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Allosteric transcription stimulation by super elongation complex

ABSTRACT: The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and a subcomplex, ELL2-EAF1, which stimulates transcription elongation by RNA polymerase II (Pol II). Here we report the cryo-EM structure of ELL2-EAF1 bound to a Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the Pol II lobe, explaining how SEC delivers P-TEFb to Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that elongation stimulation requires the dimerization module and an ELL2 protein linker that tethers this module to the Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a further closed conformation of the Pol II active center cleft.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Sus Scrofa Domesticus (domestic Pig)

SUBMITTER: Momchil Ninov

LAB HEAD: Henning Urlaub

PROVIDER: PXD024457 | Pride | 2021-07-15

REPOSITORIES: Pride

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			Action	DRS
	SEARCH_RESULTS_pLink_SEC-EC.zip	Other
	SEC-EC_Exploris_R1_pSEC_1.raw	Raw
	SEC-EC_Exploris_R1_pSEC_10.raw	Raw
	SEC-EC_Exploris_R1_pSEC_11.raw	Raw
	SEC-EC_Exploris_R1_pSEC_12.raw	Raw

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Targeting Processive Transcription Elongation via SEC Disruption for MYC-Induced Cancer Therapy

Project description:The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.

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The little elongation complex (LEC) regulates small nuclear RNA transcription

Project description:The Eleven-nineteen Lysine-rich Leukemia (ELL)-containing Super Elongation Complex (SEC) containing P-TEFb is a key regulator in the expression of HOX genes in Mixed Lineage Leukemia (MLL)-based leukemia. We have identified an SEC-like complex in Drosophila, as well as a distinct ELL-containing complex that lacks P-TEFb and other components of SEC named the "little elongation complex" (LEC). LEC subunits are highly enriched at RNA Polymerase II (Pol II) transcribed small nuclear RNA (snRNA) genes and the loss of LEC results in decreased snRNA expression in both flies and mammals. The discovery of specificity of SEC and LEC complexes for mRNA and snRNA containing genes, respectively, suggest the presence of specific classes of elongation factors for each class of genes transcribed by RNA polymerase II. Examination of genome-wide binding profiles for ELL, Ice1, Lilli, and Pol II in D. melanogaster and by ChIP-seq. Identification of differentially expressed genes in ELL-RNAi and Ice1-RNAi in D. melanogaster by RNA-seq. Examination of genome-wide binding profiles for ELL in M. musculus. Identification of differentially expressed genes in ELL-RNAi in M. musculus by RNA-seq.

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Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute 5 protein-depletion approach, prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Mechanistically, loss of SPT6 results in loss of PAF1 complex (PAF1C) from RNA Pol II, leading to NELF-bound RNA Pol II release into the gene bodies. Furthermore, SPT6 and/or PAF1 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through the recruitment of PAF1C during the early elongation.

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AFF4 globally affects the release of paused RNA polymerase II in HEL cells [ChIP-seq]

Project description:P-TEFb, a heterodimer of kinase, CDK9, and Cyclin T1, is a critical regulator of promoter-proximal pause release of Pol II in metazoans. It is capable of forming three larger multiprotein complexes, including the super elongation complex (SEC), the BRD4/P-TEFb complex and the 7SK snRNP. In the SEC or the BRD4/P-TEFb, P-TEFb is enzymatically active, while in the 7SK snRNP, its activity is inhibited. The SEC consists of AFF1 or 4, ENL or AF9, ELL1, 2 or 3 and EAF1 or 2 in addition to P-TEFb, the only subunit with catalytic activity, and the noncatalytic subunits have been found to be able to regulate pause release through P-TEFb. One recent study showed that in human DLD-1 cells, the SEC only regulates pause release of heat shock (HS) genes, whereas the BRD4/P-TEFb complex regulates pause release of the rest of the genes. In this study, we found that AFF4 knockdown in human HEL cells decreased not only cellular level but also global chromatin occupancy of CTD serine 2 phosphorylated Pol II, and notably, increased promoter-proximal pause of Pol II on several hundred HS and thousands of non-HS genes. Mechanistically, AFF4 facilitates pause release likely by facilitating the binding of P-TEFb to Pol II. These results suggest that the extent of impact of AFF4 on pause release is likely to be context-dependent or cell-type dependent.

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RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II

Project description:Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation. Comparison of the chromatin occupancy of [1] PAF1, CDC73, LEO1, CTR9, total Pol II, and CTD serine 2-phosphorylated Pol II by ChIP-seq in THP1 cells; [2] PAF1, Pol II, Pol II (ser-5p), CDK12, and CDK9 by ChIP-seq in control and PAF1 knockdown cells; [3] LEO1 and Pol II by ChIP-seq in control and flavopiridol treated THP1 cells.

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Schizosaccharomyces pombe Pol II Transcription Elongation Factor ELL Functions as Part of a Rudimentary Super Elongation Complex

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Project description:Transcriptional regulation of developmentally controlled genes is at the heart of differentiation and organogenesis. In this study, we have performed global genomic analyses in murine embryonic stem (ES) cells and in human cells in response to activation signals. We have identified an essential role for the ELL/P-TEFbcontaining Super Elongation Complex (SEC) in the regulation of gene expression including several genes bearing paused RNA polymerase II (Pol II). Paused Pol II has been proposed to be associated with loci that respond rapidly to environmental stimuli. However, our studies in ES cells have also identified a requirement for SEC at genes without preloaded Pol II, which also respond dynamically to differentiation signals. Our findings suggest that SEC is a major class of active P-TEFb-containing complexes required for transcriptional activation in response to environmental cues such as differentiation signals. Examination of ELL2, AFF4, and Pol II before and after activation signals in two cell types.

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