Canonical Wnt/ß-catenin activity and differential epigenetic marks direct sexually dimorphic regulation of Irx3 and Irx5 in developing mouse gonads.

Members of the Iroquois B (IrxB) homeodomain cluster genes, specifically Irx3 and Irx5, are crucial for heart, limb and bone development. Recently, we reported their importance for oocyte and follicle survival within the developing ovary. Irx3 and Irx5 expression begins after sex determination in the ovary but remains absent in the fetal testis. Mutually antagonistic molecular signals ensure ovary versus testis differentiation with canonical Wnt/ß-catenin signals paramount for promoting the ovary pathway. Notably, few direct downstream targets have been identified. We report that Wnt/ß-catenin signaling directly stimulates Irx3 and Irx5 transcription in the developing ovary. Using in silico analysis of ATAC- and ChIP-Seq databases in conjunction with mouse gonad explant transfection assays, we identified TCF/LEF-binding sequences within two distal enhancers of the IrxB locus that promote ß-catenin-responsive ovary expression. Meanwhile, Irx3 and Irx5 transcription is suppressed within the developing testis by the presence of H3K27me3 on these same sites. Thus, we resolved sexually dimorphic regulation of Irx3 and Irx5 via epigenetic and ß-catenin transcriptional control where their ovarian presence promotes oocyte and follicle survival vital for future ovarian health.

Dynamic enhancers control skeletal muscle identity and reprogramming.

Skeletal muscles consist of fibers of differing metabolic activities and contractility, which become remodeled in response to chronic exercise, but the epigenomic basis for muscle identity and adaptation remains poorly understood. Here, we used chromatin immunoprecipitation sequencing of dimethylated histone 3 lysine 4 and acetylated histone 3 lysine 27 as well as transposase-accessible chromatin profiling to dissect cis-regulatory networks across muscle groups. We demonstrate that in vivo enhancers specify muscles in accordance with myofiber composition, show little resemblance to cultured myotube enhancers, and identify glycolytic and oxidative muscle-specific regulators. Moreover, we find that voluntary wheel running and muscle-specific peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc1a) transgenic (mTg) overexpression, which stimulate endurance performance in mice, result in markedly different changes to the epigenome. Exercise predominantly leads to enhancer hypoacetylation, whereas mTg causes hyperacetylation at different sites. Integrative analysis of regulatory regions and gene expression revealed that exercise and mTg are each associated with myocyte enhancer factor (MEF) 2 and estrogen-related receptor (ERR) signaling and transcription of genes promoting oxidative metabolism. However, exercise was additionally associated with regulation by retinoid X receptor (RXR), jun proto-oncogene (JUN), sine oculis homeobox factor (SIX), and other factors. Overall, our work defines the unique enhancer repertoires of skeletal muscles in vivo and reveals that divergent exercise-induced or PGC1α-driven epigenomic programs direct partially convergent transcriptional networks.

CBX2 is required to stabilize the testis pathway by repressing Wnt signaling.

XX and XY fetal gonads are initially bipotential, poised between the ovary and testis fate. Multiple lines of evidence suggest that commitment to testis fate requires the repression of genes associated with ovary fate. It was previously shown that loss of CBX2, the subunit of the Polycomb Repressive Complex 1 (PRC1) that binds H3K27me3 and mediates silencing, leads to ovary development in XY mice and humans. While it had been proposed that CBX2 is an activator of the testis-determining gene Sry, we investigated the alternative possibility that CBX2 has a direct role as a repressor of the antagonistic ovary-promoting pathway. To investigate this possibility, we developed a quantitative genome-wide profile of the repressive histone mark H3K27me3 and its active counterpart H3K4me3 in isolated XY and XX gonadal supporting cells before and after sex determination. We show that testis and ovary sex-determining (SD) genes are bivalent before sex determination, providing insight into how the bipotential state of the gonad is established at the epigenetic level. After sex determination, many SD genes of the alternate pathway remain bivalent, possibly contributing to the ability of these cells to transdifferentiate even in adults. The finding that many genes in the Wnt signaling pathway were targeted for H3K27me3-mediated repression in Sertoli cells led us to test whether deletion of Wnt4 could rescue testis development in Cbx2 mutants. We show that Sry expression and testis development were rescued in XY Cbx2-/-;Wnt4-/- mice. Furthermore, we show that CBX2 directly binds the downstream Wnt signaler Lef1, an ovary-promoting gene that remains bivalent in Sertoli cells. Our results suggest that stabilization of the testis fate requires CBX2-mediated repression of bivalent ovary-determining genes, which would otherwise block testis development.

Gonadal supporting cells acquire sex-specific chromatin landscapes during mammalian sex determination.

Cis-regulatory elements are critical for the precise spatiotemporal regulation of genes during development. However, identifying functional regulatory sites that drive cell differentiation in vivo has been complicated by the high numbers of cells required for whole-genome epigenetic assays. Here, we identified putative regulatory elements during sex determination by performing ATAC-seq and ChIP-seq for H3K27ac in purified XX and XY gonadal supporting cells before and after sex determination in mice. We show that XX and XY supporting cells initiate sex determination with similar chromatin landscapes and acquire sex-specific regulatory elements as they commit to the male or female fate. To validate our approach, we identified a functional gonad-specific enhancer downstream of Bmp2, an ovary-promoting gene. This work increases our understanding of the complex regulatory network underlying mammalian sex determination and provides a powerful resource for identifying non-coding regulatory elements that could harbor mutations that lead to Disorders of Sexual Development.

Transcription is required to establish maternal imprinting at the Prader-Willi syndrome and Angelman syndrome locus.

The Prader-Willi syndrome (PWS [MIM 17620]) and Angelman syndrome (AS [MIM 105830]) locus is controlled by a bipartite imprinting center (IC) consisting of the PWS-IC and the AS-IC. The most widely accepted model of IC function proposes that the PWS-IC activates gene expression from the paternal allele, while the AS-IC acts to epigenetically inactivate the PWS-IC on the maternal allele, thus silencing the paternally expressed genes. Gene order and imprinting patterns at the PWS/AS locus are well conserved from human to mouse; however, a murine AS-IC has yet to be identified. We investigated a potential regulatory role for transcription from the Snrpn alternative upstream exons in silencing the maternal allele using a murine transgene containing Snrpn and three upstream exons. This transgene displayed appropriate imprinted expression and epigenetic marks, demonstrating the presence of a functional AS-IC. Transcription of the upstream exons from the endogenous locus correlates with imprint establishment in oocytes, and this upstream exon expression pattern was conserved on the transgene. A transgene bearing targeted deletions of each of the three upstream exons exhibited loss of imprinting upon maternal transmission. These results support a model in which transcription from the Snrpn upstream exons directs the maternal imprint at the PWS-IC.

Transcriptional programming of translation by BCL6 controls skeletal muscle proteostasis.

Skeletal muscle is dynamically controlled by the balance of protein synthesis and degradation. Here we discover an unexpected function for the transcriptional repressor B cell lymphoma 6 (BCL6) in muscle proteostasis and strength in mice. Skeletal muscle-specific Bcl6 ablation in utero or in adult mice results in over 30% decreased muscle mass and force production due to reduced protein synthesis and increased autophagy, while it promotes a shift to a slower myosin heavy chain fibre profile. Ribosome profiling reveals reduced overall translation efficiency in Bcl6-ablated muscles. Mechanistically, tandem chromatin immunoprecipitation, transcriptomic and translational analyses identify direct BCL6 repression of eukaryotic translation initiation factor 4E-binding protein 1 (Eif4ebp1) and activation of insulin-like growth factor 1 (Igf1) and androgen receptor (Ar). Together, these results uncover a bifunctional role for BCL6 in the transcriptional and translational control of muscle proteostasis.

Epigenomic tensor predicts disease subtypes and reveals constrained tumor evolution.

Understanding the epigenomic evolution and specificity of disease subtypes from complex patient data remains a major biomedical problem. We here present DeCET (decomposition and classification of epigenomic tensors), an integrative computational approach for simultaneously analyzing hierarchical heterogeneous data, to identify robust epigenomic differences among tissue types, differentiation states, and disease subtypes. Applying DeCET to our own data from 21 uterine benign tumor (leiomyoma) patients identifies distinct epigenomic features discriminating normal myometrium and leiomyoma subtypes. Leiomyomas possess preponderant alterations in distal enhancers and long-range histone modifications confined to chromatin contact domains that constrain the evolution of pathological epigenomes. Moreover, we demonstrate the power and advantage of DeCET on multiple publicly available epigenomic datasets representing different cancers and cellular states. Epigenomic features extracted by DeCET can thus help improve our understanding of disease states, cellular development, and differentiation, thereby facilitating future therapeutic, diagnostic, and prognostic strategies.

The transmembrane protein TMEM16A is required for normal development of the murine trachea.

Pathological collapsibility of the upper airways, caused by many different genetic and environmental insults, is known as tracheomalacia in humans. We determined that Tmem16a, a member of an evolutionarily conserved family of predicted transmembrane proteins, is expressed in the developing trachea. We report that all mice homozygous for a null allele of Tmem16a died within one month of birth and exhibited severe tracheomalacia with gaps in the tracheal cartilage rings along the entire length of the trachea. In addition, the development of the trachealis muscle that spans the dorsal aspect of the trachea was abnormal in Tmem16a mutants. Since the chondrogenic mesenchyme does not express Tmem16a at any time, we propose that the cartilage ring defect observed in Tmem16a mutants is secondary to an expansion of the embryonic trachea that might result from improper stratification of the embryonic tracheal epithelium or the abnormal trachealis muscle. Our data identify Tmem16a as a novel regulator of epithelial and smooth muscle cell organization in murine development. This mutant, the first knockout of a vertebrate TMEM16 family member, provides a mouse model of tracheomalacia.

Dynamic repression by BCL6 controls the genome-wide liver response to fasting and steatosis.

Transcription is tightly regulated to maintain energy homeostasis during periods of feeding or fasting, but the molecular factors that control these alternating gene programs are incompletely understood. Here, we find that the B cell lymphoma 6 (BCL6) repressor is enriched in the fed state and converges genome-wide with PPARα to potently suppress the induction of fasting transcription. Deletion of hepatocyte Bcl6 enhances lipid catabolism and ameliorates high-fat-diet-induced steatosis. In Ppara-null mice, hepatocyte Bcl6 ablation restores enhancer activity at PPARα-dependent genes and overcomes defective fasting-induced fatty acid oxidation and lipid accumulation. Together, these findings identify BCL6 as a negative regulator of oxidative metabolism and reveal that alternating recruitment of repressive and activating transcription factors to shared cis-regulatory regions dictates hepatic lipid handling.

Loss of Transcriptional Repression by BCL6 Confers Insulin Sensitivity in the Setting of Obesity.

Accumulation of visceral adiposity is directly linked to the morbidity of obesity, while subcutaneous body fat is considered more benign. We have identified an unexpected role for B cell lymphoma 6 (BCL6), a critical regulator of immunity, in the developmental expansion of subcutaneous adipose tissue. In adipocyte-specific knockout mice (Bcl6AKO), we found that Bcl6 deletion results in strikingly increased inguinal, but not perigonadal, adipocyte size and tissue mass in addition to marked insulin sensitivity. Genome-wide RNA expression and DNA binding analyses revealed that BCL6 controls gene networks involved in cell growth and fatty acid biosynthesis. Using deuterium label incorporation and comprehensive adipokine and lipid profiling, we discovered that ablation of adipocyte Bcl6 enhances subcutaneous adipocyte lipogenesis, increases levels of adiponectin and fatty acid esters of hydroxy fatty acids (FAHFAs), and prevents steatosis. Thus, our studies identify BCL6 as a negative regulator of subcutaneous adipose tissue expansion and metabolic health.

A human imprinting centre demonstrates conserved acquisition but diverged maintenance of imprinting in a mouse model for Angelman syndrome imprinting defects.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by the loss of imprinted gene expression from chromosome 15q11-q13. Imprinted gene expression in the region is regulated by a bipartite imprinting centre (IC), comprising the PWS-IC and the AS-IC. The PWS-IC is a positive regulatory element required for bidirectional activation of a number of paternally expressed genes. The function of the AS-IC appears to be to suppress PWS-IC function on the maternal chromosome through a methylation imprint acquired during female gametogenesis. Here we have placed the entire mouse locus under the control of a human PWS-IC by targeted replacement of the mouse PWS-IC with the equivalent human region. Paternal inheritance of the human PWS-IC demonstrates for the first time that a positive regulatory element in the PWS-IC has diverged. These mice show postnatal lethality and growth deficiency, phenotypes not previously attributed directly to the affected genes. Following maternal inheritance, the human PWS-IC is able to acquire a methylation imprint in mouse oocytes, suggesting that acquisition of the methylation imprint is conserved. However, the imprint is lost in somatic cells, showing that maintenance has diverged. This maternal imprinting defect results in expression of maternal Ube3a-as and repression of Ube3a in cis, providing evidence that Ube3a is regulated by its antisense and creating the first reported mouse model for AS imprinting defects.