Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS.

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.

Functional Enhancers Shape Extrachromosomal Oncogene Amplifications.

Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.

Robust Hi-C Maps of Enhancer-Promoter Interactions Reveal the Function of Non-coding Genome in Neural Development and Diseases.

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.

Convergence of case-specific epigenetic alterations identify a confluence of genetic vulnerabilities tied to opioid overdose.

Opioid use disorder is a highly heterogeneous disease driven by a variety of genetic and environmental risk factors which have yet to be fully elucidated. Opioid overdose, the most severe outcome of opioid use disorder, remains the leading cause of accidental death in the United States. We interrogated the effects of opioid overdose on the brain using ChIP-seq to quantify patterns of H3K27 acetylation in dorsolateral prefrontal cortical neurons isolated from 51 opioid-overdose cases and 51 accidental death controls. Among opioid cases, we observed global hypoacetylation and identified 388 putative enhancers consistently depleted for H3K27ac. Machine learning on H3K27ac patterns predicted case-control status with high accuracy. We focused on case-specific regulatory alterations, revealing 81,399 hypoacetylation events, uncovering vast inter-patient heterogeneity. We developed a strategy to decode this heterogeneity based on convergence analysis, which leveraged promoter-capture Hi-C to identify five genes over-burdened by alterations in their regulatory network or "plexus": ASTN2, KCNMA1, DUSP4, GABBR2, ENOX1. These convergent loci are enriched for opioid use disorder risk genes and heritability for generalized anxiety, number of sexual partners, and years of education. Overall, our multi-pronged approach uncovers neurobiological aspects of opioid use disorder and captures genetic and environmental factors perpetuating the opioid epidemic.

Transcription elongation factors represent in vivo cancer dependencies in glioblastoma.

Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of 'cancer dependencies' not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.

Enhancers: bridging the gap between gene control and human disease.

Enhancers are a class of regulatory elements essential for precise spatio-temporal control of gene expression during development and in terminally differentiated cells. This review highlights signature features of enhancer elements as well as new advances that provide mechanistic insights into enhancer-mediated gene control in the context of three-dimensional chromatin. We detail the various ways in which non-coding mutations can instigate aberrant gene control and cause a variety of Mendelian disorders, common diseases and cancer.

Combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits.

DNA variants (SNPs) that predispose to common traits often localize within noncoding regulatory elements such as enhancers. Moreover, loci identified by genome-wide association studies (GWAS) often contain multiple SNPs in linkage disequilibrium (LD), any of which may be causal. Thus, determining the effect of these multiple variant SNPs on target transcript levels has been a major challenge. Here, we provide evidence that for six common autoimmune disorders (rheumatoid arthritis, Crohn's disease, celiac disease, multiple sclerosis, lupus, and ulcerative colitis), the GWAS association arises from multiple polymorphisms in LD that map to clusters of enhancer elements active in the same cell type. This finding suggests a "multiple enhancer variant" hypothesis for common traits, where several variants in LD impact multiple enhancers and cooperatively affect gene expression. Using a novel method to delineate enhancer-gene interactions, we show that multiple enhancer variants within a given locus typically target the same gene. Using available data from HapMap and B lymphoblasts as a model system, we provide evidence at numerous loci that multiple enhancer variants cooperatively contribute to altered expression of their gene targets. The effects on target transcript levels tend to be modest and can be either gain- or loss-of-function. Additionally, the genes associated with multiple enhancer variants encode proteins that are often functionally related and enriched in common pathways. Overall, the multiple enhancer variant hypothesis offers a new paradigm by which noncoding variants can confer susceptibility to common traits.

Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing.

Trithorax (TRX) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a 'cellular memory' of active transcription of PcG-regulated genes. The mechanisms underlying these TRX functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. We report that the SET domains of TRX and TRX-related (TRR) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of TRX and Tyr2404 of TRR prevent them from being trimethyltransferases. The trx(Z11) missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3K4me1 but not the H3K4me3 level in vivo. trx(Z11) also suppresses the impaired silencing phenotypes of the Pc(3) mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by TRX-dependent H3K27 acetylation by CREB-binding protein (CBP). We show that perturbation of Polycomb silencing by TRX overexpression requires CBP. We also show that TRX and TRR are each physically associated with CBP in vivo, that TRX binds directly to the CBP KIX domain, and that the chromatin binding patterns of TRX and TRR are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of TRX and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory.

CHD7 and retinoic acid signaling cooperate to regulate neural stem cell and inner ear development in mouse models of CHARGE syndrome.

CHARGE syndrome is a multiple congenital anomaly disorder that leads to life-threatening birth defects, such as choanal atresia and cardiac malformations as well as multiple sensory impairments, that affect hearing, vision, olfaction and balance. CHARGE is caused by heterozygous mutations in CHD7, which encodes an ATP-dependent chromatin remodeling enzyme. Identification of the mechanisms underlying neurological and sensory defects in CHARGE is a first step toward developing treatments for CHARGE individuals. Here, we used mouse models of Chd7 deficiency to explore the function of CHD7 in the development of the subventricular zone (SVZ) neural stem cell niche and inner ear, structures that are important for olfactory bulb neurogenesis and hearing and balance, respectively. We found that loss of Chd7 results in cell-autonomous proliferative, neurogenic and self-renewal defects in the perinatal and mature mouse SVZ stem cell niche. Modulation of retinoic acid (RA) signaling prevented in vivo inner ear and in vitro neural stem cell defects caused by Chd7 deficiency. Our findings demonstrate critical, cooperative roles for RA and CHD7 in SVZ neural stem cell function and inner ear development, suggesting that altered RA signaling may be an effective method for treating Chd7 deficiency.

12th International CHARGE syndrome conference proceedings.

The CHARGE Syndrome Foundation holds an International conference for families and professionals every other summer. In July, 2015, the 12th meeting was held in Schaumburg, Illinois, at the Renaissance Schaumburg Hotel. Day one of the 4-day conference was dedicated to professionals caring for and researching various aspects of CHARGE, including education, medical management, animal models, and stem cell-based approaches to understanding and treating individuals with CHARGE. Here, we summarize presentations from the meeting, including a synopsis of each of the three different breakout sessions (Medical/Clinical, Basic Science/CHD7, and Education), followed by a list of abstracts and authors for both platform and poster presentations.

Twist1 mediates repression of chondrogenesis by ß-catenin to promote cranial bone progenitor specification.

The bones of the mammalian skull vault form through intramembranous ossification. Skull bones ossify directly, in a process regulated by ß-catenin, instead of passing through a cartilage intermediate. We tested whether ß-catenin is necessary for fate selection of intramembranous bone progenitors in the skull. Here, we show in mice that removal of ß-catenin from skull bone progenitors results in the near complete transformation of the skull bones to cartilage, whereas constitutive ß-catenin activation inhibits skull bone fate selection. ß-catenin directly activated Twist1 expression in skull progenitors, conditional Twist1 deletion partially phenocopied the absence of ß-catenin, and Twist1 deletion partially restored bone formation in the presence of constitutive ß-catenin activation. Finally, Twist1 bound robustly to the 3'UTR of Sox9, the central initiator of chondrogenesis, suggesting that Twist1 might directly repress cartilage formation through Sox9. These findings provide insight into how ß-catenin signaling via Twist1 actively suppresses the formation of cartilage and promotes intramembranous ossification in the skull.

Mutations in the noncoding genome.

PURPOSE OF REVIEW: Clinical diagnostic sequencing currently focuses on identifying causal mutations in the exome, wherein most disease-causing mutations are known to occur. The rest of the genome is mostly comprised of regulatory elements that control gene expression, but these have remained largely unexplored in clinical diagnostics due to the high cost of whole genome sequencing and interpretive challenges. The purpose of this review is to illustrate examples of diseases caused by mutations in regulatory elements and introduce the diagnostic potential for whole genome sequencing. Different classes of functional elements and chromatin structure are described to provide the clinician with a foundation for understanding the basis of these mutations. RECENT FINDINGS: The utilization of whole-genome sequence data, epigenomic maps and induced pluripotent stem (IPS) cell technologies facilitated the discovery that mutations in the pancreas-specific transcription factor 1a enhancer can cause isolated pancreatic agenesis. High resolution array comparative genomic hybridisation (CGH), whole-genome sequencing, maps of 3-D chromatin architecture, and mouse models generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas were used to show that disruption of topological-associated domain boundary elements cause limb defects. Structural variants that reposition enhancers in somatic cells have also been described in cancer. SUMMARY: Although not ready for diagnostics, new technologies, epigenomic maps, and improved knowledge of chromatin architecture will soon enable a better understanding and diagnostic solutions for currently unexplained genetic disorders.

Knockdown of fbxl10/kdm2bb rescues chd7 morphant phenotype in a zebrafish model of CHARGE syndrome.

CHARGE syndrome is a sporadic autosomal-dominant genetic disorder characterized by a complex array of birth defects so named for its cardinal features of ocular coloboma, heart defects, choanal atresia, growth retardation, genital abnormalities, and ear abnormalities. Approximately two-thirds of individuals clinically diagnosed with CHARGE syndrome have heterozygous loss-of-function mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7), an ATP-dependent chromatin remodeler. To examine the role of Chd7 in development, a zebrafish model was generated through morpholino (MO)-mediated targeting of the zebrafish chd7 transcript. High doses of chd7 MO induce lethality early in embryonic development. However, low dose-injected embryos are viable, and by 4 days post-fertilization, morphant fish display multiple defects in organ systems analogous to those affected in humans with CHARGE syndrome. The chd7 morphants show elevated expression of several potent cell-cycle inhibitors including ink4ab (p16/p15), p21 and p27, accompanied by reduced cell proliferation. We also show that Chd7 is required for proper organization of neural crest-derived craniofacial cartilage structures. Strikingly, MO-mediated knockdown of the jumonji domain-containing histone demethylase fbxl10/kdm2bb, a repressor of ribosomal RNA (rRNA) genes, rescues cell proliferation and cartilage defects in chd7 morphant embryos and can lead to complete rescue of the CHARGE syndrome phenotype. These results indicate that CHARGE-like phenotypes in zebrafish can be mitigated through modulation of fbxl10 levels and implicate FBXL10 as a possible therapeutic target in CHARGE syndrome.

Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions.

Epigenetic regulation of gene enhancer elements is important for establishing and maintaining the identity of cells. Gene enhancer elements are thought to exist in either active or poised states distinguishable by chromatin features, but a complete understanding of the regulation of enhancers is lacking. Here, by using mouse embryonic stem cells and their differentiated derivatives, as well as terminally differentiated cells, we report the coexistence of multiple, defined classes of enhancers that serve distinct cellular functions. Specifically, we found that active enhancers can be subclassified based on varying levels of H3K4me1, H3K27ac, and H3K36me3 and the pSer2/5 forms of RNA polymerase II. The abundance of these histone modifications positively correlates with the expression of associated genes and cellular functions consistent with the identity of the cell type. Poised enhancers can also be subclassified based on presence or absence of H3K27me3 and H3K9me3, conservation, genomic location, expression levels of associated genes, and predicted function of associated genes. These findings not only refine the repertoire of histone modifications at both active and poised gene enhancer elements but also raise the possibility that enhancers associated with distinct cellular functions are partitioned based on specific combinations of histone modifications.

Deep congenic analysis identifies many strong, context-dependent QTLs, one of which, Slc35b4, regulates obesity and glucose homeostasis.

Although central to many studies of phenotypic variation and disease susceptibility, characterizing the genetic architecture of complex traits has been unexpectedly difficult. For example, most of the susceptibility genes that contribute to highly heritable conditions such as obesity and type 2 diabetes (T2D) remain to be identified despite intensive study. We took advantage of mouse models of diet-induced metabolic disease in chromosome substitution strains (CSSs) both to characterize the genetic architecture of diet-induced obesity and glucose homeostasis and to test the feasibility of gene discovery. Beginning with a survey of CSSs, followed with genetic and phenotypic analysis of congenic, subcongenic, and subsubcongenic strains, we identified a remarkable number of closely linked, phenotypically heterogeneous quantitative trait loci (QTLs) on mouse chromosome 6 that have unexpectedly large phenotypic effects. Although fine-mapping reduced the genomic intervals and gene content of these QTLs over 3000-fold, the average phenotypic effect on body weight was reduced less than threefold, highlighting the "fractal" nature of genetic architecture in mice. Despite this genetic complexity, we found evidence for 14 QTLs in only 32 recombination events in less than 3000 mice, and with an average of four genes located within the three body weight QTLs in the subsubcongenic strains. For Obrq2a1, genetic and functional studies collectively identified the solute receptor Slc35b4 as a regulator of obesity, insulin resistance, and gluconeogenesis. This work demonstrated the unique power of CSSs as a platform for studying complex genetic traits and identifying QTLs.

Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells.

Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions into a pure population of highly expandable OPCs in 10 d. These OPCs robustly differentiate into myelinating oligodendrocytes in vitro and in vivo. Our results demonstrate that mouse pluripotent stem cells provide a pure population of myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development and drug screening.

The chromatin fingerprint of gene enhancer elements.

Different cell types within a single organism are generally distinguished by strikingly different patterns of gene expression, which are dynamic throughout development and adult life. Distal enhancer elements are key drivers of spatiotemporal specificity in gene regulation. Often located tens of kilobases from their target promoters and functioning in an orientation-independent manner, the identification of bona fide enhancers has proved a formidable challenge. With the development of ChIP-seq, global cataloging of putative enhancers has become feasible. Here, we review the current understanding of the chromatin landscape at enhancers and how these chromatin features enable robust identification of tissue-specific enhancers.

Role of canonical Wnt signaling/ß-catenin via Dermo1 in cranial dermal cell development.

Cranial dermis develops from cephalic mesoderm and neural crest cells, but what signal(s) specifies the dermal lineage is unclear. Using genetic tools to fate map and manipulate a cranial mesenchymal progenitor population in the supraorbital region, we show that the dermal progenitor cells beneath the surface ectoderm process canonical Wnt signaling at the time of specification. We show that Wnt signaling/ß-catenin is absolutely required and sufficient for Dermo1 expression and dermal cell identity in the cranium. The absence of the Wnt signaling cue leads to formation of cartilage in craniofacial and ventral trunk regions at the expense of dermal and bone lineages. Dermo1 can be a direct transcription target and may mediate the functional role of Wnt signaling in dermal precursors. This study reveals a lineage-specific role of canonical Wnt signaling/ß-catenin in promoting dermal cell fate in distinct precursor populations.

Integrative genomic analysis of human ribosomal DNA.

The transcription of ribosomal RNA (rRNA) is critical to life. Despite its importance, ribosomal DNA (rDNA) is not included in current genome assemblies and, consequently, genomic analyses to date have excluded rDNA. Here, we show that short sequence reads can be aligned to a genome assembly containing a single rDNA repeat. Integrated analysis of ChIP-seq, DNase-seq, MNase-seq and RNA-seq data reveals several novel findings. First, the coding region of active rDNA is contained within nucleosome-depleted open chromatin that is highly transcriptionally active. Second, histone modifications are located not only at the rDNA promoter but also at novel sites within the intergenic spacer. Third, the distributions of active modifications are more similar within and between different cell types than repressive modifications. Fourth, UBF, a positive regulator of rRNA transcription, binds to sites throughout the genome. Lastly, the insulator binding protein CTCF associates with the spacer promoter of rDNA, suggesting that transcriptional insulation plays a role in regulating the transcription of rRNA. Taken together, these analyses confirm and expand the results of previous ChIP studies of rDNA and provide novel avenues for exploration of chromatin-mediated regulation of rDNA.

CHD7 targets active gene enhancer elements to modulate ES cell-specific gene expression.

CHD7 is one of nine members of the chromodomain helicase DNA-binding domain family of ATP-dependent chromatin remodeling enzymes found in mammalian cells. De novo mutation of CHD7 is a major cause of CHARGE syndrome, a genetic condition characterized by multiple congenital anomalies. To gain insights to the function of CHD7, we used the technique of chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-Seq) to map CHD7 sites in mouse ES cells. We identified 10,483 sites on chromatin bound by CHD7 at high confidence. Most of the CHD7 sites show features of gene enhancer elements. Specifically, CHD7 sites are predominantly located distal to transcription start sites, contain high levels of H3K4 mono-methylation, found within open chromatin that is hypersensitive to DNase I digestion, and correlate with ES cell-specific gene expression. Moreover, CHD7 co-localizes with P300, a known enhancer-binding protein and strong predictor of enhancer activity. Correlations with 18 other factors mapped by ChIP-seq in mouse ES cells indicate that CHD7 also co-localizes with ES cell master regulators OCT4, SOX2, and NANOG. Correlations between CHD7 sites and global gene expression profiles obtained from Chd7(+/+), Chd7(+/-), and Chd7(-/-) ES cells indicate that CHD7 functions at enhancers as a transcriptional rheostat to modulate, or fine-tune the expression levels of ES-specific genes. CHD7 can modulate genes in either the positive or negative direction, although negative regulation appears to be the more direct effect of CHD7 binding. These data indicate that enhancer-binding proteins can limit gene expression and are not necessarily co-activators. Although ES cells are not likely to be affected in CHARGE syndrome, we propose that enhancer-mediated gene dysregulation contributes to disease pathogenesis and that the critical CHD7 target genes may be subject to positive or negative regulation.