Publications by authors named "Neil Brockdorff"

88 Publications

The PWWP2A Histone Deacetylase Complex Represses Intragenic Spurious Transcription Initiation in mESCs.

iScience 2020 Nov 29;23(11):101741. Epub 2020 Oct 29.

Development Epigenetics, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

Transcriptional fidelity depends on accurate promoter selection and initiation from the correct sites. In yeast, H3K36me3-mediated recruitment of the Rpd3S HDAC complex to gene bodies suppresses spurious transcription initiation. Here we describe an equivalent pathway in metazoans. PWWP2A/B is an H3K36me3 reader that forms a stable complex with HDAC1/2. We used CAGE-seq to profile all transcription initiation sites in wild-type mESCs and cells lacking PWWP2A/B. Loss of PWWP2A/B enhances spurious initiation from intragenic sites present in wild-type mESCs, and this effect is associated with increased levels of initiating Pol-II and histone acetylation. Spurious initiation events in DKO mESCs do not overlap in genomic location or chromatin features with spurious sites that arise in KO mESCs, previously reported to function in the suppression of intragenic transcriptional initiation, suggesting these pathways function cooperatively in maintaining the fidelity of transcription initiation in metazoans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.isci.2020.101741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670215PMC
November 2020

Progress toward understanding chromosome silencing by Xist RNA.

Genes Dev 2020 06;34(11-12):733-744

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.

The X inactive-specific transcript () gene is the master regulator of X chromosome inactivation in mammals. produces a long noncoding (lnc)RNA that accumulates over the entire length of the chromosome from which it is transcribed, recruiting factors to modify underlying chromatin and silence X-linked genes in Recent years have seen significant progress in identifying important functional elements in Xist RNA, their associated RNA-binding proteins (RBPs), and the downstream pathways for chromatin modification and gene silencing. In this review, we summarize progress in understanding both how these pathways function in Xist-mediated silencing and the complex interplay between them.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.337196.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263139PMC
June 2020

The many faces of Polycomb regulation by RNA.

Curr Opin Genet Dev 2020 04 11;61:53-61. Epub 2020 May 11.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom. Electronic address:

Many intricate pathways contribute to the timely control of gene expression during development. Polycomb repressive complexes (PRC1 and PRC2) and long non-coding RNAs (lncRNAs) are players associated with gene repression in various developmental processes such as X chromosome inactivation (XCI) and genomic imprinting. Historically, lncRNAs were proposed to directly recruit PRC2. However, recent evidence suggests that promiscuous interactions between PRC2 and RNA fine-tune the function of the complex through a multiplicity of mechanisms. A PRC2-recruitment model was definitively overturned in the paradigm of XCI by Xist RNA, being replaced by a novel mechanism which puts PRC1 in the spotlight. This review focuses on these recent advances in understanding the interplay between RNA and Polycomb complexes for gene expression control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.gde.2020.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653676PMC
April 2020

The role of the Xist 5' m6A region and RBM15 in X chromosome inactivation.

Wellcome Open Res 2020 17;5:31. Epub 2020 Feb 17.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

: X chromosome inactivation in mammals is regulated by the non-coding (nc) RNA, Xist, which represses the chromosome from which it is transcribed.  High levels of the N6-methyladenosine (m6A) RNA modification occur within Xist exon I, close to the 5' end of the transcript, and also further 3', in Xist exon VII. The m6A modification is catalysed by the METTL3/14 complex that is directed to specific targets, including Xist, by the RNA binding protein RBM15/15B. m6A modification of Xist RNA has been reported to be important for Xist-mediated gene silencing.  : We use CRISPR/Cas9 mediated mutagenesis to delete sequences around the 5' m6A region in interspecific XX mouse embryonic stem cells (mESCs).  Following induction of Xist RNA expression, we assay chromosome silencing using allelic RNA-seq and Xist m6A distribution using m6A-seq. Additionally, we use Xist RNA FISH to analyse the effect of deleting the 5' m6A region on the function of the endogenous Xist promoter. We purify epitope tagged RBM15 from mESCs, and then apply MS/MS analysis to define the RBM15 interactome. : We show that a deletion encompassing the entire Xist 5' m6A region results in a modest reduction in Xist-mediated silencing, and that the 5' m6A region overlaps essential DNA elements required for activation of the endogenous Xist promoter. Deletion of the Xist A-repeat, to which RBM15 binds, entirely abolishes deposition of m6A in the Xist 5' m6A region without affecting the modification in exon VII. We show that in mESCs, RBM15 interacts with the m6A complex, the SETD1B histone modifying complex, and several proteins linked to RNA metabolism. : Our findings support that RBM15 binding to the Xist A-repeat recruits the m6A complex to the 5' Xist m6A region and that this region plays a role in Xist-mediated chromosome silencing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.12688/wellcomeopenres.15711.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7097882PMC
February 2020

Localized accumulation of Xist RNA in X chromosome inactivation.

Authors:
Neil Brockdorff

Open Biol 2019 12 4;9(12):190213. Epub 2019 Dec 4.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

The non-coding RNA Xist regulates the process of X chromosome inactivation, in which one of the two X chromosomes present in cells of early female mammalian embryos is selectively and coordinately shut down. Remarkably Xist RNA functions , affecting only the chromosome from which it is transcribed. This feature is attributable to the unique propensity of Xist RNA to accumulate over the territory of the chromosome on which it is synthesized, contrasting with the majority of RNAs that are rapidly exported out of the cell nucleus. In this review I provide an overview of the progress that has been made towards understanding localized accumulation of Xist RNA, drawing attention to evidence that some other non-coding RNAs probably function in a highly analogous manner. I describe a simple model for localized accumulation of Xist RNA and discuss key unresolved questions that need to be addressed in future studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1098/rsob.190213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936258PMC
December 2019

Systematic allelic analysis defines the interplay of key pathways in X chromosome inactivation.

Nat Commun 2019 07 16;10(1):3129. Epub 2019 Jul 16.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing. Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/m6A-methyltransferase complex make only minor contributions to gene silencing. Together our results provide a comprehensive model for Xist-mediated chromosome silencing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11171-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635394PMC
July 2019

Selective Roles of Vertebrate PCF11 in Premature and Full-Length Transcript Termination.

Mol Cell 2019 04 25;74(1):158-172.e9. Epub 2019 Feb 25.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. Electronic address:

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3' mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2019.01.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458999PMC
April 2019

The non-canonical SMC protein SmcHD1 antagonises TAD formation and compartmentalisation on the inactive X chromosome.

Nat Commun 2019 01 3;10(1):30. Epub 2019 Jan 3.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including A/B compartments and partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07907-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318279PMC
January 2019

m6A modification of non-coding RNA and the control of mammalian gene expression.

Biochim Biophys Acta Gene Regul Mech 2019 03 11;1862(3):310-318. Epub 2018 Dec 11.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

The biology of non-coding RNA (ncRNA) and the regulation of mammalian gene expression is a rapidly expanding field. In this review, we consider how recent advances in technology, enabling the precise mapping of modifications to RNA transcripts, has provided new opportunities to dissect post-transcriptional gene regulation. With this has come the realisation that in the absence of translation, the modification of ncRNAs may play a fundamental role in their regulation, protein interactome and subsequent downstream effector functions. We focus upon modification of RNA by N-methyladenosine (m6A); its readers, writers and erasers, before considering the differing role of m6A modified lncRNAs MALAT1 and Xist. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagrm.2018.12.002DOI Listing
March 2019

Smchd1 Targeting to the Inactive X Is Dependent on the Xist-HnrnpK-PRC1 Pathway.

Cell Rep 2018 11;25(7):1912-1923.e9

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Melbourne, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Melbourne, VIC 3010, Australia; Department of Genetics, University of Melbourne, Melbourne, VIC 3010, Australia. Electronic address:

We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.10.044DOI Listing
November 2018

Local Tandem Repeat Expansion in RNA as a Model for the Functionalisation of ncRNA.

Authors:
Neil Brockdorff

Noncoding RNA 2018 Oct 19;4(4). Epub 2018 Oct 19.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

, the master regulator of the X chromosome inactivation in mammals, is a 17 kb lncRNA that acts to silence the majority of genes along the chromosome from which it is transcribed. The two key processes required for RNA function, localisation and recruitment of silencing factors, are genetically separable, at least in part. Recent studies have identified RNA sequences and associated RNA-binding proteins (RBPs) that are important for these processes. Notably, several of the key RNA elements correspond to local tandem repeats. In this review, I use examples to illustrate different modes whereby tandem repeat amplification has been exploited to allow orthodox RBPs to confer new functions for -mediated chromosome inactivation. I further discuss the potential generality of tandem repeat expansion in the evolution of functional long non-coding RNAs (lncRNAs).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ncrna4040028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316617PMC
October 2018

A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes.

Nat Commun 2018 09 18;9(1):3798. Epub 2018 Sep 18.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, United Kingdom.

Transcriptional regulation by chromatin is a highly dynamic process directed through the recruitment and coordinated action of epigenetic modifiers and readers of these modifications. Using an unbiased proteomic approach to find interactors of H3K36me3, a modification enriched on active chromatin, here we identify PWWP2A and HDAC2 among the top interactors. PWWP2A and its paralog PWWP2B form a stable complex with NuRD subunits MTA1/2/3:HDAC1/2:RBBP4/7, but not with MBD2/3, p66α/β, and CHD3/4. PWWP2A competes with MBD3 for binding to MTA1, thus defining a new variant NuRD complex that is mutually exclusive with the MBD2/3 containing NuRD. In mESCs, PWWP2A/B is most enriched at highly transcribed genes. Loss of PWWP2A/B leads to increases in histone acetylation predominantly at highly expressed genes, accompanied by decreases in Pol II elongation. Collectively, these findings suggest a role for PWWP2A/B in regulating transcription through the fine-tuning of histone acetylation dynamics at actively transcribed genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-06235-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143588PMC
September 2018

Unbiased Genetic Screen to Identify Factors Involved in X-Chromosome Inactivation Using a Pooled Bar-Coded shRNA Library.

Methods Mol Biol 2018 ;1861:19-36

Developmental Epigenetics, Department of Biochemistry, University of Oxford, Oxford, UK.

In mammals, a dosage compensation mechanism exists to equalize gene expression levels between male and female. This process is initiated by Xist RNA, a long noncoding RNA that mediates the transcriptional silencing of a complete chromosome. The kinetics of events occurring on the future inactive X-chromosome has been described in detail over the last 20 years. More recently, parallel studies using advanced biochemical assays and genetic screens identified key factors critical for the silencing cascade. Here, we describe the procedure adopted in one of these studies, an shRNA-based loss-of-function screen in mouse embryonic stem cells (mESCs).The screen made use of a reporter cell line in which Xist-mediated silencing could be monitored by changes in GFP fluorescence. Loss of function was achieved using a custom made bar-coded pooled shRNA library. The screen aimed to identify shRNAs that lessen Xist mediated repression of the GFP reporter. The methods that were applied are of potential relevance for the development of related screens, for example to better understand how specific repressors silence one or several genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-8766-5_2DOI Listing
June 2019

hnRNPK Recruits PCGF3/5-PRC1 to the Xist RNA B-Repeat to Establish Polycomb-Mediated Chromosomal Silencing.

Mol Cell 2017 Dec;68(5):955-969.e10

Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

The Polycomb-repressive complexes PRC1 and PRC2 play a key role in chromosome silencing induced by the non-coding RNA Xist. Polycomb recruitment is initiated by the PCGF3/5-PRC1 complex, which catalyzes chromosome-wide H2A lysine 119 ubiquitylation, signaling recruitment of other PRC1 complexes, and PRC2. However, the molecular mechanism for PCGF3/5-PRC1 recruitment by Xist RNA is not understood. Here we define the Xist RNA Polycomb Interaction Domain (XR-PID), a 600 nt sequence encompassing the Xist B-repeat element. Deletion of XR-PID abolishes Xist-dependent Polycomb recruitment, in turn abrogating Xist-mediated gene silencing and reversing Xist-induced chromatin inaccessibility. We identify the RNA-binding protein hnRNPK as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1. Accordingly, synthetically tethering hnRNPK to Xist RNA lacking XR-PID is sufficient for Xist-dependent Polycomb recruitment. Our findings define a key pathway for Polycomb recruitment by Xist RNA, providing important insights into mechanisms of chromatin modification by non-coding RNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2017.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735038PMC
December 2017

Polycomb complexes in X chromosome inactivation.

Authors:
Neil Brockdorff

Philos Trans R Soc Lond B Biol Sci 2017 Nov;372(1733)

Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

Identifying the critical RNA binding proteins (RBPs) that elicit mediated silencing has been a key goal in X inactivation research. Early studies implicated the Polycomb proteins, a family of factors linked to one of two major multiprotein complexes, PRC1 and PRC2 (Wang 2001 , 371-375 (doi:10.1038/ng574); Silva 2003 , 481-495 (doi:10.1016/S1534-5807(03)00068-6); de Napoles 2004 , 663-676 (doi:10.1016/j.devcel.2004.10.005); Plath 2003 , 131-135 (doi:10.1126/science.1084274)). PRC1 and PRC2 complexes catalyse specific histone post-translational modifications (PTMs), ubiquitylation of histone H2A at position lysine 119 (H2AK119u1) and methylation of histone H3 at position lysine 27 (H3K27me3), respectively, and accordingly, these modifications are highly enriched over the length of the inactive X chromosome (Xi). A key study proposed that PRC2 subunits bind directly to RNA A-repeat element, a region located at the 5' end of the transcript known to be required for mediated silencing (Zhao 2008 , 750-756 (doi:10.1126/science.1163045)). Subsequent recruitment of PRC1 was assumed to occur via recognition of PRC2 mediated H3K27me3 by the CBX subunit of PRC1, as has been shown to be the case at other Polycomb target loci (Cao 2002 , 1039-1043 (doi:10.1126/science.1076997)). More recently, several reports have questioned aspects of the prevailing view, both in relation to the mechanism for Polycomb recruitment by RNA and the contribution of the Polycomb pathway to mediated silencing. In this article I provide an overview of our recent progress towards resolving these discrepancies.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1098/rstb.2017.0021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627167PMC
November 2017

Preface.

Philos Trans R Soc Lond B Biol Sci 2017 Nov;372(1733)

Department of Biochemistry, University of Oxford, Oxford, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1098/rstb.2016.0353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627156PMC
November 2017

PCGF3/5-PRC1 initiates Polycomb recruitment in X chromosome inactivation.

Science 2017 06;356(6342):1081-1084

Developmental Epigenetics, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.

Recruitment of the Polycomb repressive complexes PRC1 and PRC2 by Xist RNA is an important paradigm for chromatin regulation by long noncoding RNAs. Here, we show that the noncanonical Polycomb group RING finger 3/5 (PCGF3/5)-PRC1 complex initiates recruitment of both PRC1 and PRC2 in response to Xist RNA expression. PCGF3/5-PRC1-mediated ubiquitylation of histone H2A signals recruitment of other noncanonical PRC1 complexes and of PRC2, the latter leading to deposition of histone H3 lysine 27 methylation chromosome-wide. gene knockout results in female-specific embryo lethality and abrogates Xist-mediated gene repression, highlighting a key role for Polycomb in Xist-dependent chromosome silencing. Our findings overturn existing models for Polycomb recruitment by Xist RNA and establish precedence for H2AK119u1 in initiating Polycomb domain formation in a physiological context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aal2512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522364PMC
June 2017

The nuclear matrix protein CIZ1 facilitates localization of Xist RNA to the inactive X-chromosome territory.

Genes Dev 2017 05 25;31(9):876-888. Epub 2017 May 25.

Department of Biology, University of York, York YO10 5DD, United Kingdom.

The nuclear matrix protein Cip1-interacting zinc finger protein 1 (CIZ1) promotes DNA replication in association with cyclins and has been linked to adult and pediatric cancers. Here we show that CIZ1 is highly enriched on the inactive X chromosome (Xi) in mouse and human female cells and is retained by interaction with the RNA-dependent nuclear matrix. CIZ1 is recruited to Xi in response to expression of X inactive-specific transcript (Xist) RNA during the earliest stages of X inactivation in embryonic stem cells and is dependent on the C-terminal nuclear matrix anchor domain of CIZ1 and the E repeats of CIZ1-null mice, although viable, display fully penetrant female-specific lymphoproliferative disorder. Interestingly, in mouse embryonic fibroblast cells derived from CIZ1-null embryos, Xist RNA localization is disrupted, being highly dispersed through the nucleoplasm rather than focal. Focal localization is reinstated following re-expression of CIZ1. Focal localization of Xist RNA is also disrupted in activated B and T cells isolated from CIZ1-null animals, suggesting a possible explanation for female-specific lymphoproliferative disorder. Together, these findings suggest that CIZ1 has an essential role in anchoring to the nuclear matrix in specific somatic lineages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.295907.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458755PMC
May 2017

Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2.

Nat Commun 2016 11 28;7:13661. Epub 2016 Nov 28.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

The Polycomb repressive complexes PRC1 and PRC2 play a central role in developmental gene regulation in multicellular organisms. PRC1 and PRC2 modify chromatin by catalysing histone H2A lysine 119 ubiquitylation (H2AK119u1), and H3 lysine 27 methylation (H3K27me3), respectively. Reciprocal crosstalk between these modifications is critical for the formation of stable Polycomb domains at target gene loci. While the molecular mechanism for recognition of H3K27me3 by PRC1 is well defined, the interaction of PRC2 with H2AK119u1 is poorly understood. Here we demonstrate a critical role for the PRC2 cofactor Jarid2 in mediating the interaction of PRC2 with H2AK119u1. We identify a ubiquitin interaction motif at the amino-terminus of Jarid2, and demonstrate that this domain facilitates PRC2 localization to H2AK119u1 both in vivo and in vitro. Our findings ascribe a critical function to Jarid2 and define a key mechanism that links PRC1 and PRC2 in the establishment of Polycomb domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms13661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133711PMC
November 2016

Control of Chromosomal Localization of Xist by hnRNP U Family Molecules.

Dev Cell 2016 10;39(1):11-12

RNA Biology Laboratory, RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan; RNA Biology Laboratory, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan. Electronic address:

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2016.09.022DOI Listing
October 2016

Ordered chromatin changes and human X chromosome reactivation by cell fusion-mediated pluripotent reprogramming.

Nat Commun 2016 08 10;7:12354. Epub 2016 Aug 10.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.

Erasure of epigenetic memory is required to convert somatic cells towards pluripotency. Reactivation of the inactive X chromosome (Xi) has been used to model epigenetic reprogramming in mouse, but human studies are hampered by Xi epigenetic instability and difficulties in tracking partially reprogrammed iPSCs. Here we use cell fusion to examine the earliest events in the reprogramming-induced Xi reactivation of human female fibroblasts. We show that a rapid and widespread loss of Xi-associated H3K27me3 and XIST occurs in fused cells and precedes the bi-allelic expression of selected Xi-genes by many heterokaryons (30-50%). After cell division, RNA-FISH and RNA-seq analyses confirm that Xi reactivation remains partial and that induction of human pluripotency-specific XACT transcripts is rare (1%). These data effectively separate pre- and post-mitotic events in reprogramming-induced Xi reactivation and reveal a complex hierarchy of epigenetic changes that are required to reactivate the genes on the human Xi chromosome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms12354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987517PMC
August 2016

Functional analysis of AEBP2, a PRC2 Polycomb protein, reveals a Trithorax phenotype in embryonic development and in ESCs.

Development 2016 08 17;143(15):2716-23. Epub 2016 Jun 17.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK

The Polycomb repressive complexes PRC1 and PRC2 are key mediators of heritable gene silencing in multicellular organisms. Here, we characterise AEBP2, a known PRC2 co-factor which, in vitro, has been shown to stimulate PRC2 activity. We show that AEBP2 localises specifically to PRC2 target loci, including the inactive X chromosome. Proteomic analysis confirms that AEBP2 associates exclusively with PRC2 complexes. However, analysis of embryos homozygous for a targeted mutation of Aebp2 unexpectedly revealed a Trithorax phenotype, normally linked to antagonism of Polycomb function. Consistent with this, we observe elevated levels of PRC2-mediated histone H3K27 methylation at target loci in Aebp2 mutant embryonic stem cells (ESCs). We further demonstrate that mutant ESCs assemble atypical hybrid PRC2 subcomplexes, potentially accounting for enhancement of Polycomb activity, and suggesting that AEBP2 normally plays a role in defining the mutually exclusive composition of PRC2 subcomplexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.123935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004903PMC
August 2016

MicroRNAs of the miR-290-295 Family Maintain Bivalency in Mouse Embryonic Stem Cells.

Stem Cell Reports 2016 05 14;6(5):635-642. Epub 2016 Apr 14.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK; Epigenetics Section, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK. Electronic address:

Numerous developmentally regulated genes in mouse embryonic stem cells (ESCs) are marked by both active (H3K4me3)- and polycomb group (PcG)-mediated repressive (H3K27me3) histone modifications. This bivalent state is thought to be important for transcriptional poising, but the mechanisms that regulate bivalent genes and the bivalent state remain incompletely understood. Examining the contribution of microRNAs (miRNAs) to the regulation of bivalent genes, we found that the miRNA biogenesis enzyme DICER was required for the binding of the PRC2 core components EZH2 and SUZ12, and for the presence of the PRC2-mediated histone modification H3K27me3 at many bivalent genes. Genes that lost bivalency were preferentially upregulated at the mRNA and protein levels. Finally, reconstituting Dicer-deficient ESCs with ESC miRNAs restored bivalent gene repression and PRC2 binding at formerly bivalent genes. Therefore, miRNAs regulate bivalent genes and the bivalent state itself.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2016.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939759PMC
May 2016

RNA binding proteins implicated in Xist-mediated chromosome silencing.

Semin Cell Dev Biol 2016 08 24;56:58-70. Epub 2016 Jan 24.

Dept. of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

Chromosome silencing by Xist RNA occurs in two steps; localisation in cis within the nuclear matrix to form a domain that corresponds to the territory of the inactive X chromosome elect, and transduction of silencing signals from Xist RNA to the underlying chromatin. Key factors that mediate these processes have been identified in a series of recent studies that harnessed comprehensive proteomic or genetic screening strategies. In this review we discuss these findings in light of prior knowledge both of Xist-mediated silencing and known functions/properties of the novel factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.semcdb.2016.01.029DOI Listing
August 2016

The interplay of histone modifications - writers that read.

EMBO Rep 2015 Nov 15;16(11):1467-81. Epub 2015 Oct 15.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, Oxford, UK.

Histones are subject to a vast array of posttranslational modifications including acetylation, methylation, phosphorylation, and ubiquitylation. The writers of these modifications play important roles in normal development and their mutation or misregulation is linked with both genetic disorders and various cancers. Readers of these marks contain protein domains that allow their recruitment to chromatin. Interestingly, writers often contain domains which can read chromatin marks, allowing the reinforcement of modifications through a positive feedback loop or inhibition of their activity by other modifications. We discuss how such positive reinforcement can result in chromatin states that are robust and can be epigenetically maintained through cell division. We describe the implications of these regulatory systems in relation to modifications including H3K4me3, H3K79me3, and H3K36me3 that are associated with active genes and H3K27me3 and H3K9me3 that have been linked to transcriptional repression. We also review the crosstalk between active and repressive modifications, illustrated by the interplay between the Polycomb and Trithorax histone-modifying proteins, and discuss how this may be important in defining gene expression states during development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.201540945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641500PMC
November 2015

Independent Mechanisms Target SMCHD1 to Trimethylated Histone H3 Lysine 9-Modified Chromatin and the Inactive X Chromosome.

Mol Cell Biol 2015 Dec 21;35(23):4053-68. Epub 2015 Sep 21.

Department of Biochemistry, University of Oxford, Oxford, United Kingdom

The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together, our results provide key insights into SMCHD1 function and target site selection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.00432-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628070PMC
December 2015

A Pooled shRNA Screen Identifies Rbm15, Spen, and Wtap as Factors Required for Xist RNA-Mediated Silencing.

Cell Rep 2015 Jul 16;12(4):562-72. Epub 2015 Jul 16.

Developmental Epigenetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. Electronic address:

X-chromosome inactivation is the process that evolved in mammals to equalize levels of X-linked gene expression in XX females relative to XY males. Silencing of a single X chromosome in female cells is mediated by the non-coding RNA Xist. Although progress has been made toward identifying factors that function in the maintenance of X inactivation, the primary silencing factors are largely undefined. We developed an shRNA screening strategy to produce a ranked list of candidate primary silencing factors. Validation experiments performed on several of the top hits identified the SPOC domain RNA binding proteins Rbm15 and Spen and Wtap, a component of the m6A RNA methyltransferase complex, as playing an important role in the establishment of Xist-mediated silencing. Localization analysis using super-resolution 3D-SIM microscopy demonstrates that these factors co-localize with Xist RNA within the nuclear matrix subcompartment, consistent with a direct interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.06.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534822PMC
July 2015

Jarid2 Coordinates Nanog Expression and PCP/Wnt Signaling Required for Efficient ESC Differentiation and Early Embryo Development.

Cell Rep 2015 Jul 16;12(4):573-86. Epub 2015 Jul 16.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK. Electronic address:

Jarid2 is part of the Polycomb Repressor complex 2 (PRC2) responsible for genome-wide H3K27me3 deposition. Unlike other PRC2-deficient embryonic stem cells (ESCs), however, Jarid2-deficient ESCs show a severe differentiation block, altered colony morphology, and distinctive patterns of deregulated gene expression. Here, we show that Jarid2(-/-) ESCs express constitutively high levels of Nanog but reduced PCP signaling components Wnt9a, Prickle1, and Fzd2 and lowered β-catenin activity. Depletion of Wnt9a/Prickle1/Fzd2 from wild-type ESCs or overexpression of Nanog largely phenocopies these cellular defects. Co-culture of Jarid2(-/-) with wild-type ESCs restores variable Nanog expression and β-catenin activity and can partially rescue the differentiation block of mutant cells. In addition, we show that ESCs lacking Jarid2 or Wnt9a/Prickle1/Fzd2 or overexpressing Nanog induce multiple ICM formation when injected into normal E3.5 blastocysts. These data describe a previously unrecognized role for Jarid2 in regulating a core pluripotency and Wnt/PCP signaling circuit that is important for ESC differentiation and for pre-implantation development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.06.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534826PMC
July 2015

Dosage compensation in mammals.

Cold Spring Harb Perspect Biol 2015 Mar 2;7(3):a019406. Epub 2015 Mar 2.

School of Cancer Sciences, Institute of Biomedical Research, University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom.

Many organisms show major chromosomal differences between sexes. In mammals, females have two copies of a large, gene-rich chromosome, the X, whereas males have one X and a small, gene-poor Y. The imbalance in expression of several hundred genes is lethal if not dealt with by dosage compensation. The male-female difference is addressed by silencing of genes on one female X early in development. However, both males and females now have only one active X chromosome. This is compensated by twofold up-regulation of genes on the active X. This complex system continues to provide important insights into mechanisms of epigenetic regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/cshperspect.a019406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355265PMC
March 2015