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1.
Cell ; 187(3): 733-749.e16, 2024 Feb 01.
Article in English | MEDLINE | ID: mdl-38306984

ABSTRACT

Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.


Subject(s)
Autoantibodies , Autoimmune Diseases , RNA, Long Noncoding , Animals , Female , Humans , Male , Mice , Autoantibodies/genetics , Autoimmune Diseases/genetics , Autoimmunity/genetics , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , X Chromosome/genetics , X Chromosome/metabolism , X Chromosome Inactivation , Sex Characteristics
2.
Cell ; 184(25): 6174-6192.e32, 2021 12 09.
Article in English | MEDLINE | ID: mdl-34813726

ABSTRACT

The lncRNA Xist forms ∼50 diffraction-limited foci to transcriptionally silence one X chromosome. How this small number of RNA foci and interacting proteins regulate a much larger number of X-linked genes is unknown. We show that Xist foci are locally confined, contain ∼2 RNA molecules, and nucleate supramolecular complexes (SMACs) that include many copies of the critical silencing protein SPEN. Aggregation and exchange of SMAC proteins generate local protein gradients that regulate broad, proximal chromatin regions. Partitioning of numerous SPEN molecules into SMACs is mediated by their intrinsically disordered regions and essential for transcriptional repression. Polycomb deposition via SMACs induces chromatin compaction and the increase in SMACs density around genes, which propagates silencing across the X chromosome. Our findings introduce a mechanism for functional nuclear compartmentalization whereby crowding of transcriptional and architectural regulators enables the silencing of many target genes by few RNA molecules.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Mitochondrial Proteins/metabolism , RNA, Long Noncoding/metabolism , X Chromosome/metabolism , Animals , Cell Line , Embryonic Stem Cells , Fibroblasts , Gene Silencing , Humans , Mice , Protein Binding , X Chromosome Inactivation
3.
Annu Rev Biochem ; 89: 255-282, 2020 06 20.
Article in English | MEDLINE | ID: mdl-32259458

ABSTRACT

Facultative heterochromatin (fHC) concerns the developmentally regulated heterochromatinization of different regions of the genome and, in the case of the mammalian X chromosome and imprinted loci, of only one allele of a homologous pair. The formation of fHC participates in the timely repression of genes, by resisting strong trans activators. In this review, we discuss the molecular mechanisms underlying the establishment and maintenance of fHC in mammals using a mouse model. We focus on X-chromosome inactivation (XCI) as a paradigm for fHC but also relate it to genomic imprinting and homeobox (Hox) gene cluster repression. A vital role for noncoding transcription and/or transcripts emerges as the general principle of triggering XCI and canonical imprinting. However, other types of fHC are established through an unknown mechanism, independent of noncoding transcription (Hox clusters and noncanonical imprinting). We also extensively discuss polycomb-group repressive complexes (PRCs), which frequently play a vital role in fHC maintenance.


Subject(s)
Gene Expression Regulation, Developmental , Genomic Imprinting , Heterochromatin/metabolism , Polycomb-Group Proteins/genetics , X Chromosome Inactivation , X Chromosome/metabolism , Animals , Chromatin Assembly and Disassembly , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Embryo, Mammalian , Female , Gene Silencing , Heterochromatin/chemistry , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Histones/genetics , Histones/metabolism , Humans , Male , Oocytes/cytology , Oocytes/growth & development , Oocytes/metabolism , Polycomb-Group Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Spermatozoa/cytology , Spermatozoa/growth & development , Spermatozoa/metabolism , X Chromosome/chemistry
4.
Cell ; 182(1): 127-144.e23, 2020 07 09.
Article in English | MEDLINE | ID: mdl-32502394

ABSTRACT

Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. Here, we show that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.


Subject(s)
Histones/metabolism , Lysine/metabolism , Transcriptional Activation/genetics , Acetylation , Animals , Base Sequence , Chromosome Segregation/genetics , Conserved Sequence , Dosage Compensation, Genetic , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Embryo, Nonmammalian/metabolism , Evolution, Molecular , Female , Genome , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Male , Mammals/genetics , Mice , Mutation/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleosomes/metabolism , Oocytes/metabolism , Promoter Regions, Genetic , RNA Polymerase II/metabolism , X Chromosome/metabolism , Zygote/metabolism
5.
Cell ; 176(1-2): 182-197.e23, 2019 01 10.
Article in English | MEDLINE | ID: mdl-30595450

ABSTRACT

During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing.


Subject(s)
Chromatin/metabolism , X Chromosome Inactivation/genetics , X Chromosome Inactivation/physiology , Acetylation , Animals , Chromatin/genetics , Embryonic Stem Cells , Epigenomics/methods , Female , Gene Silencing , Histone Deacetylases/metabolism , Histones/metabolism , Mice , Polycomb-Group Proteins/metabolism , Protein Processing, Post-Translational , RNA, Long Noncoding/metabolism , Transcription, Genetic , Ubiquitination , X Chromosome/metabolism
6.
Annu Rev Biochem ; 87: 323-350, 2018 06 20.
Article in English | MEDLINE | ID: mdl-29668306

ABSTRACT

X chromosome regulation represents a prime example of an epigenetic phenomenon where coordinated regulation of a whole chromosome is required. In flies, this is achieved by transcriptional upregulation of X chromosomal genes in males to equalize the gene dosage differences in females. Chromatin-bound proteins and long noncoding RNAs (lncRNAs) constituting a ribonucleoprotein complex known as the male-specific lethal (MSL) complex or the dosage compensation complex mediate this process. MSL complex members decorate the male X chromosome, and their absence leads to male lethality. The male X chromosome is also enriched with histone H4 lysine 16 acetylation (H4K16ac), indicating that the chromatin compaction status of the X chromosome also plays an important role in transcriptional activation. How the X chromosome is specifically targeted and how dosage compensation is mechanistically achieved are central questions for the field. Here, we review recent advances, which reveal a complex interplay among lncRNAs, the chromatin landscape, transcription, and chromosome conformation that fine-tune X chromosome gene expression.


Subject(s)
Dosage Compensation, Genetic , X Chromosome/genetics , Animals , Chromatin/genetics , Chromatin/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Epigenesis, Genetic , Female , Genes, X-Linked , Histone Code/genetics , Humans , Male , Models, Genetic , Models, Molecular , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , X Chromosome/metabolism
7.
Mol Cell ; 84(10): 1870-1885.e9, 2024 May 16.
Article in English | MEDLINE | ID: mdl-38759625

ABSTRACT

How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing.


Subject(s)
G-Quadruplexes , Histones , Polycomb Repressive Complex 2 , RNA, Long Noncoding , X Chromosome Inactivation , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Mice , Polycomb Repressive Complex 2/metabolism , Polycomb Repressive Complex 2/genetics , Histones/metabolism , Histones/genetics , Mouse Embryonic Stem Cells/metabolism , Chromatin/metabolism , Chromatin/genetics , X Chromosome/genetics , X Chromosome/metabolism , Gene Silencing , RNA Folding , Protein Binding
8.
Mol Cell ; 82(22): 4202-4217.e5, 2022 11 17.
Article in English | MEDLINE | ID: mdl-36302374

ABSTRACT

Condensins are evolutionarily conserved molecular motors that translocate along DNA and form loops. To address how DNA topology affects condensin translocation, we applied auxin-inducible degradation of topoisomerases I and II and analyzed the binding and function of an interphase condensin that mediates X chromosome dosage compensation in C. elegans. TOP-2 depletion reduced long-range spreading of condensin-DC (dosage compensation) from its recruitment sites and shortened 3D DNA contacts measured by Hi-C. TOP-1 depletion did not affect long-range spreading but resulted in condensin-DC accumulation within expressed gene bodies. Both TOP-1 and TOP-2 depletion resulted in X chromosome derepression, indicating that condensin-DC translocation at both scales is required for its function. Together, the distinct effects of TOP-1 and TOP-2 suggest two distinct modes of condensin-DC association with chromatin: long-range DNA loop extrusion that requires decatenation/unknotting of DNA and short-range translocation across genes that requires resolution of transcription-induced supercoiling.


Subject(s)
Adenosine Triphosphatases , Caenorhabditis elegans , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Adenosine Triphosphatases/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , X Chromosome/genetics , X Chromosome/metabolism , Chromosomes/metabolism
9.
Cell ; 159(7): 1681-97, 2014 Dec 18.
Article in English | MEDLINE | ID: mdl-25525883

ABSTRACT

Reprogramming to iPSCs resets the epigenome of somatic cells, including the reversal of X chromosome inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X inactivation, whereas others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming.


Subject(s)
Cellular Reprogramming , Epigenesis, Genetic , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , X Chromosome/metabolism , Animals , Cdh1 Proteins/metabolism , DNA Methylation , Homeodomain Proteins/metabolism , Mice , Nanog Homeobox Protein , RNA, Long Noncoding/metabolism
10.
Genes Dev ; 35(13-14): 1055-1070, 2021 07 01.
Article in English | MEDLINE | ID: mdl-34140353

ABSTRACT

The dosage compensation complex (DCC) of Drosophila identifies its X-chromosomal binding sites with exquisite selectivity. The principles that assure this vital targeting are known from the D. melanogaster model: DCC-intrinsic specificity of DNA binding, cooperativity with the CLAMP protein, and noncoding roX2 RNA transcribed from the X chromosome. We found that in D. virilis, a species separated from melanogaster by 40 million years of evolution, all principles are active but contribute differently to X specificity. In melanogaster, the DCC subunit MSL2 evolved intrinsic DNA-binding selectivity for rare PionX sites, which mark the X chromosome. In virilis, PionX motifs are abundant and not X-enriched. Accordingly, MSL2 lacks specific recognition. Here, roX2 RNA plays a more instructive role, counteracting a nonproductive interaction of CLAMP and modulating DCC binding selectivity. Remarkably, roX2 triggers a stable chromatin binding mode characteristic of DCC. Evidently, X-specific regulation is achieved by divergent evolution of protein, DNA, and RNA components.


Subject(s)
Drosophila Proteins , Drosophila melanogaster , Animals , Dosage Compensation, Genetic , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Sex Chromosomes/metabolism , Transcription Factors/metabolism , X Chromosome/genetics , X Chromosome/metabolism
11.
Cell ; 153(7): 1537-51, 2013 Jun 20.
Article in English | MEDLINE | ID: mdl-23791181

ABSTRACT

In mammals, dosage compensation between XX and XY individuals occurs through X chromosome inactivation (XCI). The noncoding Xist RNA is expressed and initiates XCI only when more than one X chromosome is present. Current models invoke a dependency on the X-to-autosome ratio (X:A), but molecular factors remain poorly defined. Here, we demonstrate that molecular titration between an X-encoded RNA and an autosomally encoded protein dictates Xist induction. In pre-XCI cells, CTCF protein represses Xist transcription. At the onset of XCI, Jpx RNA is upregulated, binds CTCF, and extricates CTCF from one Xist allele. We demonstrate that CTCF is an RNA-binding protein and is titrated away from the Xist promoter by Jpx RNA. Thus, Jpx activates Xist by evicting CTCF. The functional antagonism via molecular titration reveals a role for long noncoding RNA in epigenetic regulation.


Subject(s)
RNA, Long Noncoding/metabolism , Repressor Proteins/metabolism , Up-Regulation , X Chromosome Inactivation , Animals , CCCTC-Binding Factor , Chromosomes, Mammalian/metabolism , Embryonic Stem Cells/metabolism , Female , Male , Mice , Promoter Regions, Genetic , RNA, Long Noncoding/genetics , X Chromosome/metabolism
12.
Nature ; 589(7840): 137-142, 2021 01.
Article in English | MEDLINE | ID: mdl-33208948

ABSTRACT

Confinement of the X chromosome to a territory for dosage compensation is a prime example of how subnuclear compartmentalization is used to regulate transcription at the megabase scale. In Drosophila melanogaster, two sex-specific non-coding RNAs (roX1 and roX2) are transcribed from the X chromosome. They associate with the male-specific lethal (MSL) complex1, which acetylates histone H4 lysine 16 and thereby induces an approximately twofold increase in expression of male X-linked genes2,3. Current models suggest that X-over-autosome specificity is achieved by the recognition of cis-regulatory DNA high-affinity sites (HAS) by the MSL2 subunit4,5. However, HAS motifs are also found on autosomes, indicating that additional factors must stabilize the association of the MSL complex with the X chromosome. Here we show that the low-complexity C-terminal domain (CTD) of MSL2 renders its recruitment to the X chromosome sensitive to roX non-coding RNAs. roX non-coding RNAs and the MSL2 CTD form a stably condensed state, and functional analyses in Drosophila and mammalian cells show that their interactions are crucial for dosage compensation in vivo. Replacing the CTD of mammalian MSL2 with that from Drosophila and expressing roX in cis is sufficient to nucleate ectopic dosage compensation in mammalian cells. Thus, the condensing nature of roX-MSL2CTD is the primary determinant for specific compartmentalization of the X chromosome in Drosophila.


Subject(s)
Cell Compartmentation , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/cytology , Drosophila/genetics , RNA/metabolism , Transcription Factors/metabolism , X Chromosome/genetics , X Chromosome/metabolism , Animals , Cell Compartmentation/genetics , Cell Line , DNA-Binding Proteins/chemistry , Drosophila/metabolism , Drosophila Proteins/chemistry , Female , Humans , Male , Mice , Nucleic Acid Conformation , RNA/genetics , Transcription Factors/chemistry
13.
Mol Cell ; 74(1): 101-117.e10, 2019 04 04.
Article in English | MEDLINE | ID: mdl-30827740

ABSTRACT

During X-inactivation, Xist RNA spreads along an entire chromosome to establish silencing. However, the mechanism and functional RNA elements involved in spreading remain undefined. By performing a comprehensive endogenous Xist deletion screen, we identify Repeat B as crucial for spreading Xist and maintaining Polycomb repressive complexes 1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unexpectedly, spreading of these three factors is inextricably linked. Deleting Repeat B or its direct binding partner, HNRNPK, compromises recruitment of PRC1 and PRC2. In turn, ablating PRC1 or PRC2 impairs Xist spreading. Therefore, Xist and Polycomb complexes require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading causes failure of de novo Xi silencing, with partial compensatory downregulation of the active X, and also disrupts topological Xi reconfiguration. Thus, Repeat B is a multifunctional element that integrates interdependent Xist/Polycomb spreading, silencing, and changes in chromosome architecture.


Subject(s)
Fibroblasts/metabolism , Gene Deletion , Gene Silencing , Mouse Embryonic Stem Cells/metabolism , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 2/genetics , RNA, Long Noncoding/genetics , X Chromosome Inactivation , X Chromosome/genetics , Animals , Cell Line, Transformed , Female , Gene Expression Regulation, Developmental , Heterogeneous-Nuclear Ribonucleoprotein K , Male , Mice , Nucleotide Motifs , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 2/metabolism , Protein Binding , RNA, Long Noncoding/metabolism , Repetitive Sequences, Nucleic Acid , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , X Chromosome/metabolism
14.
Development ; 150(21)2023 11 01.
Article in English | MEDLINE | ID: mdl-37818613

ABSTRACT

The transcriptional co-regulator SIN3 influences gene expression through multiple interactions that include histone deacetylases. Haploinsufficiency and mutations in SIN3 are the underlying cause of Witteveen-Kolk syndrome and related intellectual disability and autism syndromes, emphasizing its key role in development. However, little is known about the diversity of its interactions and functions in developmental processes. Here, we show that loss of SIN-3, the single SIN3 homolog in Caenorhabditis elegans, results in maternal-effect sterility associated with de-regulation of the germline transcriptome, including de-silencing of X-linked genes. We identify at least two distinct SIN3 complexes containing specific histone deacetylases and show that they differentially contribute to fertility. Single-cell, single-molecule fluorescence in situ hybridization reveals that in sin-3 mutants the X chromosome becomes re-expressed prematurely and in a stochastic manner in individual germ cells, suggesting a role for SIN-3 in its silencing. Furthermore, we identify histone residues whose acetylation increases in the absence of SIN-3. Together, this work provides a powerful framework for the in vivo study of SIN3 and associated proteins.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Histone Deacetylases , Sin3 Histone Deacetylase and Corepressor Complex , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Germ Cells/metabolism , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Histones/metabolism , In Situ Hybridization, Fluorescence , X Chromosome/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Sin3 Histone Deacetylase and Corepressor Complex/genetics , Sin3 Histone Deacetylase and Corepressor Complex/metabolism
15.
RNA ; 30(3): 240-255, 2024 Feb 16.
Article in English | MEDLINE | ID: mdl-38164599

ABSTRACT

XIST noncoding RNA promotes the initiation of X chromosome silencing by recruiting the protein SPEN to one X chromosome in female mammals. The SPEN protein is also called SHARP (SMRT and HDAC-associated repressor protein) and MINT (Msx-2 interacting nuclear target) in humans. SPEN recruits N-CoR2 and HDAC3 to initiate histone deacetylation on the X chromosome, leading to the formation of repressive chromatin marks and silencing gene expression. We dissected the contributions of different RNA and protein regions to the formation of a human XIST-SPEN complex in vitro and identified novel sequence and structure determinants that may contribute to X chromosome silencing initiation. Binding of SPEN to XIST RNA requires RRM 4 of the protein, in contrast to the requirement of RRM 3 and RRM 4 for specific binding to SRA RNA. Measurements of SPEN binding to full-length, dimeric, trimeric, or other truncated versions of the A-repeat region revealed that high-affinity binding of XIST to SPEN in vitro requires a minimum of four A-repeat segments. SPEN binding to XIST A-repeat RNA changes the accessibility of the RNA at specific nucleotide sequences, as indicated by changes in RNA reactivity through chemical structure probing. Based on computational modeling, we found that inter-repeat duplexes formed by multiple A-repeats can present an unpaired adenosine in the context of a double-stranded region of RNA. The presence of this specific combination of sequence and structural motifs correlates with high-affinity SPEN binding in vitro. These data provide new information on the molecular basis of the XIST and SPEN interaction.


Subject(s)
RNA, Long Noncoding , RNA-Binding Proteins , Female , Humans , Chromatin , DNA-Binding Proteins/genetics , Gene Silencing , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Untranslated , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , X Chromosome/metabolism , X Chromosome Inactivation/genetics
16.
EMBO Rep ; 25(5): 2258-2277, 2024 May.
Article in English | MEDLINE | ID: mdl-38654121

ABSTRACT

X chromosome inactivation (XCI) in mammals is mediated by Xist RNA which functions in cis to silence genes on a single X chromosome in XX female cells, thereby equalising levels of X-linked gene expression relative to XY males. XCI progresses over a period of several days, with some X-linked genes silencing faster than others. The chromosomal location of a gene is an important determinant of silencing rate, but uncharacterised gene-intrinsic features also mediate resistance or susceptibility to silencing. In this study, we examine mouse embryonic stem cell lines with an inducible Xist allele (iXist-ChrX mESCs) and integrate allele-specific data of gene silencing and decreasing inactive X (Xi) chromatin accessibility over time courses of Xist induction with cellular differentiation. Our analysis reveals that motifs bound by the transcription factor YY1 are associated with persistently accessible regulatory elements, including many promoters and enhancers of slow-silencing genes. We further show that YY1 is evicted relatively slowly from target sites on Xi, and that silencing of X-linked genes is increased upon YY1 degradation. Together our results suggest that YY1 acts as a barrier to Xist-mediated silencing until the late stages of the XCI process.


Subject(s)
Gene Silencing , RNA, Long Noncoding , X Chromosome Inactivation , YY1 Transcription Factor , Animals , Female , Male , Mice , Alleles , Cell Differentiation/genetics , Cell Line , Chromatin/metabolism , Chromatin/genetics , Mouse Embryonic Stem Cells/metabolism , Promoter Regions, Genetic , Protein Binding , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , X Chromosome/genetics , X Chromosome/metabolism , X Chromosome Inactivation/genetics , YY1 Transcription Factor/metabolism , YY1 Transcription Factor/genetics
17.
Cell ; 145(3): 447-58, 2011 Apr 29.
Article in English | MEDLINE | ID: mdl-21529716

ABSTRACT

Random X inactivation represents a paradigm for monoallelic gene regulation during early ES cell differentiation. In mice, the choice of X chromosome to inactivate in XX cells is ensured by monoallelic regulation of Xist RNA via its antisense transcription unit Tsix/Xite. Homologous pairing events have been proposed to underlie asymmetric Tsix expression, but direct evidence has been lacking owing to their dynamic and transient nature. Here we investigate the live-cell dynamics and outcome of Tsix pairing in differentiating mouse ES cells. We find an overall increase in genome dynamics including the Xics during early differentiation. During pairing, however, Xic loci show markedly reduced movements. Upon separation, Tsix expression becomes transiently monoallelic, providing a window of opportunity for monoallelic Xist upregulation. Our findings reveal the spatiotemporal choreography of the X chromosomes during early differentiation and indicate a direct role for pairing in facilitating symmetry-breaking and monoallelic regulation of Xist during random X inactivation.


Subject(s)
Cell Differentiation , Chromosome Pairing , Embryonic Stem Cells/metabolism , X Chromosome Inactivation , X Chromosome/metabolism , Animals , Embryonic Stem Cells/cytology , Female , Mice , RNA, Long Noncoding , RNA, Untranslated/genetics , Time-Lapse Imaging
18.
Proc Natl Acad Sci U S A ; 120(52): e2313200120, 2023 Dec 26.
Article in English | MEDLINE | ID: mdl-38113263

ABSTRACT

In female mice, the gene dosage from X chromosomes is adjusted by a process called X chromosome inactivation (XCI) that occurs in two steps. An imprinted form of XCI (iXCI) that silences the paternally inherited X chromosome (Xp) is initiated at the 2- to 4-cell stages. As extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI require the lncRNA Xist, which is expressed from the X to be inactivated. The X-linked E3 ubiquitin ligase Rlim (Rnf12) in conjunction with its target protein Rex1 (Zfp42), a critical repressor of Xist, have emerged as major regulators of iXCI. However, their roles in rXCI remain controversial. Investigating early mouse development, we show that the Rlim-Rex1 axis is active in pre-implantation embryos. Upon implantation Rex1 levels are downregulated independently of Rlim specifically in epiblast cells. These results provide a conceptual framework of how the functional dynamics between Rlim and Rex1 ensures regulation of iXCI but not rXCI in female mice.


Subject(s)
RNA, Long Noncoding , X Chromosome Inactivation , Animals , Female , Mice , Embryo, Mammalian/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , X Chromosome/genetics , X Chromosome/metabolism , X Chromosome Inactivation/genetics
19.
Development ; 149(15)2022 08 01.
Article in English | MEDLINE | ID: mdl-35831949

ABSTRACT

Stable silencing of the inactive X chromosome (Xi) in female mammals is crucial for the development of embryos and their postnatal health. SmcHD1 is essential for stable silencing of the Xi, and its functional deficiency results in derepression of many X-inactivated genes. Although SmcHD1 has been suggested to play an important role in the formation of higher-order chromatin structure of the Xi, the underlying mechanism is largely unknown. Here, we explore the epigenetic state of the Xi in SmcHD1-deficient epiblast stem cells and mouse embryonic fibroblasts in comparison with their wild-type counterparts. The results suggest that SmcHD1 underlies the formation of H3K9me3-enriched blocks on the Xi, which, although the importance of H3K9me3 has been largely overlooked in mice, play a crucial role in the establishment of the stably silenced state. We propose that the H3K9me3 blocks formed on the Xi facilitate robust heterochromatin formation in combination with H3K27me3, and that the substantial loss of H3K9me3 caused by SmcHD1 deficiency leads to aberrant distribution of H3K27me3 on the Xi and derepression of X-inactivated genes.


Subject(s)
Histones , X Chromosome Inactivation , Animals , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Female , Fibroblasts/metabolism , Germ Layers/metabolism , Histones/metabolism , Mammals/genetics , Mice , X Chromosome/genetics , X Chromosome/metabolism , X Chromosome Inactivation/genetics
20.
Development ; 149(3)2022 02 01.
Article in English | MEDLINE | ID: mdl-35043944

ABSTRACT

Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.


Subject(s)
Gene Regulatory Networks/genetics , Oogenesis , TATA-Binding Protein Associated Factors/genetics , Transcription Factor TFIID/genetics , Animals , DNA Repair , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Female , Germ Cells/cytology , Germ Cells/metabolism , Meiosis , Mice , Mice, Inbred C57BL , Mice, Knockout , Oocytes/cytology , Oocytes/metabolism , Promoter Regions, Genetic , TATA-Binding Protein Associated Factors/deficiency , TATA-Binding Protein Associated Factors/metabolism , Transcription Factor TFIID/deficiency , Transcription Factor TFIID/metabolism , X Chromosome/genetics , X Chromosome/metabolism
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