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Genome-wide analysis of the distribution of the testis-specific double bromodomain protein BRDT reveals distinct roles in pachytene spermatocyte and round spermatids

ABSTRACT: BRDT, a member of the BET family of double bromodomain-containing proteins, is expressed uniquely in the testis from pachytene spermatocytes through round spermatids, and is essential for spermatogenesis in the mouse. Although BRDT is known to bind to acetylated lysines in chromatin, it is not known where in the genome BRDT binds or whether the sites vary during the complex stages of differentiation in which it is expressed. Herein, we use ChIP-Seq to identify genome-wide BRDT binding sites in chromatin from mouse male germ cells at two distinct stages of differentiation. We identified 5452 BRDT-bound genomic loci in pachytene spermatocytes and 5618 BRDT-bound genomic loci in round spermatids. Roughly two thirds of BRDT binding sites were located in genes and BRDT binding correlated with highly transcribed genes. BRDT binding sites also overlapped with several histone modifications or variants that are associated with active transcription. Motif analysis revealed that BRDT-bound regions are enriched for consensus sequences for the MYB, RFX, ETS and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE and RFX in round spermatids. Taken together, our data suggest that BRDT regulates distinct downstream target genes and may have unique functions in the meiotic and spermiogenic transcription programs.

ORGANISM(S): Mus musculus

PROVIDER: GSE98489 | GEO | 2021/01/26

REPOSITORIES: GEO

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Project description:Pachytene piRNAs are PIWI-interacting small RNAs abundantly expressed in pachytene spermatocytes and spermatids in adult mouse testes. Both MIWI and MILI-bound pachytene piRNAs have been found enriched in round spermatids. Miwi-null male mice are sterile due to spermiogenic arrest. In C. elegans, sperm-borne piRNAs appear to have an epigenetic role during fertilization and development because progeny of offspring derived from piRNA-deficient sperm display a progressive fertility loss after several generations. In mice, it remains unknown whether MIWI-bound pachytene piRNA-deficient round spermatids can produce offspring, and whether the progeny of offspring derived from MIWI-bound pachytene piRNA-deficient round spermatids also exhibit transgenerational loss of fertility. Here, we report that Miwi KO round spermatids could fertilize both wild type (WT) and Miwi KO oocytes through round spermatid microinjection (ROSI), and produce healthy and fertile offspring despite the aberrant pachytene piRNA profiles in those Miwi KO spermatids. Progeny of ROSI-derived heterozygotes, both male and female, displayed normal fertility for at least three generations when bred with either WT or Miwi KO females. Our data indicate that aberrant MIWI-bound pachytene piRNAs profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting a normal pachytene piRNAs profile is not required for paternal transgenerational epigenetic inheritance in mice. Method: Round spermatids were purified from WT and Miwi KO adult testes using a mini-STA-PUT method[Methods Enzymol 1993; 225:84-113.]，the purity of round spermatids was >90% based on our previous report[ J Biol Chem 2012; 287:25173-25190.]. Small RNA was isolated from round spermatids using the mirVana RNA isolation kit (Ambion) according to the manufacturer’s instructions. RNA quality and quantity were assessed using the Agilent 2100 Bioanalyzer. Small RNA-Seq was performed on an Ion Proton sequencer (Life Technologies). Libraries were prepared using the Ion Total RNA-Seq Kit v2 (Invitrogen) with biological triplicates for WT and Miwi KO samples. Resutls:Our data indicate that aberrant MIWI-bound pachytene piRNAs profiles in spermatids do not affect fertilization, early embryonic development, or fertility of the offspring, suggesting a normal pachytene piRNAs profile is not required for paternal transgenerational epigenetic inheritance in mice.

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Genome-wide analysis of the distribution of the testis-specific double bromodomain protein BRDT reveals distinct roles in pachytene spermatocyte and round spermatids

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