Publications

2022
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.
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.
Jennifer M. Luppino, Andrew Field, Son C. Nguyen, Daniel S. Park, Parisha P. Shah, Yemin Lan, Rebecca Yunker, Rajan Jain, Karen Adelman, and Eric F. Joyce. 2022. “NIPBL and WAPL balance cohesin activity to regulate chromatin folding and gene expression.” bioRxiv. Publisher's VersionAbstract
The relationship between cohesin-mediated chromatin looping and gene expression remains unclear. We investigated the roles of NIPBL and WAPL, two regulators of cohesin activity, in chromatin folding and transcription in human cells. Consistent with their opposing roles in cohesin regulation, depletion of these factors showed opposite effects on levels of chromatin-bound cohesin and spatial insulation of neighboring domains. We find that NIPBL or WAPL depletion each alter the expression of  2,000 genes, most of which are uniquely sensitive to either regulator. We find that each set of differentially expressed genes are enriched at chromatin loop anchors and clustered within the genome, suggesting there are genomic regions sensitive to either more or less cohesin. Remarkably, co-depletion of both regulators rescued chromatin misfolding and gene misexpression compared to either single knockdown. Taken together, we present a model in which the relative, rather than absolute, levels of NIPBL and WAPL are required to balance cohesin activity in chromatin folding to regulate transcription.Competing Interest StatementThe authors have declared no competing interest.
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.
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. Publisher's VersionAbstract
Summary 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.
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.
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.
Nan Liu, Shuqian Xu, Qiuming Yao, Qian Zhu, Yan Kai, Jonathan Y. Hsu, Phraew Sakon, Luca Pinello, Guo-Cheng Yuan, Daniel E. Bauer, and Stuart H. Orkin. 2021. “Transcription factor competition at the γ-globin promoters controls hemoglobin switching.” Nature Genetics, Pp. 1–10. Publisher's VersionAbstract
BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2β2). To uncover how BCL11A initiates repression, we used CRISPR–Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)–globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the \textgreater50-kb β-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.
Katerina Cermakova, Jonas Demeulemeester, Vanda Lux, Monika Nedomova, Seth R. Goldman, Eric A. Smith, Pavel Srb, Rozalie Hexnerova, Milan Fabry, Marcela Madlikova, Magdalena Horejsi, Jan De Rijck, Zeger Debyser, Karen Adelman, H. Courtney Hodges, and Vaclav Veverka. 2021. “A ubiquitous disordered protein interaction module orchestrates transcription elongation.” Science, 374, 6571, Pp. 1113–1121. Publisher's VersionAbstract
Organized by unstructured motifs The high degree of conservation in protein sequences thought to be unstructured has hinted that these regions may have important biological functions. Although unstructured regions are widely viewed to be crucial for protein signaling, localization, and stability, their roles in many other settings have remained mysterious. Cermakova et al . discovered that prominent members of the transcription elongation machinery are linked through a network of interactions involving transcription elongation factor TFIIS N-terminal domains (TNDs) and conserved unstructured sequences called “TND-interacting motifs” (TIMs). The researchers found that mutation of a single TIM in a central organizing protein of this network abolished key protein interactions and induced widespread defects in transcription elongation dynamics. —DJ , The transcription elongation machinery is linked through a network of conserved unstructured motifs. , During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.
2020
Cristina Santoriello, Audrey Sporrij, Song Yang, Ryan A. Flynn, Telmo Henriques, Bilguujin Dorjsuren, Eugenia Custo Greig, Wyatt McCall, Meredith E. Stanhope, Maurizio Fazio, Michael Superdock, Asher Lichtig, Isaac Adatto, Brian J. Abraham, Marian Kalocsay, Michael Jurynec, Yi Zhou, Karen Adelman, Eliezer Calo, and Leonard I. Zon. 2020. “RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells.” Nature Cell Biology, 22, 4, Pp. 372–379. Publisher's VersionAbstract
The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.
2019
Jean-Pierre Etchegaray, Lei Zhong, Catherine Li, Telmo Henriques, Eileen Ablondi, Tomoyoshi Nakadai, Capucine Van Rechem, Christina Ferrer, Kenneth N Ross, Jee-Eun Choi, Ann Samarakkody, Fei Ji, Andrew Chang, Ruslan I Sadreyev, Sridhar Ramaswamy, Sergei Nechaev, Johnathan R Whetstine, Robert G Roeder, Karen Adelman, Alon Goren, and Raul Mostoslavsky. 2019. “The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing.” Mol Cell, 75, 4, Pp. 683-699.e7.Abstract
Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.
Erdem Sendinc, David Valle-Garcia, Abhinav Dhall, Hao Chen, Telmo Henriques, Jose Navarrete-Perea, Wanqiang Sheng, Steven P. Gygi, Karen Adelman, and Yang Shi. 2019. “PCIF1 Catalyzes m6Am mRNA Methylation to Regulate Gene Expression.” Molecular Cell. Publisher's VersionAbstract
Summary mRNA modifications play important roles in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5′ ends of mRNAs. Furthermore, PCIF1 catalyzes only 5′ m6Am methylation of capped mRNAs but not internal m6A methylation in vitro and in vivo. To study the biological role of m6Am, we developed a robust methodology (m6Am-Exo-Seq) to map its transcriptome-wide distribution, which revealed no global crosstalk between m6Am and m6A under assayed conditions, suggesting that m6Am is functionally distinct from m6A. Importantly, we find that m6Am does not alter mRNA transcription or stability but negatively impacts cap-dependent translation of methylated mRNAs. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation.