The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

Integrative analysis of 111 reference human epigenomes

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

Sexual reproduction is an ancient feature of life on earth, and the familiar X and Y chromo- somes in humans and other model species have led to the impression that sex determination mecha- nisms are old and conserved. In fact, males and females are deter- mined by diverse mechanisms that evolve rapidly in many taxa. Yet this diversity in primary sex-deter- mining signals is coupled with conserved molecular pathways that trigger male or female develop- ment. Conflicting selection on dif- ferent parts of the genome and on the two sexes may drive many of these transitions, but few systems with rapid turnover of sex determi- nation mechanisms have been rig- orously studied. Here we survey our current understanding of how and why sex determination evolves in animals and plants and identify important gaps in our knowledge that present exciting research op- portunities to characterize the evo- lutionary forces and molecular pathways underlying the evolution of sex determination.

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Sex Determination: Why So Many Ways of Doing It?

Allergic asthma is caused by the aberrant expansion in the lung of T helper cells that produce type 2 (TH2) cytokines and is characterized by infiltration of eosinophils and bronchial hyperreactivity. This disease is often triggered by mast cells activated by immunoglobulin E (IgE)-mediated allergic challenge. Activated mast cells release various chemical mediators, including prostaglandin D2 (PGD2), whose role in allergic asthma has now been investigated by the generation of mice deficient in the PGD receptor (DP). Sensitization and aerosol challenge of the homozygous mutant (DP-/-) mice with ovalbumin (OVA) induced increases in the serum concentration of IgE similar to those in wild-type mice subjected to this model of asthma. However, the concentrations of TH2 cytokines and the extent of lymphocyte accumulation in the lung of OVA-challenged DP-/- mice were greatly reduced compared with those in wild-type animals. Moreover, DP-/- mice showed only marginal infiltration of eosinophils and failed to develop airway hyperreactivity. Thus, PGD2 functions as a mast cell-derived mediator to trigger asthmatic responses.

Prostaglandin D_2 as a Mediator of Allergic Asthma.

The pivotal point of vertebrate sex determination is the development of the gonad into a testis or ovary, which governs phenotypic sex through the production of hormones. The identification of Sry, the genetic switch that controls testis development in mammals, fuelled the race for the discovery of downstream pathways. Comparative analyses of XY versus XX gonadogenesis in both mouse and human genetic mutants have uncovered a burgeoning network of intra- and extra-cellular pathways. Here, we review the old and new players that are involved in the initial steps of testis and ovary development.

One tissue, two fates: molecular genetic events that underlie testis versus ovary development.

The new concept of mammalian sex maintenance establishes that particular key genes must remain active in the differentiated gonads to avoid genetic sex reprogramming, as described in adult ovaries after Foxl2 ablation. Dmrt1 plays a similar role in postnatal testes, but the mechanism of adult testis maintenance remains mostly unknown. Sox9 and Sox8 are required for postnatal male fertility, but their role in the adult testis has not been investigated. Here we show that after ablation of Sox9 in Sertoli cells of adult, fertile Sox8(-/-) mice, testis-to-ovary genetic reprogramming occurs and Sertoli cells transdifferentiate into granulosa-like cells. The process of testis regression culminates in complete degeneration of the seminiferous tubules, which become acellular, empty spaces among the extant Leydig cells. DMRT1 protein only remains in non-mutant cells, showing that SOX9/8 maintain Dmrt1 expression in the adult testis. Also, Sox9/8 warrant testis integrity by controlling the expression of structural proteins and protecting Sertoli cells from early apoptosis. Concluding, this study shows that, in addition to its crucial role in testis development, Sox9, together with Sox8 and coordinately with Dmrt1, also controls adult testis maintenance.

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Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration

We investigated the origin of XX sex reversal in the insectivorous mole Talpa occidentalis. Cytogenetic, histological and hormonal studies indicate that all XX individuals analyzed from two different populations are true hermaphrodites, with ovotestes. This suggests that XX sex reversal may be the norm in this species. The intersexes are functional fertile females and the trait is transmitted and maintained in the population. Intersexes lack the Y chromosome gene SRY (sex determining region Y gene), shown to be the testis determining gene. These results suggest that XX intersex moles may have arisen from a mutation of a gene located downstream from SRY/TDY in the testis determining pathway.

Fertile females of the mole Talpa occidentalis are phenotypic intersexes with ovotestes

The biological significance of nucleoli and mitotic Ag-staining NOR's has been the cause of controversy for several years. Whereas several authors state or assume a direct correlation between transcriptional activity and Ag-staining, others deny this hypothesis or claim a correlation between Ag-staining and decondensation of the NOR chromatin. Our results show that a decondensed state of the NOR chromatin is necessary but not sufficient to permit silver impregnation, allowing us to conclude that transcriptional NOR activity in the previous interphase is additionally required for silver staining.

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A study of the Ag-staining significance in mitotic NOR's.

In-situ hybridization with a rDNA probe has demonstrated the presence of non-transcribed ribosomal genes in the B chromosomes of the black rat Rattus rattus. To test whether methylation of ribosomal DNA present in the B chromosomes could account for their inactivation, we performed in-situ digestions and Southern analyses of DNA digested with the isoschizomers MspI and HpaII. Our results suggest that the accessory chromosomes of this species have originated from one of the smaller NOR-carrying chromosome pairs. In the course of evolution, repetitive sequences invaded this supernumerary element and its ribosomal DNA content was dispersed throughout the chromosome and inactivated by heterochromatinization and methylation.

Inactive ribosomal cistrons are spread throughout the B chromosomes of Rattus rattus (Rodentia, Muridae). Implications for their origin and evolution.

In mammals, sex differentiation depends on gonad development, which is controlled by two groups of sex-determining genes that promote one gonadal sex and antagonize the opposite one. SOX9 plays a key role during testis development in all studied vertebrates, whereas it is kept inactive in the XX gonad at the critical time of sex determination, otherwise, ovary-to-testis gonadal sex reversal occurs. However, molecular mechanisms underlying repression of Sox9 at the beginning of ovarian development, as well as other important aspects of gonad organogenesis, remain largely unknown. Because there is indirect evidence that micro-RNAs (miRNA) are necessary for testicular function, the possible involvement of miRNAs in mammalian sex determination deserved further research. Using microarray technology, we have identified 22 miRNAs showing sex-specific expression in the developing gonads during the critical period of sex determination. Bioinformatics analyses led to the identification of miR-124 as the candidate gene for ovarian development. We knocked down or overexpressed miR-124 in primary gonadal cell cultures and observed that miR-124 is sufficient to induce the repression of both SOX9 translation and transcription in ovarian cells. Our results provide the first evidence of the involvement of a miRNA in the regulation of the gene controlling gonad development and sex determination. The miRNA microarray data reported here will help promote further research in this field, to unravel the role of other miRNAs in the genetic control of mammalian sex determination.

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Miguel Burgos

Papers

Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration

Fertile females of the mole Talpa occidentalis are phenotypic intersexes with ovotestes

A study of the Ag-staining significance in mitotic NOR's.

Inactive ribosomal cistrons are spread throughout the B chromosomes of Rattus rattus (Rodentia, Muridae). Implications for their origin and evolution.

A MicroRNA (mmu-miR-124) Prevents Sox9 Expression in Developing Mouse Ovarian Cells