Final Report Summary - REPLACOMB (A Direct Role for Nucleosome Turnover in Polycomb Response Element Function)
REPLACOMB – Dynamic organization of Polycomb Response Elements
During development the acquisition of distinct cell identity for each cell lineage involves the establishment and maintenance of conditional gene regulation in each cell type. The loss of the maintenance of such regulation often results in pathology. Pioneer work using Drosophila genetics enabled to identify factors guiding context-specific genetic programs such as Polycomb protein complexes that specifically repress target genes in different cell types (Lewis, 1978). In Drosophila, this Polycomb dependent repression requires the activity of cis-regulatory elements - that recruit sequence-specific transcription factors - also called Polycomb Response Elements (PREs) (Muller & Bienz, 1991; Simon et al., 1993; Orlando & Paro, 1993). PREs can be conditionally repressive or inactive in each cell type (Müller & Kassis, 2006), yet the transcription factors they bind are ubiquitously expressed. This poses a fundamental paradox: how to turn ON and OFF a regulatory element in a cell type-specific manner when this element depends on general factors? To address this issue will not only shed light on Polycomb repression mechanisms, but will also provide clues as to how conditional activation and repression of genes is ensured in normal cells and turned awry in pathological situations.
To address these issues, our project REPLACOMB used a combination of genetics, biochemistry and novel genome-wide chromatin analysis methodologies. Leveraging expertise from three groups (G.Almouzni K.Ahmad and S.Henikoff) and bridged by the fellow (G.Orsi) we aimed at (i) determining what characterizes the active and repressive states of PREs, and (ii) understanding how nucleosome dynamics affects this organization. Our joint efforts allowed us to make key contributions. First, we established that Polycomb proteins are directly anchored to PRE DNA through interaction with transcription factors. These transcription factors likely need to cooperate in order to recruit Polycomb complexes, characterizing repressive vs. non-repressive situations. Our latest results suggest that histone deposition factors represent structural component of PREs. Finally, our initial data in mammals indicate this overall organization is conserved, highlighting the general implication of our work in Drosophila.
Of note, during the course of our research, we developed a novel method for base pair-resolution analysis of DNA binding proteins from native chromatin that can have broad applications.
Development of a high-resolution method to map DNA-binding factors from native chromatin.
Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is by far the most widely used method to map proteins bound to DNA, as exemplified by its massive use in large consortia aiming at elucidating the epigenome, such as modENCODE, ENCODE and the Epigenome Roadmap project. However, current ChIP-seq methods have largely relied on the use of crosslinked material, exploiting formaldehyde as a crosslinking reagent, which can cause artifactual signal (Teytelman et al., 2013; Park et al., 2013). These maps are relatively low resolution and provide limited information on the nature and organization of the chromatin particles. We thus identified a need for alternative methods that could override these limitations.
Our fruitful collaboration with S.Henikoff’s laboratory enabled us to develop a simple, inexpensive method based on the use of Micrococcal Nuclease (MNase) termed ORGANIC (Organization of Regions in Genomes from Affinity-purified Naturally Isolated Chromatin)(Kasinathan et al., 2014; Orsi et al., 2015). In this method, MNase digests unprotected DNA yielding Transcription factor-DNA complexes of minimal size. These can be solubilized and immuno-purified in the absence of crosslinking reagents. We demonstrated that ORGANIC is a high-resolution method that preferentially selects for biochemically strong/stable binding sites compared to crosslinking-dependent methods (Kasinathan et al., 2014). We further showed that careful analysis of MNase-digested particles coud provide important structural information on the nature of the chromatin particle (nucleosomal, solitary transcription factors, simultaneously-bound factors).
Cooperative Organization of Polycomb Response Elements
We thus applied ORGANIC to analyze the binding sites of Polycomb proteins in Drosophila cells. We found that Polycomb is directly recruited at PREs through its interaction with transcription factors (Orsi et al., 2014). We further discovered that a previously unsuspected factor, Adf1 (Adh regulatory factor 1), is a major structural component of PREs. We provided evidence that cooperative transcription factor binding is required for Polycomb anchoring to PREs. Importantly, we showed that factors only cooperate at repressive PREs, and only these cooperative structures stably recruit Polycomb.
These findings solve the paradox in which PREs can switch their activity state in specific cells while all required factors are ubiquitous. We proposed a new model whereby transcription factor binding per se is not sufficient for the switching function, but rather the stabilization of a cooperative interaction. Consistent with these findings, we also find that nucleosome turnover is high at PREs (as at other regulatory elements) (Mito et al., 2007; Deal et al., 2010). This is in line with the detection of nucleosome assembly factors that we found localized at PREs. Finally, our preliminary ORGANIC experiments in mouse PREs suggest a possible conserved mechanism in mammals.
With novel approaches to dissect the organization of critical DNA regulatory elements, we could provide insight into how the dynamic organization of factors and nucleosomes at PREs account for their repressive function. Three publications and participation in several international conferences allowed dissemination of this work, in parallel several outreach actions enabled to raise public awareness to the importance of the topic with its societal impact.
Along with the goal of producing significant scientific results, the project was a success in terms of training of the fellow with the spirit a trainee is a trainer and helping the fellow to gain supervision skills. The fellow was thus actively engaged in (i) teaching activities through giving lectures; (ii) but also by following courses, workshops and seminars; (iii) mentoring through advising technical personnel and trainees; (iv) managing of the project’s strategy and resources; (v) networking through participation in 6 international conferences. A major outcome of this program has been his recent appointment at the INSERM in France, through a highly competitive recruitment procedure. Together, this constitutes a durable contribution to European scientific excellence through vectoring of new methods, know-how and trained personnel, as well as establishment of fruitful collaborations with outstanding international partners.
References:
Deal RB, Henikoff JG, Henikoff S. Science. 2010 May 28;328(5982):1161-4.
Kasinathan S, Orsi GA, Zentner GE, Ahmad K, Henikoff S. Nat Methods. 2014 Feb;11(2):203.
Lewis EB. Nature. 1978 Dec 7;276(5688):565-70.
Mito Y, Henikoff JG, Henikoff S. Science. 2007 Mar 9;315(5817):1408-11.
Müller J, Bienz M. EMBO J. 1991 Nov;10(11):3147-55.
Müller J, Kassis JA. Curr Opin Genet Dev. 2006 Oct;16(5):476-84.
Orlando V, Paro R. Cell. 1993 Dec 17;75(6):1187-98.
Orsi GA, Kasinathan S, Zentner GE, Henikoff S, Ahmad K. Curr Protoc Mol Biol. 2015 Apr 1;110:21.31.1-21.31.25.
Orsi GA, Kasinathan S, Hughes KT, Saminadin S, Henikoff S, Ahmad K. Genome Res. 2014 May;24(5):809-20.
Park D, Lee Y, Bhupindersingh G, Iyer VR. PLoS One. 2013 Dec 9;8(12):e83506.
Simon J, Chiang A, Bender W, Shimell MJ, O'Connor M. Dev Biol. 1993 Jul;158(1):131-44.
Teytelman L, Thurtle DM, Rine J, van Oudenaarden A. Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18602-7.
During development the acquisition of distinct cell identity for each cell lineage involves the establishment and maintenance of conditional gene regulation in each cell type. The loss of the maintenance of such regulation often results in pathology. Pioneer work using Drosophila genetics enabled to identify factors guiding context-specific genetic programs such as Polycomb protein complexes that specifically repress target genes in different cell types (Lewis, 1978). In Drosophila, this Polycomb dependent repression requires the activity of cis-regulatory elements - that recruit sequence-specific transcription factors - also called Polycomb Response Elements (PREs) (Muller & Bienz, 1991; Simon et al., 1993; Orlando & Paro, 1993). PREs can be conditionally repressive or inactive in each cell type (Müller & Kassis, 2006), yet the transcription factors they bind are ubiquitously expressed. This poses a fundamental paradox: how to turn ON and OFF a regulatory element in a cell type-specific manner when this element depends on general factors? To address this issue will not only shed light on Polycomb repression mechanisms, but will also provide clues as to how conditional activation and repression of genes is ensured in normal cells and turned awry in pathological situations.
To address these issues, our project REPLACOMB used a combination of genetics, biochemistry and novel genome-wide chromatin analysis methodologies. Leveraging expertise from three groups (G.Almouzni K.Ahmad and S.Henikoff) and bridged by the fellow (G.Orsi) we aimed at (i) determining what characterizes the active and repressive states of PREs, and (ii) understanding how nucleosome dynamics affects this organization. Our joint efforts allowed us to make key contributions. First, we established that Polycomb proteins are directly anchored to PRE DNA through interaction with transcription factors. These transcription factors likely need to cooperate in order to recruit Polycomb complexes, characterizing repressive vs. non-repressive situations. Our latest results suggest that histone deposition factors represent structural component of PREs. Finally, our initial data in mammals indicate this overall organization is conserved, highlighting the general implication of our work in Drosophila.
Of note, during the course of our research, we developed a novel method for base pair-resolution analysis of DNA binding proteins from native chromatin that can have broad applications.
Development of a high-resolution method to map DNA-binding factors from native chromatin.
Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is by far the most widely used method to map proteins bound to DNA, as exemplified by its massive use in large consortia aiming at elucidating the epigenome, such as modENCODE, ENCODE and the Epigenome Roadmap project. However, current ChIP-seq methods have largely relied on the use of crosslinked material, exploiting formaldehyde as a crosslinking reagent, which can cause artifactual signal (Teytelman et al., 2013; Park et al., 2013). These maps are relatively low resolution and provide limited information on the nature and organization of the chromatin particles. We thus identified a need for alternative methods that could override these limitations.
Our fruitful collaboration with S.Henikoff’s laboratory enabled us to develop a simple, inexpensive method based on the use of Micrococcal Nuclease (MNase) termed ORGANIC (Organization of Regions in Genomes from Affinity-purified Naturally Isolated Chromatin)(Kasinathan et al., 2014; Orsi et al., 2015). In this method, MNase digests unprotected DNA yielding Transcription factor-DNA complexes of minimal size. These can be solubilized and immuno-purified in the absence of crosslinking reagents. We demonstrated that ORGANIC is a high-resolution method that preferentially selects for biochemically strong/stable binding sites compared to crosslinking-dependent methods (Kasinathan et al., 2014). We further showed that careful analysis of MNase-digested particles coud provide important structural information on the nature of the chromatin particle (nucleosomal, solitary transcription factors, simultaneously-bound factors).
Cooperative Organization of Polycomb Response Elements
We thus applied ORGANIC to analyze the binding sites of Polycomb proteins in Drosophila cells. We found that Polycomb is directly recruited at PREs through its interaction with transcription factors (Orsi et al., 2014). We further discovered that a previously unsuspected factor, Adf1 (Adh regulatory factor 1), is a major structural component of PREs. We provided evidence that cooperative transcription factor binding is required for Polycomb anchoring to PREs. Importantly, we showed that factors only cooperate at repressive PREs, and only these cooperative structures stably recruit Polycomb.
These findings solve the paradox in which PREs can switch their activity state in specific cells while all required factors are ubiquitous. We proposed a new model whereby transcription factor binding per se is not sufficient for the switching function, but rather the stabilization of a cooperative interaction. Consistent with these findings, we also find that nucleosome turnover is high at PREs (as at other regulatory elements) (Mito et al., 2007; Deal et al., 2010). This is in line with the detection of nucleosome assembly factors that we found localized at PREs. Finally, our preliminary ORGANIC experiments in mouse PREs suggest a possible conserved mechanism in mammals.
With novel approaches to dissect the organization of critical DNA regulatory elements, we could provide insight into how the dynamic organization of factors and nucleosomes at PREs account for their repressive function. Three publications and participation in several international conferences allowed dissemination of this work, in parallel several outreach actions enabled to raise public awareness to the importance of the topic with its societal impact.
Along with the goal of producing significant scientific results, the project was a success in terms of training of the fellow with the spirit a trainee is a trainer and helping the fellow to gain supervision skills. The fellow was thus actively engaged in (i) teaching activities through giving lectures; (ii) but also by following courses, workshops and seminars; (iii) mentoring through advising technical personnel and trainees; (iv) managing of the project’s strategy and resources; (v) networking through participation in 6 international conferences. A major outcome of this program has been his recent appointment at the INSERM in France, through a highly competitive recruitment procedure. Together, this constitutes a durable contribution to European scientific excellence through vectoring of new methods, know-how and trained personnel, as well as establishment of fruitful collaborations with outstanding international partners.
References:
Deal RB, Henikoff JG, Henikoff S. Science. 2010 May 28;328(5982):1161-4.
Kasinathan S, Orsi GA, Zentner GE, Ahmad K, Henikoff S. Nat Methods. 2014 Feb;11(2):203.
Lewis EB. Nature. 1978 Dec 7;276(5688):565-70.
Mito Y, Henikoff JG, Henikoff S. Science. 2007 Mar 9;315(5817):1408-11.
Müller J, Bienz M. EMBO J. 1991 Nov;10(11):3147-55.
Müller J, Kassis JA. Curr Opin Genet Dev. 2006 Oct;16(5):476-84.
Orlando V, Paro R. Cell. 1993 Dec 17;75(6):1187-98.
Orsi GA, Kasinathan S, Zentner GE, Henikoff S, Ahmad K. Curr Protoc Mol Biol. 2015 Apr 1;110:21.31.1-21.31.25.
Orsi GA, Kasinathan S, Hughes KT, Saminadin S, Henikoff S, Ahmad K. Genome Res. 2014 May;24(5):809-20.
Park D, Lee Y, Bhupindersingh G, Iyer VR. PLoS One. 2013 Dec 9;8(12):e83506.
Simon J, Chiang A, Bender W, Shimell MJ, O'Connor M. Dev Biol. 1993 Jul;158(1):131-44.
Teytelman L, Thurtle DM, Rine J, van Oudenaarden A. Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18602-7.