Genome-wide Polycomb localization in Drosophila whole testes and purified germline precursor cells.
ABSTRACT: The Drosophila spermatogenesis cell differentiation pathway involves the activation of a large set of genes in primary spermatocytes. Most of these genes are activated by testis-specific TATA-binding protein associated factors (tTAFs). In the current model for the activation mechanism, Polycomb plays a key role silencing these genes in the germline precursors, and tTAF-dependent activation in primary spermatocytes involves the displacement of Polycomb from gene promoters. We investigated the genome-wide binding of Polycomb in wild type and tTAF mutant testes. According to the model we expected to see a clear enhancement in Polycomb binding at tTAF-dependent spermatogenesis genes in tTAF mutant testes. However, we find little evidence for such an enhancement in tTAF mutant testes compared to wild type. To avoid problems arising from cellular heterogeneity in whole testis analysis, we further tested the model by analysing Polycomb binding in purified germline precursors, representing cells before tTAF-dependent gene activation. Although we find Polycomb associated with its canonical targets, we find little or no evidence of Polycomb at spermatogenesis genes. The lack of Polycomb at tTAF-dependent spermatogenesis genes in precursor cells argues against a model where Polycomb displacement is the mechanism of spermatogenesis gene activation. This genome-wide ChIP-array study investigates the binding of Polycomb in three biological samples: wild type (WT) whole testes, tTAF (can) mutant whole testes, and FACS-sorted germline precursor cells. We performed two biological replicates for each sample, except wild type whole testes where we performed three. For all ChIP-array experiments, input chromatin was used as the reference control to assay ChIP enrichment. We used Cy3/Cy5-labelled ChIP and input DNA for hybridisation onto Nimblegen arrays, and we performed a Cy3/Cy5 dye swap for one biological replicate of each sample (see supplementary file: GSE39935_README.txt).
Project description:The Drosophila spermatogenesis cell differentiation pathway involves the activation of a large set of genes in primary spermatocytes. Most of these genes are activated by testis-specific TATA-binding protein associated factors (tTAFs). In the current model for the activation mechanism, Polycomb plays a key role silencing these genes in the germline precursors, and tTAF-dependent activation in primary spermatocytes involves the displacement of Polycomb from gene promoters. We investigated the genome-wide binding of Polycomb in wild type and tTAF mutant testes. According to the model we expected to see a clear enhancement in Polycomb binding at tTAF-dependent spermatogenesis genes in tTAF mutant testes. However, we find little evidence for such an enhancement in tTAF mutant testes compared to wild type. To avoid problems arising from cellular heterogeneity in whole testis analysis, we further tested the model by analysing Polycomb binding in purified germline precursors, representing cells before tTAF-dependent gene activation. Although we find Polycomb associated with its canonical targets, we find little or no evidence of Polycomb at spermatogenesis genes. The lack of Polycomb at tTAF-dependent spermatogenesis genes in precursor cells argues against a model where Polycomb displacement is the mechanism of spermatogenesis gene activation.
Project description:Spermatogenesis is a complex cellular-differentiation process that relies on the precise regulation of gene expression in spermatogonia, meiotic, and postmeiotic germ cells. The Ring 1 and YY1 binding protein (Rybp) is a member of the mammalian polycomb-group (PcG) protein family that plays multifunctional roles in development. Previous findings indicate that Rybp may function as an important regulator of meiosis. However, its expression in the testes and function in spermatogenesis have not been examined. In this study, we investigated Rybp expression in postnatal mouse testes using qRT-PCR and immunohistochemistry. We also examined the function of Rybp in spermatogenesis by using a conditional-knockout approach. Results showed that the relative expression of Rybp mRNA was significantly upregulated in the testes of postnatal day (PD) 6 mice. Immunofluorescent staining revealed that Rybp was enriched in the spermatocytes. Surprisingly, a conditional deletion of Rybp in fetal germ cells did not affect the fertility or normal development of spermatogenic cells. Further analysis revealed that Rybp deletion resulted in a decreased expression of meiosis-related genes, but that meiosis progression was normal. Together, these findings suggest that Rybp expression was enriched in spermatocytes, but that it was not required for spermatogenesis.
Project description:In higher eukaryotes, histone methylation is involved in the maintenance of cellular identity during somatic development. During spermatogenesis, most nucleosomes are replaced by protamines. Therefore, it is unclear if histone modifications function in paternal transmission of epigenetic information. Here we show that active H3K4 di-methylation (H3K4me2) and repressive H3K27 tri-methylation (H3K27me3), two modifications important for Trithorax and Polycomb-mediated gene regulation, are present in chromatin of human spermatozoa and show methylation-specific distributions at regulatory regions. H3K4me2-marked promoters control gene functions in spermatogenesis and cellular homeostasis suggesting that this mark reflects germline transcription. In contrast, H3K27me3 marks promoters of key developmental regulators in sperm as in soma. Many H3K27me3-marked genes are never expressed in the male and female germline, and in early “totipotent” embryos, suggesting a function for Polycomb in repressing somatic determinants across generations. Targets of H3K4me2 and H3K27me3 are also modified in mouse spermatozoa, implicating an evolutionary conserved role for histone methylation in chromatin inheritance via the male germline. Chromatin immuno precipitation (ChIP) was performed on sperm samples obtained from 9 normospermic donors. DNA associated with H3K4me2 or H3K27me3 was precipitated using specific antibodies. Input and precipitated DNA were amplified and hybridized to a tiling microarray (NimbleGen Systems Inc.) representing 18029 promoter regions (2200bp upstream to 500bp downstream of transcription start sites) of all RefSeq annotated human genes. For each modification 3 independent ChIP experiments were performed of which one was hybridized in a dye swap configuration.
Project description:Investigation of whole genome gene expression level changes of testes in the meiotic drive system in aedes aegypti during spermatogenesis compared to non drive strain. The meiotic drive system in Aedes aegypti causes the female determining chromosome to fragment during spermatogenesis. A six chip study using total RNA from three separately extracted non driving strain testes of Aedes aegypti and three separately extracted meiotic drive strain testes of Aedes aegypti.
Project description:While apoptosis is essential for male germ cell development, improper activation of apoptosis in the testis can affect spermatogenesis and cause reproduction defects. Members of the MAGE-A (melanoma antigen family A) gene family are frequently clustered in mammalian genomes and are exclusively expressed in the testes of normal animals but abnormally activated in a wide variety of cancers. We investigated the potential roles of these genes in spermatogenesis by generating a mouse model with a 210-kb genomic deletion encompassing six members of the Magea gene cluster (Magea1, Magea2, Magea3, Magea5, Magea6 and Magea8). Male mice carrying the deletion displayed smaller testes from 2 months old with a marked increase in apoptotic germ cells in the first wave of spermatogenesis. Furthermore, we found that Magea genes prevented stress-induced spermatogenic apoptosis after N-ethyl-N-nitrosourea (ENU) treatment during the adult stage. Mechanistically, deletion of the Magea gene cluster resulted in a dramatic increase in apoptotic germ cells, predominantly spermatocytes, with activation of p53 and induction of Bax in the testes. These observations demonstrate that the Magea genes are crucial in maintaining normal testicular size and protecting germ cells from excessive apoptosis under genotoxic stress.
Project description:During spermatogenesis, a large number of germline genes essential for male fertility are coordinately activated. However, it remains unknown how timely activation of this group of germline genes is accomplished. Here we show that Polycomb-repressive complex 1 (PRC1) directs timely activation of germline genes during spermatogenesis. Inactivation of PRC1 in male germ cells results in the gradual loss of a stem cell population and severe differentiation defects, leading to male infertility. In the stem cell population, RNF2, the dominant catalytic subunit of PRC1, activates transcription of Sall4, which codes for a transcription factor essential for subsequent spermatogenic differentiation. Furthermore, RNF2 and SALL4 together occupy transcription start sites of germline genes in the stem cell population. Once differentiation commences, these germline genes are activated to enable the progression of spermatogenesis. Our study identifies a novel mechanism by which Polycomb directs the developmental process by activating a group of lineage-specific genes.
Project description:DDX3 subfamily DEAD-box RNA helicases are essential developmental regulators of RNA metabolism in eukaryotes. belle, the single DDX3 ortholog in Drosophila, is required for fly viability, fertility, and germline stem cell maintenance. Belle is involved both in translational activation and repression of target mRNAs in different tissues; however, direct targets of Belle in the testes are essentially unknown. Here we showed that belle RNAi knockdown in testis cyst cells caused a disruption of adhesion between germ and cyst cells and generation of tumor-like clusters of stem-like germ cells. Ectopic expression of ?-integrin in cyst cells rescued early stages of spermatogenesis in belle knockdown testes, indicating that integrin adhesion complexes are required for the interaction between somatic and germ cells in a cyst. To address Belle functions in spermatogenesis in detail we performed cross-linking immunoprecipitation and sequencing (CLIP-seq) analysis and identified multiple mRNAs that interacted with Belle in the testes. The set of Belle targets includes transcripts of proteins that are essential for preventing the tumor-like clusters of germ cells and for sustaining spermatogenesis. By our hypothesis, failures in the translation of a number of mRNA targets additively contribute to developmental defects observed in the testes with belle knockdowns both in cyst cells and in the germline.
Project description:To understand mechanisms of spermatogenesis, the proteome and the phosphoproteome in prepubertal and pubertal swamp buffalo (Bubalus bubalis) testes were analyzed using tandem mass tag (TMT) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In prepubertal testes, 80 proteins were overexpressed, 148 proteins were underexpressed, and 139 and 142 protein sites had higher and lower phosphorylation, respectively, compared to the levels in pubertal testes. Several of these proteins were associated with reproductive processes such as sexual reproduction, spermatogenesis, fertilization, and spermatid development. In particular, outer dense fiber protein 1 (ODF1), protein maelstrom homolog (MAEL), actin-like protein 7B (ACTL7B), tyrosine-(Y)-phosphorylation regulated (CABYR), and tripartite motif containing 36 (TRIM36) were upregulated with age at both the proteome and phosphoproteome levels. Combining proteome and phosphoproteome analysis can be effectively applied to study the protein/phosphorylation patterns of buffalo testes. These data provide new regulatory candidates and evidence for a complex network in spermatogenesis in buffalo testes, and serve as an important resource for exploring the physiological mechanism of spermatogenesis in mammals.
Project description:Sertoli cell-only (SCO) syndrome is a severe form of human male infertility seemingly characterized by the lack all spermatogenic cells. However, tubules of some SCO testes contain small patches of active spermatogenesis and thus spermatogonial stem cells. We hypothesized that these stem cells cannot replicate and seed spermatogenesis in barren areas of tubule because as-of-yet unrecognized deficits in Sertoli cell gene expression disable most stem cell niches. Performing the first thorough comparison of the transcriptomes of human testes exhibiting complete spermatogenesis with the transcriptomes of testes with SCO syndrome, we defined transcripts that are both predominantly expressed by Sertoli cells and expressed at aberrant levels in SCO testes. Some of these transcripts encode proteins required for the proper assembly of adherent and gap junctions at sites of contact with other cells, including spermatogonial stem cells (SSCs). Other transcripts encode GDNF, FGF8 and BMP4, known regulators of mouse SSCs. Thus, most SCO Sertoli cells can neither organize junctions at normal sites of cell-cell contact nor stimulate SSCs with adequate levels of growth factors. We propose that the critical deficits in Sertoli cell gene expression we have identified contribute to the inability of spermatogonial stem cells within small patches of spermatogenesis in some SCO testes to seed spermatogenesis to adjacent areas of tubule that are barren of spermatogenesis. Furthermore, we predict that one or more of these deficits in gene expression are primary causes of human SCO syndrome.
Project description:In higher eukaryotes, histone methylation is involved in the maintenance of cellular identity during somatic development. During spermatogenesis, Since most nucleosomes are replaced by protamines during spermatogenesis . Iit is therefore unclear whether if histone modifications function in paternal transmission of epigenetic information. Here we show that H3K4 di-methylation (H3K4me2) and H3K27 tri-methylation (H3K27me3), two modifications important for Trithorax and Polycomb-mediated gene regulation, display methylation-specific distributions at regulatory regions in human spermatozoa. H3K4 dimethylation H3K4me2-marksed promoters of genes relevant control gene functions in spermatogenesis and cellular homeostasis suggesting that this mark reflects germline transcription. In contrast, H3K27 trimethylation (H3K27me3) marks promoters of key developmental regulators in sperm like in somatic cells. Promoters of orthologous genes are similarly modified in mouse spermatozoa. Further, particularly genes with extensive H3K27me3 coverage around transcriptional start sites are never expressed during male and female gametogenesis, nor in pre-implantation embryos. These data are compatible with a function for Polycomb in repressing somatic determinants across generations. Importantly, however, we observe only modest selective retention of nucleosomes at regulatory regions in human sperm suggesting that paternal transmission of H3K27me3-encoded epigenetic information may be subjected to variegation. Identification of nucleosome containing regions in 6 human sperm samples