Expression profiling of Ythdc2 ht and Ythdc2 iKO pachytene spermatocytes in mouse.
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ABSTRACT:
PROVIDER: PRJNA701283 | ENA |
REPOSITORIES: ENA
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Project description:We report the differentially expressed genes caused by inducible deletion of YTHDC2 in pachytene spermatocytes. A large number of differentially epxressed genes were identified.
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Project description:Post-transcriptional regulation of gene expression by RNA-binding proteins helps facilitate fast, clean transitions from one cell state to the next during germ cell differentiation. Previously we showed that the RNA helicase YTHDC2 is required for germ cells to properly switch from mitosis to meiosis (Bailey et al., 2017). While YTHDC2 protein is first expressed as male germ cells enter meiosis, when it is needed to shut down the mitotic program, YTHDC2 expression continues to increase and reaches its highest levels later in meiotic prophase, in pachytene spermatocytes. Here we show that YTHDC2 has an additional essential role regulating meiotic progression in late spermatocytes during mouse germ cell differentiation. Inducing conditional knockout of Ythdc2 during the first wave of spermatogenesis, after the germ cells have already initiated meiotic prophase, allowed Ythdc2-deficient germ cells to successfully reach the pachytene stage and properly express many meiotic markers. However, instead of continuing through meiotic prophase and initiating the meiotic divisions, late pachytene spermatocytes failed to transition to the diplotene stage and quickly died. Loss of function of Ythdc2 in spermatocytes resulted in changes in transcript levels for a number of genes by RNA-sequencing compared to control spermatocytes. Our findings suggest that YTHDC2 facilitates proper progression of germ cells through multiple steps of meiosis, potentially via several mechanisms of post-transcriptional RNA regulation.
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Project description:Expression profiling of isolated populations of prepachytene spermatocytes (LP), pachytene spermatocytes (RP) and spermatids (ST) from PWD and B6 was performed to study the genome wide variation in gene expression between two mouse subspecies. To evaluate the transcriptional difference between B6 and PWD in during meiosis, we compared their transcriptomes in sorted populations of pre-pachytene primary spermatocytes (Leptonema, Zygotene and Pachytene), pachytene spermatocytes (Mid-late pachytene and diplotene) and spermatids.
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Project description:Expression profiling of isolated populations of prepachytene spermatocytes (LP), pachytene spermatocytes (RP) and spermatids (ST) from PWD and B6 was performed to study the genome wide variation in gene expression between two mouse subspecies. To evaluate the transcriptional difference between B6 and PWD in during meiosis, we compared their transcriptomes in sorted populations of pre-pachytene primary spermatocytes (Leptonema, Zygotene and Pachytene), pachytene spermatocytes (Mid-late pachytene and diplotene) and spermatids. Populations of pre-pachytene spermatocytes, pachytene spermatocytes and spermatids were isolated from PWD and B6 mice. All populations were isolated in duplicates from two animals. Hence, RNA was prepared from 12 samples (= 2 strains x 3 tissues x 2 replicas) and was then subject to labeling and hybridization on microarray chips.
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Project description:MiRNA expression profiling of isolated populations of prepachytene spermatocytes (LP), pachytene spermatocytes (RP) and spermatids (ST) from PWD and B6 was performed to study the genome wide variation in miRNA expression between two mouse subspecies. Populations of pre-pachytene spermatocytes, pachytene spermatocytes and spermatids were isolated from PWD and B6 mice. All populations were isolated in triplicates from two bilogical replicas Hence, RNA was prepared from 18 samples and was then subject to labeling and hybridization on microarray chips.
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Project description:MiRNA expression profiling of isolated populations of prepachytene spermatocytes (LP), pachytene spermatocytes (RP) and spermatids (ST) from PWD and B6 was performed to study the genome wide variation in miRNA expression between two mouse subspecies.
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Project description:The DSB-machinery, which induces the programmed DNA double-strand breaks (DSBs) in leptotene and zygotene stages during meiosis, needs to be kept in silence after the initiation of pachytene stage to prevent the activation of DSB checkpoint that may lead to meiotic arrest or apoptosis of germ cells. However, the mechanisms underlying this repression remain largely unknown. Here, we report that ZFP541, a germ cell-specific zinc finger protein, is responsible for the suppression of DSBs formation at late pachytene. Lack of Zfp541 in mice leads to generation of DSBs in late pachytene spermatocytes by DSB formation related-proteins and causes male infertility due to meiotic failure. Plated-based scRNA-seq of Zfp541-/- spermatocytes revealed that ZFP541 negatively regulates many meiotic prophase genes, including genes for DSB formation and their upstream transcriptional regulators, in late pachytene spermatocytes. These results were confirmed by 10x single-cell RNA-seq data on spermatogenesis of Zfp541-/- testes, which suggested that Zfp541 is required for repressing the activation of pre-pachytene gene expression programs from early to late pachytene. ZFP541 ChIP-seq on pachytene and diplotene spermatocytes demonstrated that ZFP541 occupies the promoters of meiosis initiators (e.g., Meiosin and Rxra) and a subset of their downstream genes to repress their transcription, and thus prevent the reactivation of pre-pachytene gene expression programs in pachytene spermatocytes. Thus, our results not only revealed the role of ZFP541 in maintaining the repression of pre-pachytene transcriptional programs in pachytene spermatocytes but also provide new insight into the regulation of meiotic progression by timely turning off pre-pachytene genes.
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Project description:The DSB-machinery, which induces the programmed DNA double-strand breaks (DSBs) in leptotene and zygotene stages during meiosis, needs to be kept in silence after the initiation of pachytene stage to prevent the activation of DSB checkpoint that may lead to meiotic arrest or apoptosis of germ cells. However, the mechanisms underlying this repression remain largely unknown. Here, we report that ZFP541, a germ cell-specific zinc finger protein, is responsible for the suppression of DSBs formation at late pachytene. Lack of Zfp541 in mice leads to generation of DSBs in late pachytene spermatocytes by DSB formation related-proteins and causes male infertility due to meiotic failure. Plated-based scRNA-seq of Zfp541-/- spermatocytes revealed that ZFP541 negatively regulates many meiotic prophase genes, including genes for DSB formation and their upstream transcriptional regulators, in late pachytene spermatocytes. These results were confirmed by 10x single-cell RNA-seq data on spermatogenesis of Zfp541-/- testes, which suggested that Zfp541 is required for repressing the activation of pre-pachytene gene expression programs from early to late pachytene. ZFP541 ChIP-seq on pachytene and diplotene spermatocytes demonstrated that ZFP541 occupies the promoters of meiosis initiators (e.g., Meiosin and Rxra) and a subset of their downstream genes to repress their transcription, and thus prevent the reactivation of pre-pachytene gene expression programs in pachytene spermatocytes. Thus, our results not only revealed the role of ZFP541 in maintaining the repression of pre-pachytene transcriptional programs in pachytene spermatocytes but also provide new insight into the regulation of meiotic progression by timely turning off pre-pachytene genes.
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