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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 regulates RNAPII occupancy and processivity of NELF-target genes.(a) Box plots showing the variations in RNAPII occupancy (ChIP-Seq reads over gene bodies; see Methods) between control and INTS11- (upper panel) or INTS3- (lower panel) -depleted cells (see Supplementary Fig. 4A for the knockdown efficiency) as a function of NELF-E/Spt5, INTS3 and/or INTS11 binding or not (as indicated on top). P-values, wilcoxon pair-wise tests. (b) Box plot showing RNAPII occupancy over gene bodies normalised to TSSs (see Methods) between control and INTS11-depleted cells depending on NELF-E/Spt5, INTS3 and/or INTS11 binding or not (as indicated on top). P-values, pair-wise wilcoxon tests. (c) Box plots showing the variations in RNAPII occupancy (see panel B) upon INTS11-KD for groups of up-regulated genes (upon INTS11-KD and/or of NELF-KD) and control genes (no change in expression; boxes in grey). P-values, wilcoxon pair-wise tests. 68/32%, percentages of genes up-regulated upon INTS11-KD that were also up-regulated upon NELF-KD (68%) or not (32%)(see Fig. 3a; Supplementary Dataset 2 for a list). (d) Average profiles of RNASeq ‘+’ reads in control-, INTS11− or NELF-E- depleted cells over exons (left) or transcription ends (right) for ‘direct targets’ (genes bound by NELF-E/INTS3/INTS11) and that were up-regulated upon INTS11-KD as compared to control. Y axis, normalised ChIP-Seq reads (see Methods). (e) Scatter plot showing the percentage of INTS11 (x axis) and of NELF (y axis) ChIP-Seq peaks harbouring a given consensus motif. Binding motifs were obtained through systematic search among all ChIP-Seq peaks of INTS11 and NELF using RSAT (see Methods). Only the motifs showing significant intersections with INTS11 or NELF peaks (fisher exact test<1e−3) were analysed (see Supplementary Dataset 5 for a list). (f) Scatter plot showing the enrichment of genes harbouring a given motif (+/− 250 bp from TSSs; see Methods) among DE genes (up- or down- regulated upon INTS11-KD; x axis and y axis, respectively; in Log2 P-values). Note that TAR motif are enriched among up-regulated genes (see Supplementary Dataset 5 for a list of all motifs).
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f4: Integrator regulates RNAPII occupancy and processivity of NELF-target genes.(a) Box plots showing the variations in RNAPII occupancy (ChIP-Seq reads over gene bodies; see Methods) between control and INTS11- (upper panel) or INTS3- (lower panel) -depleted cells (see Supplementary Fig. 4A for the knockdown efficiency) as a function of NELF-E/Spt5, INTS3 and/or INTS11 binding or not (as indicated on top). P-values, wilcoxon pair-wise tests. (b) Box plot showing RNAPII occupancy over gene bodies normalised to TSSs (see Methods) between control and INTS11-depleted cells depending on NELF-E/Spt5, INTS3 and/or INTS11 binding or not (as indicated on top). P-values, pair-wise wilcoxon tests. (c) Box plots showing the variations in RNAPII occupancy (see panel B) upon INTS11-KD for groups of up-regulated genes (upon INTS11-KD and/or of NELF-KD) and control genes (no change in expression; boxes in grey). P-values, wilcoxon pair-wise tests. 68/32%, percentages of genes up-regulated upon INTS11-KD that were also up-regulated upon NELF-KD (68%) or not (32%)(see Fig. 3a; Supplementary Dataset 2 for a list). (d) Average profiles of RNASeq ‘+’ reads in control-, INTS11− or NELF-E- depleted cells over exons (left) or transcription ends (right) for ‘direct targets’ (genes bound by NELF-E/INTS3/INTS11) and that were up-regulated upon INTS11-KD as compared to control. Y axis, normalised ChIP-Seq reads (see Methods). (e) Scatter plot showing the percentage of INTS11 (x axis) and of NELF (y axis) ChIP-Seq peaks harbouring a given consensus motif. Binding motifs were obtained through systematic search among all ChIP-Seq peaks of INTS11 and NELF using RSAT (see Methods). Only the motifs showing significant intersections with INTS11 or NELF peaks (fisher exact test<1e−3) were analysed (see Supplementary Dataset 5 for a list). (f) Scatter plot showing the enrichment of genes harbouring a given motif (+/− 250 bp from TSSs; see Methods) among DE genes (up- or down- regulated upon INTS11-KD; x axis and y axis, respectively; in Log2 P-values). Note that TAR motif are enriched among up-regulated genes (see Supplementary Dataset 5 for a list of all motifs).

Mentions: To further assess how INTS3/11 may influence RNAPII occupancy, ChIP-Seq analyses of RNAPII were performed upon depletion of either subunit (see Supplementary Fig. 7C for knockdown efficiency). INTS11 depletion increased RNAPII occupancy over gene bodies (Fig. 4a), that is, the number of ChIP-Seq reads found in gene bodies (see Methods). Such increase in reads was specific for direct targets whose promoters were bound by NELF and INTS3 (Fig. 4a, upper panel; compare boxes 1 and 2 with 6 and 7; P-values of 1e−23 and 1, respectively). In stark contrast, INTS3-depletion led to the opposite effect, namely the decrease of RNAPII occupancy over NELF-regulated genes (Fig. 4a, lower panel, P~1e−6 and 1, respectively). Further normalisation of ChIP-Seq reads in gene bodies by reads over TSSs showed a specific decrease of RNAPII occupancy for the subset of genes that are bound by INTScom subunits and NELF (Fig. 4b). Such decrease was detected for the genes that were up-regulated upon INTS11-KD and NELF-KD (Fig. 4c).


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 regulates RNAPII occupancy and processivity of NELF-target genes.(a) Box plots showing the variations in RNAPII occupancy (ChIP-Seq reads over gene bodies; see Methods) between control and INTS11- (upper panel) or INTS3- (lower panel) -depleted cells (see Supplementary Fig. 4A for the knockdown efficiency) as a function of NELF-E/Spt5, INTS3 and/or INTS11 binding or not (as indicated on top). P-values, wilcoxon pair-wise tests. (b) Box plot showing RNAPII occupancy over gene bodies normalised to TSSs (see Methods) between control and INTS11-depleted cells depending on NELF-E/Spt5, INTS3 and/or INTS11 binding or not (as indicated on top). P-values, pair-wise wilcoxon tests. (c) Box plots showing the variations in RNAPII occupancy (see panel B) upon INTS11-KD for groups of up-regulated genes (upon INTS11-KD and/or of NELF-KD) and control genes (no change in expression; boxes in grey). P-values, wilcoxon pair-wise tests. 68/32%, percentages of genes up-regulated upon INTS11-KD that were also up-regulated upon NELF-KD (68%) or not (32%)(see Fig. 3a; Supplementary Dataset 2 for a list). (d) Average profiles of RNASeq ‘+’ reads in control-, INTS11− or NELF-E- depleted cells over exons (left) or transcription ends (right) for ‘direct targets’ (genes bound by NELF-E/INTS3/INTS11) and that were up-regulated upon INTS11-KD as compared to control. Y axis, normalised ChIP-Seq reads (see Methods). (e) Scatter plot showing the percentage of INTS11 (x axis) and of NELF (y axis) ChIP-Seq peaks harbouring a given consensus motif. Binding motifs were obtained through systematic search among all ChIP-Seq peaks of INTS11 and NELF using RSAT (see Methods). Only the motifs showing significant intersections with INTS11 or NELF peaks (fisher exact test<1e−3) were analysed (see Supplementary Dataset 5 for a list). (f) Scatter plot showing the enrichment of genes harbouring a given motif (+/− 250 bp from TSSs; see Methods) among DE genes (up- or down- regulated upon INTS11-KD; x axis and y axis, respectively; in Log2 P-values). Note that TAR motif are enriched among up-regulated genes (see Supplementary Dataset 5 for a list of all motifs).
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Related In: Results  -  Collection

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Show All Figures
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f4: Integrator regulates RNAPII occupancy and processivity of NELF-target genes.(a) Box plots showing the variations in RNAPII occupancy (ChIP-Seq reads over gene bodies; see Methods) between control and INTS11- (upper panel) or INTS3- (lower panel) -depleted cells (see Supplementary Fig. 4A for the knockdown efficiency) as a function of NELF-E/Spt5, INTS3 and/or INTS11 binding or not (as indicated on top). P-values, wilcoxon pair-wise tests. (b) Box plot showing RNAPII occupancy over gene bodies normalised to TSSs (see Methods) between control and INTS11-depleted cells depending on NELF-E/Spt5, INTS3 and/or INTS11 binding or not (as indicated on top). P-values, pair-wise wilcoxon tests. (c) Box plots showing the variations in RNAPII occupancy (see panel B) upon INTS11-KD for groups of up-regulated genes (upon INTS11-KD and/or of NELF-KD) and control genes (no change in expression; boxes in grey). P-values, wilcoxon pair-wise tests. 68/32%, percentages of genes up-regulated upon INTS11-KD that were also up-regulated upon NELF-KD (68%) or not (32%)(see Fig. 3a; Supplementary Dataset 2 for a list). (d) Average profiles of RNASeq ‘+’ reads in control-, INTS11− or NELF-E- depleted cells over exons (left) or transcription ends (right) for ‘direct targets’ (genes bound by NELF-E/INTS3/INTS11) and that were up-regulated upon INTS11-KD as compared to control. Y axis, normalised ChIP-Seq reads (see Methods). (e) Scatter plot showing the percentage of INTS11 (x axis) and of NELF (y axis) ChIP-Seq peaks harbouring a given consensus motif. Binding motifs were obtained through systematic search among all ChIP-Seq peaks of INTS11 and NELF using RSAT (see Methods). Only the motifs showing significant intersections with INTS11 or NELF peaks (fisher exact test<1e−3) were analysed (see Supplementary Dataset 5 for a list). (f) Scatter plot showing the enrichment of genes harbouring a given motif (+/− 250 bp from TSSs; see Methods) among DE genes (up- or down- regulated upon INTS11-KD; x axis and y axis, respectively; in Log2 P-values). Note that TAR motif are enriched among up-regulated genes (see Supplementary Dataset 5 for a list of all motifs).
Mentions: To further assess how INTS3/11 may influence RNAPII occupancy, ChIP-Seq analyses of RNAPII were performed upon depletion of either subunit (see Supplementary Fig. 7C for knockdown efficiency). INTS11 depletion increased RNAPII occupancy over gene bodies (Fig. 4a), that is, the number of ChIP-Seq reads found in gene bodies (see Methods). Such increase in reads was specific for direct targets whose promoters were bound by NELF and INTS3 (Fig. 4a, upper panel; compare boxes 1 and 2 with 6 and 7; P-values of 1e−23 and 1, respectively). In stark contrast, INTS3-depletion led to the opposite effect, namely the decrease of RNAPII occupancy over NELF-regulated genes (Fig. 4a, lower panel, P~1e−6 and 1, respectively). Further normalisation of ChIP-Seq reads in gene bodies by reads over TSSs showed a specific decrease of RNAPII occupancy for the subset of genes that are bound by INTScom subunits and NELF (Fig. 4b). Such decrease was detected for the genes that were up-regulated upon INTS11-KD and NELF-KD (Fig. 4c).

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