Publications

2023
Andrew L. Spencley, Shiran Bar, Tomek Swigut, Ryan A. Flynn, Cameron H. Lee, Liang-Fu Chen, Michael C. Bassik, and Joanna Wysocka. 2023. “Co-transcriptional genome surveillance by HUSH is coupled to termination machinery.” Molecular Cell, 83, 10, Pp. 1623–1639.e8. Publisher's VersionAbstract
The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and—via its component MPP8—with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.
Brittany A. Townley, Luke Buerer, Ning Tsao, Albino Bacolla, Fadhel Mansoori, Timur Rusanov, Nathaniel E. Clark, Negar Goodarzi, Nicolas Schmidt, Sridhar Nonavinkere Srivatsan, Hua Sun, Reilly A. Sample, Joshua R. Brickner, Drew McDonald, Miaw-Sheue Tsai, Matthew J. Walter, David F. Wozniak, Alex S. Holehouse, Vladimir Pena, John A. Tainer, William G. Fairbrother, and Nima Mosammaparast. 2023. “A functional link between lariat debranching enzyme and the intron-binding complex is defective in non-photosensitive trichothiodystrophy.” Molecular Cell, 83, 13, Pp. 2258–2275.e11. Publisher's VersionAbstract
The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.
Hannah J. Uckelmann, Elena L. Haarer, Reina Takeda, Eric M. Wong, Charlie Hatton, Christian Marinaccio, Florian Perner, Masooma Rajput, Noa J.C. Antonissen, Yanhe Wen, Lu Yang, Lorenzo Brunetti, Chun-Wei Chen, and Scott A. Armstrong. 2023. “Mutant NPM1 Directly Regulates Oncogenic Transcription in Acute Myeloid Leukemia.” Cancer Discovery, 13, 3, Pp. 746–765. Publisher's VersionAbstract
The dysregulation of developmental and stem cell–associated genes is a common phenomenon during cancer development. Around half of patients with acute myeloid leukemia (AML) express high levels of HOXA cluster genes and MEIS1. Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncoprotein mislocalized from the nucleolus to the cytoplasm. How NPM1c expression in hematopoietic cells leads to its characteristic gene-expression pattern remains unclear. Here, we show that NPM1c directly binds to specific chromatin targets, which are co-occupied by the histone methyltransferase KMT2A (MLL1). Targeted degradation of NPM1c leads to a rapid decrease in gene expression and loss of RNA polymerase II, as well as activating histone modifications at its targets. We demonstrate that NPM1c directly regulates oncogenic gene expression in collaboration with the MLL1 complex and define the mechanism by which MLL1–Menin small-molecule inhibitors produce clinical responses in patients with NPM1-mutated AML.We uncovered an important functional role of mutant NPM1 as a crucial direct driver of oncogenic gene expression in AML. NPM1c can bind to chromatin and cooperate with the MLL complex, providing the first functional insight into the mechanism of Menin–MLL inhibition in NPM1c leukemias.See related article by Wang et al., p. 724.This article is highlighted in the In This Issue feature, p. 517
Megan L. Insco, Brian J. Abraham, Sara J. Dubbury, Ines H. Kaltheuner, Sofia Dust, Constance Wu, Kevin Y. Chen, David Liu, Stanislav Bellaousov, Anna M. Cox, Benjamin J. E. Martin, Tongwu Zhang, Calvin G. Ludwig, Tania Fabo, Rodsy Modhurima, Dakarai E. Esgdaille, Telmo Henriques, Kevin M. Brown, Stephen J. Chanock, Matthias Geyer, Karen Adelman, Phillip A. Sharp, Richard A. Young, Paul L. Boutz, and Leonard I. Zon. 2023. “Oncogenic CDK13 mutations impede nuclear RNA surveillance.” Science, 380, 6642, Pp. eabn7625. Publisher's VersionAbstract
RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 (CDK13) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.
Xavier Rambout, Hana Cho, Roméo Blanc, Qing Lyu, Joseph M. Miano, Joe V. Chakkalakal, Geoffrey M. Nelson, Hari K. Yalamanchili, Karen Adelman, and Lynne E. Maquat. 2023. “PGC-1α senses the CBC of pre-mRNA to dictate the fate of promoter-proximally paused RNAPII.” Molecular Cell, 83, 2, Pp. 186–202.e11. Publisher's VersionAbstract
PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway.
Gongwei Wu, Noriaki Yoshida, Jihe Liu, Xiaoyang Zhang, Yuan Xiong, Tayla B. Heavican-Foral, Elisa Mandato, Huiyun Liu, Geoffrey M. Nelson, Lu Yang, Renee Chen, Katherine A. Donovan, Marcus K. Jones, Mikhail Roshal, Yanming Zhang, Ran Xu, Ajit J. Nirmal, Salvia Jain, Catharine Leahy, Kristen L. Jones, Kristen E. Stevenson, Natasha Galasso, Nivetha Ganesan, Tiffany Chang, Wen-Chao Wu, Abner Louissaint, Lydie Debaize, Hojong Yoon, Paola Dal Cin, Wing C. Chan, Shannan J. Ho Sui, Samuel Y. Ng, Andrew L. Feldman, Steven M. Horwitz, Karen Adelman, Eric S. Fischer, Chun-Wei Chen, David M. Weinstock, and Myles Brown. 2023. “TP63 fusions drive multicomplex enhancer rewiring, lymphomagenesis, and EZH2 dependence.” Science Translational Medicine, 15, 714, Pp. eadi7244. Publisher's VersionAbstract
Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T and B cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here, we show that TP63 fusions act as bona fide oncogenes and are essential for fusion-positive lymphomas. Transgenic mice expressing TBL1XR1::TP63, the most common TP63 fusion, develop diverse lymphomas that recapitulate multiple human T and B cell lymphomas. Here, we identify that TP63 fusions coordinate the recruitment of two epigenetic modifying complexes, the nuclear receptor corepressor (NCoR)—histone deacetylase 3 (HDAC3) by the N-terminal TP63 fusion partner and the lysine methyltransferase 2D (KMT2D) by the C-terminal TP63 component, which are both required for fusion-dependent survival. TBL1XR1::TP63 localization at enhancers drives a unique cell state that involves up-regulation of MYC and the polycomb repressor complex 2 (PRC2) components EED and EZH2. Inhibiting EZH2 with the therapeutic agent valemetostat is highly effective at treating transgenic lymphoma murine models, xenografts, and patient-derived xenografts harboring TP63 fusions. One patient with TP63 -rearranged lymphoma showed a rapid response to valemetostat treatment. In summary, TP63 fusions link partner components that, together, coordinate multiple epigenetic complexes, resulting in therapeutic vulnerability to EZH2 inhibition. , TP63 fusions coordinate multiple epigenetic complexes, drive lymphomagenesis, and create a therapeutic vulnerability to EZH2 inhibition. , Editor’s summary Patients with T and B cell lymphomas with gene fusions involving tumor protein p63 gene (TP63) often have poor overall survival. To investigate these fusions, Wu et al . evaluated the mechanism behind the promotion of tumor growth and how to inhibit it. The authors found that these fusions drove tumor survival through EZH2 and, through inhibition of EZH2 with valemetostat, were able to treat patient-derived xenografts harboring TP63 fusions. In addition, one patient with a TP63-rearranged lymphoma responded to valemetostat treatment, suggesting a potential targeted therapy that requires further investigation. —Dorothy Hallberg
2022
Jennifer M. Luppino, Andrew Field, Son C. Nguyen, Daniel S. Park, Parisha P. Shah, Richard J. Abdill, Yemin Lan, Rebecca Yunker, Rajan Jain, Karen Adelman, and Eric F. Joyce. 2022. “Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression.” PLOS Genetics, 18, 11, Pp. e1010528. Publisher's VersionAbstract
The relationship between cohesin-mediated chromatin looping and gene expression remains unclear. NIPBL and WAPL are two opposing regulators of cohesin activity; depletion of either is associated with changes in both chromatin folding and transcription across a wide range of cell types. However, a direct comparison of their individual and combined effects on gene expression in the same cell type is lacking. We find that NIPBL or WAPL depletion in human HCT116 cells each alter the expression of \textasciitilde2,000 genes, with only \textasciitilde30% of the genes shared between the conditions. We find that clusters of differentially expressed genes within the same topologically associated domain (TAD) show coordinated misexpression, suggesting some genomic domains are especially sensitive to both more or less cohesin. Finally, co-depletion of NIPBL and WAPL restores the majority of gene misexpression as compared to either knockdown alone. A similar set of NIPBL-sensitive genes are rescued following CTCF co-depletion. Together, this indicates that altered transcription due to reduced cohesin activity can be functionally offset by removal of either its negative regulator (WAPL) or the physical barriers (CTCF) that restrict loop-extrusion events.
Jing Luan, Marit W. Vermunt, Camille M. Syrett, Allison Cote, Jacob M. Tome, Haoyue Zhang, Anran Huang, Jennifer M. Luppino, Cheryl A. Keller, Belinda M. Giardine, Shiping Zhang, Margaret C. Dunagin, Zhe Zhang, Eric F. Joyce, John T. Lis, Arjun Raj, Ross C. Hardison, and Gerd A. Blobel. 2022. “CTCF blocks antisense transcription initiation at divergent promoters.” Nature structural & molecular biology, 29, 11, Pp. 1136–1144.Abstract
Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is not well understood. Here, by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters. Primary transcript RNA-FISH shows that CTCF lowers burst fraction but not burst intensity of uasTrx and that co-bursting of sense and antisense transcripts is disfavored. Genome editing, chromatin conformation studies and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its architectural function. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription. Luan et al. find that CTCF shapes the transcriptional landscape in part by suppressing the initiation of upstream antisense transcription at hundreds of divergent gene promoters.
Wenqi Xu, Chenxi He, Emily G. Kaye, Jiahui Li, Mandi Mu, Geoffrey M. Nelson, Li Dong, Jiahua Wang, Feizhen Wu, Yujiang Geno Shi, Karen Adelman, Fei Lan, Yang Shi, and Hongjie Shen. 2022. “Dynamic control of chromatin-associated m6A methylation regulates nascent RNA synthesis.” Molecular Cell, 82, 6, Pp. 1156-1168.e7. Publisher's VersionAbstract
Summary N6-methyladenosine (m6A) methylation is co-transcriptionally deposited on mRNA, but a possible role of m6A on transcription remains poorly understood. Here, we demonstrate that the METTL3/METTL14/WTAP m6A methyltransferase complex (MTC) is localized to many promoters and enhancers and deposits the m6A modification on nascent transcripts, including pre-mRNAs, promoter upstream transcripts (PROMPTs), and enhancer RNAs. PRO-seq analyses demonstrate that nascent RNAs originating from both promoters and enhancers are significantly decreased in the METTL3-depleted cells. Furthermore, genes targeted by the Integrator complex for premature termination are depleted of METTL3, suggesting a potential antagonistic relationship between METTL3 and Integrator. Consistently, we found the Integrator complex component INTS11 elevated at promoters and enhancers upon loss of MTC or nuclear m6A binders. Taken together, our findings suggest that MTC-mediated m6A modification protects nascent RNAs from Integrator-mediated termination and promotes productive transcription, thus unraveling an unexpected layer of gene regulation imposed by RNA m6A modification.
Chad B Stein, Andrew R Field, Claudia A Mimoso, ChenCheng Zhao, Kai-Lieh Huang, Eric J Wagner, and Karen Adelman. 2022. “Integrator endonuclease drives promoter-proximal termination at all RNA polymerase II-transcribed loci.” Molecular Cell, 82, 22, Pp. 4232–4245.
Joseph M. Replogle, Reuben A. Saunders, Angela N. Pogson, Jeffrey A. Hussmann, Alexander Lenail, Alina Guna, Lauren Mascibroda, Eric J. Wagner, Karen Adelman, Gila Lithwick-Yanai, Nika Iremadze, Florian Oberstrass, Doron Lipson, Jessica L. Bonnar, Marco Jost, Thomas M. Norman, and Jonathan S. Weissman. 2022. “Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq.” Cell, 185, 14, Pp. 2559-2575.e28. Publisher's VersionAbstract
Summary A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena—from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.
Sarah Naomi Olsen, Laura Godfrey, James P. Healy, Yoolim A. Choi, Yan Kai, Charles Hatton, Florian Perner, Elena L. Haarer, Behnam Nabet, Guo-Cheng Yuan, and Scott A. Armstrong. 2022. “MLL::AF9 degradation induces rapid changes in transcriptional elongation and subsequent loss of an active chromatin landscape.” Molecular Cell, 82, 6, Pp. 1140-1155.e11. Publisher's VersionAbstract
Summary MLL rearrangements produce fusion oncoproteins that drive leukemia development, but the direct effects of MLL-fusion inactivation remain poorly defined. We designed models with degradable MLL::AF9 where treatment with small molecules induces rapid degradation. We leveraged the kinetics of this system to identify a core subset of MLL::AF9 target genes where MLL::AF9 degradation induces changes in transcriptional elongation within 15 minutes. MLL::AF9 degradation subsequently causes loss of a transcriptionally active chromatin landscape. We used this insight to assess the effectiveness of small molecules that target members of the MLL::AF9 multiprotein complex, specifically DOT1L and MENIN. Combined DOT1L/MENIN inhibition resembles MLL::AF9 degradation, whereas single-agent treatment has more modest effects on MLL::AF9 occupancy and gene expression. Our data show that MLL::AF9 degradation leads to decreases in transcriptional elongation prior to changes in chromatin landscape at select loci and that combined inhibition of chromatin complexes releases the MLL::AF9 oncoprotein from chromatin globally.
Hannah J. Uckelmann, Elena L. Haarer, Reina Takeda, Eric M. Wong, Charlie Hatton, Christian Marinaccio, Florian Perner, Masooma Rajput, Noa J.C. Antonissen, Yanhe Wen, Lu Yang, Lorenzo Brunetti, Chun-Wei Chen, and Scott A. Armstrong. 2022. “Mutant NPM1 directly regulates oncogenic transcription in acute myeloid leukemia.” Cancer Discovery. Publisher's VersionAbstract
The dysregulation of developmental and stem cell associated genes is a common phenomenon during cancer development. Around half of acute myeloid leukemia (AML) patients express high levels of HOXA cluster genes and MEIS1. Most of these AML cases harbor an NPM1 mutation (NPM1c), which encodes for an oncogene mislocalized from the nucleolus to the cytoplasm. How NPM1c expression in hematopoietic cells leads to its characteristic gene expression pattern remains unclear. Here, we show that NPM1c directly binds to specific chromatin targets, which are co-occupied by the histone methyltransferase KMT2A (MLL1). Targeted degradation of NPM1c leads to a rapid decrease in gene expression and loss of RNA Polymerase II, as well as activating histone modifications at its targets. We demonstrate that NPM1c directly regulates oncogenic gene expression in collaboration with the MLL1 complex and define the mechanism by which MLL1-Menin small molecule inhibitors produce clinical responses in patients with NPM1-mutated AML.
Wenchao Wu, Geoffrey M. Nelson, Raphael Koch, Katherine A. Donovan, Radosław P. Nowak, Tayla B. Heavican-Foral, Ajit J. Nirmal, Huiyun Liu, Lei Yang, Jessica Duffy, Foster Powers, Kristen E. Stevenson, Marcus Kenneth Jones, Samuel Y. Ng, Gongwei Wu, Salvia Jain, Ran Xu, Sam Amaka, Christopher Trevisani, Nicholas L. Donaldson, Patrick R. Hagner, Laurence de Leval, Philippe Gaulard, Javeed Iqbal, Anjan Thakurta, Eric S. Fischer, Karen Adelman, and David M. Weinstock. 2022. “Overcoming IMiD resistance in T-cell lymphomas through potent degradation of ZFP91 and IKZF1.” Blood, 139, 13, Pp. 2024-2037. Publisher's VersionAbstract
Immunomodulatory (IMiD) agents like lenalidomide and pomalidomide induce the recruitment of IKZF1 and other targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and degradation. These agents are highly active in B-cell lymphomas and a subset of myeloid diseases but have compromised effects in T-cell lymphomas (TCLs). Here, we show that 2 factors determine resistance to IMiDs among TCLs. First, limited CRBN expression reduces IMiD activity in TCLs but can be overcome by newer-generation degrader CC-92480. Using mass spectrometry, we show that CC-92480 selectively degrades IKZF1 and ZFP91 in TCL cells with greater potency than pomalidomide. As a result, CC-92480 is highly active against multiple TCL subtypes and showed greater efficacy than pomalidomide across 4 in vivo TCL models. Second, we demonstrate that ZFP91 functions as a bona fide transcription factor that coregulates cell survival with IKZF1 in IMiD-resistant TCLs. By activating keynote genes from WNT, NF-kB, and MAP kinase signaling, ZFP91 directly promotes resistance to IKZF1 loss. Moreover, lenalidomide-sensitive TCLs can acquire stable resistance via ZFP91 rewiring, which involves casein kinase 2–mediated c-Jun inactivation. Overall, these findings identify a critical transcription factor network within TCLs and provide clinical proof of concept for the novel therapy using next-generation degraders.
Stuti Mehta, Altantsetseg Buyanbat, Yan Kai, Ozge Karayel, Seth Raphael Goldman, Davide Seruggia, Kevin Zhang, Yuko Fujiwara, Katherine A. Donovan, Qian Zhu, Huan Yang, Behnam Nabet, Nathanael S. Gray, Matthias Mann, Eric S. Fischer, Karen Adelman, and Stuart H. Orkin. 2022. “Temporal resolution of gene derepression and proteome changes upon PROTAC-mediated degradation of BCL11A protein in erythroid cells.” Cell chemical biology, 29, 8, Pp. 1273–1287.e8.Abstract
Reactivation of fetal hemoglobin expression by the downregulation of BCL11A is a promising treatment for β-hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of γ-globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR-Cas9 gene editing. We leveraged the dTAG PROTAC degradation platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by nascent transcriptomics, proteomics, chromatin accessibility, and histone profiling. Among 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in <2 h. Robust HBG1/2 reactivation upon acute BCL11A depletion occurred without the loss of promoter 5-methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci. [Display omitted] •PROTAC-mediated degradation of BCL11A reactivates high-level γ-globin expression•BCL11A represses <31 primary target genes; HBG, HBZ most induced upon BCL11A loss•Presence of BCL11A is the major barrier to γ-globin promoter activation•Upon BCL11A loss, increased chromatin accessibility closely follows transcription Reactivation of γ-globin by the downregulation of BCL11A expression is a promising strategy for the treatment of sickle cell disease. In this issue of Cell Chemical Biology, Mehta et al. use PROTAC-mediated depletion of BCL11A to identify a small set of target genes and chart the kinetics of γ-globin induction.
Haining Zhou, Chad B Stein, Tiasha A Shafiq, Gergana Shipkovenska, Marian Kalocsay, Joao A Paulo, Jiuchun Zhang, Zhenhua Luo, Steven P Gygi, Karen Adelman, and Danesh Moazed. 2022. “Rixosomal RNA degradation contributes to silencing of Polycomb target genes.” Nature (London), 604, 7904, Pp. 167–174. Publisher's VersionAbstract
Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate . Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex . Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5' monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells.
Hanneke Vlaming, Claudia A Mimoso, Andrew R Field, Benjamin JE Martin, and Karen Adelman. 2022. “Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential.” Nature structural & molecular biology, 29, 6, Pp. 613–620. Publisher's VersionAbstract
Precise regulation of transcription by RNA polymerase II (RNAPII) is critical for organismal growth and development. However, what determines whether an engaged RNAPII will synthesize a full-length transcript or terminate prematurely is poorly understood. Notably, RNAPII is far more susceptible to termination when transcribing non-coding RNAs than when synthesizing protein-coding mRNAs, but the mechanisms underlying this are unclear. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in non-coding RNAs, rather than specific sequence motifs, drives RNAPII termination. Further, we demonstrate that 5' splice sites autonomously stimulate processive transcription, even in the absence of polyadenylation signals. Our results reveal a potent role for the transcribed sequence in dictating gene output and demonstrate the power of INSERT-seq toward illuminating these contributions.
2021
Andrea J. Kriz, David Colognori, Hongjae Sunwoo, Behnam Nabet, and Jeannie T. Lee. 2021. “Balancing cohesin eviction and retention prevents aberrant chromosomal interactions, Polycomb-mediated repression, and X-inactivation.” Molecular Cell, 81, 9, Pp. 1970–1987.e9. Publisher's VersionAbstract
Depletion of architectural factors globally alters chromatin structure but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Cohesin loss disrupts the Xi superstructure, unveiling superloops between escapee genes with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure, compromises spreading of Xist RNA-Polycomb complexes, and attenuates Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss creates an Xi superloop, and cohesin retention creates Xi megadomains on the active X chromosome. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X inactivation and inter-chromosomal interactions across the genome.
Michael Rosenberg, Roy Blum, Barry Kesner, Eric Aeby, Jean-Michel Garant, Attila Szanto, and Jeannie T. Lee. 2021. “Motif-driven interactions between RNA and PRC2 are rheostats that regulate transcription elongation.” Nature Structural & Molecular Biology, 28, 1, Pp. 103–117. Publisher's VersionAbstract
Although polycomb repressive complex 2 (PRC2) is now recognized as an RNA-binding complex, the full range of binding motifs and why PRC2–RNA complexes often associate with active genes have not been elucidated. Here, we identify high-affinity RNA motifs whose mutations weaken PRC2 binding and attenuate its repressive function in mouse embryonic stem cells. Interactions occur at promoter-proximal regions and frequently coincide with pausing of RNA polymerase II (POL-II). Surprisingly, while PRC2-associated nascent transcripts are highly expressed, ablating PRC2 further upregulates expression via loss of pausing and enhanced transcription elongation. Thus, PRC2-nascent RNA complexes operate as rheostats to fine-tune transcription by regulating transitions between pausing and elongation, explaining why PRC2–RNA complexes frequently occur within active genes. Nascent RNA also targets PRC2 in cis and downregulates neighboring genes. We propose a unifying model in which RNA specifically recruits PRC2 to repress genes through POL-II pausing and, more classically, trimethylation of histone H3 at Lys27.
Daniel C. L. Robinson, Morten Ritso, Geoffrey M. Nelson, Zeinab Mokhtari, Kiran Nakka, Hina Bandukwala, Seth R. Goldman, Peter J. Park, Rémi Mounier, Bénédicte Chazaud, Marjorie Brand, Michael A. Rudnicki, Karen Adelman, and F. Jeffrey Dilworth. 2021. “Negative elongation factor regulates muscle progenitor expansion for efficient myofiber repair and stem cell pool repopulation.” Developmental Cell, 56, 7, Pp. 1014–1029.e7. Publisher's VersionAbstract
Negative elongation factor (NELF) is a critical transcriptional regulator that stabilizes paused RNA polymerase to permit rapid gene expression changes in response to environmental cues. Although NELF is essential for embryonic development, its role in adult stem cells remains unclear. In this study, through a muscle-stem-cell-specific deletion, we showed that NELF is required for efficient muscle regeneration and stem cell pool replenishment. In mechanistic studies using PRO-seq, single-cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of regeneration whereby it modulates p53 signaling to permit massive expansion of muscle progenitors. Strikingly, transplantation experiments indicated that these progenitors are also necessary for stem cell pool repopulation, implying that they are able to return to quiescence. Thus, we identified a critical role for NELF in the expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to the stem cell pool repopulation.

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