Project description:Mammalian genomes harbour hundreds of thousands of RNA Polymerase II (Pol II) landing pads, enhancers and promoters, from which transcription can initiate bidirectionally. Nevertheless, processive transcription is largely restricted to the small gene-containing fraction of the genome. A highly conserved and essential metazoan complex, Restrictor, composed of WDR82 and ZC3H4, restrains processive Pol II activity at thousands of extragenic transcription units, thus representing a critical enforcer of genome utilization. However, because of the widespread recruitment of Restrictor to both genic and non-genic transcription sites, the identification of the mechanistic basis for its exquisite selectivity for extragenic transcription has remained elusive thus far. Here, we show that while WDR82 tethers Restrictor to sites of transcription initiation, the three C3H1-type zinc fingers of ZC3H4 make sequence-specific interactions with short motifs selectively enriched at the 5’ end of extragenic transcripts, with such interactions being required for transcription termination. Hence, while Restrictor recruitment relies on WDR82-dependent tethering to the initiating Pol II, its selectivity mainly arises from sequence-specific RNA recognition.
Project description:Mammalian genomes harbour hundreds of thousands of RNA Polymerase II (Pol II) landing pads, enhancers and promoters, from which transcription can initiate bidirectionally. Nevertheless, processive transcription is largely restricted to the small gene-containing fraction of the genome. A highly conserved and essential metazoan complex, Restrictor, composed of WDR82 and ZC3H4, restrains processive Pol II activity at thousands of extragenic transcription units, thus representing a critical enforcer of genome utilization. However, because of the widespread recruitment of Restrictor to both genic and non-genic transcription sites, the identification of the mechanistic basis for its exquisite selectivity for extragenic transcription has remained elusive thus far. Here, we show that while WDR82 tethers Restrictor to sites of transcription initiation, the three C3H1-type zinc fingers of ZC3H4 make sequence-specific interactions with short motifs selectively enriched at the 5’ end of extragenic transcripts, with such interactions being required for transcription termination. Hence, while Restrictor recruitment relies on WDR82-dependent tethering to the initiating Pol II, its selectivity mainly arises from sequence-specific RNA recognition.
Project description:Mammalian genomes harbour hundreds of thousands of RNA Polymerase II (Pol II) landing pads, enhancers and promoters, from which transcription can initiate bidirectionally. Nevertheless, processive transcription is largely restricted to the small gene-containing fraction of the genome. A highly conserved and essential metazoan complex, Restrictor, composed of WDR82 and ZC3H4, restrains processive Pol II activity at thousands of extragenic transcription units, thus representing a critical enforcer of genome utilization. However, because of the widespread recruitment of Restrictor to both genic and non-genic transcription sites, the identification of the mechanistic basis for its exquisite selectivity for extragenic transcription has remained elusive thus far. Here, we show that while WDR82 tethers Restrictor to sites of transcription initiation, the three C3H1-type zinc fingers of ZC3H4 make sequence-specific interactions with short motifs selectively enriched at the 5’ end of extragenic transcripts, with such interactions being required for transcription termination. Hence, while Restrictor recruitment relies on WDR82-dependent tethering to the initiating Pol II, its selectivity mainly arises from sequence-specific RNA recognition.
Project description:Mammalian genomes harbour hundreds of thousands of RNA Polymerase II (Pol II) landing pads, enhancers and promoters, from which transcription can initiate bidirectionally. Nevertheless, processive transcription is largely restricted to the small gene-containing fraction of the genome. A highly conserved and essential metazoan complex, Restrictor, composed of WDR82 and ZC3H4, restrains processive Pol II activity at thousands of extragenic transcription units, thus representing a critical enforcer of genome utilization. However, because of the widespread recruitment of Restrictor to both genic and non-genic transcription sites, the identification of the mechanistic basis for its exquisite selectivity for extragenic transcription has remained elusive thus far. Here, we show that while WDR82 tethers Restrictor to sites of transcription initiation, the three C3H1-type zinc fingers of ZC3H4 make sequence-specific interactions with short motifs selectively enriched at the 5’ end of extragenic transcripts, with such interactions being required for transcription termination. Hence, while Restrictor recruitment relies on WDR82-dependent tethering to the initiating Pol II, its selectivity mainly arises from sequence-specific RNA recognition.
Project description:Mammalian genomes harbour hundreds of thousands of RNA Polymerase II (Pol II) landing pads, enhancers and promoters, from which transcription can initiate bidirectionally. Nevertheless, processive transcription is largely restricted to the small gene-containing fraction of the genome. A highly conserved and essential metazoan complex, Restrictor, composed of WDR82 and ZC3H4, restrains processive Pol II activity at thousands of extragenic transcription units, thus representing a critical enforcer of genome utilization. However, because of the widespread recruitment of Restrictor to both genic and non-genic transcription sites, the identification of the mechanistic basis for its exquisite selectivity for extragenic transcription has remained elusive thus far. Here, we show that while WDR82 tethers Restrictor to sites of transcription initiation, the three C3H1-type zinc fingers of ZC3H4 make sequence-specific interactions with short motifs selectively enriched at the 5’ end of extragenic transcripts, with such interactions being required for transcription termination. Hence, while Restrictor recruitment relies on WDR82-dependent tethering to the initiating Pol II, its selectivity mainly arises from sequence-specific RNA recognition.
Project description:Mammalian genomes harbour hundreds of thousands of RNA Polymerase II (Pol II) landing pads, enhancers and promoters, from which transcription can initiate bidirectionally. Nevertheless, processive transcription is largely restricted to the small gene-containing fraction of the genome. A highly conserved and essential metazoan complex, Restrictor, composed of WDR82 and ZC3H4, restrains processive Pol II activity at thousands of extragenic transcription units, thus representing a critical enforcer of genome utilization. However, because of the widespread recruitment of Restrictor to both genic and non-genic transcription sites, the identification of the mechanistic basis for its exquisite selectivity for extragenic transcription has remained elusive thus far. Here, we show that while WDR82 tethers Restrictor to sites of transcription initiation, the three C3H1-type zinc fingers of ZC3H4 make sequence-specific interactions with short motifs selectively enriched at the 5’ end of extragenic transcripts, with such interactions being required for transcription termination. Hence, while Restrictor recruitment relies on WDR82-dependent tethering to the initiating Pol II, its selectivity mainly arises from sequence-specific RNA recognition.
Project description:Mammalian genomes are pervasively transcribed outside mapped protein-coding genes. One class of extragenic transcription products is represented by long non-coding RNAs (lncRNAs), some of which result from Pol_II transcription of bona-fide RNA genes. Whether all lncRNAs described insofar are products of RNA genes, however, is still unclear. Here we have characterized transcription sites located outside protein-coding genes in a highly regulated response, macrophage activation by endotoxin. Using chromatin signatures, we could unambiguously classify extragenic Pol_II transcription sites as belonging to either canonical RNA genes or transcribed enhancers. Unexpectedly, 70% of extragenic Pol_II peaks were associated with genomic regions with a canonical chromatin signature of enhancers. Enhancer-associated extragenic transcription was frequently adjacent to inducible inflammatory genes, was regulated in response to endotoxin stimulation and generated very low abundance transcripts. Moreover, transcribed enhancers were under purifying selection and contained binding sites for inflammatory transcription factors, thus suggesting their functionality. These data demonstrate that a large fraction of extragenic Pol_II transcription sites can be ascribed to cis-regulatory genomic regions rather than to autonomous RNA genes. Discrimination between lncRNAs generated by canonical RNA genes and products of transcribed enhancers will provide a framework for experimental approaches to lncRNAs and help complete the annotation of mammalian genomes. Chromatin immunoprecipitation of RNA polymerase II phosphorylated in ser5 followed by multiparallel sequencing was performed in bone marrow-derived macrophages. The experiment was also carried out in cells treated for 2hrs with lipopolysaccharide (LPS).
Project description:Mammalian genomes are pervasively transcribed outside mapped protein-coding genes. One class of extragenic transcription products is represented by long non-coding RNAs (lncRNAs), some of which result from Pol_II transcription of bona-fide RNA genes. Whether all lncRNAs described insofar are products of RNA genes, however, is still unclear. Here we have characterized transcription sites located outside protein-coding genes in a highly regulated response, macrophage activation by endotoxin. Using chromatin signatures, we could unambiguously classify extragenic Pol_II transcription sites as belonging to either canonical RNA genes or transcribed enhancers. Unexpectedly, 70% of extragenic Pol_II peaks were associated with genomic regions with a canonical chromatin signature of enhancers. Enhancer-associated extragenic transcription was frequently adjacent to inducible inflammatory genes, was regulated in response to endotoxin stimulation and generated very low abundance transcripts. Moreover, transcribed enhancers were under purifying selection and contained binding sites for inflammatory transcription factors, thus suggesting their functionality. These data demonstrate that a large fraction of extragenic Pol_II transcription sites can be ascribed to cis-regulatory genomic regions rather than to autonomous RNA genes. Discrimination between lncRNAs generated by canonical RNA genes and products of transcribed enhancers will provide a framework for experimental approaches to lncRNAs and help complete the annotation of mammalian genomes. Analysis of nuclear RNA in bone marrow-derive macrophages.
Project description:Mammalian genomes are pervasively transcribed outside mapped protein-coding genes. One class of extragenic transcription products is represented by long non-coding RNAs (lncRNAs), some of which result from Pol_II transcription of bona-fide RNA genes. Whether all lncRNAs described insofar are products of RNA genes, however, is still unclear. Here we have characterized transcription sites located outside protein-coding genes in a highly regulated response, macrophage activation by endotoxin. Using chromatin signatures, we could unambiguously classify extragenic Pol_II transcription sites as belonging to either canonical RNA genes or transcribed enhancers. Unexpectedly, 70% of extragenic Pol_II peaks were associated with genomic regions with a canonical chromatin signature of enhancers. Enhancer-associated extragenic transcription was frequently adjacent to inducible inflammatory genes, was regulated in response to endotoxin stimulation and generated very low abundance transcripts. Moreover, transcribed enhancers were under purifying selection and contained binding sites for inflammatory transcription factors, thus suggesting their functionality. These data demonstrate that a large fraction of extragenic Pol_II transcription sites can be ascribed to cis-regulatory genomic regions rather than to autonomous RNA genes. Discrimination between lncRNAs generated by canonical RNA genes and products of transcribed enhancers will provide a framework for experimental approaches to lncRNAs and help complete the annotation of mammalian genomes.