Truncating mutations of chromodomain helicase DNA-binding protein 8 (CHD8), and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA sequencing) with genome-wide CHD8 binding (ChIP sequencing). Suppressing CHD8 to levels comparable with the loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8-binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (P < 10 −8) and CHD8-bound genes (P = 0.0043), which align with previously identified coexpression modules during fetal development. We also find an intriguing enrichment of cancer-related gene sets among CHD8-bound genes (P < 10 −10). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene-expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis.he genetic architecture of autism spectrum disorder (ASD) is complex and heterogeneous. A wave of recent discoveries has identified individual genes that contribute to ASD when they suffer heterozygous inactivation by coding mutation, copy number variation, or balanced chromosomal abnormalities (1-7). Many of these genes fit neatly into current biological models of ASD involving altered synaptic structure and glutamatergic neurotransmission, but others have been surprising, with a less ready biological interpretation, including genes involved in chromatin modification, DNA methylation, cell adhesion, and global transcriptional regulation. This diversity of genes predisposing to ASD suggests either that there are many pathways that independently can result in the autism phenotype or that functionally distinct ASD-risk genes can trigger consequences that converge on a limited number of shared pathways of ASD pathogenesis. Because now experimental tools are available to reduce gene expression specifically in human neural progenitor cells (NPCs), which can mimic the impact of functional hemizygosity, we have explored this question by investigating the functional genomic consequences of suppr...

Chromatin state maps were developed to elucidate sex differences in chromatin structure and their impact on sex-differential chromatin accessibility and sex-biased gene expression in mouse liver. Genes in active, inactive, and poised chromatin states exhibited differential responsiveness to ligand-activated nuclear receptors and distinct enrichments for functional gene categories. Sex-biased genes were clustered by chromatin environments and mapped to DNase-hypersensitive sites (

Reciprocal copy-number variation (CNV) of a 593 kb region of 16p11.2 is a common genetic cause of autism spectrum disorder (ASD), yet it is not completely penetrant and can manifest in a wide array of phenotypes. To explore its molecular consequences, we performed RNA sequencing of cerebral cortex from mouse models with CNV of the syntenic 7qF3 region and lymphoblast lines from 34 members of 7 multiplex ASD-affected families harboring the 16p11.2 CNV. Expression of all genes in the CNV region correlated well with their DNA copy number, with no evidence of dosage compensation. We observed effects on gene expression outside the CNV region, including apparent positional effects in cis and in trans at genomic segments with evidence of physical interaction in Hi-C chromosome conformation data. One of the most significant positional effects was telomeric to the 16p11.2 CNV and includes the previously described "distal" 16p11.2 microdeletion. Overall, 16p11.2 CNV was associated with altered expression of genes and networks that converge on multiple hypotheses of ASD pathogenesis, including synaptic function (e.g., NRXN1, NRXN3), chromatin modification (e.g., CHD8, EHMT1, MECP2), transcriptional regulation (e.g., TCF4, SATB2), and intellectual disability (e.g., FMR1, CEP290). However, there were differences between tissues and species, with the strongest effects being consistently within the CNV region itself. Our analyses suggest that through a combination of indirect regulatory effects and direct effects on nuclear architecture, alteration of 16p11.2 genes disrupts expression networks that involve other genes and pathways known to contribute to ASD, suggesting an overlap in mechanisms of pathogenesis.

Sex-differences in human liver gene expression were characterized on a genome-wide scale using a large liver sample collection, allowing for detection of small expression differences with high statistical power. 1,249 sex-biased genes were identified, 70% showing higher expression in females. Chromosomal bias was apparent, with female-biased genes enriched on chrX and male-biased genes enriched on chrY and chr19, where 11 male-biased zinc-finger KRAB-repressor domain genes are distributed in six clusters. Top biological functions and diseases significantly enriched in sex-biased genes include transcription, chromatin organization and modification, sexual reproduction, lipid metabolism and cardiovascular disease. Notably, sex-biased genes are enriched at loci associated with polygenic dyslipidemia and coronary artery disease in genome-wide association studies. Moreover, of the 8 sex-biased genes at these loci, 4 have been directly linked to monogenic disorders of lipid metabolism and show an expression profile in females (elevated expression of ABCA1, APOA5 and LDLR; reduced expression of LIPC) that is consistent with the lower female risk of coronary artery disease. Female-biased expression was also observed for CYP7A1, which is activated by drugs used to treat hypercholesterolemia. Several sex-biased drug-metabolizing enzyme genes were identified, including members of the CYP, UGT, GPX and ALDH families. Half of 879 mouse orthologs, including many genes of lipid metabolism and homeostasis, show growth hormone-regulated sex-biased expression in mouse liver, suggesting growth hormone might play a similar regulatory role in human liver. Finally, the evolutionary rate of protein coding regions for human-mouse orthologs, revealed by dN/dS ratio, is significantly higher for genes showing the same sex-bias in both species than for non-sex-biased genes. These findings establish that human hepatic sex differences are widespread and affect diverse cell metabolic processes, and may help explain sex differences in lipid profiles associated with sex differential risk of coronary artery disease.

Sex differences in liver gene expression are dictated by sex differences in circulating GH profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a global scale to discover sex-specific early GH response genes that could contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex differences characterize hepatic responses to plasma GH stimulation. Global RNA expression analysis identified two distinct classes of sex-specific mouse liver genes: genes subject to positive regulation (class I) and genes subject to negative regulation by pituitary hormones (class II). Genes activated or repressed in hypophysectomized (Hypox) mouse liver within 30-90 min of GH pulse treatment at a physiological dose were identified as putative direct targets of GH action (early response genes). Intrinsic sex differences in the GH responsiveness of a subset of these early response genes were observed. Notably, 45 male-specific genes, including five encoding transcriptional regulators that may mediate downstream sex-specific transcriptional responses, were induced by GH within 30 min in Hypox male but not Hypox female mouse liver. The early GH response genes were enriched in 29 male-specific targets of the transcription factor myocyte enhancer factor 2, whose activation in hepatic stellate cells is associated with liver fibrosis leading to hepatocellular carcinoma, a male-predominant disease. Thus, the rapid activation by GH pulses of certain sex-specific genes is modulated by intrinsic sex-specific factors, which may be associated with prior hormone exposure (epigenetic mechanisms) or genetic factors that are pituitary-independent, and could contribute to sex differences in predisposition to liver cancer or other hepatic patho-physiologies.

We have used a simple and efficient method to identify condition-specific transcriptional regulatory sites in vivo to help elucidate the molecular basis of sex-related differences in transcription, which are widespread in mammalian tissues and affect normal physiology, drug response, inflammation, and disease. To systematically uncover transcriptional regulators responsible for these differences, we used DNase hypersensitivity analysis coupled with high-throughput sequencing to produce condition-specific maps of regulatory sites in male and female mouse livers and in livers of male mice feminized by continuous infusion of growth hormone (GH). We identified 71,264 hypersensitive sites, with 1,284 showing robust sex-related differences. Continuous GH infusion suppressed the vast majority of male-specific sites and induced a subset of female-specific sites in male livers. We also identified broad genomic regions (up to ϳ100 kb) showing sex-dependent hypersensitivity and similar patterns of GH responses. We found a strong association of sex-specific sites with sex-specific transcription; however, a majority of sex-specific sites were >100 kb from sex-specific genes. By analyzing sequence motifs within regulatory regions, we identified two known regulators of liver sexual dimorphism and several new candidates for further investigation. This approach can readily be applied to mapping condition-specific regulatory sites in mammalian tissues under a wide variety of physiological conditions. Sexual dimorphism in gene expression is common in mammalian somatic tissues (23) and has broad implications for human health. Sex differences in gene expression may contribute to differences between men and women in the prevalence, extent, and progression of disease, including autoimmune diseases (54), kidney disease (37), cardiovascular disease (45), and liver diseases, such as hepatocellular carcinoma (9, 58). In addition, sex-related differences in pharmacokinetics and pharmacodynamics are common and may affect drug response (52). Sex-related differences in gene expression have been widely studied in liver, where they affect Ͼ1,000 transcripts (5, 51, 57) and impact physiological and pathophysiological functions ranging from lipid and fatty acid metabolism to xenobiotic metabolism and disease susceptibility (52). In the liver, sexrelated differences in gene expression are primarily determined by growth hormone (GH) signaling (3, 21), which shows important sex-related differences that reflect the sex-related differences in plasma GH profiles seen in many species, including rats, mice, and humans (53).The underlying mechanisms of sexual dimorphism in mammalian tissues have been only partly elucidated at the molecular level. In the male rat liver, intermittent plasma GH pulses repeatedly activate the latent cytoplasmic transcription factor STAT5b, whose activity is essential for sex-related differences in the liver (5). The more continuous, female-like pattern of pituitary GH secretion can be mimicked by continuous GH infusion in mal...

Phylogenetic footprinting was used to predict functional transcription factor binding sites (TFBS) for signal transducer and activator of transcription (STAT) 5, a GH-activated transcription factor, in the GH-responsive genes IGF-I, SOCS2, and HNF6. Each gene, including upstream (100 kb) and downstream regions (25 kb), was aligned across four species and searched for conserved STAT5-binding sites using TFBS matrices. Predicted sites were classified as paired or single and whether or not they matched the STAT5 consensus sequence TTCN(3)GAA. Fifty-seven of the predicted genomic regions were assayed by chromatin immunoprecipitation from male rat liver with high STAT5 activity. STAT5 binding was enriched (up to 24-fold) at eight genomic regions of IGF-I, including three novel regions in the second intron, and at four regions of SOCS2, including three novel upstream sites. STAT5 binding to HNF6 was modestly enriched (up to 3-fold) at one consensus site and two novel, nonconsensus sites. Overall, 14 of 17 identified sites were paired STAT5 sites. STAT5 binding to these sites was dynamic in male rat liver, cycling on and off in response to each plasma GH pulse. Moreover, sex-specific STAT5 binding was apparent; in female rat liver, where nuclear STAT5 activity is generally low, STAT5 binding to IGF-I and SOCS2 was limited to high-affinity sites. Analysis of the verified STAT5 binding sites indicated that STAT5 TFBS matrix 459 in combination with a STAT5 consensus sequence was the best predictor of STAT5 binding to these three genes. Using these criteria, multiple novel STAT5 binding sites were identified and then verified in several other GH-inducible genes, including MUP genes, where male-specific gene expression was associated with male-specific STAT5 binding to multiple low-affinity STAT5 sites.

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