bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2022‒12‒04
24 papers selected by
Connor Rogerson
University of Cambridge
  1. ERK1/2 signalling dynamics promote neural differentiation by regulating chromatin accessibility and the polycomb repressive complex.
  2. Extensive androgen receptor enhancer heterogeneity in primary prostate cancers underlies transcriptional diversity and metastatic potential.
  3. TERRA regulates DNA G-quadruplex formation and ATRX recruitment to chromatin.
  4. Structural basis of RNA polymerase II transcription on the chromatosome containing linker histone H1.
  5. Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression.
  6. Cross-regulome profiling of RNA polymerases highlights the regulatory role of polymerase III on mRNA transcription by maintaining local chromatin architecture.
  7. Single-cell multiomics reveals the complexity of TGFβ signalling to chromatin in iPSC-derived kidney organoids.
  8. Transcription elongator SPT6L regulates the occupancies of the SWI2/SNF2 chromatin remodelers SYD/BRM and nucleosomes at transcription start sites in Arabidopsis.
  9. DNMT3B overexpression downregulates genes with CpG islands, common motifs, and transcription factor binding sites that interact with DNMT3B.
  10. Chromatin accessibility dynamics dictate renal tubular epithelial cell response to injury.
  11. Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.
  12. GILoop: Robust chromatin loop calling across multiple sequencing depths on Hi-C data.
  13. Mutant NPM1 directly regulates oncogenic transcription in acute myeloid leukemia.
  14. Active DNA demethylation of developmental cis-regulatory regions predates vertebrate origins.
  15. Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis.
  16. METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets.
  17. SETD1A regulates transcriptional pause release of heme biosynthesis genes in leukemia.
  18. ARID1A loss induces polymorphonuclear myeloid-derived suppressor cell chemotaxis and promotes prostate cancer progression.
  19. Extensive germline-somatic interplay contributes to prostate cancer progression through HNF1B co-option of TMPRSS2-ERG.
  20. Human histone pre-mRNA assembles histone or canonical mRNA-processing complexes by overlapping 3'-end sequence elements.
  21. Gene activation guided by nascent RNA-bound transcription factors.
  22. Normalization benchmark of ATAC-seq datasets shows the importance of accounting for GC-content effects.
  23. Estrogen receptor α K303R mutation reorganizes its binding to forkhead box protein A1 regions and induces chromatin opening.
  24. A single N6-methyladenosine site regulates lncRNA HOTAIR function in breast cancer cells.
  1. PLoS Biol. 2022 Dec 01. 20(12): e3000221
    ERK1/2 signalling dynamics promote neural differentiation by regulating chromatin accessibility and the polycomb repressive complex.
    Claudia I Semprich, Lindsay Davidson, Adriana Amorim Torres, Harshil Patel, James Briscoe, Vicki Metzis, Kate G Storey.
      Fibroblast growth factor (FGF) is a neural inducer in many vertebrate embryos, but how it regulates chromatin organization to coordinate the activation of neural genes is unclear. Moreover, for differentiation to progress, FGF signalling must decline. Why these signalling dynamics are required has not been determined. Here, we show that dephosphorylation of the FGF effector kinase ERK1/2 rapidly increases chromatin accessibility at neural genes in mouse embryos, and, using ATAC-seq in human embryonic stem cell derived spinal cord precursors, we demonstrate that this occurs genome-wide across neural genes. Importantly, ERK1/2 inhibition induces precocious neural gene transcription, and this involves dissociation of the polycomb repressive complex from key gene loci. This takes place independently of subsequent loss of the repressive histone mark H3K27me3 and transcriptional onset. Transient ERK1/2 inhibition is sufficient for the dissociation of the repressive complex, and this is not reversed on resumption of ERK1/2 signalling. Moreover, genomic footprinting of sites identified by ATAC-seq together with ChIP-seq for polycomb protein Ring1B revealed that ERK1/2 inhibition promotes the occupancy of neural transcription factors (TFs) at non-polycomb as well as polycomb associated sites. Together, these findings indicate that ERK1/2 signalling decline promotes global changes in chromatin accessibility and TF binding at neural genes by directing polycomb and other regulators and appears to serve as a gating mechanism that provides directionality to the process of differentiation.
    DOI:  https://doi.org/10.1371/journal.pbio.3000221
  2. Nat Commun. 2022 Nov 30. 13(1): 7367
    Extensive androgen receptor enhancer heterogeneity in primary prostate cancers underlies transcriptional diversity and metastatic potential.
    Jeroen Kneppers, Tesa M Severson, Joseph C Siefert, Pieter Schol, Stacey E P Joosten, Ivan Pak Lok Yu, Chia-Chi Flora Huang, Tunç Morova, Umut Berkay Altıntaş, Claudia Giambartolomei, Ji-Heui Seo, Sylvan C Baca, Isa Carneiro, Eldon Emberly, Bogdan Pasaniuc, Carmen Jerónimo, Rui Henrique, Matthew L Freedman, Lodewyk F A Wessels, Nathan A Lack, Andries M Bergman, Wilbert Zwart.
      Androgen receptor (AR) drives prostate cancer (PCa) development and progression. AR chromatin binding profiles are highly plastic and form recurrent programmatic changes that differentiate disease stages, subtypes and patient outcomes. While prior studies focused on concordance between patient subgroups, inter-tumor heterogeneity of AR enhancer selectivity remains unexplored. Here we report high levels of AR chromatin binding heterogeneity in human primary prostate tumors, that overlap with heterogeneity observed in healthy prostate epithelium. Such heterogeneity has functional consequences, as somatic mutations converge on commonly-shared AR sites in primary over metastatic tissues. In contrast, less-frequently shared AR sites associate strongly with AR-driven gene expression, while such heterogeneous AR enhancer usage also distinguishes patients' outcome. These findings indicate that epigenetic heterogeneity in primary disease is directly informative for risk of biochemical relapse. Cumulatively, our results illustrate a high level of AR enhancer heterogeneity in primary PCa driving differential expression and clinical impact.
    DOI:  https://doi.org/10.1038/s41467-022-35135-2
  3. Nucleic Acids Res. 2022 Nov 28. pii: gkac1114. [Epub ahead of print]
    TERRA regulates DNA G-quadruplex formation and ATRX recruitment to chromatin.
    Ru-Xuan Tsai, Kuo-Chen Fang, Po-Cheng Yang, Yu-Hung Hsieh, I-Tien Chiang, Yunfei Chen, Hun-Goo Lee, Jeannie T Lee, Hsueh-Ping Catherine Chu.
      The genome consists of non-B-DNA structures such as G-quadruplexes (G4) that are involved in the regulation of genome stability and transcription. Telomeric-repeat containing RNA (TERRA) is capable of folding into G-quadruplex and interacting with chromatin remodeler ATRX. Here we show that TERRA modulates ATRX occupancy on repetitive sequences and over genes, and maintains DNA G-quadruplex structures at TERRA target and non-target sites in mouse embryonic stem cells. TERRA prevents ATRX from binding to subtelomeric regions and represses H3K9me3 formation. G4 ChIP-seq reveals that G4 abundance decreases at accessible chromatin regions, particularly at transcription start sites (TSS) after TERRA depletion; such G4 reduction at TSS is associated with elevated ATRX occupancy and differentially expressed genes. Loss of ATRX alleviates the effect of gene repression caused by TERRA depletion. Immunostaining analyses demonstrate that knockdown of TERRA diminishes DNA G4 signals, whereas silencing ATRX elevates G4 formation. Our results uncover an epigenetic regulation by TERRA that sequesters ATRX and preserves DNA G4 structures.
    DOI:  https://doi.org/10.1093/nar/gkac1114
  4. Nat Commun. 2022 Nov 26. 13(1): 7287
    Structural basis of RNA polymerase II transcription on the chromatosome containing linker histone H1.
    Rina Hirano, Haruhiko Ehara, Tomoya Kujirai, Tamami Uejima, Yoshimasa Takizawa, Shun-Ichi Sekine, Hitoshi Kurumizaka.
      In chromatin, linker histone H1 binds to nucleosomes, forming chromatosomes, and changes the transcription status. However, the mechanism by which RNA polymerase II (RNAPII) transcribes the DNA in the chromatosome has remained enigmatic. Here we report the cryo-electron microscopy (cryo-EM) structures of transcribing RNAPII-chromatosome complexes (forms I and II), in which RNAPII is paused at the entry linker DNA region of the chromatosome due to H1 binding. In the form I complex, the H1 bound to the nucleosome restricts the linker DNA orientation, and the exit linker DNA is captured by the RNAPII DNA binding cleft. In the form II complex, the RNAPII progresses a few bases ahead by releasing the exit linker DNA from the RNAPII cleft, and directly clashes with the H1 bound to the nucleosome. The transcription elongation factor Spt4/5 masks the RNAPII DNA binding region, and drastically reduces the H1-mediated RNAPII pausing.
    DOI:  https://doi.org/10.1038/s41467-022-35003-z
  5. PLoS Genet. 2022 Nov 30. 18(11): e1010528
    Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression.
    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, Eric F Joyce.
      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 ~2,000 genes, with only ~30% 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.
    DOI:  https://doi.org/10.1371/journal.pgen.1010528
  6. Genome Biol. 2022 Nov 28. 23(1): 246
    Cross-regulome profiling of RNA polymerases highlights the regulatory role of polymerase III on mRNA transcription by maintaining local chromatin architecture.
    Yongpeng Jiang, Jie Huang, Kai Tian, Xiao Yi, Haonan Zheng, Yi Zhu, Tiannan Guo, Xiong Ji.
      BACKGROUND: Mammalian cells have three types of RNA polymerases (Pols), Pol I, II, and III. However, the extent to which these polymerases are cross-regulated and the underlying mechanisms remain unclear.RESULTS: We employ genome-wide profiling after acute depletion of Pol I, Pol II, or Pol III to assess cross-regulatory effects between these Pols. We find that these enzymes mainly affect the transcription of their own target genes, while certain genes are transcribed by the other polymerases. Importantly, the most active type of crosstalk is exemplified by the fact that Pol III depletion affects Pol II transcription. Pol II genes with transcription changes upon Pol III depletion are enriched in diverse cellular functions, and Pol III binding sites are found near their promoters. However, these Pol III binding sites do not correspond to transfer RNAs. Moreover, we demonstrate that Pol III regulates Pol II transcription and chromatin binding of the facilitates chromatin transcription (FACT) complex to alter local chromatin structures, which in turn affects the Pol II transcription rate.
    CONCLUSIONS: Our results support a model suggesting that RNA polymerases show cross-regulatory effects: Pol III affects local chromatin structures and the FACT-Pol II axis to regulate the Pol II transcription rate at certain gene loci. This study provides a new perspective for understanding the dysregulation of Pol III in various tissues affected by developmental diseases.
    Keywords:  FACT complex; Local chromatin structure; RNA polymerases II and III; Transcription rate
    DOI:  https://doi.org/10.1186/s13059-022-02812-w
  7. Commun Biol. 2022 Nov 27. 5(1): 1301
    Single-cell multiomics reveals the complexity of TGFβ signalling to chromatin in iPSC-derived kidney organoids.
    Jessica L Davis, Ciaran Kennedy, Shane Clerkin, Niall J Treacy, Thomas Dodd, Catherine Moss, Alison Murphy, Derek P Brazil, Gerard Cagney, Dermot F Brougham, Rabi Murad, Darren Finlay, Kristiina Vuori, John Crean.
      TGFβ1 plays a regulatory role in the determination of renal cell fate and the progression of renal fibrosis. Here we show an association between SMAD3 and the histone methyltransferase, EZH2, during cell differentiation; ChIP-seq revealed that SMAD3 and EZH2 co-occupy the genome in iPSCs and in iPSC-derived nephron progenitors. Through integration of single cell gene expression and epigenome profiling, we identified de novo ACTA2+ve/POSTN+ve myofibroblasts in kidney organoids treated with TGFβ1, characterised by increased SMAD3-dependent cis chromatin accessibility and gene expression associated with fibroblast activation. We have identified fibrosis-associated regulons characterised by enrichment of SMAD3, AP1, the ETS family of transcription factors, and NUAK1, CREB3L1, and RARG, corresponding to enriched motifs at accessible loci identified by scATACseq. Treatment with the EZH2 specific inhibitor GSK343, blocked SMAD3-dependent cis co-accessibility and inhibited myofibroblast activation. This mechanism, through which TGFβ signals directly to chromatin, represents a critical determinant of fibrotic, differentiated states.
    DOI:  https://doi.org/10.1038/s42003-022-04264-1
  8. Nucleic Acids Res. 2022 Dec 01. pii: gkac1126. [Epub ahead of print]
    Transcription elongator SPT6L regulates the occupancies of the SWI2/SNF2 chromatin remodelers SYD/BRM and nucleosomes at transcription start sites in Arabidopsis.
    Jie Shu, Ning Ding, Jun Liu, Yuhai Cui, Chen Chen.
      Chromatin remodelers have been thought to be crucial in creating an accessible chromatin environment before transcription activation. However, it is still unclear how chromatin remodelers recognize and bind to the active regions. In this study, we found that chromatin remodelers SPLAYED (SYD) and BRAHMA (BRM) interact and co-occupy with Suppressor of Ty6-like (SPT6L), a core subunit of the transcription machinery, at thousands of the transcription start sites (TSS). The association of SYD and BRM to chromatin is dramatically reduced in spt6l and can be restored mainly by SPT6LΔtSH2, which binds to TSS in a RNA polymerase II (Pol II)-independent manner. Furthermore, SPT6L and SYD/BRM are involved in regulating the nucleosome and Pol II occupancy around TSS. The presence of SPT6L is sufficient to restore the association of the chromatin remodeler SYD to chromatin and maintain normal nucleosome occupancy. Our findings suggest that the two chromatin remodelers can form protein complexes with the core subunit of the transcription machinery and regulate nucleosome occupancy in the early transcription stage.
    DOI:  https://doi.org/10.1093/nar/gkac1126
  9. Sci Rep. 2022 Dec 02. 12(1): 20839
    DNMT3B overexpression downregulates genes with CpG islands, common motifs, and transcription factor binding sites that interact with DNMT3B.
    Jaqueline Loaeza-Loaeza, Angel Josué Cerecedo-Castillo, Hugo Alberto Rodríguez-Ruiz, Yaneth Castro-Coronel, Oscar Del Moral-Hernández, Félix Recillas-Targa, Daniel Hernández-Sotelo.
      DNA methylation is a key epigenetic modification to regulate gene expression in mammalian cells. Abnormal DNA methylation in gene promoters is common across human cancer types. DNMT3B is the main de novo methyltransferase enhanced in several primary tumors. How de novo methylation is established in genes related to cancer is poorly understood. CpG islands (CGIs), common sequences, and transcription factors (TFs) that interact with DNMT3B have been associated with abnormal de novo methylation. We initially identified cis elements associated with DNA methylation to investigate the contribution of DNMT3B overexpression to the deregulation of its possible target genes in an epithelial cell model. In a set of downregulated genes (n = 146) from HaCaT cells with DNMT3B overexpression, we found CGI, common sequences, and TFs Binding Sites that interact with DNMT3B (we called them P-down-3B). PPL1, VAV3, IRF1, and BRAF are P-down-3B genes that are downregulated and increased their methylation in DNMT3B presence. Together these findings suggest that methylated promoters aberrantly have some cis elements that could conduce de novo methylation by DNMT3B.
    DOI:  https://doi.org/10.1038/s41598-022-24186-6
  10. Nat Commun. 2022 Nov 28. 13(1): 7322
    Chromatin accessibility dynamics dictate renal tubular epithelial cell response to injury.
    Xinyi Cao, Jiuchen Wang, Tianye Zhang, Zhiheng Liu, Lijun Liu, Ying Chen, Zehua Li, Youlu Zhao, Qi Yu, Tong Liu, Jing Nie, Yuanjie Niu, Yupeng Chen, Li Yang, Lirong Zhang.
      Renal tubular epithelial cells (TECs) can initiate an adaptive response to completely recover from mild acute kidney injury (AKI), whereas severe injury often leads to persistence of maladaptive repair and progression to kidney fibrosis. Through profiling of active DNA regulatory elements by ATAC-seq, we reveal widespread, dynamic changes in the chromatin accessibility of TECs after ischemia-reperfusion injury. We show that injury-specific domains of regulatory chromatin become accessible prior to gene activation, creating poised chromatin states to activate the consequent gene expression program and injury response. We further identify RXRα as a key transcription factor in promoting adaptive repair. Activation of RXRα by bexarotene, an FDA-approved RXRα agonist, restores the chromatin state and gene expression program to protect TECs against severe kidney injury. Together, our findings elucidate a chromatin-mediated mechanism underlying differential responses of TECs to varying injuries and identify RXRα as a therapeutic target of acute kidney injury.
    DOI:  https://doi.org/10.1038/s41467-022-34854-w
  11. Cell Rep. 2022 Nov 29. pii: S2211-1247(22)01608-4. [Epub ahead of print]41(9): 111730
    Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.
    Isabel N Goronzy, Sofia A Quinodoz, Joanna W Jachowicz, Noah Ollikainen, Prashant Bhat, Mitchell Guttman.
      Mammalian genomes are organized into three-dimensional DNA structures called A/B compartments that are associated with transcriptional activity/inactivity. However, whether these structures are simply correlated with gene expression or are permissive/impermissible to transcription has remained largely unknown because we lack methods to measure DNA organization and transcription simultaneously. Recently, we developed RNA & DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization of RNA and DNA. Here we show that RD-SPRITE measures genomic structure surrounding nascent pre-mRNAs and maps their spatial contacts. We find that transcription occurs within B compartments-with multiple active genes simultaneously colocalizing within the same B compartment-and at genes proximal to nucleoli. These results suggest that localization near or within nuclear structures thought to be inactive does not preclude transcription and that active transcription can occur throughout the nucleus. In general, we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure and transcription.
    Keywords:  AB compartments; CP: Molecular biology; RNA polymerase II transcription; nuclear structure; nucleolus
    DOI:  https://doi.org/10.1016/j.celrep.2022.111730
  12. iScience. 2022 Dec 22. 25(12): 105535
    GILoop: Robust chromatin loop calling across multiple sequencing depths on Hi-C data.
    Fuzhou Wang, Tingxiao Gao, Jiecong Lin, Zetian Zheng, Lei Huang, Muhammad Toseef, Xiangtao Li, Ka-Chun Wong.
      Graph and image are two common representations of Hi-C cis-contact maps. Existing computational tools have only adopted Hi-C data modeled as unitary data structures but neglected the potential advantages of synergizing the information of different views. Here we propose GILoop, a dual-branch neural network that learns from both representations to identify genome-wide CTCF-mediated loops. With GILoop, we explore the combined strength of integrating the two view representations of Hi-C data and corroborate the complementary relationship between the views. In particular, the model outperforms the state-of-the-art loop calling framework and is also more robust against low-quality Hi-C libraries. We also uncover distinct preferences for matrix density by graph-based and image-based models, revealing interesting insights into Hi-C data elucidation. Finally, along with multiple transfer-learning case studies, we demonstrate that GILoop can accurately model the organizational and functional patterns of CTCF-mediated looping across different cell lines.
    Keywords:  Computational bioinformatics; Genomic analysis; Neural networks
    DOI:  https://doi.org/10.1016/j.isci.2022.105535
  13. Cancer Discov. 2022 Dec 01. pii: CD-22-0366. [Epub ahead of print]
    Mutant NPM1 directly regulates oncogenic transcription in acute myeloid leukemia.
    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, Scott A Armstrong.
      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.
    DOI:  https://doi.org/10.1158/2159-8290.CD-22-0366
  14. Sci Adv. 2022 Dec 02. 8(48): eabn2258
    Active DNA demethylation of developmental cis-regulatory regions predates vertebrate origins.
    Ksenia Skvortsova, Stephanie Bertrand, Danila Voronov, Paul E Duckett, Samuel E Ross, Marta Silvia Magri, Ignacio Maeso, Robert J Weatheritt, Jose Luis Gómez Skarmeta, Maria Ina Arnone, Hector Escriva, Ozren Bogdanovic.
      DNA methylation [5-methylcytosine (5mC)] is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the action of DNA methyltransferases, which deposit 5mC, and ten-eleven translocation (TET) enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers; however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in nonvertebrate lineages is lacking. By using base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of nonvertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes use TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of an unexpected deep conservation of a major gene-regulatory module.
    DOI:  https://doi.org/10.1126/sciadv.abn2258
  15. Elife. 2022 Dec 02. pii: e78342. [Epub ahead of print]11
    Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis.
    Xiaodong Li, Patrick J Gordon, John A Gaynes, Alexandra W Fuller, Randy Ringuette, Clayton P Santiago, Valerie Wallace, Seth Blackshaw, Pulin Li, Edward M Levine.
      An important question in organogenesis is how tissue-specific transcription factors interact with signaling pathways. In some cases, transcription factors define the context for how signaling pathways elicit tissue- or cell-specific responses, and in others, they influence signaling through transcriptional regulation of signaling components or accessory factors. We previously showed that during optic vesicle patterning, the Lim-homeodomain transcription factor Lhx2 has a contextual role by linking the Sonic Hedgehog (Shh) pathway to downstream targets without regulating the pathway itself. Here, we show that during early retinal neurogenesis in mice, Lhx2 is a multilevel regulator of Shh signaling. Specifically, Lhx2 acts cell autonomously to control the expression of pathway genes required for efficient activation and maintenance of signaling in retinal progenitor cells. The Shh co-receptors Cdon and Gas1 are candidate direct targets of Lhx2 that mediate pathway activation, whereas Lhx2 directly or indirectly promotes the expression of other pathway components important for activation and sustained signaling. We also provide genetic evidence suggesting that Lhx2 has a contextual role by linking the Shh pathway to downstream targets. Through these interactions, Lhx2 establishes the competence for Shh signaling in retinal progenitors and the context for the pathway to promote early retinal neurogenesis. The temporally distinct interactions between Lhx2 and the Shh pathway in retinal development illustrate how transcription factors and signaling pathways adapt to meet stage-dependent requirements of tissue formation.
    Keywords:  Lhx2; Sonic Hedgehog; development; developmental biology; genetics; genomics; mouse; retina; signaling
    DOI:  https://doi.org/10.7554/eLife.78342
  16. Genome Biol. 2022 Dec 02. 23(1): 249
    METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets.
    Kai-Wen Hsu, Joseph Chieh-Yu Lai, Jeng-Shou Chang, Pei-Hua Peng, Ching-Hui Huang, Der-Yen Lee, Yu-Cheng Tsai, Chi-Jung Chung, Han Chang, Chao-Hsiang Chang, Ji-Lin Chen, See-Tong Pang, Ziyang Hao, Xiao-Long Cui, Chuan He, Kou-Juey Wu.
      BACKGROUND: DNA N6-methyldeoxyadenosine (6mA) is rarely present in mammalian cells and its nuclear role remains elusive.RESULTS: Here we show that hypoxia induces nuclear 6mA modification through a DNA methyltransferase, METTL4, in hypoxia-induced epithelial-mesenchymal transition (EMT) and tumor metastasis. Co-expression of METTL4 and 6mA represents a prognosis marker for upper tract urothelial cancer patients. By RNA sequencing and 6mA chromatin immunoprecipitation-exonuclease digestion followed by sequencing, we identify lncRNA RP11-390F4.3 and one novel HIF-1α co-activator, ZMIZ1, that are co-regulated by hypoxia and METTL4. Other genes involved in hypoxia-mediated phenotypes are also regulated by 6mA modification. Quantitative chromatin isolation by RNA purification assay shows the occupancy of lncRNA RP11-390F4.3 on the promoters of multiple EMT regulators, indicating lncRNA-chromatin interaction. Knockdown of lncRNA RP11-390F4.3 abolishes METTL4-mediated tumor metastasis. We demonstrate that ZMIZ1 is an essential co-activator of HIF-1α.
    CONCLUSIONS: We show that hypoxia results in enriched 6mA levels in mammalian tumor cells through METTL4. This METTL4-mediated nuclear 6mA deposition induces tumor metastasis through activating multiple metastasis-inducing genes. METTL4 is characterized as a potential therapeutic target in hypoxic tumors.
    Keywords:  6mA; Hypoxia; METTL4; Metastasis; ZMIZ1; lncRNA
    DOI:  https://doi.org/10.1186/s13059-022-02819-3
  17. Cell Rep. 2022 Nov 29. pii: S2211-1247(22)01605-9. [Epub ahead of print]41(9): 111727
    SETD1A regulates transcriptional pause release of heme biosynthesis genes in leukemia.
    Takayuki Hoshii, Sarah Perlee, Sota Kikuchi, Bahityar Rahmutulla, Masaki Fukuyo, Takeshi Masuda, Sumio Ohtsuki, Tomoyoshi Soga, Behnam Nabet, Atsushi Kaneda.
      Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAPII) at the transcriptional start site (TSS), and induces the promoter-proximal pausing of RNAPII in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAPII pausing and its function in cancer.
    Keywords:  CP: Molecular biology; SETD1A; cyclin K; elongation; heme biosynthesis; leukemia
    DOI:  https://doi.org/10.1016/j.celrep.2022.111727
  18. Nat Commun. 2022 Nov 26. 13(1): 7281
    ARID1A loss induces polymorphonuclear myeloid-derived suppressor cell chemotaxis and promotes prostate cancer progression.
    Ni Li, Qiuli Liu, Ying Han, Siyu Pei, Bisheng Cheng, Junyu Xu, Xiang Miao, Qiang Pan, Hanling Wang, Jiacheng Guo, Xuege Wang, Guoying Zhang, Yannan Lian, Wei Zhang, Yi Zang, Minjia Tan, Qintong Li, Xiaoming Wang, Yichuan Xiao, Guohong Hu, Jun Jiang, Hai Huang, Jun Qin.
      Chronic inflammation and an immunosuppressive microenvironment promote prostate cancer (PCa) progression and diminish the response to immune checkpoint blockade (ICB) therapies. However, it remains unclear how and to what extent these two events are coordinated. Here, we show that ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, functions downstream of inflammation-induced IKKβ activation to shape the immunosuppressive tumor microenvironment (TME). Prostate-specific deletion of Arid1a cooperates with Pten loss to accelerate prostate tumorigenesis. We identify polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) as the major infiltrating immune cell type that causes immune evasion and reveal that neutralization of PMN-MDSCs restricts the progression of Arid1a-deficient tumors. Mechanistically, inflammatory cues activate IKKβ to phosphorylate ARID1A, leading to its degradation via β-TRCP. ARID1A downregulation in turn silences the enhancer of A20 deubiquitinase, a critical negative regulator of NF-κB signaling, and thereby unleashes CXCR2 ligand-mediated MDSC chemotaxis. Importantly, our results support the therapeutic strategy of anti-NF-κB antibody or targeting CXCR2 combined with ICB for advanced PCa. Together, our findings highlight that the IKKβ/ARID1A/NF-κB feedback axis integrates inflammation and immunosuppression to promote PCa progression.
    DOI:  https://doi.org/10.1038/s41467-022-34871-9
  19. Nat Commun. 2022 Nov 28. 13(1): 7320
    Extensive germline-somatic interplay contributes to prostate cancer progression through HNF1B co-option of TMPRSS2-ERG.
    Nikolaos Giannareas, Qin Zhang, Xiayun Yang, Rong Na, Yijun Tian, Yuehong Yang, Xiaohao Ruan, Da Huang, Xiaoqun Yang, Chaofu Wang, Peng Zhang, Aki Manninen, Liang Wang, Gong-Hong Wei.
      Genome-wide association studies have identified 270 loci conferring risk for prostate cancer (PCa), yet the underlying biology and clinical impact remain to be investigated. Here we observe an enrichment of transcription factor genes including HNF1B within PCa risk-associated regions. While focused on the 17q12/HNF1B locus, we find a strong eQTL for HNF1B and multiple potential causal variants involved in the regulation of HNF1B expression in PCa. An unbiased genome-wide co-expression analysis reveals PCa-specific somatic TMPRSS2-ERG fusion as a transcriptional mediator of this locus and the HNF1B eQTL signal is ERG fusion status dependent. We investigate the role of HNF1B and find its involvement in several pathways related to cell cycle progression and PCa severity. Furthermore, HNF1B interacts with TMPRSS2-ERG to co-occupy large proportion of genomic regions with a remarkable enrichment of additional PCa risk alleles. We finally show that HNF1B co-opts ERG fusion to mediate mechanistic and biological effects of the PCa risk-associated locus 17p13.3/VPS53/FAM57A/GEMIN4. Taken together, we report an extensive germline-somatic interaction between TMPRSS2-ERG fusion and genetic variations underpinning PCa risk association and progression.
    DOI:  https://doi.org/10.1038/s41467-022-34994-z
  20. Nucleic Acids Res. 2022 Nov 30. pii: gkac878. [Epub ahead of print]
    Human histone pre-mRNA assembles histone or canonical mRNA-processing complexes by overlapping 3'-end sequence elements.
    Francesco S Ielasi, Sara Ternifi, Emeline Fontaine, Domenico Iuso, Yohann Couté, Andrés Palencia.
      Human pre-mRNA processing relies on multi-subunit macromolecular complexes, which recognize specific RNA sequence elements essential for assembly and activity. Canonical pre-mRNA processing proceeds via the recognition of a polyadenylation signal (PAS) and a downstream sequence element (DSE), and produces polyadenylated mature mRNAs, while replication-dependent (RD) histone pre-mRNA processing requires association with a stem-loop (SL) motif and a histone downstream element (HDE), and produces cleaved but non-polyadenylated mature mRNAs. H2AC18 mRNA, a specific H2A RD histone pre-mRNA, can be processed to give either a non-polyadenylated mRNA, ending at the histone SL, or a polyadenylated mRNA. Here, we reveal how H2AC18 captures the two human pre-mRNA processing complexes in a mutually exclusive mode by overlapping a canonical PAS (AAUAAA) sequence element with a HDE. Disruption of the PAS sequence on H2AC18 pre-mRNA prevents recruitment of the canonical complex in vitro, without affecting the histone machinery. This shows how the relative position of cis-acting elements in histone pre-mRNAs allows the selective recruitment of distinct human pre-mRNA complexes, thereby expanding the capability to regulate 3' processing and polyadenylation.
    DOI:  https://doi.org/10.1093/nar/gkac878
  21. Nat Commun. 2022 Nov 28. 13(1): 7329
    Gene activation guided by nascent RNA-bound transcription factors.
    Ying Liang, Haiyue Xu, Tao Cheng, Yujuan Fu, Hanwei Huang, Wenchang Qian, Junyan Wang, Yuenan Zhou, Pengxu Qian, Yafei Yin, Pengfei Xu, Wei Zou, Baohui Chen.
      Technologies for gene activation are valuable tools for the study of gene functions and have a wide range of potential applications in bioengineering and medicine. In contrast to existing methods based on recruiting transcriptional modulators via DNA-binding proteins, we developed a strategy termed Narta (nascent RNA-guided transcriptional activation) to achieve gene activation by recruiting artificial transcription factors (aTFs) to transcription sites through nascent RNAs of the target gene. Using Narta, we demonstrate robust activation of a broad range of exogenous and endogenous genes in various cell types, including zebrafish embryos, mouse and human cells. Importantly, the activation is reversible, tunable and specific. Moreover, Narta provides better activation potency of some expressed genes than CRISPRa and, when used in combination with CRISPRa, has an enhancing effect on gene activation. Quantitative imaging illustrated that nascent RNA-directed aTFs could induce the high-density assembly of coactivators at transcription sites, which may explain the larger transcriptional burst size induced by Narta. Overall, our work expands the gene activation toolbox for biomedical research.
    DOI:  https://doi.org/10.1038/s41467-022-35041-7
  22. Cell Rep Methods. 2022 Nov 21. 2(11): 100321
    Normalization benchmark of ATAC-seq datasets shows the importance of accounting for GC-content effects.
    Koen Van den Berge, Hsin-Jung Chou, Hector Roux de Bézieux, Kelly Street, Davide Risso, John Ngai, Sandrine Dudoit.
      The assay for transposase-accessible chromatin using sequencing (ATAC-seq) allows the study of epigenetic regulation of gene expression by assessing chromatin configuration for an entire genome. Despite its popularity, there have been limited studies investigating the analytical challenges related to ATAC-seq data, with most studies leveraging tools developed for bulk transcriptome sequencing. Here, we show that GC-content effects are omnipresent in ATAC-seq datasets. Since the GC-content effects are sample specific, they can bias downstream analyses such as clustering and differential accessibility analysis. We introduce a normalization method based on smooth-quantile normalization within GC-content bins and evaluate it together with 11 different normalization procedures on 8 public ATAC-seq datasets. Accounting for GC-content effects in the normalization is crucial for common downstream ATAC-seq data analyses, improving accuracy and interpretability. Through case studies, we show that exploratory data analysis is essential to guide the choice of an appropriate normalization method for a given dataset.
    Keywords:  ATAC-seq; GC-content; clustering; differential accessibility; epigenetics; normalization
    DOI:  https://doi.org/10.1016/j.crmeth.2022.100321
  23. Mol Biol Rep. 2022 Nov 27.
    Estrogen receptor α K303R mutation reorganizes its binding to forkhead box protein A1 regions and induces chromatin opening.
    Tomoyoshi Nakadai, Liying Yang, Kohei Kumegawa, Reo Maruyama.
      BACKGROUND: Estrogen receptor alpha (ERα) is a frequently mutated gene in breast cancer (BC). While many studies have investigated molecular dysregulation by hotspot mutations at Y537 and D538, which exhibit an estrogen-independent constitutively active phenotype, the functional abnormalities of other mutations remain obscure. The K303R mutation in primary invasive BC has been implicated with endocrine resistance, tumor size, and lymph node positivity. However, the impact of the K303R mutation on the cell epigenome is yet unknown.METHODS AND RESULTS: We introduced the K303R ERα mutant in ERα-negative MDA-MB-453 cells to monitor ERα-dependent transactivation and to perform epigenomic analyses. ATAC-seq and ChIP-Seq analyses indicated that both wild-type (WT) and the K303R mutant associated with Forkhead box (Fox) protein family motif regions at similar rates, even without an ERα-binding sequence, but only the K303R mutant induced chromatin opening at those regions. Biochemical analyses demonstrated that the WT and the K303R mutant can be tethered on DNA by FoxA1 indirectly, but only the K303R/FoxA1/DNA complex can induce associations with the nuclear receptor cofactor 2 (NCOA2).
    CONCLUSIONS: These findings suggest that the K303R mutant induces chromatin opening at the Fox binding region through the FoxA1-dependent associations of the K303R mutant to NCOA2 and then probably disrupts the regulation of Fox-target genes, resulting in K303R-related BC events.
    Keywords:  Breast cancer; ERα; FoxA1; K303R; MDA-MB-453; NCOA2
    DOI:  https://doi.org/10.1007/s11033-022-08089-3
  24. PLoS Biol. 2022 Nov 28. 20(11): e3001885
    A single N6-methyladenosine site regulates lncRNA HOTAIR function in breast cancer cells.
    Allison M Porman, Justin T Roberts, Emily D Duncan, Madeline L Chrupcala, Ariel A Levine, Michelle A Kennedy, Michelle M Williams, Jennifer K Richer, Aaron M Johnson.
      N6-methyladenosine (m6A) modification of RNA regulates normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we reveal that m6A regulates the breast cancer-associated human lncRNA HOTAIR. Mapping m6A in breast cancer cell lines, we identify multiple m6A sites on HOTAIR, with 1 single consistently methylated site (A783) that is critical for HOTAIR-driven proliferation and invasion of triple-negative breast cancer (TNBC) cells. Methylated A783 interacts with the m6A "reader" YTHDC1, promoting chromatin association of HOTAIR, proliferation and invasion of TNBC cells, and gene repression. A783U mutant HOTAIR induces a unique antitumor gene expression profile and displays loss-of-function and antimorph behaviors by impairing and, in some cases, causing opposite gene expression changes induced by wild-type (WT) HOTAIR. Our work demonstrates how modification of 1 base in an lncRNA can elicit a distinct gene regulation mechanism and drive cancer-associated phenotypes.
    DOI:  https://doi.org/10.1371/journal.pbio.3001885
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