 3-dimensional organization of genes within the cell nucleus.

Technical Summary

Cohesin proteins have a well established role in sister chromatid cohesion. In addition to their structural role, new evidence implicates cohesins in the regulation of gene expression. Cohesin has been shown to colocalise with CTCF on mammalian chromosome arms. CTCF is a sequence-specific DNA binding protein known to function as a transcriptional regulator, chromatin insulator and boundary element. The functional association between CTCF and cohesin provides one way by which cohesins could influence the expression of genes.

Both inter- and intra-chromosomal CTCF-dependent chromatin loops have been described at several loci. It is likely that cohesin proteins could mediate CTCF-dependent chromatin loops due to their unique ability to establish links between chromatin fibres, as well as their colocalization to CTCF sites reported to be involved in chromatin looping. By constraining the conformation of chromatin, cohesins could affect the probability with which distal regulatory elements interact and thus gene expression. The first aim of this proposal is to address whether cohesin proteins, rather then simply behaving as architectural elements, can re-use their ability to tether chromatids to mediate chromatin loop conformations and if these conformations are important for gene function. RNAi and genetic methods will help to establish if these interactions are functional.

The binding of CTCF to chromatin is sensitive to DNA methylation and recent reports indicate that DNA methylation profiles change extensively during development. Indeed, cohesin mapping data reveals cell-type specific patterns of cohesin binding that correlate with differential methylation. The second aim of this proposal is to address whether CTCF-dependent cohesin recruitment to specific sites changes from pluripotent to committed cells. The differential localization of cohesin and CTCF proteins could function to regulate the expression of these genes, possibly through the formation of cell-type specific chromatin loops or through the recruitment of regulatory complexes whose composition changes during development.

I will use readily available molecular and computational biology techniques to study chromatin topology and cohesin localization during development, at both specific loci and genome-wide to gain insight into the role of cohesins in gene regulation.

Cohesins play a key role during cell division to prevent anueploidy. Normal cells are able to sense DNA damage or abnormal chromosome segregation and will cease to divide whereas cancer cells can override these checkpoints and accumulate abnormal DNA. Thus, having a basic understanding of the mechanisms and proteins which facilitate normal chromosome transmission and function will be directly relevant to an understanding of cancer.
 
Description EU FP7 EpiGeneSys Network of Excellence
Amount £115,000 (GBP)
Organisation Community Research and Development Information Service (CORDIS) 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 06/2012 
End 06/2015
 
Description MRC Project Grant
Amount £350,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2011 
End 12/2015
 
Title 3D-FISH and confocal microscopy 
Description we have established the FISH technique in the lab, applied it to many cell types, learned how to collect the images from these experiments and analyse them using image analysis software programs 
Type Of Material Biological samples 
Provided To Others? No  
Impact this has allowed us to move our research into single cell analysis 
 
Title 4C-Seq and MiSeq 
Description a proximity based assay complexed with a sequencing platform not previously used in our Institite. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact this technology was used in our recent publication in the EMBO J 
 
Title AID-proteosome vectors 
Description using these vectors, we will engineer ES cell lines containing tagged AID cohesin subunits with the ultimate goal of generating a new mouse strain 
Type Of Material Cell line 
Provided To Others? No  
Impact this will be a very valuable addition to our research as it will allow us to reversibly remove proteins from cells very quickly. 
 
Title Animals 
Description we have Rad21 conditional animals, SA1 conditional animals, Pds5a and Pds5b animals and Nestin-Cre animals for breeding 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact we will use these animal models to build in vivo models of our research 
 
Title Neural Stem Cell lines 
Description Differentiated NSC from conditional ES cells 
Type Of Material Cell line 
Provided To Others? No  
Impact Ability to compare different stem cell populations with increased specialization and the ability to generate post-mitotic cells from these. 
 
Title multiple conditional knockout cell lines 
Description obtained some ES cells with conditional mutations of CTCF, SA1, SA2 and Wapl. 
Type Of Material Cell line 
Provided To Others? No  
Impact these different cell lines will provide us with a panel of genetic mutants which we can use in multiple ways. 
 
Description Computational Analysis 
Organisation Weizmann Institute of Science
Department Department of Biological Regulation
Country Israel, State of 
Sector Academic/University 
PI Contribution We have provided HiC sequencing data
Collaborator Contribution teaching us how to do the analysis of these data
Impact this collaboration has resulted in a publication in the EMBO J in 2013.
Start Year 2011
 
Description FRAP analysis 
Organisation The Hebrew University of Jerusalem
Country Israel, State of 
Sector Academic/University 
PI Contribution We have been generating stably expressing ES cells with flourescent reporters for downstream FRAP studies to be performed in collaboration with Eran Meshorer in Isreal.
Collaborator Contribution My collborators will perform the downstream FRAP studies and analysis of the data.
Impact BACs with flourescent tags to our geens of interest have been obtained.
Start Year 2013
 
Description Fluorescence 3 Hybrid Assay 
Organisation Ludwig Maximilian University of Munich (LMU Munich)
Country Germany, Federal Republic of 
Sector Academic/University 
PI Contribution We will use this system to tag CTCF and cohesin and search for regulators of their direct interaction in vivo.
Collaborator Contribution They have provided the GFP-BP constructs.
Impact not yet.
Start Year 2015
 
Description In vitro derivation of neural stem cells 
Organisation University College London (UCL)
Department University College London (UCL), UCL Cancer Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We have contributed to this collaboration by learning how to grow and establish NS lines, maintain them and test their differentiation state by markers in RT-PCR expts.
Collaborator Contribution Jointly we have established neural stem cell lines from ES cultures in vitro. The expertise for this came through this collaboration.
Impact outcomes include cell lines used in downstream analysis and expts.
Start Year 2010
 
Description RNA-Seq analysis of NS and AST 
Organisation Medical Research Council (MRC)
Department Biomedical NMR Centre at NIMR
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We prepared RNA-SEQ libraries and data from both NSC and AST populations
Collaborator Contribution they further analysed our datasets
Impact My PhD student and myself were authors on the work which was published in Genes and Dev this year.
Start Year 2012
 
Description SA1 and SA2 in chromatin interactions 
Organisation Spanish Ministry of Economy and Competitiveness
Department Spanish National Cancer Research Centre (CNIO)
Country Spain, Kingdom of 
Sector Academic/University 
PI Contribution we will provide an analysis of chromatin interactions using 4C-seq in cell deficient for SA1 and SA2 complexes
Collaborator Contribution provide the SA1 and SA2 conditional animals
Impact this collaboration has been recently initiated
Start Year 2013
 
Description Sequencing capacity 
Organisation Illumina Inc.
Country United States of America 
Sector Private 
PI Contribution We have been working with Illumina to prepare and setup sequencing of our libraries.
Collaborator Contribution They have provided the reagents and the sequencing capacity for us
Impact We have prepared a number of libraries; RNA-Seq, ChIP-Seq and HiC which have been successfully sequenced and analysed.
Start Year 2011
 
Description Smc3-Ac ChIP 
Organisation University of Tokyo
Country Japan 
Sector Academic/University 
PI Contribution We have asked whether the separation of sister chromatids after transcription through a gene is correlated to the acetylation of Smc3 proteins, thought to be necessary for establishment of cohesion
Collaborator Contribution by providing us with a specialized antibody to Smc3-Ac that can be used in ChIP.
Impact currently analysing ChIP data and confirming the soecificity of the antibody.
Start Year 2011
 
Description Athena Swan Speaker Programme 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact I organized a monthly speaker programme on behalf of the Athena Swan Initiative for my Institute and for UCL. the talks were given by influential women in the UK in multiple sectors and ~ 100-150 people attend each sesion.

we aimed to inspire women to stay in academia.
Year(s) Of Engagement Activity 2013
 
Description CIRT Fundraising Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A fudraising event where my role was to speak to the audience about how my work is relevant to cancer research and answer any questions they have.

We raised soem funds to help support the cost of a confocal microscope for the Institute.
Year(s) Of Engagement Activity 2012
 
Description Hosting of highschool student for summer experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact one female high school student from Canada spent a month in our lab gaining experience in basic science.

she went on to win the science award for her school back in Canada the following term
Year(s) Of Engagement Activity 2011