A combinatorial approach to uncover an additional Integrator subunit

Offley, Sarah; Pfleiderer, Moritz M.; Zucco, Avery; Fraudeau, Angélique; Welsh, Sarah A.; Rażew, Michał; Galej, Wojciech P.; Gardini, Alessandro

doi:10.1016/j.celrep.2023.112244

Cited by 13 publications

(22 citation statements)

References 56 publications

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“…Consistent with several recent studies that identified C7ORF26 as a subunit of INT (INTS15), [45][46][47][48][49] we detected it consistently in all our INTS13ΔC MS datasets (Figure 1A 7E), which is consistent with a recent study that came to similar conclusions. 47 We next inspected the published cryoEM map (EMDB 30473) of an INT preparation, which was purified from human cells directly 30 for unexplained densities. We noticed a well resolved additional density section on a surface between INTS2-7 consistent with a short α-helix extended by an unstructured coil (so far modelled as unassigned poly-Ala chain).…”

Section: Ints15 and Dss1 Are Stable Subunits Of Intsupporting

confidence: 93%

“…55 Consistent with our data, previously published interactomes of ZMYND8, ZEB1, LEC subunit ICE1, and mouse ZNF609, contain several INTS in addition to INTS10-13-14, 36,51, [56][57][58] and conversely numerous INTS interactomes also feature ZMYND8, ZNF609, and ZNF655. 35,37,47,[59][60][61][62][63] Together these data indicate that the regulatory factors interact with the whole INT by directly binding the INTS10-13-14 module and that this function is conserved to vertebrates (Supplemental Figure 2B). We therefore propose that INTS10-13-14 form the INT TF binding module (ITFM).…”

Section: Resultsmentioning

confidence: 92%

“…44 Therefore, we set out to define the interactome of this module via tandem affinity purification followed by mass spectrometry (TAP-MS) (Figure 1). We used HEK293T- [45][46][47][48][49] was present in both interactomes suggesting that it closely interacts with the INTS10-13-14 module. Importantly, we observed many additional factors that are tightly associated to transcription and its regulation in either of the two datasets.…”

Section: Resultsmentioning

confidence: 99%

“…The following constructs were expressed separately or together in E.coli BL21 Star (DE3) cells (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50), hsZEB1 (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) were expressed in E.coli Rosetta (Sigma Aldrich). Cells were grown at 37 °C in LB medium with appropriate antibiotics until OD600 = 0.5 was reached, then 1 mM isopropyl-β -D-thiogalactopyranoside (IPTG) was added and cells were incubated for 3 hours at 37 °C.…”

Section: Recombinant Protein Expression In Ecolimentioning

confidence: 99%

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Mechanistic basis of gene-specific transcription regulation by the Integrator complex

Sabath,

Nabih,

Arnold

et al. 2024

Preprint

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The Integrator complex (INT) regulates gene expression via premature transcription termination of RNA polymerase II (RNAP2) at promoter-proximal pausing sites. This attenuation of transcription is required for cellular response to external stimuli, cell differentiation and neurodevelopment. How gene-specific regulation is achieved by INT in an inducible manner remains unclear. Here, we identify two sites on INT subunits 13/14 that serve as direct binding hubs for diverse sets of sequence-specific transcription factors (TFs) and other transcription effector complexes. The TFs co-localize with INT genome-wide, increase INT abundance on target genes and co-regulate inducible transcriptional programs. Consistently, disruption of INT-TF contacts impairs sensory cilia formation in response to glucose starvation. Structural analysis places INT's TF binding hubs upstream of the transcription bubble when attached to paused RNAP2, consistent with simultaneous TF-promoter association. Our data establish TF-mediated recruitment of INT to promoters as a widespread mechanism for targeted and inducible transcription attenuation.

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Section: Ints15 and Dss1 Are Stable Subunits Of Intsupporting

confidence: 93%

Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Recombinant Protein Expression In Ecolimentioning

confidence: 99%

See 2 more Smart Citations

Mechanistic basis of gene-specific transcription regulation by the Integrator complex

Sabath,

Nabih,

Arnold

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Biochemical and structural studies revealed several stable sub-complexes of the Integrator, including the Cleavage Module (INTS4/9/11) containing β-CASP/MBL catalytic endonuclease INTS11 [38][39][40] , as well as the INTS10/13/14 module 41,42 and its extended variant INTS10/13/14/15 27,29 referred to as the Arm module hereafter. Cryo-EM reconstructions of the higher-order Integrator-PP2A complexes revealed its overall architecture and intimate interactions between different modules as well as the structural basis of its recruitment to the paused elongating RNAPII complex (PEC) 43 and the mechanism of INTS11 endonuclease activation 20,44,45 .…”

Section: Introductionmentioning

confidence: 99%

Structural basis of the Integrator complex assembly and association with transcription factors

Razew,

Fraudeau,

Pfleiderer

et al. 2024

Preprint

Self Cite

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SummaryIntegrator is a multi-subunit protein complex responsible for premature transcription termination of coding and non-coding RNAs in Metazoans. This is achieved via Integrator’s two enzymatic activities, RNA endonuclease and protein phosphatase, acting on the promoter-proximally paused RNA Polymerase II (RNAPII). Yet, it remains unclear how Integrator assembly and recruitment are regulated and what are the functions of many of its core subunits. Here we report two cryo-EM reconstructions of large Integrator sub-complexes: INTS10/13/14/15 (Arm module) and INTS5/8/10/15, which allowed integrative modelling of the fully-assembled Integrator bound to the RNAPII paused elongating complex (PEC). INTS13/14 are positioned near the DNA upstream from the transcription pause site, suggesting a potential role in the chromatin context. Anin silicoprotein interaction screen of over 1500 transcription factors (TFs), identified Zinc Finger Protein 655 (ZNF655) as a direct interacting partner of INTS13 that associates with a fully assembled, 17-subunit Integrator complex. We propose a model wherein the Arm module acts as a platform for the recruitment of TFs that could modulate the stability of the Integrator’s association at specific loci and modulate transcription attenuation of the target genes.

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A homozygous variant in INTS11 links mitosis and neurogenesis defects to a severe neurodevelopmental disorder

Kuang,

Li,

Wang

et al. 2023

Cell Reports

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A combinatorial approach to uncover an additional Integrator subunit

Cited by 13 publications

References 56 publications

Mechanistic basis of gene-specific transcription regulation by the Integrator complex

Mechanistic basis of gene-specific transcription regulation by the Integrator complex

Structural basis of the Integrator complex assembly and association with transcription factors

A homozygous variant in INTS11 links mitosis and neurogenesis defects to a severe neurodevelopmental disorder

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