Search

During transcription, RNA polymerase II (RNAPII) faces numerous obstacles, including DNA damage, which can lead to stalling or arrest. One mechanism to contend with this situation is ubiquitylation and degradation of the largest RNAPII subunit, RPB1 - the 'last resort' pathway. This conserved, multi-step pathway was first identified in yeast, and the functional human orthologues of all but one protein, RNAPII Degradation Factor 1 (Def1), have been discovered. Here we show that following UV-irradiation, human Ubiquitin-associated protein 2 (UBAP2) or its paralogue UBAP2-like (UBAP2L) are involved in the ubiquitylation and degradation of RNAPII through the recruitment of Elongin-Cul5 ubiquitin ligase. Together, our data indicate that UBAP2 and UBAP2L are the human orthologues of yeast Def1, and so identify the key missing proteins in the human last resort pathway., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3'-end processing factors for RDH genes are enriched; however, this association's role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs' association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)-Cap binding complex (CBC)-Negative elongation factor (NELF), components of CBs, interferes with CBs' association with HLBs and 3' Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator's involvement in CBs' association with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing of RDH genes by supplying 3'-end processing factors., (© 2022. The Author(s).)

The bZIP transcription factor ATF6α is a master regulator of endoplasmic reticulum (ER) stress response genes. In this report, we identify the multifunctional RNA polymerase II transcription factor Elongin as a cofactor for ATF6α-dependent transcription activation. Biochemical studies reveal that Elongin functions at least in part by facilitating ATF6α-dependent loading of Mediator at the promoters and enhancers of ER stress response genes. Depletion of Elongin from cells leads to impaired transcription of ER stress response genes and to defects in the recruitment of Mediator and its CDK8 kinase subunit. Taken together, these findings bring to light a role for Elongin as a loading factor for Mediator during the ER stress response., Competing Interests: The authors declare no competing interest.

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD + A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling., Competing Interests: The authors declare no competing interest.

The Elongin complex was originally identified as an RNA polymerase II (RNAPII) elongation factor and subsequently as the substrate recognition component of a Cullin-RING E3 ubiquitin ligase. More recent evidence indicates that the Elongin ubiquitin ligase assembles with the Cockayne syndrome B helicase (CSB) in response to DNA damage and can target stalled polymerases for ubiquitylation and removal from the genome. In this report, we present evidence that the CSB-Elongin ubiquitin ligase pathway has roles beyond the DNA damage response in the activation of RNAPII-mediated transcription. We observed that assembly of the CSB-Elongin ubiquitin ligase is induced not just by DNA damage, but also by a variety of signals that activate RNAPII-mediated transcription, including endoplasmic reticulum (ER) stress, amino acid starvation, retinoic acid, glucocorticoids, and doxycycline treatment of cells carrying several copies of a doxycycline-inducible reporter. Using glucocorticoid receptor (GR)-regulated genes as a model, we showed that glucocorticoid-induced transcription is accompanied by rapid recruitment of CSB and the Elongin ubiquitin ligase to target genes in a step that depends upon the presence of transcribing RNAPII on those genes. Consistent with the idea that the CSB-Elongin pathway plays a direct role in GR-regulated transcription, mouse cells lacking the Elongin subunit Elongin A exhibit delays in both RNAPII accumulation on and dismissal from target genes following glucocorticoid addition and withdrawal, respectively. Taken together, our findings bring to light a new role for the CSB-Elongin pathway in RNAPII-mediated transcription., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K 1268 ), is the focal point for DNA-damage-response coordination. K 1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by accumulations of Aβ peptides. Production and fibrillation of Aβ are downregulated by BRI2 and BRI3, which are physiological inhibitors of amyloid precursor protein (APP) processing and Aβ oligomerization. Here, we identify nuclear receptor binding protein 1 (NRBP1) as a substrate receptor of a Cullin-RING ubiquitin ligase (CRL) that targets BRI2 and BRI3 for degradation. Moreover, we demonstrate that (1) dimerized NRBP1 assembles into a functional Cul2- and Cul4A-containing heterodimeric CRL through its BC-box and an overlapping cryptic H-box, (2) both Cul2 and Cul4A contribute to NRBP1 CRL function, and (3) formation of the NRBP1 heterodimeric CRL is strongly enhanced by chaperone-like function of TSC22D3 and TSC22D4. NRBP1 knockdown in neuronal cells results in an increase in the abundance of BRI2 and BRI3 and significantly reduces Aβ production. Thus, disrupting interactions between NRBP1 and its substrates BRI2 and BRI3 may provide a useful therapeutic strategy for AD., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.

The discovery of the three eukaryotic nuclear RNA polymerases paved the way for serious biochemical investigations of eukaryotic transcription and the identification of eukaryotic transcription factors. Here we describe this adventure from our vantage point, with a focus on the hunt for factors that regulate elongation by RNA polymerase II.

FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3'-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility. In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Elongin A binds to Elongins B and C to form the RNA polymerase II transcription elongation factor Elongin. It also functions as the substrate recognition subunit of a ubiquitin ligase that is formed by binding of Elongin to Cullin protein CUL5 and RING finger protein RBX2 and that targets RNA polymerase II for ubiquitination. In this article, we describe use of acceptor photobleaching fluorescence resonance energy transfer (AP-FRET) and laser microirradiation-based assays to study regulated assembly of the Elongin ubiquitin ligase and its recruitment to regions of localized DNA damage., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In a recent publication in Science, Kujirai et al. (2018) use single-particle cryo-EM to resolve several Pol II-nucleosome interactions, shedding new light on transcription elongation in a native chromatin environment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

ELL family transcription factors activate the overall rate of RNA polymerase II (Pol II) transcription elongation by binding directly to Pol II and suppressing its tendency to pause. In metazoa, ELL regulates Pol II transcription elongation as part of a large multisubunit complex referred to as the Super Elongation Complex (SEC), which includes P-TEFb and EAF, AF9 or ENL, and an AFF family protein. Although orthologs of ELL and EAF have been identified in lower eukaryotes including Schizosaccharomyces pombe, it has been unclear whether SEC-like complexes function in lower eukaryotes. In this report, we describe isolation from S. pombe of an ELL-containing complex with features of a rudimentary SEC. This complex includes S. pombe Ell1, Eaf1, and a previously uncharacterized protein we designate Ell1 binding protein 1 (Ebp1), which is distantly related to metazoan AFF family members. Like the metazoan SEC, this S. pombe ELL complex appears to function broadly in Pol II transcription. Interestingly, it appears to have a particularly important role in regulating genes involved in cell separation.

Co-transcriptional capping of RNA polymerase II (Pol II) transcripts by capping enzyme proceeds orders of magnitude more efficiently than capping of free RNA. Previous studies brought to light a role for the phosphorylated Pol II carboxyl-terminal domain (CTD) in activation of co-transcriptional capping; however, CTD phosphorylation alone could not account for the observed magnitude of activation. Here, we exploit a defined Pol II transcription system that supports both CTD phosphorylation and robust activation of capping to dissect the mechanism of co-transcriptional capping. Taken together, our findings identify a CTD-independent, but Pol II-mediated, mechanism that functions in parallel with CTD-dependent processes to ensure optimal capping, and they support a "tethering" model for the mechanism of activation.

Although cell metabolism has been established as a major regulator of eukaryotic gene expression, the mechanisms underlying this regulation are still being uncovered. Recent years have seen great advances in our understanding of biochemical mechanisms of metabolic regulation of transcription and chromatin. Prime examples include insights into how nutrients and cellular energy status regulate synthesis of ribosomal RNAs by RNA polymerases I and III during ribosome biogenesis and how a variety of enzymes that catalyze modifications of histones in chromatin are regulated by the levels of certain metabolites. This volume of the Annual Review of Biochemistry includes a set of reviews describing these and other advances in understanding aspects of the metabolic regulation of RNA polymerases I and III transcription and chromatin.

The human males absent on the first (MOF)-containing histone acetyltransferase nonspecific lethal (NSL) complex comprises nine subunits including the O -linked N -acetylglucosamine ( O -GlcNAc) transferase, isoform 1 (OGT1). However, whether the O -GlcNAc transferase activity of OGT1 controls histone acetyltransferase activity of the NSL complex and whether OGT1 physically interacts with the other NSL complex subunits remain unclear. Here, we demonstrate that OGT1 regulates the activity of the NSL complex by mainly acetylating histone H4 Lys-16, Lys-5, and Lys-8 via O -GlcNAcylation and stabilization of the NSL complex subunit NSL3. Knocking down or overexpressing OGT1 in human cells remarkably affected the global acetylation of histone H4 residues Lys-16, Lys-5, and Lys-8. Because OGT1 is a subunit of the NSL complex, we also investigated the function of OGT1 in this complex. Co-transfection/co-immunoprecipitation experiments combined with in vitro O -GlcNAc transferase assays confirmed that OGT1 specifically binds to and O -GlcNAcylates NSL3. In addition, wheat germ agglutinin affinity purification verified the occurrence of O -GlcNAc modification on NSL3 in cells. Moreover, O -GlcNAcylation of NSL3 by wild-type OGT1 (OGT1-WT) stabilized NSL3. This stabilization was lost after co-transfection of NSL3 with an OGT1 mutant, OGT1 C964A , that lacks O -GlcNAc transferase activity. Furthermore, stabilization of NSL3 by OGT1-WT significantly increased the global acetylation levels of H4 Lys-5, Lys-8, and Lys-16 in cells. These results suggest that OGT1 regulates the activity of the NSL complex by stabilizing NSL3., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and α-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and α-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator-RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.

Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved "hinge" in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

In order to facilitate the identification of factors and pathways in the cellular response to UV-induced DNA damage, several descriptive proteomic screens and a functional genomics screen were performed in parallel. Numerous factors could be identified with high confidence when the screen results were superimposed and interpreted together, incorporating biological knowledge. A searchable database, bioLOGIC, which provides access to relevant information about a protein or process of interest, was established to host the results and facilitate data mining. Besides uncovering roles in the DNA damage response for numerous proteins and complexes, including Integrator, Cohesin, PHF3, ASC-1, SCAF4, SCAF8, and SCAF11, we uncovered a role for the poorly studied, melanoma-associated serine/threonine kinase 19 (STK19). Besides effectively uncovering relevant factors, the multiomic approach also provides a systems-wide overview of the diverse cellular processes connected to the transcription-related DNA damage response., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although DNA double-strand break (DSB) repair is mediated by numerous proteins accumulated at DSB sites, how DNA repair proteins are assembled into damaged chromatin has not been fully elucidated. Here we show that a member of the tripartite motif protein family, TRIM29, is a histone-binding protein responsible for DNA damage response (DDR). We found that TRIM29 interacts with BRCA1-associated surveillance complex, cohesion, DNA-PKcs and components of TIP60 complex. The dynamics of the TRIM29-containing complex on H2AX nucleosomes is coordinated by a cross-talk between histone modifications. TRIM29 binds to modified histone H3 and H4 tails in the context of nucleosomes. Furthermore, chromatin binding of TRIM29 is required for the phosphorylation of H2AX and cell viability in response to ionizing radiation. Our results suggest that TRIM29 functions as a scaffold protein to assemble DNA repair proteins into chromatin followed by efficient activation of DDR.

Elongin A performs dual functions in cells as a component of RNA polymerase II (Pol II) transcription elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that has been shown to target Pol II stalled at sites of DNA damage. Here we investigate the mechanism(s) governing conversion of the Elongin complex from its elongation factor to its ubiquitin ligase form. We report the discovery that assembly of the Elongin A ubiquitin ligase is a tightly regulated process. In unstressed cells, Elongin A is predominately present as part of Pol II elongation factor Elongin. Assembly of Elongin A into the ubiquitin ligase is strongly induced by genotoxic stress; by transcriptional stresses that lead to accumulation of stalled Pol II; and by other stimuli, including endoplasmic reticulum and nutrient stress and retinoic acid signaling, that activate Elongin A-dependent transcription. Taken together, our findings shed new light on mechanisms that control the Elongin A ubiquitin ligase and suggest that it may play a role in Elongin A-dependent transcription., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Regulation of transcription elongation by RNA polymerase II (Pol II) is a key regulatory step in gene transcription. Recently, the little elongation complex (LEC)-which contains the transcription elongation factor ELL/EAF-was found to be required for the transcription of Pol II-dependent small nuclear RNA (snRNA) genes. Here we show that the human Mediator subunit MED26 plays a role in the recruitment of LEC to a subset of snRNA genes through direct interaction of EAF and the N-terminal domain (NTD) of MED26. Loss of MED26 in cells decreases the occupancy of LEC at a subset of snRNA genes and results in a reduction in their transcription. Our results suggest that the MED26-NTD functions as a molecular switch in the exchange of TBP-associated factor 7 (TAF7) for LEC to facilitate the transition from initiation to elongation during transcription of a subset of snRNA genes.

The study of conserved protein interaction networks seeks to better understand the evolution and regulation of protein interactions. Here, we present a quantitative proteomic analysis of 18 orthologous baits from three distinct chromatin-remodeling complexes in Saccharomyces cerevisiae and Homo sapiens. We demonstrate that abundance levels of orthologous proteins correlate strongly between the two organisms and both networks have highly similar topologies. We therefore used the protein abundances in one species to cross-predict missing protein abundance levels in the other species. Lastly, we identified a novel conserved low-abundance subnetwork further demonstrating the value of quantitative analysis of networks., (© 2014 The Authors.)

CSB/ERCC6 (Cockayne syndrome B protein/excision repair cross-complementation group 6), a member of a subfamily of SWI2/SNF2 (SWItch/sucrose nonfermentable)-related chromatin remodelers, plays crucial roles in gene expression and the maintenance of genome integrity. Here, we report the mechanism of the autoregulation of Rhp26, which is the homolog of CSB/ERCC6 in Schizosaccharomyces pombe. We identified a novel conserved protein motif, termed the "leucine latch," at the N terminus of Rhp26. The leucine latch motif mediates the autoinhibition of the ATPase and chromatin-remodeling activities of Rhp26 via its interaction with the core ATPase domain. Moreover, we found that the C terminus of the protein counteracts this autoinhibition and that both the N- and C-terminal regions of Rhp26 are needed for its proper function in DNA repair in vivo. The presence of the leucine latch motif in organisms ranging from yeast to humans suggests a conserved mechanism for the autoregulation of CSB/ERCC6 despite the otherwise highly divergent nature of the N- and C-terminal regions.

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex's chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

SNF2 family ATPases are ATP-dependent motors that often function in multisubunit complexes to regulate chromatin structure. Although the central role of SNF2 ATPases in chromatin biology is well established, mechanisms by which their catalytic activities are regulated by additional subunits of chromatin-remodeling complexes are less well understood. Here we present evidence that the human Inositol auxotrophy 80 (Ino80) SNF2 ATPase is subject to regulation at multiple levels in the INO80 chromatin-remodeling complex. The zinc finger histidine triad domain-containing protein Ies2 (Ino Eighty Subunit 2) functions as a potent activator of the intrinsic catalytic activity of the Ino80 ATPase, whereas the YL-1 family Ies6 (Ino Eighty Subunit 6) and actin-related Arp5 proteins function together to promote binding of the Ino80 ATPase to nucleosomes. These findings support the idea that both substrate recognition and the intrinsic catalytic activities of SNF2 ATPases have evolved as important sites for their regulation.

hMOF (MYST1), a histone acetyltransferase (HAT), forms at least two distinct multiprotein complexes in human cells. The male specific lethal (MSL) HAT complex plays a key role in dosage compensation in Drosophila and is responsible for histone H4K16ac in vivo. We and others previously described a second hMOF-containing HAT complex, the non-specific lethal (NSL) HAT complex. The NSL complex has a broader substrate specificity, can acetylate H4 on K16, K5, and K8. The WD (tryptophan-aspartate) repeat domain 5 (WDR5) and host cell factor 1 (HCF1) are shared among members of the MLL/SET (mixed-lineage leukemia/set-domain containing) family of histone H3K4 methyltransferase complexes. The presence of these shared subunits raises the possibility that there are functional links between these complexes and the histone modifications they catalyze; however, the degree to which NSL and MLL/SET influence one another's activities remains unclear. Here, we present evidence from biochemical assays and knockdown/overexpression approaches arguing that the NSL HAT promotes histone H3K4me2 by MLL/SET complexes by an acetylation-dependent mechanism. In genomic experiments, we identified a set of genes including ANKRD2, that are affected by knockdown of both NSL and MLL/SET subunits, suggested they are co-regulated by NSL and MLL/SET complexes. In ChIP assays, we observe that depletion of the NSL subunits hMOF or NSL1 resulted in a significant reduction of both H4K16ac and H3K4me2 in the vicinity of the ANKRD2 transcriptional start site proximal region. However, depletion of RbBP5 (a core component of MLL/SET complexes) only reduced H3K4me2 marks, but not H4K16ac in the same region of ANKRD2, consistent with the idea that NSL acts upstream of MLL/SET to regulate H3K4me2 at certain promoters, suggesting coordination between NSL and MLL/SET complexes is involved in transcriptional regulation of certain genes. Taken together, our results suggest a crosstalk between the NSL and MLL/SET complexes in cells., Competing Interests: The authors have declared that no competing interests exist.

Transcription factor ATF6α functions as a master regulator of endoplasmic reticulum (ER) stress response genes. In response to ER stress, ATF6α translocates from its site of latency in the ER membrane to the nucleus, where it activates RNA polymerase II transcription of ER stress response genes upon binding sequence-specifically to ER stress response enhancer elements (ERSEs) in their promoter-regulatory regions. In a recent study, we demonstrated that ATF6α activates transcription of ER stress response genes by a mechanism involving recruitment to ERSEs of the multisubunit Mediator and several histone acetyltransferase (HAT) complexes, including Spt-Ada-Gcn5 (SAGA) and Ada-Two-A-containing (ATAC) (Sela, D., Chen, L., Martin-Brown, S., Washburn, M.P., Florens, L., Conaway, J.W., and Conaway, R.C. (2012) J. Biol. Chem. 287, 23035-23045). In this study, we extend our investigation of the mechanism by which ATF6α supports recruitment of Mediator to ER stress response genes. We present findings arguing that Mediator subunit MED25 plays a critical role in this process and identify a MED25 domain that serves as a docking site on Mediator for the ATF6α transcription activation domain.

Elongin A was shown previously to be capable of potently activating the rate of RNA polymerase II (RNAPII) transcription elongation in vitro by suppressing transient pausing by the enzyme at many sites along DNA templates. The role of Elongin A in RNAPII transcription in mammalian cells, however, has not been clearly established. In this report, we investigate the function of Elongin A in RNAPII transcription. We present evidence that Elongin A associates with the IIO form of RNAPII at sites of newly transcribed RNA and is relocated to dotlike domains distinct from those containing RNAPII when cells are treated with the kinase inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole. Significantly, Elongin A is required for maximal induction of transcription of the stress response genes ATF3 and p21 in response to several stimuli. Evidence from structure-function studies argues that Elongin A transcription elongation activity, but not its ubiquitination activity, is most important for its function in induction of transcription of ATF3 and p21. Taken together, our data provide new insights into the function of Elongin A in RNAPII transcription and bring to light a previously unrecognized role for Elongin A in the regulation of stress response genes.

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a pre-eminent complex in eukaryotic transcription regulation. We used macromolecular EM and biochemistry to investigate the subunit organization, structure and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator's middle module interferes with CTD-dependent RNAPII binding to a previously unknown middle-module CTD-binding site and with the holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the mechanism of transcription regulation by Mediator.

Background: Mediator is an evolutionarily conserved multisubunit RNA polymerase II (Pol II) coregulatory complex. Although Mediator was initially found to play a critical role in the regulation of the initiation of Pol II transcription, recent studies have brought to light an expanded role for Mediator at post-initiation stages of transcription., Scope of Review: We provide a brief description of the structure of Mediator and its function in the regulation of Pol II transcription initiation, and we summarize recent findings implicating Mediator in the regulation of various stages of Pol II transcription elongation., Major Conclusions: Emerging evidence is revealing new roles for Mediator in nearly all stages of Pol II transcription, including initiation, promoter escape, elongation, pre-mRNA processing, and termination., General Significance: Mediator plays a central role in the regulation of gene expression by impacting nearly all stages of mRNA synthesis. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation., (Copyright © 2012. Published by Elsevier B.V.)

In this chapter, we describe a purification scheme designed to isolate multisubunit protein complexes gently and quickly from crude extracts of mammalian cells using immunoaffinity purification of epitope tagged proteins and the multisubunit complexes with which they associate. As an example we describe isolation of the mammalian Mediator complex from HeLa S3 cells.

The human ALC1/CHD1L oncogene encodes an SNF2 family ATPase with a macrodomain that binds poly(ADP-ribose) (PAR). We and others previously showed that ALC1 possesses a cryptic ATP-dependent nucleosome remodeling activity that is potently activated in the presence of PARP1 and NAD(+), its substrate for PAR synthesis. In this work, we dissected the mechanism by which PARP1 and NAD(+) activate ALC1 nucleosome remodeling. We demonstrate that ALC1 activation depends on the formation of a stable ALC1·PARylated PARP1·nucleosome intermediate. In addition, by exploiting a novel PAR footprinting assay, we obtained evidence that the ALC1 macrodomain remains stably associated with PAR on autoPARylated PARP1 during the course of nucleosome remodeling reactions. Taken together, our findings are consistent with the model that PAR present on PARylated PARP1 acts as an allosteric effector of ALC1 nucleosome remodeling activity.

Elongin A increases the rate of RNA polymerase II (pol II) transcript elongation by suppressing transient pausing by the enzyme. Elongin A also acts as a component of a cullin-RING ligase that can target stalled pol II for ubiquitylation and proteasome-dependent degradation. It is not known whether these activities of Elongin A are functionally interdependent in vivo. Here, we demonstrate that Elongin A-deficient (Elongin A(-/-)) embryos exhibit abnormalities in the formation of both cranial and spinal nerves and that Elongin A(-/-) embryonic stem cells (ESCs) show a markedly decreased capacity to differentiate into neurons. Moreover, we identify Elongin A mutations that selectively inactivate one or the other of the aforementioned activities and show that mutants that retain the elongation stimulatory, but not pol II ubiquitylation, activity of Elongin A rescue neuronal differentiation and support retinoic acid-induced upregulation of a subset of neurogenesis-related genes in Elongin A(-/-) ESCs., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

The basic leucine zipper transcription factor ATF6α functions as a master regulator of endoplasmic reticulum (ER) stress response genes. Previous studies have established that, in response to ER stress, ATF6α translocates to the nucleus and activates transcription of ER stress response genes upon binding sequence specifically to ER stress response enhancer elements in their promoters. In this study, we investigate the biochemical mechanism by which ATF6α activates transcription. By exploiting a combination of biochemical and multidimensional protein identification technology-based mass spectrometry approaches, we have obtained evidence that ATF6α functions at least in part by recruiting to the ER stress response enhancer elements of ER stress response genes a collection of RNA polymerase II coregulatory complexes, including the Mediator and multiple histone acetyltransferase complexes, among which are the Spt-Ada-Gcn5 acetyltransferase (SAGA) and Ada-Two-A-containing (ATAC) complexes. Our findings shed new light on the mechanism of action of ATF6α, and they outline a straightforward strategy for applying multidimensional protein identification technology mass spectrometry to determine which RNA polymerase II transcription factors and coregulators are recruited to promoters and other regulatory elements to control transcription.

The human tumour antigen PRAME (preferentially expressed antigen in melanoma) is frequently overexpressed during oncogenesis, and high PRAME levels are associated with poor clinical outcome in a variety of cancers. However, the molecular pathways in which PRAME is implicated are not well understood. We recently characterized PRAME as a BC-box subunit of a Cullin2-based E3 ubiquitin ligase. In this study, we mined the PRAME interactome to a deeper level and identified specific interactions with OSGEP and LAGE3, which are human orthologues of the ancient EKC/KEOPS complex. By characterizing biochemically the human EKC complex and its interactions with PRAME, we show that PRAME recruits a Cul2 ubiquitin ligase to EKC. Moreover, EKC subunits associate with PRAME target sites on chromatin. Our data reveal a novel link between the oncoprotein PRAME and the conserved EKC complex and support a role for both complexes in the same pathways.

The Mediator is a large, multisubunit RNA polymerase II transcriptional regulator that was first identified in Saccharomyces cerevisiae as a factor required for responsiveness of Pol II and the general initiation factors to DNA binding transactivators. Since its discovery in yeast, Mediator has been shown to be an integral and highly evolutionarily conserved component of the Pol II transcriptional machinery with critical roles in multiple stages of transcription, from regulation of assembly of the Pol II initiation complex to regulation of Pol II elongation. Here we provide a brief overview of the evolutionary origins of Mediator, its subunit composition, and its remarkably diverse collection of activities in Pol II transcription., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The human tumour antigen PRAME (preferentially expressed antigen of melanoma) is frequently overexpressed in tumours. High PRAME levels correlate with poor clinical outcome of several cancers, but the mechanisms by which PRAME could be involved in tumourigenesis remain largely elusive. We applied protein-complex purification strategies and identified PRAME as a substrate recognition subunit of a Cullin2-based E3 ubiquitin ligase. PRAME can be recruited to DNA in vitro, and genome-wide chromatin immunoprecipitation experiments revealed that PRAME is specifically enriched at transcriptionally active promoters that are also bound by NFY and at enhancers. Our results are consistent with a role for the PRAME ubiquitin ligase complex in NFY-mediated transcriptional regulation.

Promoter-proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II correlates with recruitment of super-elongation complexes (SECs) containing ELL/EAF family members, P-TEFb, and other proteins, but the mechanism of their recruitment is an unanswered question. Here, we present evidence for a role of human Mediator subunit MED26 in this process. We identify in the conserved N-terminal domain of MED26 overlapping docking sites for SEC and a second ELL/EAF-containing complex, as well as general initiation factor TFIID. In addition, we present evidence consistent with the model that MED26 can function as a molecular switch that interacts first with TFIID in the Pol II initiation complex and then exchanges TFIID for complexes containing ELL/EAF and P-TEFb to facilitate transition of Pol II into the elongation stage of transcription., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We previously identified and purified a human ATP-dependent chromatin remodeling complex with similarity to the Saccharomyces cerevisiae INO80 complex (Jin, J., Cai, Y., Yao, T., Gottschalk, A. J., Florens, L., Swanson, S. K., Gutierrez, J. L., Coleman, M. K., Workman, J. L., Mushegian, A., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 41207-41212) and demonstrated that it is composed of (i) a Snf2 family ATPase (hIno80) related in sequence to the S. cerevisiae Ino80 ATPase; (ii) seven additional evolutionarily conserved subunits orthologous to yeast INO80 complex subunits; and (iii) six apparently metazoan-specific subunits. In this report, we present evidence that the human INO80 complex is composed of three modules that assemble with three distinct domains of the hIno80 ATPase. These modules include (i) one that is composed of the N terminus of the hIno80 protein and all of the metazoan-specific subunits and is not required for ATP-dependent nucleosome remodeling; (ii) a second that is composed of the hIno80 Snf2-like ATPase/helicase and helicase-SANT-associated/post-HSA (HSA/PTH) domain, the actin-related proteins Arp4 and Arp8, and the GLI-Kruppel family transcription factor YY1; and (iii) a third that is composed of the hIno80 Snf2 ATPase domain, the Ies2 and Ies6 proteins, the AAA(+) ATPases Tip49a and Tip49b, and the actin-related protein Arp5. Through purification and characterization of hINO80 complex subassemblies, we demonstrate that ATP-dependent nucleosome remodeling by the hINO80 complex is catalyzed by a core complex comprising the hIno80 protein HSA/PTH and Snf2 ATPase domains acting in concert with YY1 and the complete set of its evolutionarily conserved subunits. Taken together, our findings shed new light on the structure and function of the INO80 chromatin-remodeling complex.

Over the past few years, advances in biochemical and genetic studies of the structure and function of the Mediator complex have shed new light on its subunit architecture and its mechanism of action in transcription by RNA polymerase II (pol II). The development of improved methods for reconstitution of recombinant Mediator subassemblies is enabling more in-depth analyses of basic features of the mechanisms by which Mediator interacts with and controls the activity of pol II and the general initiation factors. The discovery and characterization of multiple, functionally distinct forms of Mediator characterized by the presence or absence of the Cdk8 kinase module have led to new insights into how Mediator functions in both Pol II transcription activation and repression. Finally, progress in studies of the mechanisms by which the transcriptional activation domains (ADs) of DNA binding transcription factors target Mediator have brought to light unexpected complexities in the way Mediator participates in signal transduction., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The human epigenetic cell-cycle regulator HCF-1 undergoes an unusual proteolytic maturation process resulting in stably associated HCF-1(N) and HCF-1(C) subunits that regulate different aspects of the cell cycle. Proteolysis occurs at six centrally located HCF-1(PRO)-repeat sequences and is important for activation of HCF-1(C)-subunit functions in M phase progression. We show here that the HCF-1(PRO) repeat is recognized by O-linked β-N-acetylglucosamine transferase (OGT), which both O-GlcNAcylates the HCF-1(N) subunit and directly cleaves the HCF-1(PRO) repeat. Replacement of the HCF-1(PRO) repeats by a heterologous proteolytic cleavage signal promotes HCF-1 proteolysis but fails to activate HCF-1(C)-subunit M phase functions. These results reveal an unexpected role of OGT in HCF-1 proteolytic maturation and an unforeseen nexus between OGT-directed O-GlcNAcylation and proteolytic maturation in HCF-1 cell-cycle regulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Chromosomal translocations involving the MLL gene are associated with infant acute lymphoblastic and mixed lineage leukemia. There are a large number of translocation partners of MLL that share very little sequence or seemingly functional similarities; however, their translocations into MLL result in the pathogenesis of leukemia. To define the molecular reason why these translocations result in the pathogenesis of leukemia, we purified several of the commonly occurring MLL chimeras. We have identified super elongation complex (SEC) associated with all chimeras purified. SEC includes ELL, P-TEFb, AFF4, and several other factors. AFF4 is required for SEC stability and proper transcription by poised RNA polymerase II in metazoans. Knockdown of AFF4 in leukemic cells shows reduction in MLL chimera target gene expression, suggesting that AFF4/SEC could be a key regulator in the pathogenesis of leukemia through many of the MLL partners.