The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment

Yang, Wei; Lee, Yun-Hwa; Jones, Amanda E.; Woolnough, Jessica L.; Zhou, Dewang; Dai, Qian; Wu, Qiang; Giles, Keith E.; Townes, Tim M.; Wang, Hengbin

doi:10.1038/ncomms4818

Cited by 70 publications

(87 citation statements)

References 54 publications

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“…5E and Dataset S3). These data indicate that Usp16 regulates the expression of many genes through an indirect mechanism, consistent with our previous study in mouse ESCs (27).…”

Section: Usp16supporting

confidence: 81%

“…Constitutive knockout of Usp16 in mice causes embryonic lethality by preventing ESC lineage commitment (27). To determine the tissue-specific function of Usp16, we generated the Usp16 conditional knockout mouse model, in which Usp16 exons 5 and 6 are flanked with LoxP sites.…”

Section: Resultsmentioning

confidence: 99%

“…mouse embryonic stem cells (ESCs), and differentiated ESCs (27,28). These observations suggest that Usp16 could be the primary enzyme that antagonizes the H2A ubiquitination function of PRC1.…”

Section: Significancementioning

confidence: 95%

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The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

Jones

Yang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

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“…5E and Dataset S3). These data indicate that Usp16 regulates the expression of many genes through an indirect mechanism, consistent with our previous study in mouse ESCs (27).…”

Section: Usp16supporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

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The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

Jones

Yang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The groupBy output was visualized using a line graph in Microsoft Excel. Binding profile heatmaps were gen-erated essentially as previously described, except without binning the genes (56). Briefly, the coverage of each nucleotide across the features was determined as described above, and outputs were converted to data matrices using a custom script and then visualized using conditional formatting options in Microsoft Excel 2013.…”

Section: Discussionmentioning

confidence: 99%

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Atwood

Woolnough

Lefèvre

et al. 2016

Journal of Biological Chemistry

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The primary role of the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation within the nucleus. To further our understanding of this role, we have identified chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene. The location of these sites was mirrored by the positions of AGO2 cross-linking sites identified via PAR-CLIP-seq. AGO2 binding to the rRNA within the nucleus was confirmed by RNA immunoprecipitation and quantitative-PCR. To explore a possible mechanism by which AGO2 could be recruited to the rRNA, we identified 1174 regions within the 45S rRNA transcript that have the ability to form a perfect duplex with position 2-6 (seed sequence) of each microRNA expressed in HEK293T cells. Of these potential AGO2 binding sites, 479 occurred within experimentally verified AGO2-rRNA crosslinking sites. The ability of AGO2 to cross-link to rRNA was almost completely lost in a DICER knock-out cell line. The transfection of miR-92a-2-3p into the noDICE cell line facilitated AGO2 cross-linking at a region of the rRNA that has a perfect seed match at positions 3-8, including a single G-U base pair. Knockdown of AGO2 within HEK293T cells causes a slight, but statistically significant increase in the overall rRNA synthesis rate but did not impact the ratio of processing intermediates or the recruitment of the Pol I transcription factor UBTF.The RNAi machinery has many functions in the eukaryotic cell, and aspects of the RNAi molecular mechanism are highly conserved between yeast and humans (1). Essentially, a small RNA is bound by a member of the Argonaute family of proteins and contributes sequence specificity to a larger protein complex. In the cytoplasm, the RNAi machinery uses Watson-Crick base pairing to target the RNA-induced silencing complex to a specific mRNA and facilitate its degradation. A related process is well established in the nucleus of Schizosaccharomyces pombe, where instead of targeting cytoplasmic mRNAs for destruction, a small RNA targets the RNA-induced transcriptional silencing complex to the pericentromeric regions of each chromosome and facilitates the generation of heterochromatin (2, 3).Work in a chicken-human hybrid cell line supports the possibility that the RNAi machinery is responsible for centromeric chromatin structure in vertebrates as well (4). Indeed, when Dicer is conditionally inactivated, transcription of ␣-satellite DNA from human chromosome 21 increases. Furthermore, the loss of Dicer results in a loss of siRNAs originating from these repeat regions, a delocalization of HP1, and disruption of mitosis (4). The RNAi machinery is also implicated in the creation and/or maintenance of heterochromatin at various sites throughout the genome, in addition to the centromeric regions. Transfection of a siRNA homologous to the EF1a promoter in human cells silences the endogenous gene (5). In a related study, human Argonaute 1 (A...

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“…The groupBy output was visualized using a line graph in Microsoft Excel. Binding profile heat maps were generated essentially as previously described, except without binning the genes (52). Briefly, the coverage of each nucleotide across the tRNA genes was determined using coverageBed against a BED file of the tRNAscan-SE tRNA gene annotations and the "-d" option of the BEDTools.…”

Section: Rt-qpcrmentioning

confidence: 99%

Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

Woolnough

Atwood

Giles

2015

Molecular and Cellular Biology

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To further our understanding of the RNAi machinery within the human nucleus, we analyzed the chromatin and RNA binding of Argonaute 2 (AGO2) within human cancer cell lines. Our data indicated that AGO2 binds directly to nascent tRNA and 5S rRNA, and to the genomic loci from which these RNAs are transcribed, in a small RNA-and DICER-independent manner. AGO2 chromatin binding was not observed at non-TFIIIC-dependent RNA polymerase III (Pol III) genes or at extra-TFIIIC (ETC) sites, indicating that the interaction is specific for TFIIIC-dependent Pol III genes. A genome-wide analysis indicated that loss of AGO2 caused a global increase in mRNA expression level among genes that flank AGO2-bound tRNA genes. This effect was shown to be distinct from that of the disruption of DICER, DROSHA, or CTCF. We propose that AGO2 binding to tRNA genes has a novel and important regulatory role in human cells. The RNA interference (RNAi) machinery has several important functions that are conserved from fission yeast to humans. In the cytoplasm of eukaryotic cells, Argonaute proteins promote degradation and/or translational repression of specific mRNAs through tethering by a complementary small RNA (1). The nuclear functions of the RNAi machinery appear to be diverse across species and include roles in promoting heterochromatin formation, regulating alternative splicing, and promoting insulator function (2-5). The ability of both Argonaute 1 (AGO1) and AGO2 to alter splicing in human cells depends upon small RNAs derived from exonic sequence, which tether Argonaute proteins to nascent transcripts (3, 6). Tethering of each Argonaute protein was shown to be capable of recruiting a histone lysine methyltransferase, which altered the RNA polymerase II (Pol II) elongation rate and thus facilitated alternative splicing. This interaction between Argonaute proteins and the transcriptional apparatus is conserved in Drosophila melanogaster (7).Despite the similarities between Argonaute proteins, AGO2 is the only human Argonaute protein believed to have the endonuclease activity (Slicer) due to a unique amino-terminal region (8-10), and Ago2 but not Ago1 gene deletions are embryonic lethal in mice (11). AGO2 was shown to promote transcriptional gene silencing in a microRNA (miRNA)-dependent manner (12) and to affect nucleosome occupancy at certain transcription start sites through interaction with the SWI/SNF complex (13). Furthermore, AGO1 has been shown to interact with actively transcribed genes and enhancer regions (14, 15). Recent work has identified expanded binding capabilities of AGO2, which include binding to longer RNAs such as pre-miRNAs (65 to 75 nucleotides [nt]) and full-length tRNAs (ϳ75 nt) (16)(17)(18)(19)(20). To wit, AGO2 was recently shown to functionally interact with DICER in human nuclei but was unable to load duplex small interfering RNA (siRNA), indicating that nuclear RNAi processes may proceed through a mechanism distinct from that observed in the cytoplasm (21). These findings indicate that AGO2 may have roles beyo...

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The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment

Cited by 70 publications

References 54 publications

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

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