Systematic discovery of Xist RNA binding proteins.

Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.

Site-specific silencing of regulatory elements as a mechanism of X inactivation.

The inactive X chromosome's (Xi) physical territory is microscopically devoid of transcriptional hallmarks and enriched in silencing-associated modifications. How these microscopic signatures relate to specific Xi sequences is unknown. Therefore, we profiled Xi gene expression and chromatin states at high resolution via allele-specific sequencing in mouse trophoblast stem cells. Most notably, X-inactivated transcription start sites harbored distinct epigenetic signatures relative to surrounding Xi DNA. These sites displayed H3-lysine27-trimethylation enrichment and DNaseI hypersensitivity, similar to autosomal Polycomb targets, yet excluded Pol II and other transcriptional hallmarks, similar to nontranscribed genes. CTCF bound X-inactivated and escaping genes, irrespective of measured chromatin boundaries. Escape from X inactivation occurred within, and X inactivation was maintained exterior to, the area encompassed by Xist in cells subject to imprinted and random X inactivation. The data support a model whereby inactivation of specific regulatory elements, rather than a simple chromosome-wide separation from transcription machinery, governs gene silencing over the Xi.

lncRNA-Induced Spread of Polycomb Controlled by Genome Architecture, RNA Abundance, and CpG Island DNA.

Long noncoding RNAs (lncRNAs) cause Polycomb repressive complexes (PRCs) to spread over broad regions of the mammalian genome. We report that in mouse trophoblast stem cells, the Airn and Kcnq1ot1 lncRNAs induce PRC-dependent chromatin modifications over multi-megabase domains. Throughout the Airn-targeted domain, the extent of PRC-dependent modification correlated with intra-nuclear distance to the Airn locus, preexisting genome architecture, and the abundance of Airn itself. Specific CpG islands (CGIs) displayed characteristics indicating that they nucleate the spread of PRCs upon exposure to Airn. Chromatin environments surrounding Xist, Airn, and Kcnq1ot1 suggest common mechanisms of PRC engagement and spreading. Our data indicate that lncRNA potency can be tightly linked to lncRNA abundance and that within lncRNA-targeted domains, PRCs are recruited to CGIs via lncRNA-independent mechanisms. We propose that CGIs that autonomously recruit PRCs interact with lncRNAs and their associated proteins through three-dimensional space to nucleate the spread of PRCs in lncRNA-targeted domains.

INO80 regulates chromatin accessibility to facilitate suppression of sex-linked gene expression during mouse spermatogenesis.

The INO80 protein is the main catalytic subunit of the INO80-chromatin remodeling complex, which is critical for DNA repair and transcription regulation in murine spermatocytes. In this study, we explored the role of INO80 in silencing genes on meiotic sex chromosomes in male mice. INO80 immunolocalization at the XY body in pachytene spermatocytes suggested a role for INO80 in the meiotic sex body. Subsequent deletion of Ino80 resulted in high expression of sex-linked genes. Furthermore, the active form of RNA polymerase II at the sex chromosomes of Ino80-null pachytene spermatocytes indicates incomplete inactivation of sex-linked genes. A reduction in the recruitment of initiators of meiotic sex chromosome inhibition (MSCI) argues for INO80-facilitated recruitment of DNA repair factors required for silencing sex-linked genes. This role of INO80 is independent of a common INO80 target, H2A.Z. Instead, in the absence of INO80, a reduction in chromatin accessibility at DNA repair sites occurs on the sex chromosomes. These data suggest a role for INO80 in DNA repair factor localization, thereby facilitating the silencing of sex-linked genes during the onset of pachynema.

Spt6 Association with RNA Polymerase II Directs mRNA Turnover During Transcription.

Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 also associates with RNA polymerase II (RNAPII) via a tandem Src2 homology domain. However, the significance of Spt6-RNAPII interaction is not well understood. Here, we show that Spt6 recruitment to genes and the nucleosome reassembly functions of Spt6 can still occur in the absence of its association with RNAPII. Surprisingly, we found that Spt6-RNAPII association is required for efficient recruitment of the Ccr4-Not de-adenylation complex to transcribed genes for essential degradation of a range of mRNAs, including mRNAs required for cell-cycle progression. These findings reveal an unexpected control mechanism for mRNA turnover during transcription facilitated by a histone chaperone.

INO80 requires a polycomb subunit to regulate the establishment of poised chromatin in murine spermatocytes.

INO80 is the catalytic subunit of the INO80-chromatin remodeling complex that is involved in DNA replication, repair and transcription regulation. Ino80 deficiency in murine spermatocytes (Ino80cKO) results in pachytene arrest of spermatocytes due to incomplete synapsis and aberrant DNA double-strand break repair, which leads to apoptosis. RNA-seq on Ino80cKO spermatocytes revealed major changes in transcription, indicating that an aberrant transcription program arises upon INO80 depletion. In Ino80WT spermatocytes, genome-wide analysis showed that INO80-binding sites were mostly promoter proximal and necessary for the regulation of spermatogenic gene expression, primarily of premeiotic and meiotic genes. Furthermore, most of the genes poised for activity, as well as those genes that are active, shared INO80 binding. In Ino80cKO spermatocytes, most poised genes demonstrated de-repression due to reduced H3K27me3 enrichment and, in turn, showed increased expression levels. INO80 interacts with the core PRC2 complex member SUZ12 and promotes its recruitment. Furthermore, INO80 mediates H2A.Z incorporation at the poised promoters, which was reduced in Ino80cKO spermatocytes. Taken together, INO80 is emerging as a major regulator of the meiotic transcription program by mediating poised chromatin establishment through SUZ12 binding.

HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer.

Defects in transcriptional regulators of pancreatic exocrine differentiation have been implicated in pancreatic tumorigenesis, but the molecular mechanisms are poorly understood. The locus encoding the transcription factor HNF1A harbors susceptibility variants for pancreatic ductal adenocarcinoma (PDAC), while KDM6A, encoding Lysine-specific demethylase 6A, carries somatic mutations in PDAC. Here, we show that pancreas-specific Hnf1a null mutant transcriptomes phenocopy those of Kdm6a mutations, and both defects synergize with KrasG12D to cause PDAC with sarcomatoid features. We combine genetic, epigenomic, and biochemical studies to show that HNF1A recruits KDM6A to genomic binding sites in pancreatic acinar cells. This remodels the acinar enhancer landscape, activates differentiated acinar cell programs, and indirectly suppresses oncogenic and epithelial-mesenchymal transition genes. We also identify a subset of non-classical PDAC samples that exhibit the HNF1A/KDM6A-deficient molecular phenotype. These findings provide direct genetic evidence that HNF1A deficiency promotes PDAC. They also connect the tumor-suppressive role of KDM6A deficiency with a cell-specific molecular mechanism that underlies PDAC subtype definition.

The mutant mouse resource and research center (MMRRC) consortium: the US-based public mouse repository system.

Now in its 25th year, the Mutant Mouse Resource and Research Center (MMRRC) consortium continues to serve the United States and international biomedical scientific community as a public repository and distribution archive of laboratory mouse models of human disease for research. Supported by the National Institutes of Health (NIH), the MMRRC consists of 4 regionally distributed and dedicated vivaria, offices, and specialized laboratory facilities and an Informatics Coordination and Service Center (ICSC). The overarching purpose of the MMRRC is to facilitate groundbreaking biomedical research by offering an extensive repertoire of mutant mice that are essential for advancing the understanding of human physiology and disease. The function of the MMRRC is to identify, acquire, evaluate, characterize, cryopreserve, and distribute mutant mouse strains to qualified biomedical investigators around the nation and the globe. Mouse strains accepted from the research community are held to the highest scientific standards to optimize reproducibility and enhance scientific rigor and transparency. All submitted strains are thoroughly reviewed, documented, and validated using extensive scientific quality control measures. In addition, the MMRRC conducts resource-related research on cryopreservation, mouse genetics, environmental conditions, and other topics that enhance operations of the MMRRC. Today, the MMRRC maintains an archive of mice, cryopreserved embryos and sperm, embryonic stem (ES) cell lines, and murine hybridomas for nearly 65,000 alleles. Since its inception, the MMRRC has fulfilled more than 20,000 orders from 13,651 scientists at 8441 institutions worldwide. The MMRRC also provides numerous services to assist researchers, including scientific consultation, technical assistance, genetic assays, microbiome analysis, analytical phenotyping, pathology, cryorecovery, husbandry, breeding and colony management, infectious disease surveillance, and disease modeling. The ICSC coordinates MMRRC operations, interacts with researchers, and manages the website (mmrrc.org) and online catalogue. Researchers benefit from an expansive list of well-defined mouse models of disease that meet the highest scientific standards while submitting investigators benefit by having their mouse strains cryopreserved, protected, and distributed in compliance with NIH policies.

T Follicular Helper Cell-Dependent Clearance of a Persistent Virus Infection Requires T Cell Expression of the Histone Demethylase UTX.

Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection.

The KMT2D Kabuki syndrome histone methylase controls neural crest cell differentiation and facial morphology.

Kabuki syndrome (KS) is a congenital craniofacial disorder resulting from mutations in the KMT2D histone methylase (KS1) or the UTX histone demethylase (KS2). With small cohorts of KS2 patients, it is not clear whether differences exist in clinical manifestations relative to KS1. We mutated KMT2D in neural crest cells (NCCs) to study cellular and molecular functions in craniofacial development with respect to UTX. Similar to UTX, KMT2D NCC knockout mice demonstrate hypoplasia with reductions in frontonasal bone lengths. We have traced the onset of KMT2D and UTX mutant NCC frontal dysfunction to a stage of altered osteochondral progenitor differentiation. KMT2D NCC loss-of-function does exhibit unique phenotypes distinct from UTX mutation, including fully penetrant cleft palate, mandible hypoplasia and deficits in cranial base ossification. KMT2D mutant NCCs lead to defective secondary palatal shelf elevation with reduced expression of extracellular matrix components. KMT2D mutant chondrocytes in the cranial base fail to properly differentiate, leading to defective endochondral ossification. We conclude that KMT2D is required for appropriate cranial NCC differentiation and KMT2D-specific phenotypes may underlie differences between Kabuki syndrome subtypes.

An Interactive Database for the Assessment of Histone Antibody Specificity.

Access to high-quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut the Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloging the behavior of widely used, commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community that routinely uses these antibodies as detection reagents for a wide range of applications.

Histone H3.3 maintains genome integrity during mammalian development.

Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans and has been implicated in many important biological processes, including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components or in H3.3 encoding genes have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3-null mouse models through classical genetic approaches. We found that H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres, and pericentromeric regions of chromosomes, leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development.

Mammalian SWI/SNF collaborates with a polycomb-associated protein to regulate male germline transcription in the mouse.

A deficiency in BRG1, the catalytic subunit of the SWI/SNF chromatin remodeling complex, results in a meiotic arrest during spermatogenesis. Here, we explore the causative mechanisms. BRG1 is preferentially enriched at active promoters of genes essential for spermatogonial pluripotency and meiosis. In contrast, BRG1 is also associated with the repression of somatic genes. Chromatin accessibility at these target promoters is dependent upon BRG1. These results favor a model in which BRG1 coordinates spermatogenic transcription to ensure meiotic progression. In spermatocytes, BRG1 interacts with SCML2, a testis-specific PRC1 factor that is associated with the repression of somatic genes. We present evidence to suggest that BRG1 and SCML2 concordantly regulate genes during meiosis. Furthermore, BRG1 is required for the proper localization of SCML2 and its associated deubiquitylase, USP7, to the sex chromosomes during pachynema. SCML2-associated mono-ubiquitylation of histone H2A lysine 119 (H2AK119ub1) and acetylation of histone lysine 27 (H3K27ac) are elevated in Brg1cKO testes. Coincidentally, the PRC1 ubiquitin ligase RNF2 is activated while a histone H2A/H2B deubiquitylase USP3 is repressed. Thus, BRG1 impacts the male epigenome by influencing the localization and expression of epigenetic modifiers. This mechanism highlights a novel paradigm of cooperativity between SWI/SNF and PRC1.

The Mutant Mouse Resource and Research Center (MMRRC): the NIH-supported National Public Repository and Distribution Archive of Mutant Mouse Models in the USA.

The Mutant Mouse Resource and Research Center (MMRRC) Program is the pre-eminent public national mutant mouse repository and distribution archive in the USA, serving as a national resource of mutant mice available to the global scientific community for biomedical research. Established more than two decades ago with grants from the National Institutes of Health (NIH), the MMRRC Program supports a Consortium of regionally distributed and dedicated vivaria, laboratories, and offices (Centers) and an Informatics Coordination and Service Center (ICSC) at three academic teaching and research universities and one non-profit genetic research institution. The MMRRC Program accepts the submission of unique, scientifically rigorous, and experimentally valuable genetically altered and other mouse models donated by academic and commercial scientists and organizations for deposition, maintenance, preservation, and dissemination to scientists upon request. The four Centers maintain an archive of nearly 60,000 mutant alleles as live mice, frozen germplasm, and/or embryonic stem (ES) cells. Since its inception, the Centers have fulfilled 13,184 orders for mutant mouse models from 9591 scientists at 6626 institutions around the globe. Centers also provide numerous services that facilitate using mutant mouse models obtained from the MMRRC, including genetic assays, microbiome analysis, analytical phenotyping and pathology, cryorecovery, mouse husbandry, infectious disease surveillance and diagnosis, and disease modeling. The ICSC coordinates activities between the Centers, manages the website (mmrrc.org) and online catalog, and conducts communication, outreach, and education to the research community. Centers preserve, secure, and protect mutant mouse lines in perpetuity, promote rigor and reproducibility in scientific experiments using mice, provide experiential training and consultation in the responsible use of mice in research, and pursue cutting edge technologies to advance biomedical studies using mice to improve human health. Researchers benefit from an expansive list of well-defined mouse models of disease that meet the highest standards of rigor and reproducibility, while donating investigators benefit by having their mouse lines preserved, protected, and distributed in compliance with NIH policies.

Repression of the soma-specific transcriptome by Polycomb-repressive complex 2 promotes male germ cell development.

Polycomb-repressive complex 2 (PRC2) catalyzes the methylation of histone H3 Lys27 (H3K27) and functions as a critical epigenetic regulator of both stem cell pluripotency and somatic differentiation, but its role in male germ cell development is unknown. Using conditional mutagenesis to remove the core PRC2 subunits EED and SUZ12 during male germ cell development, we identified a requirement for PRC2 in both mitotic and meiotic germ cells. We observed a paucity of mutant spermatogonial stem cells (SSCs), which appears independent of repression of the known cell cycle inhibitors Ink4a/Ink4b/Arf. Moreover, mutant spermatocytes exhibited ectopic expression of somatic lamins and an abnormal distribution of SUN1 proteins on the nuclear envelope. These defects were coincident with abnormal chromosome dynamics, affecting homologous chromosome pairing and synapsis. We observed acquisition of H3K27me3 on stage-specific genes during meiotic progression, indicating a requirement for PRC2 in regulating the meiotic transcriptional program. Together, these data demonstrate that transcriptional repression of soma-specific genes by PRC2 facilitates homeostasis and differentiation during mammalian spermatogenesis.

UTX-guided neural crest function underlies craniofacial features of Kabuki syndrome.

Kabuki syndrome, a congenital craniofacial disorder, manifests from mutations in an X-linked histone H3 lysine 27 demethylase (UTX/KDM6A) or a H3 lysine 4 methylase (KMT2D). However, the cellular and molecular etiology of histone-modifying enzymes in craniofacial disorders is unknown. We now establish Kabuki syndrome as a neurocristopathy, whereby the majority of clinical features are modeled in mice carrying neural crest (NC) deletion of UTX, including craniofacial dysmorphism, cardiac defects, and postnatal growth retardation. Female UTX NC knockout (FKO) demonstrates enhanced phenotypic severity over males (MKOs), due to partial redundancy with UTY, a Y-chromosome demethylase-dead homolog. Thus, NC cells may require demethylase-independent UTX activity. Consistently, Kabuki causative point mutations upstream of the JmjC domain do not disrupt UTX demethylation. We have isolated primary NC cells at a phenocritical postmigratory timepoint in both FKO and MKO mice, and genome-wide expression and histone profiling have revealed UTX molecular function in establishing appropriate chromatin structure to regulate crucial NC stem-cell signaling pathways. However, the majority of UTX regulated genes do not experience aberrations in H3K27me3 or H3K4me3, implicating alternative roles for UTX in transcriptional control. These findings are substantiated through demethylase-dead knockin mutation of UTX, which supports appropriate facial development.

A statistical method for joint estimation of cis-eQTLs and parent-of-origin effects under family trio design.

RNA sequencing allows one to study allelic imbalance of gene expression, which may be due to genetic factors or genomic imprinting (i.e., higher expression of maternal or paternal allele). It is desirable to model both genetic and parent-of-origin effects simultaneously to avoid confounding and to improve the power to detect either effect. In studies of genetically tractable model organisms, separation of genetic and parent-of-origin effects can be achieved by studying reciprocal cross of two inbred strains. In contrast, this task is much more challenging in outbred populations such as humans. To address this challenge, we propose a new framework to combine experimental strategies and novel statistical methods. Specifically, we propose to study genetic and imprinting effects in family trios with RNA-seq data from the children and genotype data from both parents and children, and quantify genetic effects by cis-eQTLs. Towards this end, we have extended our method that studies the eQTLs of RNA-seq data (Sun, Biometrics 2012, 68(1): 1-11) to model both cis-eQTL and parent-of-origin effects, and evaluated its performance using extensive simulations. Since sample size may be limited in family trios, we have developed a data analysis pipeline that borrows information from external data of unrelated individuals for cis-eQTL mapping. We have also collected RNA-seq data from the children of 30 family trios, applied our method to analyze this dataset, and identified some previously reported imprinted genes as well as some new candidates of imprinted genes.

Topoisomerases facilitate transcription of long genes linked to autism.

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders.

The histone demethylase Kdm6b regulates a mature gene expression program in differentiating cerebellar granule neurons.

The histone H3 lysine 27 (H3K27) demethylase Kdm6b (Jmjd3) can promote cellular differentiation, however its physiological functions in neurons remain to be fully determined. We studied the expression and function of Kdm6b in differentiating granule neurons of the developing postnatal mouse cerebellum. At postnatal day 7, Kdm6b is expressed throughout the layers of the developing cerebellar cortex, but its expression is upregulated in newborn cerebellar granule neurons (CGNs). Atoh1-Cre mediated conditional knockout of Kdm6b in CGN precursors either alone or in combination with Kdm6a did not disturb the gross morphological development of the cerebellum. Furthermore, RNAi-mediated knockdown of Kdm6b in cultured CGN precursors did not alter the induced expression of early neuronal marker genes upon cell cycle exit. By contrast, knockdown of Kdm6b significantly impaired the induction of a mature neuronal gene expression program, which includes gene products required for functional synapse maturation. Loss of Kdm6b also impaired the ability of Brain-Derived Neurotrophic Factor (BDNF) to induce expression of Grin2c and Tiam1 in maturing CGNs. Taken together, these data reveal a previously unknown role for Kdm6b in the postmitotic stages of CGN maturation and suggest that Kdm6b may work, at least in part, by a transcriptional mechanism that promotes gene sensitivity to regulation by BDNF.