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Homo sapiens

ABSTRACT: DND1 PAR-CLIP in human HEK293 and mouse germ line cells

PROVIDER: PRJNA352073 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5224320.fastq.gz	Fastqsanger.gz
	SRR5224330.fastq.gz	Fastqsanger.gz
	SRR5224340.fastq.gz	Fastqsanger.gz
	SRR5224350.fastq.gz	Fastqsanger.gz
	SRR5224360.fastq.gz	Fastqsanger.gz

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Project description:Specification of germ cell fate establishes the germline development during early embryogenesis, yet the underlying mechanisms remain largely unknown in humans. Here we focus on the functional roles of the RNA-binding protein (RBP) DND1 in human germline specification. We deleted the whole genomic region of DND1 in human embryonic stem cells (hESCs), based on which we generated human primordial germ cell-like cells (hPGCLCs). Interestingly, we discovered an increased percentage of hPGCLCs induced from DND1 deleted hESCs, suggesting that DND1 may restrict the specification of human germ cell lineage. Mechanistic investigation reveals that DND1 forms a complex with another RBP NANOS3, in which DND1 facilitates the binding of NANOS3 to their target mRNAs. Furthermore, by analyzing the mRNAs bound by DND1 and NANOS3, we identified SOX4 mRNAs as the key downstream factor for DND1 and NANOS3 complex to restrict the induction of hPGCLCs. Interestingly, DND1 and NANOS3 function in processing bodies (P-bodies) to repress the translation of SOX4 mRNAs, where NANOS3 bridges the interaction between DND1 and the translational repressor 4E-T. Altogether, these findings identify the RBPs DND1 and NANOS3 as “break system” to restrict the entry of germ cell fate in humans.

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The RNA Binding Protein Dead End1 Is Differentially Expressed in Male Germ Cells During Late Gestation and Targets Chromatin Modifiers and Translational Machinery

Project description:DND1 is essential to maintain germ cell identity. Loss of Dnd1 function results in trans-differentiation of germ cells to somatic fates in zebrafish or the formation of teratomas in mice. To explore the mechanistic role of DND1, we recently developed a transgenic mouse line in which a functional fusion protein between DND1 and GFP is expressed from the endogenous locus (Dnd1GFP). Surprisingly, we found that this reporter distinguishes two male germ cell populations (MGCs) during late gestation cell cycle arrest (G0). Most MGCs express low levels of DND1-GFP, but 5-12% of the population express high levels of DND1-GFP. An RNA-seq time course during late gestation revealed that Dnd1 transcript levels as well as transcript levels for multiple epigenetic regulators are 5-10-fold higher in DND1-GFP-hi cells. Furthermore, using antibodies against DND1-GFP for RNA immunoprecipitation (RIP) time course sequencing during late gestation, we identified multiple epigenetic and translational regulators that are binding targets of DND1 during G0. Among these targets are DNA methyltransferases (Dnmts), the enzyme Setdb1 that imposes the nuclear lamina associated repressive histone mark (H3K9me3), five Tudor domain proteins (Tdrds), four actin dependent regulators (Smarcs), and a group of ribosomal and Golgi proteins. These data suggest that DND1 binds to transcripts of a group of epigenetic enzymes and gates their translation during MGC G0 arrest in late gestation.

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Project description:AGO-PAR-CLIP was employed to identify microRNA binding sites in BCBL-1, a Kaposi's sarcoma-associated herpesvirus (KSHV) infected B-cell line and DG75, a KSHV negative B-cell line as a control. By using our novel computational method (PARma) and differential analysis of PAR-CLIP data, highly accurate target sites of KSHV microRNAs can be defined.

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