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Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes.

Stadelmayer B, Micas G, Gamot A, Martin P, Malirat N, Koval S, Raffel R, Sobhian B, Severac D, Rialle S, Parrinello H, Cuvier O, Benkirane M - Nat Commun (2014)

Bottom Line: The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits.Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity.Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Human Genetics, CNRS UPR1142, Laboratory of Molecular Virology; MGX-Montpellier GenomiX, 141 rue de la Cardonille, Montpellier 34396, France [2] LBME-CNRS, Cell Cycle Chromatin Dynamics Laboratory. University Paul Sabatier, Toulouse 31061, France [3] INRA, TOXALIM (Research Centre in Food Toxicology), Toulouse 31300, France [4] IGF, MGX-Montpellier GenomiX, France.

ABSTRACT
RNA polymerase II (RNAPII) pausing/termination shortly after initiation is a hallmark of gene regulation. Here, we show that negative elongation factor (NELF) interacts with Integrator complex subunits (INTScom), RNAPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both human immunodeficiency virus type 1 promoter and genome-wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits. Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity. Finally, INTScom target genes are enriched in human immunodeficiency virus type 1 transactivation response element/NELF binding element and in a 3' box sequence required for small nuclear RNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.

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Integrator complex regulates NELF-mediated RNAPII pausing at coding genes.(a) Venn diagram showing the intersection among differentially expressed (DE) genes identified by microarray analysis (n=3) upon depletion of −INTS11, −INTS3− or −NELF-E (see Methods; P<0.001). (b) Genomic view of ChIP-Seq data over the protein coding MTF2 gene for: H3K4me3, total RNAPII, NELF-E, SPT5, INTS3 and INTS11. (c) Heat maps showing the binding of NELF-E, RNAPII, INTS3 and INTS11 by ranking the top 10,000 genes according to NELF binding (+/− 250 bp from TSSs; see Methods). (d) Heat map showing the intersection analyses between DE (down- or up- regulated) genes and the corresponding TSS bound or not (+/−) by NELF-E, INTS3 and INTS11 (see Methods). Differential expression (DE) was analysed in R using limma (adjusted P<0.01; see Supplementary Dataset 2B–D; see Methods). The enrichment between these two lists is indicated in Log P-value (see colour bar). (e) Averaged RNA RNAPII profiles as measured by ChIP-Seq as a function of NELF-E-, Spt5-, INTS3 and INTS11 binding. The ChIP-Seq profiles of ‘High/Low pausing’ are indicated for comparison (RNAPII profiles of highly paused (blue line)- or active genes (light blue line), respectively, as previously described22). Genes bound by −NELF, SPT5 and INTS3 with or without INTS11 (purple and orange line, respectively) or by −NELF and SPT5 with or without INTS3 (yellow and orange line, respectively) or by SPT5, INTS3 and INTS11 (green line). (f) Box plots showing the RNAPII pausing indices depending on NELF/SPT5/INTS3 and INTS11 binding or not (+/−) to the corresponding TSS. P-values, Wilcoxon pair-wise test (see also Supplementary Fig. 7A).
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f3: Integrator complex regulates NELF-mediated RNAPII pausing at coding genes.(a) Venn diagram showing the intersection among differentially expressed (DE) genes identified by microarray analysis (n=3) upon depletion of −INTS11, −INTS3− or −NELF-E (see Methods; P<0.001). (b) Genomic view of ChIP-Seq data over the protein coding MTF2 gene for: H3K4me3, total RNAPII, NELF-E, SPT5, INTS3 and INTS11. (c) Heat maps showing the binding of NELF-E, RNAPII, INTS3 and INTS11 by ranking the top 10,000 genes according to NELF binding (+/− 250 bp from TSSs; see Methods). (d) Heat map showing the intersection analyses between DE (down- or up- regulated) genes and the corresponding TSS bound or not (+/−) by NELF-E, INTS3 and INTS11 (see Methods). Differential expression (DE) was analysed in R using limma (adjusted P<0.01; see Supplementary Dataset 2B–D; see Methods). The enrichment between these two lists is indicated in Log P-value (see colour bar). (e) Averaged RNA RNAPII profiles as measured by ChIP-Seq as a function of NELF-E-, Spt5-, INTS3 and INTS11 binding. The ChIP-Seq profiles of ‘High/Low pausing’ are indicated for comparison (RNAPII profiles of highly paused (blue line)- or active genes (light blue line), respectively, as previously described22). Genes bound by −NELF, SPT5 and INTS3 with or without INTS11 (purple and orange line, respectively) or by −NELF and SPT5 with or without INTS3 (yellow and orange line, respectively) or by SPT5, INTS3 and INTS11 (green line). (f) Box plots showing the RNAPII pausing indices depending on NELF/SPT5/INTS3 and INTS11 binding or not (+/−) to the corresponding TSS. P-values, Wilcoxon pair-wise test (see also Supplementary Fig. 7A).

Mentions: We next asked whether the function of the INTScom in regulating RNAPII pausing at the HIV-1 promoter also applies to cellular coding genes. Supporting this idea, microarray analyses showed that INTS3 and INTS11depletion (see Supplementary Fig. 4A for knockdown efficiency) from HeLa cells led to changes in the expression of thousands of genes (Fig. 3a and Supplementary Dataset 2a–d). The impact on gene expression could not be attributed to the loss of U1 or U2 snRNA, since no significant changes in their expression were observed within the time frame of these experiments (data not shown) or to an effect on cell cycle (Supplementary Fig. 4B). Interestingly, a majority of these genes (2,164 genes) were commonly identified as differentially expressed (DE) upon depletion of NELF (Fig. 3a and Supplementary Dataset 2a). Such significant overlap (P-value <1e−300) highlighted a novel possible function of INTScom subunits in co-regulating NELF-dependent coding genes.


Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes.

Stadelmayer B, Micas G, Gamot A, Martin P, Malirat N, Koval S, Raffel R, Sobhian B, Severac D, Rialle S, Parrinello H, Cuvier O, Benkirane M - Nat Commun (2014)

Integrator complex regulates NELF-mediated RNAPII pausing at coding genes.(a) Venn diagram showing the intersection among differentially expressed (DE) genes identified by microarray analysis (n=3) upon depletion of −INTS11, −INTS3− or −NELF-E (see Methods; P<0.001). (b) Genomic view of ChIP-Seq data over the protein coding MTF2 gene for: H3K4me3, total RNAPII, NELF-E, SPT5, INTS3 and INTS11. (c) Heat maps showing the binding of NELF-E, RNAPII, INTS3 and INTS11 by ranking the top 10,000 genes according to NELF binding (+/− 250 bp from TSSs; see Methods). (d) Heat map showing the intersection analyses between DE (down- or up- regulated) genes and the corresponding TSS bound or not (+/−) by NELF-E, INTS3 and INTS11 (see Methods). Differential expression (DE) was analysed in R using limma (adjusted P<0.01; see Supplementary Dataset 2B–D; see Methods). The enrichment between these two lists is indicated in Log P-value (see colour bar). (e) Averaged RNA RNAPII profiles as measured by ChIP-Seq as a function of NELF-E-, Spt5-, INTS3 and INTS11 binding. The ChIP-Seq profiles of ‘High/Low pausing’ are indicated for comparison (RNAPII profiles of highly paused (blue line)- or active genes (light blue line), respectively, as previously described22). Genes bound by −NELF, SPT5 and INTS3 with or without INTS11 (purple and orange line, respectively) or by −NELF and SPT5 with or without INTS3 (yellow and orange line, respectively) or by SPT5, INTS3 and INTS11 (green line). (f) Box plots showing the RNAPII pausing indices depending on NELF/SPT5/INTS3 and INTS11 binding or not (+/−) to the corresponding TSS. P-values, Wilcoxon pair-wise test (see also Supplementary Fig. 7A).
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getmorefigures.php?uid=PMC4263189&req=5

f3: Integrator complex regulates NELF-mediated RNAPII pausing at coding genes.(a) Venn diagram showing the intersection among differentially expressed (DE) genes identified by microarray analysis (n=3) upon depletion of −INTS11, −INTS3− or −NELF-E (see Methods; P<0.001). (b) Genomic view of ChIP-Seq data over the protein coding MTF2 gene for: H3K4me3, total RNAPII, NELF-E, SPT5, INTS3 and INTS11. (c) Heat maps showing the binding of NELF-E, RNAPII, INTS3 and INTS11 by ranking the top 10,000 genes according to NELF binding (+/− 250 bp from TSSs; see Methods). (d) Heat map showing the intersection analyses between DE (down- or up- regulated) genes and the corresponding TSS bound or not (+/−) by NELF-E, INTS3 and INTS11 (see Methods). Differential expression (DE) was analysed in R using limma (adjusted P<0.01; see Supplementary Dataset 2B–D; see Methods). The enrichment between these two lists is indicated in Log P-value (see colour bar). (e) Averaged RNA RNAPII profiles as measured by ChIP-Seq as a function of NELF-E-, Spt5-, INTS3 and INTS11 binding. The ChIP-Seq profiles of ‘High/Low pausing’ are indicated for comparison (RNAPII profiles of highly paused (blue line)- or active genes (light blue line), respectively, as previously described22). Genes bound by −NELF, SPT5 and INTS3 with or without INTS11 (purple and orange line, respectively) or by −NELF and SPT5 with or without INTS3 (yellow and orange line, respectively) or by SPT5, INTS3 and INTS11 (green line). (f) Box plots showing the RNAPII pausing indices depending on NELF/SPT5/INTS3 and INTS11 binding or not (+/−) to the corresponding TSS. P-values, Wilcoxon pair-wise test (see also Supplementary Fig. 7A).
Mentions: We next asked whether the function of the INTScom in regulating RNAPII pausing at the HIV-1 promoter also applies to cellular coding genes. Supporting this idea, microarray analyses showed that INTS3 and INTS11depletion (see Supplementary Fig. 4A for knockdown efficiency) from HeLa cells led to changes in the expression of thousands of genes (Fig. 3a and Supplementary Dataset 2a–d). The impact on gene expression could not be attributed to the loss of U1 or U2 snRNA, since no significant changes in their expression were observed within the time frame of these experiments (data not shown) or to an effect on cell cycle (Supplementary Fig. 4B). Interestingly, a majority of these genes (2,164 genes) were commonly identified as differentially expressed (DE) upon depletion of NELF (Fig. 3a and Supplementary Dataset 2a). Such significant overlap (P-value <1e−300) highlighted a novel possible function of INTScom subunits in co-regulating NELF-dependent coding genes.

Bottom Line: The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits.Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity.Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Human Genetics, CNRS UPR1142, Laboratory of Molecular Virology; MGX-Montpellier GenomiX, 141 rue de la Cardonille, Montpellier 34396, France [2] LBME-CNRS, Cell Cycle Chromatin Dynamics Laboratory. University Paul Sabatier, Toulouse 31061, France [3] INRA, TOXALIM (Research Centre in Food Toxicology), Toulouse 31300, France [4] IGF, MGX-Montpellier GenomiX, France.

ABSTRACT
RNA polymerase II (RNAPII) pausing/termination shortly after initiation is a hallmark of gene regulation. Here, we show that negative elongation factor (NELF) interacts with Integrator complex subunits (INTScom), RNAPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both human immunodeficiency virus type 1 promoter and genome-wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits. Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity. Finally, INTScom target genes are enriched in human immunodeficiency virus type 1 transactivation response element/NELF binding element and in a 3' box sequence required for small nuclear RNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.

Show MeSH
Related in: MedlinePlus