ABSTRACT: ZSCAN5B and its primate-specific paralogs bind RNA polymerase III genes and extra-TFIIIC (ETC) sites to modulate mitotic progression [Mouse Placenta ChIP-Seq data set]
Project description:ZSCAN5B and its primate-specific paralogs bind RNA polymerase III genes and extra-TFIIIC (ETC) sites to modulate mitotic progression
Project description:Particularly in the context of differentiation and development, the importance of three-dimensional chromatin architecture to gene regulatory mechanisms is becoming increasingly clear. The most ancient known mechanism of chromatin organization involves TFIIIC, a transcription factor (TF) that recruits RNA polymerase III (Pol III) for transcription of tRNA and other types of non-coding RNA genes. From yeast to mammals, TFIIIC binds to tRNA genes (tDNAs) and scattered “extra-TFIIIC” (ETC) loci and serves to tether these loci together as anchors of chromatin loops. TFIIIC activities are modulated by MAF, MYC, and other TF proteins that are still unidentified. Here we identify the ZSCAN5 TF family - including mammalian ZSCAN5B and its primate-specific paralogs - as proteins that occupy mammalian Pol III promoters and ETC sites. We show that ZSCAN5B binds with high specificity to a conserved subset of tDNA loci and other Pol III genes in human and mouse and that primate-specific ZSCAN5A and ZSCAN5D also bind Pol III genes, although ZSCAN5D preferentially localizes to MIR SINE- and LINE2-associated ETC sites. ZSCAN5 genes are expressed in proliferating cell populations and are cell-cycle regulated, and gene expression data suggested that they might cooperate to regulate basic cellular functions including mitotic progression. Consistent with this predicted role, ZSCAN5A knockdown led to increasing numbers of cells in mitotic cells and aneuploidy in cultured cells. Together these data implicate ZSCAN5 genes in regulation of Pol III gene transcription and nearby Pol II genes, ultimately influencing cell cycle progression and differentiation in a variety of tissues.
Project description:Particularly in the context of differentiation and development, the importance of three-dimensional chromatin architecture to gene regulatory mechanisms is becoming increasingly clear. The most ancient known mechanism of chromatin organization involves TFIIIC, a transcription factor (TF) that recruits RNA polymerase III (Pol III) for transcription of tRNA and other types of non-coding RNA genes. From yeast to mammals, TFIIIC binds to tRNA genes (tDNAs) and scattered “extra-TFIIIC” (ETC) loci and serves to tether these loci together as anchors of chromatin loops. TFIIIC activities are modulated by MAF, MYC, and other TF proteins that are still unidentified. Here we identify the ZSCAN5 TF family - including mammalian ZSCAN5B and its primate-specific paralogs - as proteins that occupy mammalian Pol III promoters and ETC sites. We show that ZSCAN5B binds with high specificity to a conserved subset of tDNA loci and other Pol III genes in human and mouse and that primate-specific ZSCAN5A and ZSCAN5D also bind Pol III genes, although ZSCAN5D preferentially localizes to MIR SINE- and LINE2-associated ETC sites. ZSCAN5 genes are expressed in proliferating cell populations and are cell-cycle regulated, and gene expression data suggested that they might cooperate to regulate basic cellular functions including mitotic progression. Consistent with this predicted role, ZSCAN5A knockdown led to increasing numbers of cells in mitotic cells and aneuploidy in cultured cells. Together these data implicate ZSCAN5 genes in regulation of Pol III gene transcription and nearby Pol II genes, ultimately influencing cell cycle progression and differentiation in a variety of tissues.
Project description:Particularly in the context of differentiation and development, the importance of three-dimensional chromatin architecture to gene regulatory mechanisms is becoming increasingly clear. The most ancient known mechanism of chromatin organization involves TFIIIC, a transcription factor (TF) that recruits RNA polymerase III (Pol III) for transcription of tRNA and other types of non-coding RNA genes. From yeast to mammals, TFIIIC binds to tRNA genes (tDNAs) and scattered “extra-TFIIIC” (ETC) loci and serves to tether these loci together as anchors of chromatin loops. TFIIIC activities are modulated by MAF, MYC, and other TF proteins that are still unidentified. Here we identify the ZSCAN5 TF family - including mammalian ZSCAN5B and its primate-specific paralogs - as proteins that occupy mammalian Pol III promoters and ETC sites. We show that ZSCAN5B binds with high specificity to a conserved subset of tDNA loci and other Pol III genes in human and mouse and that primate-specific ZSCAN5A and ZSCAN5D also bind Pol III genes, although ZSCAN5D preferentially localizes to MIR SINE- and LINE2-associated ETC sites. ZSCAN5 genes are expressed in proliferating cell populations and are cell-cycle regulated, and gene expression data suggested that they might cooperate to regulate basic cellular functions including mitotic progression. Consistent with this predicted role, ZSCAN5A knockdown led to increasing numbers of cells in mitotic cells and aneuploidy in cultured cells. Together these data implicate ZSCAN5 genes in regulation of Pol III gene transcription and nearby Pol II genes, ultimately influencing cell cycle progression and differentiation in a variety of tissues.
Project description:ZSCAN5B and its primate-specific paralogs bind RNA polymerase III genes and extra-TFIIIC (ETC) sites to modulate mitotic progression [SiRNA RNA-Seq data set]
Project description:ZSCAN5B and its primate-specific paralogs bind RNA polymerase III genes and extra-TFIIIC (ETC) sites to modulate mitotic progression [Human cell line ChIP-Seq data set]
Project description: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 (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 the 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.
Project description: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 (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 the 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. ChIP-seq for AGO2 was performed from K562 cells using 2 commercially available monoclonal antibodies (mAbs) and 5 replicates. Replicates 1-3 were performed with Millipore 04-642, and replicates 4 and 5 were performed with Abcam 57113. RNA-seq was carried out on 2 replicates of shMock and 2 replicates of shAGO2. Lentiviral vectors GIPZ Lentiviral #RHS4531-EG271611.
Project description:MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (RNAPII). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of RNAPII promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of RNAPII. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC and RAD21 with N-MYC during S-phase, blocking N-MYC-dependent release of RNAPII from the promoter. Inhibition of Aurora-A in S-phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.
Project description:MYC proteins bind to virtually all active promoters transcribed by RNA polymerase II (RNAPII), but whether they interact with the three-dimensional chromatin architecture is unknown. Here we used HiChIP sequencing of the MYCN oncoprotein and found that MYCN localizes to three-dimensional hubs formed by active promoters and enhancers. In these hubs, MYCN interacts with TFIIIC, an architectural protein complex. MYCN recruits TFIIIC to promoters when transcription elongation is inhibited, and the complex of both proteins induces premature transcription termination. Termination correlates closely with the TFIIIC-dependent removal of MYCN from promoter hubs and with corresponding alterations in the three-dimensional interactions of cohesin complexes. This limits DNA damage by removing RNAPII that stalls proximal to double-strand breaks. Binding of TFIIIC to MYCN is limited by competition with Aurora-A and this protects genes involved in mRNA processing from termination, arguing that MYCN contributes to the unusual proliferative capacity of neuroblastoma cells via removing stalled RNAPII from promoter hubs and via increasing the capacity for RNA processing.