Project description:Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. PAS-Seq and CAGE in mouse testes
Project description:Spermatogenesis in MILI-KO mice was blocked completely at early prophase of the first meiosis during the zygotene and early pachytene stage. To analyze the molecular basis of MILI function, we examined the difference between gene expression for MILI-/- and MILI+/- testes. Keywords: total RNAs of day10 testes
Project description:Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome. 3' end sequencing of poly (A) + RNAs in mouse ES cells with and without U1 snRNP inhibition using antisense morpholino oligonucleotides (AMO). Each with two biological replicates.
Project description:Spermatogenesis in MILI-KO mice was blocked completely at early prophase of the first meiosis during the zygotene and early pachytene stage. To analyze the molecular basis of MILI function, we examined the difference between gene expression for MILI-/- and MILI+/- testes. Experiment Overall Design: To exclude secondary effects, we prepared mRNA from day10 mice. Because the apoptotic cells in KO testis were observed at day11 after birth.
Project description:Microarray experiment to identify changes in gene expression in 16 day post partum prepubertal Tex19.1-/- mouse testes. Tex19.1 is expressed in germ cells in testes. Tex19.1-/- mice have spermatogenic defects and errors in progression through meiosis. Data provides insight into the changes in gene expression in developing testes at the time when meiotic defects first start to become apparent.
Project description:Meiosis is a specialized division that is specifically engaged in germ cells. This program is finely and dichotomically regulated: germ cells enter meiosis at fetal life in ovaries (13.5 dpc) and only at post-natal life in testes (8 dpp). We used microarrays to determine the gene expression modifications in Meioc mutant gonads displaying early meiotic defects.