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Elongin a Regulates Transcription In&Nbsp RESEARCH ARTICLE Elongin A regulates transcription in vivo through enhanced RNA polymerase processivity Received for publication, September 2, 2020, and in revised form, December 3, 2020 Published, Papers in Press, December 9, 2020, https://doi.org/10.1074/jbc.RA120.015876 Yating Wang1 , Liming Hou1 , M. Behfar Ardehali2,3 , Robert E. Kingston2,3, and Brian D. Dynlacht1,* From the 1Department of Pathology, New York University School of Medicine, New York, New York, USA; 2Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA; and 3Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA Edited by Karin Musier-Forsyth Elongin is an RNA polymerase II (RNAPII)-associated factor well as negative elongation factor. The release of paused that has been shown to stimulate transcriptional elongation RNAPII into productive elongation is mediated by positive in vitro. The Elongin complex is thought to be required for transcription elongation factor b and cyclin-dependent kinase transcriptional induction in response to cellular stimuli and to 12, which phosphorylates DRB sensitivity-inducing factor, ubiquitinate RNAPII in response to DNA damage. Yet, the negative elongation factor, and serine 2 (Ser2) on the C-ter- impact of the Elongin complex on transcription in vivo has not minal domain of the largest subunit of RNAPII (1, 2). Tran- been well studied. Here, we performed comprehensive studies scription initiation has been recognized as a critical regulatory of the role of Elongin A, the largest subunit of the Elongin point during the transcription cycle, but recently, a growing complex, on RNAPII transcription genome-wide. Our results body of evidence suggests that transcription elongation can also suggest that Elongin A localizes to actively transcribed regions be an important rate-limiting step. Although transcription and potential enhancers, and the level of recruitment corre- elongation rates on individual genes vary widely (3), maintaining lated with transcription levels. We also identified a large group a proper elongation rate is critically important. Recent studies of factors involved in transcription as Elongin A–associated suggest that misregulation of transcription elongation can cause factors. In addition, we found that loss of Elongin A leads to defects in transcription directionality, cotranscriptional chro- dramatically reduced levels of serine2-phosphorylated, but not matin modifications, and mRNA splicing (4, 5). A mouse strain total, RNAPII, and cells depleted of Elongin A show stronger harboring a slow RNAPII mutant shows embryonic lethality, promoter RNAPII pausing, suggesting that Elongin A may be suggesting that proper RNAPII elongation is also critical for involved in the release of paused RNAPII. Our RNA-seq studies mouse development (6). suggest that loss of Elongin A did not alter global transcription, Several factors have been shown to mediate proper tran- and unlike prior in vitro studies, we did not observe a dramatic scription elongation, including PAF1 complex, SPT6, and impact on RNAPII elongation rates in our cell-based nascent SPT5 (7–9). Another factor that is potentially involved in RNA-seq experiments upon Elongin A depletion. Taken regulating RNAPII elongation is the Elongin complex (10), together, our studies provide the first comprehensive analysis which was identified biochemically as one of the factors that of the role of Elongin A in regulating transcription in vivo. Our can stimulate RNAPII elongation on DNA templates in vitro. studies also revealed that unlike prior in vitro findings, deple- Previous studies suggested that Elongin can stimulate RNAPII tion of Elongin A has little impact on global transcription elongation in vitro, potentially by overcoming the transient profiles and transcription elongation in vivo. pausing of RNAPII on a DNA template. However, the role of this complex during transcription elongation has not been tested in vivo. The synthesis of mRNA by RNA polymerase II (RNAPII) The Elongin complex is a trimer consisting of Elongin A, B requires the highly orchestrated action of a large cohort of and C, and Elongin A is the largest subunit. The N-terminus factors. During the transcription cycle, general transcription of Elongin A shares homology with elongation factor tran- factors recruit RNAPII to promoters to initiate transcription, scription elongation factor IIS, and the C-terminus of Elon- and the RNAPII complex pauses 50 to 100 nt downstream of gin A contains a conserved motif, BC-box, that allows the transcription start sites (TSSs). Pausing is enforced by 5,6- Elongin B and C subunits to bind. Elongin A is the dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) transcription-stimulatory subunit, and Elongin B and C can sensitivity-inducing factor, which includes SPT4 and SPT5, as enhance the activity of Elongin A (10, 11). Apart from Elongin A, a large group of proteins also contains a BC-box This article contains supporting information. motif and can associate with Elongin B and C. These pro- * For correspondence: Brian D. Dynlacht, [email protected]. teins can be linked to a Cullin-RING ligase through the Present address for Liming Hou: Department of Animal Genetics, Breeding Elongin BC complex to function as an E3 ligase complex. The and Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210000, China. Cullin-RING ligase in these cases contains Cul2 or Cul5 and J. Biol. Chem. (2021) 296 100170 1 © 2020 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Elongin A regulates transcription in vivo Rbx1 or Rbx2 (12–14). Upon UV irradiation, the mammalian ribosomal proteins, mRNA processing, ciliary, and VEGF Elongin complex can assemble with the Cul5/Rbx2 module signaling pathway proteins, as well as translation factors are to act as the ubiquitin ligase for RNAPII subunit, RPB1 (15). among the top enriched categories (Fig. S1C). It was also shown that the yeast homologs of Elongin A and C Next, we performed RNA-seq and RNAPII ChIP-seq anal- are responsible for the ubiquitination of RNAPII under ysis and compared these results with our Elongin A occupancy conditions of DNA damage (16). data. We found that Elongin A was enriched on actively The Elongin complex was initially identified as a potential transcribed genes and binding was highly correlated with elongation factor in in vitro studies more than 2 decades ago. active transcription (Fig. 1A and Fig. S1D). We found that Since then, several studies have been performed to investigate ChIP-seq peaks were also observed over some regions that the role of Elongin in vivo. It was shown that Elongin A is exhibited strong Elongin A enrichment in CUT&RUN exper- required for transcriptional induction in response to multiple iments (Fig. S1D). Metagene analysis of annotated Refseq stimuli in mammalian cells (17, 18), and Elongin A–deficient genes suggests that Elongin A is enriched at TSS regions, gene mouse embryo exhibit developmental abnormalities (18). bodies, and transcript end sites (TESs) (Fig. 1B). Interestingly, Cellular immunofluorescence studies suggest that Elongin A we found that we were more likely to observe Elongin A colocalizes with the hyperphosphorylated form of RNAPII, and enrichment on gene bodies and regions downstream of the chromatin immunoprecipitation (ChIP)-quantitative PCR ex- TES on short genes, whereas on longer genes, we were more periments show Elongin A associates with stress-response genes likely to observe Elongin A enrichment only at TSS-proximal upon induction (17). However, although it has been generally regions (Fig. 1A, Fig. S1E). These results suggest that Elongin assumed that Elongin also functions as a transcription elonga- A may only travel with the RNAPII complex up to a certain tion factor in vivo, none of these prior studies has confirm this distance downstream of TSS or that Elongin A has more hypothesis. In addition, there have been no genome-wide important roles near TSS. Interestingly, when we plotted studies of Elongin, and the impact of Elongin on different as- Elongin A CUT&RUN signal in the immediate vicinity of the pects of the transcription cycle has not been systematically TSS, we found that Elongin A enrichment is stronger imme- investigated. Here, we investigated the genome-wide localiza- diately upstream of the TSS than downstream (Fig. S1F). Our tion of Elongin A and the impact of its loss on RNAPII distri- analysis also suggested that Elongin A enrichment correlated bution, as well as the production of nascent and mature with gene expression levels. We ranked Refseq genes based on transcripts. Our studies suggest that Elongin A localizes to their expression levels (fragments per kilobase per million actively transcribed genomic regions, including annotated mapped reads [FPKM]) and divided them into four groups. We transcripts and potential enhancer regions. Concordantly, we then quantified average Elongin A enrichment on each group found that Elongin A associates with a large group of factors of genes and found that genes with higher expression levels involved in transcription. Interestingly, we showed that Elongin tended to show stronger Elongin A enrichment (Fig. 1C). A depletion has a dramatic impact on levels of the Ser2- Plotting individual genes on heat maps likewise showed a clear phosphorylated
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