Chromatin organization in NPP-13 knockdown C. elegans embryos
Ontology highlight
ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
SUBMITTER: Kohta Ikegami
PROVIDER: E-GEOD-50488 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13. Genome-wide ChIP-seq and ChIP-chip for nuclear membrane protein LEM-2, RNA Polymerase II (AMA-1) and H3K4me3 were performed in mixed-stage C. elegans embryos depleted for NPP-13. As a control, ChIP was also performed in wild-type embryos treated with empty vector.
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Project description:Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-M-CM-^_ IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans. Genome-wide ChIP-seq and ChIP-chip were performed in mixed-stage C. elegans embryos for nuclear pore proteins NPP-13, NPP-3, IMB-1 and chromatin proteins Pol III (RPC-1), TBP-1, TFC-1 (SFC-1), TFC-4 (TAG-315), and Pol II (AMA-1). For RPC-1 and TBP-1 ChIP-seq, embryos depleted for NPP-13 were also used. Total RNAs from wild-type, NPP-13 RNAi, and IMB-1 RNAi embryos were analyzed by RNA-seq.
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Project description:Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-M-CM-^_ IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans. Genome-wide ChIP-seq and ChIP-chip were performed in mixed-stage C. elegans embryos for nuclear pore proteins NPP-13, NPP-3, IMB-1 and chromatin proteins Pol III (RPC-1), TBP-1, TFC-1 (SFC-1), TFC-4 (TAG-315), and Pol II (AMA-1). For RPC-1 and TBP-1 ChIP-seq, embryos depleted for NPP-13 were also used. Total RNAs from wild-type, NPP-13 RNAi, and IMB-1 RNAi embryos were analyzed by RNA-seq.
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Project description:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13. Genome-wide ChIP-seq and ChIP-chip for nuclear membrane protein LEM-2, RNA Polymerase II (AMA-1) and H3K4me3 were performed in mixed-stage C. elegans embryos depleted for NPP-13. As a control, ChIP was also performed in wild-type embryos treated with empty vector.
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2012-04-07 | E-GEOD-37083 | biostudies-arrayexpress
Project description:NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in C. elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparative expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical coregulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa. Synchronized populations of L1 larvae were plated with two sets of HT115 bacteria, one that had been transformed with the RNAi vector only (L4440 plasmid) and another that had been transformed with a vector targeting nhr-23 (clone 5174) [Proc Natl Acad Sci USA 98 (2001) 7360-7365]. Worms were kept on 2% agarose plates for 21 hr at 20C, collected, and approximately 200ml of worms resuspended in PBS were used in each individual experiment. Total RNA was isolated from frozen pellets using a Mixer-Mill (Miller-Mill 300) following an RNeasy Mini Kit (Qiagen, Germantown, MD) according to manufacturer protocol. Aliquots of cultures used for RNA isolation were kept on nhr-23 RNAi plates to confirm the knockdown phenotypic changes occurred during subsequent molts.
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