Presence of a bi-directional S phase-specific transcription regulatory element in the promoter shared by testis-specific TH2A and TH2B histone genes

, 19, pp. 93–98 (1991) EMBL accession nos X59961–X5996

Study of dynamics of histone H3 variants and H3 proteolysis during mouse spermatogenesis

Spermatogenesis is a complex differentiation process in which male gametes, known as spermatozoa, are produced from spermatogonial stem cells in the seminiferous tubules of the testis. The spermatogenesis process is typically divided into three phases: a mitotic phase, a meiotic phase and post-meiotic spermiogenesis. During mammalian spermiogenesis, haploid round spermatids undergo remarkable morphological changes and an extensive reorganization of chromatin to differentiate into mature spermatozoa. As part of the chromatin reorganization, most histones in round spermatids are replaced by transition proteins and subsequently by protamines. This histone-to-protamine exchange is required for efficient compaction of paternal genome into the sperm head and implicated in male fertility. Nonetheless, previous studies found that 1-10 % histones are still retained at specific loci, particularly at unmethylated CpG-rich promoters, in mouse and human sperm. How spermatid chromatin is reorganized genome-wide during spermiogenesis while some loci are exempted from histone eviction is still elusive. Our previous study has shown that the residual nucleosomes in mouse sperm largely contain the histone H3 variant, H3.3. The study also revealed differential histone turnover of canonical and variant H3 in round spermatids, which may underlie the final histone composition in mature sperm. In order to determine the dynamics of H3 variants during mouse spermatogenesis, I analyze protein expression of canonical and variant H3 proteins at different stages of male germ cells by triton-acetic acid-urea gel-Western blotting. Surprisingly, I find that mouse testis-specific H3 variant (H3t), not canonical H3, is the most abundant H3 protein from spermatogonia to spermatids and that most canonical H3 is replaced by H3.3 during meiosis. I further observe that a relatively large portion of H3t is removed from chromatin during the process of histone-to-protamine exchange compared with H3.3, which is consistent with that H3.3 is the predominant H3 in residual sperm nucleosomes. Taken together, the first part of my thesis reveals important findings on chromatin composition and dynamics of histone H3 variants during mouse spermatogenesis. In the second part of my thesis, I describe the discovery that histone H3 is cleaved at its N-terminal tail by a serine protease activity in nuclei of the late-stage mouse spermatids. Arginine 26 and lysine 27 on H3 are important to the H3 protease activity. This proteolytic cleavage of H3 tail may result in nucleosome destabilization and then contribute to nucleosome eviction during spermiogenesis. Interestingly, I find that the acetylation on H3 can prevent H3 from proteolytic cleavage in vitro and that the genome-wide distribution of H3 lysine 27 acetylation (H3K27Ac) is positively correlated to the occupancy of nucleosomes containing transcriptionally active mark in sperm, suggesting that the inhibition of H3 cleavage by acetylated lysine 27 in late-stage spermatids may lead to the nucleosome retention at specific loci in mature sperm. Overall, these findings provide novel insights into the mechanism of nucleosome eviction and retention during spermiogenesis through the regulation of H3 proteolytic cleavage

Deciphering the Transcriptional Mechanisms and Function of SOX3 in the Developing Embryonic Mouse Brain and Postnatal Testes

SOX3 is a transcription factor found within neural progenitor cells (NPC) of the developing and adult vertebrate central nervous system. SOX3 is also found in other tissues, most notably the spermatogonial progenitor/stem cell populations in the testes. Normal brain development in both humans and mice, and sperm production in mice, is reliant on the correct expression and dosage of SOX3. The function of SOX3 has been explored through a number of different cell and mouse model based techniques, however, the mechanisms through which SOX3 acts remain largely unknown. This thesis explores the genome wide DNA binding profile of SOX3 in both NPCs and postnatal testes, two very different sources of SOX3 expressing cells. We identified 8064 binding sites within NPCs derived from cultured mouse embryonic stem cells, linking SOX3 to a number of different neural development pathways. Additionally, we identified 778 SOX3 binding sites within postnatal day 7 mouse testes, linking SOX3 to the control of histones and histone variants, most of which was also true for NPCs. We utilised our Sox3 null mouse model and a number of different marker genes of spermatogenesis to identify that SOX3 is found within the committed progenitor fraction of the undifferentiated spermatogonial pool. We identified that SOX3 is required for the transition from a GFRα1+ state to a NGN3+ committed progenitor state, and in the absence of SOX3 GFRα1+ cells accumulate and spermatogonia fail to differentiate, leading to empty testes with no mature sperm. We provide further evidence that Ngn3 is a direct target of SOX3 in both NPCs and the testes albeit thought different regulatory regions. We have generated two invaluable genome wide ChIP-seq datasets that will deepen our understanding of mechanisms by which SOX3 controls context-specific differentiation. Taken together, the data presented in this thesis expand our knowledge of the genomic regions bound by SOX3 and its role in neurogenesis and spermatogenesis.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

Presence of a bi-directional S phase-specific transcription regulatory element in the promoter shared by testis-specific TH2A and TH2B histone genes

During mammalian spermatogenesis, somatic histones are replaced by testis-specific variants. The synthesis of the variants occurs primarily in the germ cells undergoing meiosis in the absence of DNA replication. We have cloned the genes encoding rat somatic and testis-specific H2A (TH2A) histones. The two genes share 300 bp of 5' upstream region with respective H2B genes: somatic H2A with somatic H2B and testis-specific TH2A with testis-specific TH2B gene. The deduced amino acid sequences show that H2A and TH2A histones have eight amino acid differences in the first half of the molecules and three consecutive changes in the C-terminal region. TH2A gene is expressed only in testis. Although synthesis of TH2A and TH2B histones is independent of DNA replication and insensitive to inhibitors of DNA synthesis in testis, the regulatory region shared by the two genes contain a bi-directional S phase-specific transcription regulatory element. In addition, TH2A gene, like TH2B gene, contains the consensus sequence element in the 3' non-coding region which is involved in the S phase-specific stabilization of histone mRNA.11Nsciescopu

Presence of a bi-directional S phase-specific transcription regulatory element in the promoter shared by testis-specific TH2A and TH2B histone genes.

During mammalian spermatogenesis, somatic histones are replaced by testis-specific variants. The synthesis of the variants occurs primarily in the germ cells undergoing meiosis in the absence of DNA replication. We have cloned the genes encoding rat somatic and testis-specific H2A (TH2A) histones. The two genes share 300 bp of 5' upstream region with respective H2B genes: somatic H2A with somatic H2B and testis-specific TH2A with testis-specific TH2B gene. The deduced amino acid sequences show that H2A and TH2A histones have eight amino acid differences in the first half of the molecules and three consecutive changes in the C-terminal region. TH2A gene is expressed only in testis. Although synthesis of TH2A and TH2B histones is independent of DNA replication and insensitive to inhibitors of DNA synthesis in testis, the regulatory region shared by the two genes contain a bi-directional S phase-specific transcription regulatory element. In addition, TH2A gene, like TH2B gene, contains the consensus sequence element in the 3' non-coding region which is involved in the S phase-specific stabilization of histone mRNA