TRA-1 ChIP-seq reveals regulators of sexual differentiation and multilevel feedback in nematode sex determination

Sex-determining genes have been identified in many animals, but how they impose sex specificity on development is poorly understood. We ask how the nematode sex-determining transcription factor Transformer 1 (TRA-1) regulates sex by identifying where in the genome TRA-1 binds and which nearby genes may be affected by this binding. We find that TRA-1 promotes female development primarily by preventing the expression of genes involved in male development. Among the genes repressed by TRA-1 are a number that control the timing of developmental events and also several that function upstream of TRA-1 in the global sex-determination pathway. The suite of TRA-1 targets presented here provides a resource to continue uncovering the basis of sex-specific development

Sequence determinants in human polyadenylation site selection

BACKGROUND: Differential polyadenylation is a widespread mechanism in higher eukaryotes producing mRNAs with different 3' ends in different contexts. This involves several alternative polyadenylation sites in the 3' UTR, each with its specific strength. Here, we analyze the vicinity of human polyadenylation signals in search of patterns that would help discriminate strong and weak polyadenylation sites, or true sites from randomly occurring signals. RESULTS: We used human genomic sequences to retrieve the region downstream of polyadenylation signals, usually absent from cDNA or mRNA databases. Analyzing 4956 EST-validated polyadenylation sites and their -300/+300 nt flanking regions, we clearly visualized the upstream (USE) and downstream (DSE) sequence elements, both characterized by U-rich (not GU-rich) segments. The presence of a USE and a DSE is the main feature distinguishing true polyadenylation sites from randomly occurring A(A/U)UAAA hexamers. While USEs are indifferently associated with strong and weak poly(A) sites, DSEs are more conspicuous near strong poly(A) sites. We then used the region encompassing the hexamer and DSE as a training set for poly(A) site identification by the ERPIN program and achieved a prediction specificity of 69 to 85% for a sensitivity of 56%. CONCLUSION: The availability of complete genomes and large EST sequence databases now permit large-scale observation of polyadenylation sites. Both U-rich sequences flanking both sides of poly(A) signals contribute to the definition of "true" sites. However, the downstream U-rich sequences may also play an enhancing role. Based on this information, poly(A) site prediction accuracy was moderately but consistently improved compared to the best previously available algorithm

SPE-44 Implements Sperm Cell Fate

The sperm/oocyte decision in the hermaphrodite germline of Caenorhabditis elegans provides a powerful model for the characterization of stem cell fate specification and differentiation. The germline sex determination program that governs gamete fate has been well studied, but direct mediators of cell-type-specific transcription are largely unknown. We report the identification of spe-44 as a critical regulator of sperm gene expression. Deletion of spe-44 causes sperm-specific defects in cytokinesis, cell cycle progression, and organelle assembly resulting in sterility. Expression of spe-44 correlates precisely with spermatogenesis and is regulated by the germline sex determination pathway. spe-44 is required for the appropriate expression of several hundred sperm-enriched genes. The SPE-44 protein is restricted to the sperm-producing germline, where it localizes to the autosomes (which contain sperm genes) but is excluded from the transcriptionally silent X chromosome (which does not). The orthologous gene in other Caenorhabditis species is similarly expressed in a sex-biased manner, and the protein likewise exhibits autosome-specific localization in developing sperm, strongly suggestive of an evolutionarily conserved role in sperm gene expression. Our analysis represents the first identification of a transcriptional regulator whose primary function is the control of gamete-type-specific transcription in this system

A Novel Signaling Pathway Mediated by the Nuclear Targeting of C-Terminal Fragments of Mammalian Patched 1

Background: Patched 1 (Ptc1) is a polytopic receptor protein that is essential for growth and differentiation. Its extracellular domains accept its ligand, Sonic Hedgehog, while the function of its C-terminal intracellular domain is largely obscure. Principal Findings: In this study, we stably expressed human Ptc1 protein in HeLa cells and found that it is subjected to proteolytic cleavage at the C-terminus, resulting in the generation of soluble C-terminal fragments. These fragments accumulated in the nucleus, while the N-terminal region of Ptc1 remained in the cytoplasmic membrane fractions. Using an anti-Ptc1 C-terminal domain antibody, we provide conclusive evidence that C-terminal fragments of endogenous Ptc1 accumulate in the nucleus of C3H10T1/2 cells. Similar nuclear accumulation of endogenous C-terminal fragments was observed not only in C3H10T1/2 cells but also in mouse embryonic primary cells. Importantly, the C-terminal fragments of Ptc1 modulate transcriptional activity of Gli1. Conclusions: Although Ptc1 protein was originally thought to be restricted to cell membrane fractions, our findings sugges

Transcriptome Analysis of Female and Male Xiphophorus maculatus Jp 163 A

Background: Xiphophorus models are important for melanoma, sex determination and differentiation, ovoviviparity and evolution. To gain a global view of the molecular mechanism(s) whereby gene expression may influence sexual dimorphism in Xiphophorus and to develop a database for future studies, we performed a large-scale transcriptome study. Methodology/Principal Findings: The 454-FLX massively parallel DNA sequencing platform was employed to obtain 742,771 and 721,543 reads from 2 normalized cDNA libraries generated from whole adult female and male X. maculatus Jp 163 A, respectively. The reads assembled into 45,538 contigs (here, a "contig" is a set of contiguous sequences), of which, 11,918 shared homology to existing protein sequences. These numbers estimate that the contigs may cover 53% of the total number of Xiphophorus transcriptome. Putative translations were obtained for 11,918 cDNA contigs, of which, 3,049 amino acid sequences contain Pfam domains and 11,064 contigs encode secretory proteins. A total of 3,898 contigs were associated with 2,781 InterPro (IPR) entries and 5,411 contigs with 132 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. There were 10,446 contigs annotated with 69,778 gene ontology (GO) terms and the three corresponding organizing principles. Fifty-four potential sex differentially expressed genes have been identified from these contigs. Eight and nine of these contigs were confirmed by real-time PCR as female and male predominantly expressed genes respectively. Based on annotation results, 34 contigs were predicted to be differentially expressed in male and female and 17 of them were also confirmed by real-time PCR. Conclusions/Significance: This is the first report of an annotated overview of the transcriptome of X. maculatus and identification of sex differentially expressed genes. These data will be of interest to researchers using the Xiphophorus model. This work also provides an archive for future studies in molecular mechanisms of sexual dimorphism and evolution, and can be used in comparative studies of other fish

Sex Determination in the Squalius alburnoides Complex: An Initial Characterization of Sex Cascade Elements in the Context of a Hybrid Polyploid Genome

BACKGROUND:Sex determination processes vary widely among different vertebrate taxa, but no group offers as much diversity for the study of the evolution of sex determination as teleost fish. However, the knowledge about sex determination gene cascades is scarce in this species-rich group and further difficulties arise when considering hybrid fish taxa, in which mechanisms exhibited by parental species are often disrupted. Even though hybridisation is frequent among teleosts, gene based approaches on sex determination have seldom been conducted in hybrid fish. The hybrid polyploid complex of Squalius alburnoides was used as a model to address this question. METHODOLOGY/PRINCIPAL FINDINGS:We have initiated the isolation and characterization of regulatory elements (dmrt1, wt1, dax1 and figla) potentially involved in sex determination in S. alburnoides and in the parental species S. pyrenaicus and analysed their expression patterns by in situ hybridisation. In adults, an overall conservation in the cellular localization of the gene transcripts was observed between the hybrids and parental species. Some novel features emerged, such as dmrt1 expression in adult ovaries, and the non-dimorphic expression of figla, an ovarian marker in other species, in gonads of both sexes in S. alburnoides and S. pyrenaicus. The potential contribution of each gene to the sex determination process was assessed based on the timing and location of expression. Dmrt1 and wt1 transcripts were found at early stages of male development in S. alburnoides and are most likely implicated in the process of gonad development. CONCLUSIONS/SIGNIFICANCE:For the first time in the study of this hybrid complex, it was possible to directly compare the gene expression patterns between the bisexual parental species and the various hybrid forms, for an extended set of genes. The contribution of these genes to gonad integrity maintenance and functionality is apparently unaltered in the hybrids, suggesting that no abrupt shifts in gene expression occurred as a result of hybridisation

Widespread Presence of Human BOULE Homologs among Animals and Conservation of Their Ancient Reproductive Function

Sex-specific traits that lead to the production of dimorphic gametes, sperm in males and eggs in females, are fundamental for sexual reproduction and accordingly widespread among animals. Yet the sex-biased genes that underlie these sex-specific traits are under strong selective pressure, and as a result of adaptive evolution they often become divergent. Indeed out of hundreds of male or female fertility genes identified in diverse organisms, only a very small number of them are implicated specifically in reproduction in more than one lineage. Few genes have exhibited a sex-biased, reproductive-specific requirement beyond a given phylum, raising the question of whether any sex-specific gametogenesis factors could be conserved and whether gametogenesis might have evolved multiple times. Here we describe a metazoan origin of a conserved human reproductive protein, BOULE, and its prevalence from primitive basal metazoans to chordates. We found that BOULE homologs are present in the genomes of representative species of each of the major lineages of metazoans and exhibit reproductive-specific expression in all species examined, with a preponderance of male-biased expression. Examination of Boule evolution within insect and mammalian lineages revealed little evidence for accelerated evolution, unlike most reproductive genes. Instead, purifying selection was the major force behind Boule evolution. Furthermore, loss of function of mammalian Boule resulted in male-specific infertility and a global arrest of sperm development remarkably similar to the phenotype in an insect boule mutation. This work demonstrates the conservation of a reproductive protein throughout eumetazoa, its predominant testis-biased expression in diverse bilaterian species, and conservation of a male gametogenic requirement in mice. This shows an ancient gametogenesis requirement for Boule among Bilateria and supports a model of a common origin of spermatogenesis