MDC1 maintains active elongation complexes of RNA polymerase II
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PROVIDER: PRJNA891669 | ENA |
REPOSITORIES: ENA
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Project description:The role of MDC1 in the DNA damage response has been extensively studied, however, its impact on other cellular processes is not well understood. Here, we describe a role for MDC1 in transcription by regulating the activity of the RNAPolymerase II (RNAPII). Depletion of MDC1 caused a genome-wide reduction in the abundance of actively engaged RNAPII elongation complexes throughout the gene body of protein coding genes under unperturbed conditions. Decreased engaged RNAPII subsequently alters the assembly of the spliceosome complex on chromatin, leading to defects in pre-mRNA splicing. Mechanistically, the S/TQ domain of MDC1 modulates RNAPII-mediated transcription. Upon genotoxic stress, MDC1 promotes the abundance of engaged RNAPII complexes at DNA breaks, thereby stimulating nascent transcription at the damaged sites. Of clinical relevance, cancer cells lacking MDC1 display hypersensitivity to RNAPII inhibitors. Overall, we unveil a previously uncharacterized role of MDC1 in RNAPII-mediated transcription with potential implications for cancer treatment.
2022-11-24 | GSE215990 | GEO
Project description:Elongation factor-specific capture of RNA polymerase II complexes
| PRJNA856248 | ENA
Project description:Transcription of protein-coding genes is regulated by dynamic association of co-factors with RNA polymerase II (RNAPII). The function of these factors and their relationship with RNAPII is often poorly understood. Here, we present an approach for elongation factor-specific mNET capture (EMCAP) of RNA polymerase II complexes for sequencing and mass spectrometry analysis, for investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply EMCAP to the RNAPII-associated proteins SCAF4 and SCAF8, which share an essential role as mRNA anti-terminators but have individual roles at the 3’end of genes. Mass spectrometry analysis shows that both SCAF4 and SCAF8 are part of RNAPII elongation complexes containing 3’end processing factors but depleted of splicing components. Importantly, the EMCAP-seq profiles of SCAF4- and SCAF8-RNAPII complexes reflect their function as mRNA-anti-terminators and their competing functions at the end of genes where they prevent or promote transcriptional readthrough.
2022-12-08 | GSE207568 | GEO
Project description:MDC1 is a large, modular phospho-protein scaffold that mediates recruitment of signaling and repair complexes to sites of DNA double-strand breaks (DSBs). MDC1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with phosphorylated H2AX (γH2AX), where it recruits downstream factors via direct phosphorylation-dependent protein-protein interactions. Here, using unbiased proteomic analyses we identify a highly conserved protein interaction surface near the MDC1 N-terminus for the DNA damage response protein TOPBP1. We show that TOPBP1 directly binds to two residues in MDC1 that are phosphorylated by CK2. Interestingly, we find that TOPBP1 recruitment to DSBs depends on direct interaction with MDC1 only during mitosis. Furthermore, we demonstrate that disrupting MDC1-TOPBP1 binding causes hypersensitivity to ionizing radiation in mitosis, as well as increased micronuclei and chromosomal instability. Thus, our results highlight an important and hitherto unnoticed function for MDC1 and TOPBP1 in maintaining genome stability during cell division.
2019-03-25 | PXD011850 | Pride
Project description:Streptococcus equinus strain:MDC1 Genome sequencing
| PRJNA647691 | ENA
Project description:In all organisms, the multi-subunit RNA polymerases (RNAPs) that synthesize messenger RNAs bind multiple accessory proteins to regulate transcript elongation rate, transcriptional pausing, and termination. However, the dynamics of regulatory protein association/dissociation and how different regulators influence one another’s function is unclear. We used single-molecule multi-wavelength fluorescence colocalization techniques to directly observe the dynamics of the elongation regulators NusA and σ70 with E. coli RNAP in vitro. Contrary to previous proposals, NusA was observed to repeatedly bind to and release from elongation complexes (ECs) during synthesis of a single RNA. However, elongation complexes that retained bound σ70 did not bind NusA and the EC-bound σ70 was largely retained even in the presence of physiological (µM) concentrations of competing elongation factors NusA and/or NusG. Factor occupancy of elongation complexes was non-equilibrium, with substantial amounts of σ70ECs even in the absence of free σ70, because composition was controlled by slow σ70 dissociation from σ70ECs. The experiments show that at steady state the same gene is transcribed by two distinct types of elongation complexes that are (ECs) or are not (σ70ECs) capable of binding NusA. Consistent with the known regulatory effects of NusA, the two types are observed to have different elongation rates, pausing dynamics, and efficiency of termination at an intrinsic terminator. During cellular transcription, these non-equilibrium populations of ECs are predicted to cause selective EC loss at intergenic transcriptional attenuators, traffic jams and altered pausing. This is an example of a potentially general “two-body” mechanism in which a functionally silent protein fine-tunes gene expression by modulating a second, functionally consequential regulator.
2020-08-20 | GSE110956 | GEO
Project description:The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and is expressed in a strictly growth factor-dependent manner in non-tumor cells. Here we show that MYC directly binds SPT5, a subunit of the RNA polymerase II (POL2) elongation factor DSIF. MYC recruits SPT5 to genes and enables the CDK7-dependent transfer of SPT5 onto POL2. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. In addition, MYC increases the directionality of promoters by stimulating sense transcription and suppressing the synthesis of antisense RNAs. Our results argue that MYC controls the productive assembly of POL2 with general elongation factors to form processive elongation complexes. The high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, slows transcription at growth-suppressive genes and promotes uncontrolled cellular growth.
2019-03-27 | GSE115365 | GEO
Project description:We identified MDC1 as a putative novel transcriptional co-regulator of estrogen receptor alpha (ER) in models of invasive lobular carcinoma. In this study, our goal was to define the contribution of MDC1 to regulation of the ER transcriptome in ILC cell lines versus invasive ductal carcinoma cells.
2021-05-13 | GSE171364 | GEO
Project description:Male germ cells establish a unique heterochromatin domain, the XY-body, early in meiosis. How this domain is maintained through the end of meiosis and into post-meiotic germ cell differentiation is poorly understood. ADAD2 is a late meiotic male germ cell specific RNA binding protein, loss of which leads to post-meiotic germ cell defects. Analysis of ribosome association in Adad2 mutants revealed defective translation of Mdc1, a key regulator of XY-body formation, late in meiosis. As a result, Adad2 mutants show normal establishment but failed maintenance of the XY-body. Observed XY-body defects are concurrent with abnormal autosomal heterochromatin and ultimately lead to severely perturbed post-meiotic germ cell heterochromatin and cell death. These findings highlight the requirement of ADAD2 for Mdc1 translation, the role of MDC1 in maintaining meiotic male germ cell heterochromatin, and the importance of late meiotic heterochromatin for normal post-meiotic germ cell differentiation.
2021-12-11 | GSE190525 | GEO
Project description:Spt5 is a highly conserved RNA polymerase II (Pol II)-associated pausing and elongation factor. However, its impact on global elongation and Pol II processivity in mammalian cells has not been clarified. Here, we show that depleting Spt5 in mouse embryonic fibroblasts (MEFs) does not cause global elongation defects or decreased elongation rates. Instead, in the absence of Spt5, a fraction of Pol II molecules are dislodged during elongation thus decreasing the number of Pol II complexes that complete the transcription cycle. Most strikingly, this decrease is restricted to a narrow window between 15-20 kb from the promoter, a distance which coincides with the stage where accelerating Pol II attains maximum elongation speed and processivity. Consequently, long genes show a greater dependency on Spt5 for optimal elongation efficiency and overall gene expression than short genes. We propose that an important role of Spt5 in mammalian elongation is to promote the processivity of those Pol II complexes that are transitioning towards maximum elongation speed 15-20 kb from the promoter.
2018-03-06 | GSE106313 | GEO