Dataset Information

Alkbh1 and Tzfp repress a non-repeat piRNA cluster in pachytene spermatocytes.

ABSTRACT: Piwi proteins and Piwi-interacting small RNAs (piRNAs) have known functions in transposon silencing in the male germline of fetal and newborn mice. Both are also present in adult testes; however, their function here remains a mystery. Here, we confirm that most piRNAs in meiotic spermatocytes originate from clusters in non-repeat intergenic regions of DNA. The regulation of these piRNA clusters, including the processing of the precursor transcripts into individual piRNAs, is accomplished through mostly unknown processes. We present a possible regulatory mechanism for one such cluster, named cluster 1082B, located on chromosome 7 in the mouse genome. The 1082B precursor transcript and its 788 unique piRNAs are repressed by the Alkbh1 dioxygenase and the testis-specific transcription repressor Tzfp. We observe a remarkable >1000-fold upregulation of individual piRNAs in pachytene spermatocytes isolated from Alkbh1- and Tzfp-deficient murine testes. Repression of cluster 1082B is further supported by the identification of a 10-bp Tzfp recognition sequence contained within the precursor transcript. Downregulation of LINE1 and IAP transcripts in the Alkbh1- and Tzfp-deficient mice leads us to propose a potential role for the 1082B-encoded piRNAs in transposon control.

SUBMITTER: Nordstrand LM

PROVIDER: S-EPMC3505970 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Alkbh1 and Tzfp repress a non-repeat piRNA cluster in pachytene spermatocytes.

Nordstrand Line M LM Furu Kari K Paulsen Jonas J Rognes Torbjørn T Klungland Arne A

Nucleic acids research 20120910 21

Piwi proteins and Piwi-interacting small RNAs (piRNAs) have known functions in transposon silencing in the male germline of fetal and newborn mice. Both are also present in adult testes; however, their function here remains a mystery. Here, we confirm that most piRNAs in meiotic spermatocytes originate from clusters in non-repeat intergenic regions of DNA. The regulation of these piRNA clusters, including the processing of the precursor transcripts into individual piRNAs, is accomplished through ...[more]

PMID: 22965116

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Project description:BACKGROUND:H1t is the major linker histone variant in pachytene spermatocytes, where it constitutes 50-60% of total H1. This linker histone variant was previously reported to localize in the nucleolar rDNA element in mouse spermatocytes. Our main aim was to determine the extra-nucleolar localization of this linker histone variant in pachytene spermatocytes. RESULTS:We generated H1t-specific antibodies in rabbits and validated its specificity by multiple assays like ELISA, western blot, etc. Genome-wide occupancy studies, as determined by ChIP-sequencing in P20 mouse testicular cells revealed that H1t did not closely associate with active gene promoters and open chromatin regions. Annotation of H1t-bound genomic regions revealed that H1t is depleted from DSB hotspots and TSS, but are predominantly associated with retrotransposable repeat elements like LINE and LTR in pachytene spermatocytes. These chromatin domains are repressed based on co-association of H1t observed with methylated CpGs and repressive histone marks like H3K9me3 and H4K20me3 in vivo. Mass spectrometric analysis of proteins associated with H1t-containing oligonucleosomes identified piRNA-PIWI pathway proteins, repeat repression-associated proteins and heterochromatin proteins confirming the association with repressed repeat-element genomic regions. We validated the interaction of key proteins with H1t-containing oligonucleosomes by use of ChIP-western blot assays. On the other hand, we observe majority of H1t peaks to be associated with the intergenic spacer of the rDNA element, also in association with SINE elements of the rDNA element. Thus, we have identified the genomic and chromatin features of both nucleolar and extranucleolar localization patterns of linker histone H1t in the context of pachytene spermatocytes. CONCLUSIONS:H1t-containing repeat-element LINE and LTR chromatin domains are associated with repressive marks like methylated CpGs, histone modifications H3K9me3 and H4K20me3, and heterochromatin proteins like HP1?, Trim28, PIWIL1, etc. Apart from localization of H1t at the rDNA element, we demonstrate the extranucleolar association of this linker histone variant at repeat-associated chromatin domains in pachytene spermatocytes. We hypothesize that H1t might induce local chromatin relaxation to recruit heterochromatin and repeat repression-associated protein factors necessary for TE (transposable element) repression, the final biological effect being formation of closed chromatin repressed structures.

Project description:Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs) that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod) arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type) despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger spermatocyte arrest via pathways than are genetically distinct from sex body failure-promoted apoptosis and confirm that the latter can function even when recombination-dependent arrest is inoperative. Implications of these findings for understanding the complex relationships between spermatocyte arrest and apoptosis are discussed.

Dataset Information

Alkbh1 and Tzfp repress a non-repeat piRNA cluster in pachytene spermatocytes.

Publications

Alkbh1 and Tzfp repress a non-repeat piRNA cluster in pachytene spermatocytes.

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