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Human Negative Elongation Factor Activates Transcription and Regulates Alternative Transcription Initiation

ABSTRACT: The human Negative Elongation Factor (NELF) is a four-subunit protein complex that inhibits the movement of RNA polymerase II (RNAPII) at an early elongation stage in vitro. NELF-mediated stalling of RNAPII also attenuates transcription of a number of inducible genes in human cells. To obtain a genome-wide understanding of human NELF-mediated transcriptional regulation in vivo, we carried out an exon-array study in T47D breast cancer cells with transient siRNA knockdown of individual NELF subunits. Upon depletion of NELF-A, -C, or -E, the vast majority of NELF-regulated genes were down-regulated. Many of the down-regulated genes encode proteins that play key roles in cell cycle progression. Consequently, NELF knockdown resulted in significant reduction in DNA synthesis and cell proliferation. Chromatin immunoprecipitation showed that NELF knockdown led to dissociation of RNAPII from the promoter-proximal region of the cell cycle-regulating genes. This was accompanied by decreased histone modifications associated with active transcription initiation (H3K9Ac) and elongation (H3K36Me3), as well as reduced recruitment of the general transcription factor TFIIB and increased overall histone occupancy at a subset of the down-regulated promoters. Lastly, our study indicates that NELF regulates alternative transcription initiation of Basigin gene (BSG) by differentially influencing RNAPII density at the two neighboring exons at the 5’ end of the gene. Taken together, our data suggest a diverse transcriptional consequence of NELF-mediated RNAPII pausing in the human genome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE19940 | GEO | 2010/01/20

SECONDARY ACCESSION(S): PRJNA122633

REPOSITORIES: GEO

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The Human Integrator Complex Facilitates Transcriptional Elongation by Endonucleolytic Cleavage of Nascent Transcripts

Project description:Transcription by RNA polymerase II (RNAPII) is pervasive in the human genome. However, the mechanisms controlling transcription at promoters and enhancers remain enigmatic. Here, we demonstrate that Integrator subunit 11 (INTS11), the catalytic subunit of the Integrator complex, regulates transcription at these loci through its endonuclease activity. Promoters of genes require INTS11 to cleave nascent transcripts associated with paused RNAPII and induce their premature termination in the proximity of the +1 nucleosome. The turnover of RNAPII permits the subsequent recruitment of an elongation-competent RNAPII complex, leading to productive elongation. In contrast, enhancers require INTS11 catalysis not to evict paused RNAPII but rather to terminate enhancer RNA transcription beyond the +1 nucleosome. These findings are supported by the differential occupancy of negative elongation factor (NELF), SPT5, and tyrosine-1-phosphorylated RNAPII. This study elucidates the role of Integrator in mediating transcriptional elongation at human promoters through the endonucleolytic cleavage of nascent transcripts and the dynamic turnover of RNAPII.

2020-07-21 | GSE125534 | GEO

The Human Integrator Complex Facilitates Transcriptional Elongation by Endonucleolytic Cleavage of Nascent Transcripts

2020-07-21 | GSE125535 | GEO

Translation initiation at a non-AUG start codon for mouse and human NELF-B

Project description:Negative elongation factor (NELF), a four-subunit protein complex in metazoan, plays an important role in regulating promoter-proximal pausing of RNA polymerase II (RNAPII). Genetic studies demonstrate that the B subunit of mouse NELF (NELF-B) is critical for embryonic development and homeostasis in adult tissue. We report here that both human and mouse NELF-B proteins are translated from a non-AUG codon upstream of the annotated AUG. This non-AUG codon sequence is conserved in mammalian NELF-B but not NELF-B orthologs of lower metazoan. The full-length and a truncated NELF-B that starts at the first AUG codon interact with the other three NELF subunits with comparable efficiency. Furthermore, these two forms of NELF-B have a similar impact on the transcriptomics and proliferation of mouse embryonic fibroblasts. These results strongly suggest that additional amino acid sequence upstream of the annotated AUG is dispensable for the essential function of NELF in supporting cell growth in vitro. While the majority of mouse adult tissues surveyed exclusively express the full-length NELF-B protein, mouse kidney only contains a truncated NELF-B protein with the same apparent size as the AUG-initiated version. This result raises the distinct possibility that translational initiation of mouse NELF-B is regulated in a tissue-dependent manner. triplicates for ATG-Nelf-B, triplicates for FL-Nelf-B

2014-10-11 | E-GEOD-62248 | biostudies-arrayexpress

Integrator complex regulates NELF-mediated RNA Polymerase II pause/release and processivity at coding genes [RNA-seq]

Project description:RNAPII pausing/termination shortly after initiation is a hallmark of gene regulation. However, the molecular mechanisms involved are still to be uncovered. Here, we show that NELF interacts with Integrator complex subunits (INTScom) forming a stable complex with RNPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both HIV-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 complex. Interestingly, in addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length mRNA. Finally, INTScom-target genes are enriched in HIV-1 TAR/ NELF-binding element and in a 3’box sequence required for snRNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pausing/release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle. Genome-wide expression in HeLa cells in the absence of Integrator 11, or NELF or mock (control) depleted by strand-specific RNASeq (Illumina)

2015-05-19 | E-GEOD-60585 | biostudies-arrayexpress

Total RNA Pol2 ChIP-seq in mES cells treated with flavopiridol (Cdk9 inhibitor) after DSIF and NELF knockdown

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2011-01-19 | E-GEOD-26658 | biostudies-arrayexpress

Total RNA Pol2 ChIP-seq in mES cells treated with flavopiridol (Cdk9 inhibitor) after DSIF and NELF knockdown

2011-01-19 | GSE26658 | GEO

NELF-A controls Drosophila healthspan by regulating heat-shock protein-mediated cellular protection and heterochromatin maintenance

Project description:Epigenetic alteration is a hallmark of aging, but it remains unclear if perturbation of transcription machineries may affect longevity. NELF-mediated pausing of RNA polymerase II (RNAPII) constitutes an important step in transcription regulation. We found that halving NELF-A level in heterozygous mutants or via neuronal-specific depletion of NELF-A improves locomotor activity, stress resistance and lifespan of Drosophila significantly. Mechanistically, lower NELF-A releases paused RNAPII to facilitate productive transcription of the heat-shock protein (Hsp) genes. Elevated HSPs attenuate the level of protein aggregation, reactive oxidative species, DNA damage and systemic inflammation in the brains of NELF-A depleted flies. This protection is conserved in human cells where NELF-A depletion confers improved resistance against oxidative stress. Reduced oxidative DNA damage allows the maintenance of H3K9me2-enriched heterochromatin, which represses the activation of specific retrotransposons during aging. Therefore, by regulating transcription of Hsp genes in the brains, NELF-A controls multiple aspects of aging in Drosophila.

2020-11-30 | GSE155940 | GEO

Integrator complex regulates NELF-mediated RNA Polymerase II pause/release and processivity at coding genes [ChIP-seq]

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2015-05-19 | E-GEOD-60584 | biostudies-arrayexpress

The RPB2 flap loop of human RNA polymerase II is dispensable for transcription initiation and elongation.

Project description:Deletion of the flap loop in the Rp2 subunit of human RNAPII did not alter the ability of human RNAPII to initiate transcription or elongate the initiated transcripts in vivo and in vitro, and was also dispensable for elongation factor-mediated enhancement and inhibition of transcript elongation in vitro. ChIP:chip of wild-type and flap deletion human RNAPII revealed that the mutant RNAPII was not defective for promoter binding, initiation and elongation in vivo.

2011-06-24 | GSE29734 | GEO

A CSB-PAF1 axis activates RNA polymerase II from promoter-proximal sites and restores efficient transcription elongation after DNA damage repair

Project description:The coordinated transcription of genes involves RNA polymerase II enzymes (RNAPII), which pull DNA through their active sites. DNA lesions in transcribed strands block RNAPII elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway ensures the efficient removal of transcription-blocking DNA lesions, but this is not sufficient to overcome this arrest and resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions specifically in response to DNA damage. PAF1C is dispensable for TCR-mediated repair,but is essential to resume RNA synthesis after UV irradiation, suggesting an unexpected uncoupling between DNA repair and transcription restart. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation waves throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

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