bims-crepig 2021-08-29 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2021–08–29
thirty-six papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit

Acute depletion of CTCF rewires genome-wide chromatin accessibility.
SnapHiC: a computational pipeline to identify chromatin loops from single-cell Hi-C data.
CTCF and transcription influence chromatin structure re-configuration after mitosis.
The Polycomb protein RING1B enables estrogen-mediated gene expression by promoting enhancer-promoter interaction and R-loop formation.
Chromatin landscape signals differentially dictate the activities of mSWI/SNF family complexes.
HDAC1 is required for GATA-1 transcription activity, global chromatin occupancy and hematopoiesis.
Interrogation of the dynamic properties of higher-order heterochromatin using CRISPR-dCas9.
Full methylation of H3K27 by PRC2 is dispensable for initial embryoid body formation but required to maintain differentiated cell identity.
TooManyPeaks identifies drug-resistant-specific regulatory elements from single-cell leukemic epigenomes.
Complete lung agenesis caused by complex genomic rearrangements with neo-TAD formation at the SHH locus.
Ten-eleven translocation 1 mediated-DNA hydroxymethylation is required for myelination and remyelination in the mouse brain.
OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human.
NucHMM: a method for quantitative modeling of nucleosome organization identifying functional nucleosome states distinctly associated with splicing potentiality.
Pharmacological Inhibition of Core Regulatory Circuitry Liquid-liquid Phase Separation Suppresses Metastasis and Chemoresistance in Osteosarcoma.
Histone H3K27 methylation-mediated repression of Hairy regulates insect developmental transition by modulating ecdysone biosynthesis.
A TALE/HOX code unlocks WNT signalling response towards paraxial mesoderm.
Combinations of maternal-specific repressive epigenetic marks in the endosperm control seed dormancy.
A dual role for DNA-binding by Runt in activation and repression of sloppy paired transcription.
mTORC1 Promotes ARID1A Degradation and Oncogenic Chromatin Remodeling in Hepatocellular Carcinoma.
CHD4 ensures stem cell lineage fidelity during skeletal muscle regeneration.
Unraveling the functions of uncharacterized transcription factors in Escherichia coli using ChIP-exo.
The tumor suppressor WT1 drives progenitor cell progression and epithelialization to prevent Wilms tumorigenesis in human kidney organoids.
Histone citrullination by PADI4 is required for HIF-dependent transcriptional responses to hypoxia and tumor vascularization.
An epigenetic basis of inbreeding depression in maize.
Combinatorial transcription factor profiles predict mature and functional human islet α and β cells.
Single-cell measurement of higher-order 3D genome organization with scSPRITE.
Short- and long-range cis interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis.
The transcription factor ZEB1 regulates stem cell self-renewal and cell fate in the adult hippocampus.
The related coactivator complexes SAGA and ATAC control embryonic stem cell self-renewal through acetyltransferase-independent mechanisms.
A lineage-specific requirement for YY1 Polycomb Group protein function in early T cell development.
Diverse transcriptional regulation and functional effects revealed by CRISPR/Cas9-directed epigenetic editing.
METTL14 facilitates global genome repair and suppresses skin tumorigenesis.
Epigenomic and transcriptional profiling identifies impaired glyoxylate detoxification in NAFLD as a risk factor for hyperoxaluria.
Lineage-specific control of convergent differentiation by a Forkhead repressor.
Bromodomain containing 9 (BRD9) regulates macrophage inflammatory responses by potentiating glucocorticoid receptor activity.
Identification of a KLF5-dependent program and drug development for skeletal muscle atrophy.

Genome Biol. 2021 Aug 24. 22(1): 244

Acute depletion of CTCF rewires genome-wide chromatin accessibility.

Beisi Xu, Hong Wang, Shaela Wright, Judith Hyle, Yang Zhang, Ying Shao, Mingming Niu, Yiping Fan, Wojciech Rosikiewicz, Mohamed Nadhir Djekidel, Junmin Peng, Rui Lu, Chunliang Li.

   BACKGROUND: The transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription.
RESULTS: Here, we systematically integrate multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, and CRISPR-Cas9 survival dropout screens, and time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewires genome-wide chromatin accessibility. Increased accessible chromatin regions are frequently located adjacent to CTCF-binding sites at promoter regions and insulator sites associated with enhanced transcription of nearby genes. In addition, we use CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners. We successfully identify 40 candidates, including multiple established partners. Interestingly, many CTCF co-regulators that have alterations of their respective downstream gene expression do not show changes of their own expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription.
CONCLUSIONS: This study highlights that CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation.

Keywords:  ATAC-seq; Auxin-induced degron; CTCF; Chromatin accessibility; Phosphoproteomics; Proteomics; Transcription factor

DOI:  https://doi.org/10.1186/s13059-021-02466-0
Nat Methods. 2021 Aug 26.

SnapHiC: a computational pipeline to identify chromatin loops from single-cell Hi-C data.

Miao Yu, Armen Abnousi, Yanxiao Zhang, Guoqiang Li, Lindsay Lee, Ziyin Chen, Rongxin Fang, Taylor M Lagler, Yuchen Yang, Jia Wen, Quan Sun, Yun Li, Bing Ren, Ming Hu.

Single-cell Hi-C (scHi-C) analysis has been increasingly used to map chromatin architecture in diverse tissue contexts, but computational tools to define chromatin loops at high resolution from scHi-C data are still lacking. Here, we describe Single-Nucleus Analysis Pipeline for Hi-C (SnapHiC), a method that can identify chromatin loops at high resolution and accuracy from scHi-C data. Using scHi-C data from 742 mouse embryonic stem cells, we benchmark SnapHiC against a number of computational tools developed for mapping chromatin loops and interactions from bulk Hi-C. We further demonstrate its use by analyzing single-nucleus methyl-3C-seq data from 2,869 human prefrontal cortical cells, which uncovers cell type-specific chromatin loops and predicts putative target genes for noncoding sequence variants associated with neuropsychiatric disorders. Our results indicate that SnapHiC could facilitate the analysis of cell type-specific chromatin architecture and gene regulatory programs in complex tissues.

DOI: https://doi.org/10.1038/s41592-021-01231-2
Nat Commun. 2021 Aug 27. 12(1): 5157

CTCF and transcription influence chromatin structure re-configuration after mitosis.

Haoyue Zhang, Jessica Lam, Di Zhang, Yemin Lan, Marit W Vermunt, Cheryl A Keller, Belinda Giardine, Ross C Hardison, Gerd A Blobel.

During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. We exploited the M- to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to genome re-sculpting in newborn nuclei. Depletion of CTCF during the M- to G1-phase transition alters short-range compartmentalization after mitosis. Chromatin domain boundary re-formation is impaired upon CTCF loss, but a subset of boundaries, characterized by transitions in chromatin states, is established normally. Without CTCF, structural loops fail to form, leading to illegitimate contacts between cis-regulatory elements (CREs). Transient CRE contacts that are normally resolved after telophase persist deeply into G1-phase in CTCF-depleted cells. CTCF loss-associated gains in transcription are often linked to increased, normally illegitimate enhancer-promoter contacts. In contrast, at genes whose expression declines upon CTCF loss, CTCF seems to function as a conventional transcription activator, independent of its architectural role. CTCF-anchored structural loops facilitate formation of CRE loops nested within them, especially those involving weak CREs. Transcription inhibition does not significantly affect global architecture or transcription start site-associated boundaries. However, ongoing transcription contributes considerably to the formation of gene domains, regions of enriched contacts along gene bodies. Notably, gene domains emerge in ana/telophase prior to completion of the first round of transcription, suggesting that epigenetic features in gene bodies contribute to genome reconfiguration prior to transcription. The focus on the de novo formation of nuclear architecture during G1 entry yields insights into the contributions of CTCF and transcription to chromatin architecture dynamics during the mitosis to G1-phase progression.

DOI: https://doi.org/10.1038/s41467-021-25418-5
Nucleic Acids Res. 2021 Aug 24. pii: gkab723. [Epub ahead of print]

The Polycomb protein RING1B enables estrogen-mediated gene expression by promoting enhancer-promoter interaction and R-loop formation.

Yusheng Zhang, Tong Liu, Fenghua Yuan, Liliana Garcia-Martinez, Kyutae D Lee, Stephanie Stransky, Simone Sidoli, Ramiro E Verdun, Yanbin Zhang, Zheng Wang, Lluis Morey.

Polycomb complexes have traditionally been prescribed roles as transcriptional repressors, though increasing evidence demonstrate they can also activate gene expression. However, the mechanisms underlying positive gene regulation mediated by Polycomb proteins are poorly understood. Here, we show that RING1B, a core component of Polycomb Repressive Complex 1, regulates enhancer-promoter interaction of the bona fide estrogen-activated GREB1 gene. Systematic characterization of RNA:DNA hybrid formation (R-loops), nascent transcription and RNA Pol II activity upon estrogen administration revealed a key role of RING1B in gene activation by regulating R-loop formation and RNA Pol II elongation. We also found that the estrogen receptor alpha (ERα) and RNA are both necessary for full RING1B recruitment to estrogen-activated genes. Notably, RING1B recruitment was mostly unaffected upon RNA Pol II depletion. Our findings delineate the functional interplay between RING1B, RNA and ERα to safeguard chromatin architecture perturbations required for estrogen-mediated gene regulation and highlight the crosstalk between steroid hormones and Polycomb proteins to regulate oncogenic programs.

DOI: https://doi.org/10.1093/nar/gkab723
Science. 2021 Jul 16. 373(6552): 306-315

Chromatin landscape signals differentially dictate the activities of mSWI/SNF family complexes.

Nazar Mashtalir, Hai T Dao, Akshay Sankar, Hengyuan Liu, Aaron J Corin, John D Bagert, Eva J Ge, Andrew R D'Avino, Martin Filipovski, Brittany C Michel, Geoffrey P Dann, Tom W Muir, Cigall Kadoch.

Mammalian SWI/SNF (mSWI/SNF) adenosine triphosphate-dependent chromatin remodelers modulate genomic architecture and gene expression and are frequently mutated in disease. However, the specific chromatin features that govern their nucleosome binding and remodeling activities remain unknown. We subjected endogenously purified mSWI/SNF complexes and their constituent assembly modules to a diverse library of DNA-barcoded mononucleosomes, performing more than 25,000 binding and remodeling measurements. Here, we define histone modification-, variant-, and mutation-specific effects, alone and in combination, on mSWI/SNF activities and chromatin interactions. Further, we identify the combinatorial contributions of complex module components, reader domains, and nucleosome engagement properties to the localization of complexes to selectively permissive chromatin states. These findings uncover principles that shape the genomic binding and activity of a major chromatin remodeler complex family.

DOI: https://doi.org/10.1126/science.abf8705
Nucleic Acids Res. 2021 Aug 27. pii: gkab737. [Epub ahead of print]

HDAC1 is required for GATA-1 transcription activity, global chromatin occupancy and hematopoiesis.

Bowen Yan, Jennifer Yang, Min Young Kim, Huacheng Luo, Nicholas Cesari, Tao Yang, John Strouboulis, Jiwang Zhang, Ross Hardison, Suming Huang, Yi Qiu.

The activity of hematopoietic factor GATA-1 is modulated through p300/CBP-mediated acetylation and FOG-1 mediated indirect interaction with HDAC1/2 containing NuRD complex. Although GATA-1 acetylation is implicated in GATA-1 activation, the role of deacetylation is not studied. Here, we found that the FOG-1/NuRD does not deacetylate GATA-1. However, HDAC1/2 can directly bind and deacetylate GATA-1. Two arginine residues within the GATA-1 linker region mediates direct interaction with HDAC1. The arginine to alanine mutation (2RA) blocks GATA-1 deacetylation and fails to induce erythroid differentiation. Gene expression profiling and ChIP-seq analysis further demonstrate the importance of GATA-1 deacetylation for gene activation and chromatin recruitment. GATA-12RA knock-in (KI) mice suffer mild anemia and thrombocytopenia with accumulation of immature erythrocytes and megakaryocytes in bone marrow and spleen. Single cell RNA-seq analysis of Lin- cKit+ (LK) cells further reveal a profound change in cell subpopulations and signature gene expression patterns in HSC, myeloid progenitors, and erythroid/megakaryocyte clusters in KI mice. Thus, GATA-1 deacetylation and its interaction with HDAC1 modulates GATA-1 chromatin binding and transcriptional activity that control erythroid/megakaryocyte commitment and differentiation.

DOI: https://doi.org/10.1093/nar/gkab737
Mol Cell. 2021 Aug 13. pii: S1097-2765(21)00612-2. [Epub ahead of print]

Interrogation of the dynamic properties of higher-order heterochromatin using CRISPR-dCas9.

Yuchen Gao, Mengting Han, Stephen Shang, Haifeng Wang, Lei S Qi.

  Eukaryotic chromosomes feature large regions of compact, repressed heterochromatin hallmarked by Heterochromatin Protein 1 (HP1). HP1 proteins play multi-faceted roles in shaping heterochromatin, and in cells, HP1 tethering to individual gene promoters leads to epigenetic modifications and silencing. However, emergent properties of HP1 at supranucleosomal scales remain difficult to study in cells because of a lack of appropriate tools. Here, we develop CRISPR-engineered chromatin organization (EChO), combining live-cell CRISPR imaging with inducible large-scale recruitment of chromatin proteins to native genomic targets. We demonstrate that human HP1α tiled across kilobase-scale genomic DNA form novel contacts with natural heterochromatin, integrates two distantly targeted regions, and reversibly changes chromatin from a diffuse to compact state. The compact state exhibits delayed disassembly kinetics and represses transcription across over 600 kb. These findings support a polymer model of HP1α-mediated chromatin regulation and highlight the utility of CRISPR-EChO in studying supranucleosomal chromatin organization in living cells.

Keywords:  3D genome; CRISPR; CRISPR-EChO; Cas9; HP1; chromatin conformation; epigenetics; gene regulation; heterochromatin; synthetic biology

DOI:  https://doi.org/10.1016/j.molcel.2021.07.034
Development. 2021 Apr 01. pii: dev196329. [Epub ahead of print]148(7):

Full methylation of H3K27 by PRC2 is dispensable for initial embryoid body formation but required to maintain differentiated cell identity.

Sara A Miller, Manashree Damle, Jongmin Kim, Robert E Kingston.

  Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 on lysine 27 and is required for normal development of complex eukaryotes. The nature of that requirement is not clear. H3K27me3 is associated with repressed genes, but the modification is not sufficient to induce repression and, in some instances, is not required. We blocked full methylation of H3K27 with both a small molecule inhibitor, GSK343, and by introducing a point mutation into EZH2, the catalytic subunit of PRC2, in the mouse CJ7 cell line. Cells with substantively decreased H3K27 methylation differentiate into embryoid bodies, which contrasts with EZH2 null cells. PRC2 targets had varied requirements for H3K27me3, with a subset that maintained normal levels of repression in the absence of methylation. The primary cellular phenotype of blocked H3K27 methylation was an inability of altered cells to maintain a differentiated state when challenged. This phenotype was determined by H3K27 methylation in embryonic stem cells through the first 4 days of differentiation. Full H3K27 methylation therefore was not necessary for formation of differentiated cell states during embryoid body formation but was required to maintain a stable differentiated state.

Keywords:  Differentiation; EZH2; Embryoid body; H3K27 methylation; Maintenance; PRC2

DOI:  https://doi.org/10.1242/dev.196329
Cell Rep. 2021 Aug 24. pii: S2211-1247(21)01009-3. [Epub ahead of print]36(8): 109575

TooManyPeaks identifies drug-resistant-specific regulatory elements from single-cell leukemic epigenomes.

Gregory W Schwartz, Yeqiao Zhou, Jelena Petrovic, Warren S Pear, Robert B Faryabi.

  Emerging single-cell epigenomic assays are used to investigate the heterogeneity of chromatin activity and its function. However, identifying cells with distinct regulatory elements and clearly visualizing their relationships remains challenging. To this end, we introduce TooManyPeaks to address the need for the simultaneous study of chromatin state heterogeneity in both rare and abundant subpopulations. Our analyses of existing data from three widely used single-cell assays for transposase-accessible chromatin using sequencing (scATAC-seq) show the superior performance of TooManyPeaks in delineating and visualizing pure clusters of rare and abundant subpopulations. Furthermore, the application of TooManyPeaks to new scATAC-seq data from drug-naive and drug-resistant leukemic T cells clearly visualizes relationships among these cells and stratifies a rare "resistant-like" drug-naive sub-clone with distinct cis-regulatory elements.

Keywords:  T cell leukemia; chromatin accessibility; drug resistance; single-cell ATAC-seq; visualization and clustering algorithms

DOI:  https://doi.org/10.1016/j.celrep.2021.109575
Hum Genet. 2021 Aug 26.

Complete lung agenesis caused by complex genomic rearrangements with neo-TAD formation at the SHH locus.

Uirá Souto Melo, Juliette Piard, Björn Fischer-Zirnsak, Marius-Konstantin Klever, Robert Schöpflin, Martin Atta Mensah, Manuel Holtgrewe, Francine Arbez-Gindre, Alain Martin, Virginie Guigue, Dominique Gaillard, Emilie Landais, Virginie Roze, Valerie Kremer, Rajeev Ramanah, Christelle Cabrol, Frederike L Harms, Uwe Kornak, Malte Spielmann, Stefan Mundlos, Lionel Van Maldergem.

During human organogenesis, lung development is a timely and tightly regulated developmental process under the control of a large number of signaling molecules. Understanding how genetic variants can disturb normal lung development causing different lung malformations is a major goal for dissecting molecular mechanisms during embryogenesis. Here, through exome sequencing (ES), array CGH, genome sequencing (GS) and Hi-C, we aimed at elucidating the molecular basis of bilateral isolated lung agenesis in three fetuses born to a non-consanguineous family. We detected a complex genomic rearrangement containing duplicated, triplicated and deleted fragments involving the SHH locus in fetuses presenting complete agenesis of both lungs and near-complete agenesis of the trachea, diagnosed by ultrasound screening and confirmed at autopsy following termination. The rearrangement did not include SHH itself, but several regulatory elements for lung development, such as MACS1, a major SHH lung enhancer, and the neighboring genes MNX1 and NOM1. The rearrangement incorporated parts of two topologically associating domains (TADs) including their boundaries. Hi-C of cells from one of the affected fetuses showed the formation of two novel TADs each containing SHH enhancers and the MNX1 and NOM1 genes. Hi-C together with GS indicate that the new 3D conformation is likely causative for this condition by an inappropriate activation of MNX1 included in the neo-TADs by MACS1 enhancer, further highlighting the importance of the 3D chromatin conformation in human disease.

DOI: https://doi.org/10.1007/s00439-021-02344-6
Nat Commun. 2021 08 24. 12(1): 5091

Ten-eleven translocation 1 mediated-DNA hydroxymethylation is required for myelination and remyelination in the mouse brain.

Ming Zhang, Jian Wang, Kaixiang Zhang, Guozhen Lu, Yuming Liu, Keke Ren, Wenting Wang, Dazhuan Xin, Lingli Xu, Honghui Mao, Junlin Xing, Xingchun Gao, Weilin Jin, Kalen Berry, Katsuhiko Mikoshiba, Shengxi Wu, Q Richard Lu, Xianghui Zhao.

Ten-eleven translocation (TET) proteins, the dioxygenase for DNA hydroxymethylation, are important players in nervous system development and diseases. However, their role in myelination and remyelination after injury remains elusive. Here, we identify a genome-wide and locus-specific DNA hydroxymethylation landscape shift during differentiation of oligodendrocyte-progenitor cells (OPC). Ablation of Tet1 results in stage-dependent defects in oligodendrocyte (OL) development and myelination in the mouse brain. The mice lacking Tet1 in the oligodendrocyte lineage develop behavioral deficiency. We also show that TET1 is required for remyelination in adulthood. Transcriptomic, genomic occupancy, and 5-hydroxymethylcytosine (5hmC) profiling reveal a critical TET1-regulated epigenetic program for oligodendrocyte differentiation that includes genes associated with myelination, cell division, and calcium transport. Tet1-deficient OPCs exhibit reduced calcium activity, increasing calcium activity rescues the differentiation defects in vitro. Deletion of a TET1-5hmC target gene, Itpr2, impairs the onset of OPC differentiation. Together, our results suggest that stage-specific TET1-mediated epigenetic programming and intracellular signaling are important for proper myelination and remyelination in mice.

DOI: https://doi.org/10.1038/s41467-021-25353-5
Nat Commun. 2021 Aug 26. 12(1): 5123

OCT4 cooperates with distinct ATP-dependent chromatin remodelers in naïve and primed pluripotent states in human.

Xin Huang, Kyoung-Mi Park, Paul Gontarz, Bo Zhang, Joshua Pan, Zachary McKenzie, Laura A Fischer, Chen Dong, Sabine Dietmann, Xiaoyun Xing, Pavel V Shliaha, Jihong Yang, Dan Li, Junjun Ding, Tenzin Lungjangwa, Maya Mitalipova, Shafqat A Khan, Sumeth Imsoonthornruksa, Nick Jensen, Ting Wang, Cigall Kadoch, Rudolf Jaenisch, Jianlong Wang, Thorold W Theunissen.

Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.

DOI: https://doi.org/10.1038/s41467-021-25107-3
Genome Biol. 2021 Aug 26. 22(1): 250

NucHMM: a method for quantitative modeling of nucleosome organization identifying functional nucleosome states distinctly associated with splicing potentiality.

Kun Fang, Tianbao Li, Yufei Huang, Victor X Jin.

  We develop a novel computational method, NucHMM, to identify functional nucleosome states associated with cell type-specific combinatorial histone marks and nucleosome organization features such as phasing, spacing and positioning. We test it on publicly available MNase-seq and ChIP-seq data in MCF7, H1, and IMR90 cells and identify 11 distinct functional nucleosome states. We demonstrate these nucleosome states are distinctly associated with the splicing potentiality of skipping exons. This advances our understanding of the chromatin function at the nucleosome level and offers insights into the interplay between nucleosome organization and splicing processes.

Keywords:  Hidden Markov model; Nucleosome organization; Splicing potentiality

DOI:  https://doi.org/10.1186/s13059-021-02465-1
Adv Sci (Weinh). 2021 Aug 25. e2101895

Pharmacological Inhibition of Core Regulatory Circuitry Liquid-liquid Phase Separation Suppresses Metastasis and Chemoresistance in Osteosarcoma.

Bing Lu, Changye Zou, Meiling Yang, Yangyang He, Jincan He, Chuanxia Zhang, Siyun Chen, Jiaming Yu, Kilia Yun Liu, Qi Cao, Wei Zhao.

  Liquid-liquid phase-separated (LLPS) transcriptional factor assemblies at super-enhancers (SEs) provide a conceptual framework for underlying transcriptional control in mammal cells. However, the mechanistic understanding of LLPS in aberrant transcription driven by dysregulation of SEs in human malignancies is still elusive. By integrating SE profiling and core regulatory circuitry (CRC) calling algorithm, the CRC of metastatic and chemo-resistant osteosarcoma is delineated. CRC components, HOXB8 and FOSL1, produce dense and dynamic phase-separated droplets in vitro and liquid-like puncta in cell nuclei. Disruption of CRC phase separation decreases the chromatin accessibility in SE regions and inhibits the release of RNA polymerase II from the promoter of SE-driven genes. Importantly, absence of CRC key component causes a reduction in osteosarcoma tumor growth and metastasis. Moreover, it is shown that CRC condensates can be specifically attenuated by the H3K27 demethylase inhibitor, GSK-J4. Pharmacological inhibition of the CRC phase separation results in metastasis suppression and re-sensitivity to chemotherapy drugs in patient-derived xenograft model. Taken together, this study reveals a previously unknown mechanism that CRC factors formed LLPS condensates, and provides a phase separation-based pharmacological strategy to target undruggable CRC components for the treatment of metastatic and chemo-resistant osteosarcoma.

Keywords:  HOXB8; core regulatory circuitry; liquid-liquid phase separation; osteosarcoma; super-enhancer

DOI:  https://doi.org/10.1002/advs.202101895
Proc Natl Acad Sci U S A. 2021 Aug 31. pii: e2101442118. [Epub ahead of print]118(35):

Histone H3K27 methylation-mediated repression of Hairy regulates insect developmental transition by modulating ecdysone biosynthesis.

Yan Yang, Tujing Zhao, Zheng Li, Wenliang Qian, Jian Peng, Ling Wei, Dongqin Yuan, Yaoyao Li, Qingyou Xia, Daojun Cheng.

  Insect development is cooperatively orchestrated by the steroid hormone ecdysone and juvenile hormone (JH). The polycomb repressive complex 2 (PRC2)-mediated histone H3K27 trimethylation (H3K27me3) epigenetically silences gene transcription and is essential for a range of biological processes, but the functions of H3K27 methylation in insect hormone action are poorly understood. Here, we demonstrate that H3K27 methylation-mediated repression of Hairy transcription in the larval prothoracic gland (PG) is required for ecdysone biosynthesis in Bombyx and Drosophila H3K27me3 levels in the PG are dynamically increased during the last larval instar. H3K27me3 reduction induced by the down-regulation of PRC2 activity via inhibitor treatment in Bombyx or PG-specific knockdown of the PRC2 component Su(z)12 in Drosophila diminishes ecdysone biosynthesis and disturbs the larval-pupal transition. Mechanistically, H3K27 methylation targets the JH signal transducer Hairy to repress its transcription in the PG; PG-specific knockdown or overexpression of the Hairy gene disrupts ecdysone biosynthesis and developmental transition; and developmental defects caused by PG-specific Su(z)12 knockdown can be partially rescued by Hairy down-regulation. The application of JH mimic to the PG decreases both H3K27me3 levels and Su(z)12 expression. Altogether, our study reveals that PRC2-mediated H3K27 methylation at Hairy in the PG during the larval period is required for ecdysone biosynthesis and the larval-pupal transition and provides insights into epigenetic regulation of the crosstalk between JH and ecdysone during insect development.

Keywords:  H3K27 methylation; Hairy; ecdysone biosynthesis; juvenile hormone; repression

DOI:  https://doi.org/10.1073/pnas.2101442118
Nat Commun. 2021 Aug 26. 12(1): 5136

A TALE/HOX code unlocks WNT signalling response towards paraxial mesoderm.

Luca Mariani, Xiaogang Guo, Niels Alvaro Menezes, Anna Maria Drozd, Selgin Deniz Çakal, Qinhu Wang, Elisabetta Ferretti.

One fundamental yet unresolved question in biology remains how cells interpret the same signalling cues in a context-dependent manner resulting in lineage specification. A key step for decoding signalling cues is the establishment of a permissive chromatin environment at lineage-specific genes triggering transcriptional responses to inductive signals. For instance, bipotent neuromesodermal progenitors (NMPs) are equipped with a WNT-decoding module, which relies on TCFs/LEF activity to sustain both NMP expansion and paraxial mesoderm differentiation. However, how WNT signalling activates lineage specific genes in a temporal manner remains unclear. Here, we demonstrate that paraxial mesoderm induction relies on the TALE/HOX combinatorial activity that simultaneously represses NMP genes and activates the differentiation program. We identify the BRACHYURY-TALE/HOX code that destabilizes the nucleosomes at WNT-responsive regions and establishes the permissive chromatin landscape for de novo recruitment of the WNT-effector LEF1, unlocking the WNT-mediated transcriptional program that drives NMPs towards the paraxial mesodermal fate.

DOI: https://doi.org/10.1038/s41467-021-25370-4
Elife. 2021 Aug 24. pii: e64593. [Epub ahead of print]10

Combinations of maternal-specific repressive epigenetic marks in the endosperm control seed dormancy.

Hikaru Sato, Juan Santos-González, Claudia Köhler.

  Polycomb Repressive Complex 2 (PRC2)-mediated trimethylation of histone H3 on lysine 27 (H3K27me3) and methylation of histone 3 on lysine 9 (H3K9me) are two repressive epigenetic modifications that are typically localized in distinct regions of the genome. For reasons unknown, however, they co-occur in some organisms and special tissue types. In this study, we show that maternal alleles marked by H3K27me3 in the Arabidopsis endosperm were targeted by the H3K27me3 demethylase REF6 and became activated during germination. In contrast, maternal alleles marked by H3K27me3, H3K9me2, and CHG methylation (CHGm) are likely to be protected from REF6 targeting and remained silenced. Our study unveils that combinations of different repressive epigenetic modifications time a key adaptive trait by modulating access of REF6.

Keywords:  A. thaliana; chromosomes; gene expression

DOI:  https://doi.org/10.7554/eLife.64593
Mol Biol Cell. 2021 Aug 25. mbcE20080509

A dual role for DNA-binding by Runt in activation and repression of sloppy paired transcription.

Lisa Prazak, Yasuno Iwasaki, Ah-Ram Kim, Konstantin Kozlov, Kevin King, J Peter Gergen.

This work investigates the role of DNA-binding by Runt in regulating the sloppy-paired-1 (slp1) gene, and in particular two distinct cis-regulatory elements that mediate regulation by Runt and other pair-rule transcription factors during Drosophila segmentation. We find that a DNA-binding defective form of Runt is ineffective at repressing both the distal (DESE) and proximal (PESE) early stripe elements of slp1 and is also compromised for DESE-dependent activation. The function of Runt-binding sites in DESE is further investigated using site-specific transgenesis and quantitative imaging techniques. When DESE is tested as an autonomous enhancer, mutagenesis of the Runt sites results in a clear loss of Runt-dependent repression but has little to no effect on Runt-dependent activation. Notably, mutagenesis of these same sites in the context of a reporter gene construct that also contains the PESE enhancer results in a significant reduction of DESE-dependent activation as well as the loss of repression observed for the autonomous mutant DESE enhancer. These results provide strong evidence that DNA-binding by Runt directly contributes to the regulatory interplay of interactions between these two enhancers in the early embryo.

DOI: https://doi.org/10.1091/mbc.E20-08-0509
Cancer Res. 2021 Aug 24. pii: canres.0206.2021. [Epub ahead of print]

mTORC1 Promotes ARID1A Degradation and Oncogenic Chromatin Remodeling in Hepatocellular Carcinoma.

Shanshan Zhang, Yu-Feng Zhou, Jian Cao, Stephen K Burley, Hui-Yun Wang, X F Steven Zheng.

The SWI/SNF chromatin remodeling complexes control accessibility of chromatin to transcriptional and co-regulatory machineries. Chromatin remodeling plays important roles in normal physiology and diseases, particularly cancer. The ARID1A-containing SWI/SNF complex is commonly mutated and thought to be a key tumor suppressor in hepatocellular carcinoma (HCC), but its regulation in response to oncogenic signals remains poorly understood. mTOR is a conserved central controller of cell growth and an oncogenic driver of HCC. Remarkably, cancer mutations in mTOR and SWI/SNF complex are mutually exclusive in human HCC tumors, suggesting that they share a common oncogenic function. Here we report that mTOR complex 1 (mTORC1) interact with ARID1A and regulates ubiquitination and proteasomal degradation of ARID1A protein. The mTORC1-ARID1A axis promoted oncogenic chromatin remodeling and YAP-dependent transcription, thereby enhancing liver cancer cell growth in vitro and tumor development in vivo. Conversely, excessive ARID1A expression counteracted AKT-driven liver tumorigenesis in vivo. Moreover, dysregulation of this axis conferred resistance to mTOR-targeted therapies. These findings demonstrate that the ARID1A-SWI/SNF complex is a regulatory target for oncogenic mTOR signaling, which is important for mTORC1-driven hepatocarcinogenesis with implications for therapeutic interventions in HCC.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-0206
Stem Cell Reports. 2021 Aug 16. pii: S2213-6711(21)00388-X. [Epub ahead of print]

CHD4 ensures stem cell lineage fidelity during skeletal muscle regeneration.

Krishnamoorthy Sreenivasan, Alejandra Rodríguez-delaRosa, Johnny Kim, Diana Mesquita, Jessica Segalés, Pablo Gómez-Del Arco, Isabel Espejo, Alessandro Ianni, Luciano Di Croce, Frederic Relaix, Juan Miguel Redondo, Thomas Braun, Antonio L Serrano, Eusebio Perdiguero, Pura Muñoz-Cánoves.

  Regeneration of skeletal muscle requires resident stem cells called satellite cells. Here, we report that the chromatin remodeler CHD4, a member of the nucleosome remodeling and deacetylase (NuRD) repressive complex, is essential for the expansion and regenerative functions of satellite cells. We show that conditional deletion of the Chd4 gene in satellite cells results in failure to regenerate muscle after injury. This defect is principally associated with increased stem cell plasticity and lineage infidelity during the expansion of satellite cells, caused by de-repression of non-muscle-cell lineage genes in the absence of Chd4. Thus, CHD4 ensures that a transcriptional program that safeguards satellite cell identity during muscle regeneration is maintained. Given the therapeutic potential of muscle stem cells in diverse neuromuscular pathologies, CHD4 constitutes an attractive target for satellite cell-based therapies.

Keywords:  Chd4; NuRD; lineage maintenance; muscle stem cell; regeneration; satellite cells; skeletal muscle

DOI:  https://doi.org/10.1016/j.stemcr.2021.07.022
Nucleic Acids Res. 2021 Aug 24. pii: gkab735. [Epub ahead of print]

Unraveling the functions of uncharacterized transcription factors in Escherichia coli using ChIP-exo.

Ye Gao, Hyun Gyu Lim, Hans Verkler, Richard Szubin, Daniel Quach, Irina Rodionova, Ke Chen, James T Yurkovich, Byung-Kwan Cho, Bernhard O Palsson.

Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, no TRN is fully characterized since the identity and activity of all the transcriptional regulators comprising a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in Escherichia coli K-12 MG1655, which were computationally predicted to be transcription factors (TFs). First, we used a multiplexed chromatin immunoprecipitation method combined with lambda exonuclease digestion (multiplexed ChIP-exo) assay to characterize binding sites for these candidate TFs; 34 of them were found to be DNA-binding proteins. We then compared the relative location between binding sites and RNA polymerase (RNAP). We found 48% (283/588) overlap between the TFs and RNAP. Finally, we used these data to infer potential functions for 10 of the 34 TFs with validated DNA binding sites and consensus binding motifs. Taken together, this study: (i) significantly expands the number of confirmed TFs to 276, close to the estimated total of about 280 TFs; (ii) provides putative functions for the newly discovered TFs and (iii) confirms the functions of four representative TFs through mutant phenotypes.

DOI: https://doi.org/10.1093/nar/gkab735
Stem Cell Reports. 2021 Aug 17. pii: S2213-6711(21)00389-1. [Epub ahead of print]

The tumor suppressor WT1 drives progenitor cell progression and epithelialization to prevent Wilms tumorigenesis in human kidney organoids.

Verena Waehle, Rosemarie Ungricht, Philipp S Hoppe, Joerg Betschinger.

  Wilms tumor is the most widespread kidney cancer in children and frequently associated with homozygous loss of the tumor suppressor WT1. Pediatric tumorigenesis is largely inaccessible in humans. Here, we develop a human kidney organoid model for Wilms tumor formation and show that deletion of WT1 during organoid development induces overgrowth of kidney progenitor cells at the expense of differentiating glomeruli and tubules. Functional and gene expression analyses demonstrate that absence of WT1 halts progenitor cell progression at a pre-epithelialized cell state and recapitulates the transcriptional changes detected in a subgroup of Wilms tumor patients with ectopic myogenesis. By "transplanting" WT1 mutant cells into wild-type kidney organoids, we find that their propagation requires an untransformed microenvironment. This work defines the role of WT1 in kidney progenitor cell progression and tumor suppression, and establishes human kidney organoids as a phenotypic model for pediatric tumorigenesis.

Keywords:  SIX2; WT1; Wilms tumor; disease modeling; human kidney organoid; iPS cell; kidney progenitor cell; mesenchymal-epithelial transition; pediatric tumor; tumor suppressor

DOI:  https://doi.org/10.1016/j.stemcr.2021.07.023
Sci Adv. 2021 Aug;pii: eabe3771. [Epub ahead of print]7(35):

Histone citrullination by PADI4 is required for HIF-dependent transcriptional responses to hypoxia and tumor vascularization.

Yufeng Wang, Yajing Lyu, Kangsheng Tu, Qiuran Xu, Yongkang Yang, Shaima Salman, Nguyet Le, Haiquan Lu, Chelsey Chen, Yayun Zhu, Ru Wang, Qingguang Liu, Gregg L Semenza.

Hypoxia-inducible factors (HIFs) activate transcription of target genes by recruiting coactivators and chromatin-modifying enzymes. Peptidylarginine deiminase 4 (PADI4) catalyzes the deimination of histone arginine residues to citrulline. Here, we demonstrate that PADI4 expression is induced by hypoxia in a HIF-dependent manner in breast cancer and hepatocellular carcinoma cells. PADI4, in turn, is recruited by HIFs to hypoxia response elements (HREs) and is required for HIF target gene transcription. Hypoxia induces histone citrullination at HREs that is PADI4 and HIF dependent. RNA sequencing revealed that almost all HIF target genes in breast cancer cells are PADI4 dependent. PADI4 is required for breast and liver tumor growth and angiogenesis in mice. PADI4 expression is correlated with HIF-1α expression and vascularization in human breast cancer biopsies. Thus, HIF-dependent recruitment of PADI4 to target genes and local histone citrullination are required for transcriptional responses to hypoxia.

DOI: https://doi.org/10.1126/sciadv.abe3771
Sci Adv. 2021 Aug;pii: eabg5442. [Epub ahead of print]7(35):

An epigenetic basis of inbreeding depression in maize.

Tongwen Han, Fang Wang, Qingxin Song, Wenxue Ye, Tieshan Liu, Liming Wang, Z Jeffrey Chen.

Inbreeding depression is widespread across plant and animal kingdoms and may arise from the exposure of deleterious alleles and/or loss of overdominant alleles resulting from increased homozygosity, but these genetic models cannot fully explain the phenomenon. Here, we report epigenetic links to inbreeding depression in maize. Teosinte branched1/cycloidea/proliferating cell factor (TCP) transcription factors control plant development. During successive inbreeding among inbred lines, thousands of genomic regions across TCP-binding sites (TBS) are hypermethylated through the H3K9me2-mediated pathway. These hypermethylated regions are accompanied by decreased chromatin accessibility, increased levels of the repressive histone marks H3K27me2 and H3K27me3, and reduced binding affinity of maize TCP-proteins to TBS. Consequently, hundreds of TCP-target genes involved in mitochondrion, chloroplast, and ribosome functions are down-regulated, leading to reduced growth vigor. Conversely, random mating can reverse corresponding hypermethylation sites and TCP-target gene expression, restoring growth vigor. These results support a unique role of reversible epigenetic modifications in inbreeding depression.

DOI: https://doi.org/10.1126/sciadv.abg5442
JCI Insight. 2021 Aug 24. pii: 151621. [Epub ahead of print]

Combinatorial transcription factor profiles predict mature and functional human islet α and β cells.

Shristi Shrestha, Diane C Saunders, John T Walker, Joan Camunas-Soler, Xiao-Qing Dai, Rachana Haliyur, Radhika Aramandla, Greg Poffenberger, Nripesh Prasad, Rita Bottino, Roland Stein, Jean-Philippe Cartailler, Stephen Cj Parker, Patrick E MacDonald, Shawn E Levy, Alvin C Powers, Marcela Brissova.

  Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled >40,000 cells from normal human islets by scRNA-seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells co-expressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-seq, MAFA/MAFB co-expressing β cells showed enhanced electrophysiological activity. Thus, these results indicate combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.

Keywords:  Beta cells; Bioinformatics; Cell Biology; Diabetes; Endocrinology

DOI:  https://doi.org/10.1172/jci.insight.151621
Nat Biotechnol. 2021 Aug 23.

Single-cell measurement of higher-order 3D genome organization with scSPRITE.

Mary V Arrastia, Joanna W Jachowicz, Noah Ollikainen, Matthew S Curtis, Charlotte Lai, Sofia A Quinodoz, David A Selck, Rustem F Ismagilov, Mitchell Guttman.

Although three-dimensional (3D) genome organization is central to many aspects of nuclear function, it has been difficult to measure at the single-cell level. To address this, we developed 'single-cell split-pool recognition of interactions by tag extension' (scSPRITE). scSPRITE uses split-and-pool barcoding to tag DNA fragments in the same nucleus and their 3D spatial arrangement. Because scSPRITE measures multiway DNA contacts, it generates higher-resolution maps within an individual cell than can be achieved by proximity ligation. We applied scSPRITE to thousands of mouse embryonic stem cells and detected known genome structures, including chromosome territories, active and inactive compartments, and topologically associating domains (TADs) as well as long-range inter-chromosomal structures organized around various nuclear bodies. We observe that these structures exhibit different levels of heterogeneity across the population, with TADs representing dynamic units of genome organization across cells. We expect that scSPRITE will be a critical tool for studying genome structure within heterogeneous populations.

DOI: https://doi.org/10.1038/s41587-021-00998-1
PLoS Pathog. 2021 Aug 25. 17(8): e1009875

Short- and long-range cis interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis.

Ian J Groves, Emma L A Drane, Marco Michalski, Jack M Monahan, Cinzia G Scarpini, Stephen P Smith, Giovanni Bussotti, Csilla Várnai, Stefan Schoenfelder, Peter Fraser, Anton J Enright, Nicholas Coleman.

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art 'HPV integrated site capture' (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a 'looping' mechanism by which flanking host regions become amplified. Furthermore, using our 'HPV16-specific Region Capture Hi-C' technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (<500 kbp) and long-range (>500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a 'cancer-causing gene' is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.

DOI: https://doi.org/10.1371/journal.ppat.1009875
Cell Rep. 2021 Aug 24. pii: S2211-1247(21)01022-6. [Epub ahead of print]36(8): 109588

The transcription factor ZEB1 regulates stem cell self-renewal and cell fate in the adult hippocampus.

Bhavana Gupta, Adam C Errington, Ana Jimenez-Pascual, Vasileios Eftychidis, Simone Brabletz, Marc P Stemmler, Thomas Brabletz, David Petrik, Florian A Siebzehnrubl.

  Radial glia-like (RGL) stem cells persist in the adult mammalian hippocampus, where they generate new neurons and astrocytes throughout life. The process of adult neurogenesis is well documented, but cell-autonomous factors regulating neuronal and astroglial differentiation are incompletely understood. Here, we evaluate the functions of the transcription factor zinc-finger E-box binding homeobox 1 (ZEB1) in adult hippocampal RGL cells using a conditional-inducible mouse model. We find that ZEB1 is necessary for self-renewal of active RGL cells. Genetic deletion of Zeb1 causes a shift toward symmetric cell division that consumes the RGL cell and generates pro-neuronal progenies, resulting in an increase of newborn neurons and a decrease of newly generated astrocytes. We identify ZEB1 as positive regulator of the ets-domain transcription factor ETV5 that is critical for asymmetric division.

Keywords:  Cre-loxP; EMT; animal model; astrogliogenesis; asymmetrical division; epithelial-mesenchymal transition; gliogenesis; lineage specification; neural stem cell; neurogenesis

DOI:  https://doi.org/10.1016/j.celrep.2021.109588
Cell Rep. 2021 Aug 24. pii: S2211-1247(21)01036-6. [Epub ahead of print]36(8): 109598

The related coactivator complexes SAGA and ATAC control embryonic stem cell self-renewal through acetyltransferase-independent mechanisms.

Veronique Fischer, Damien Plassard, Tao Ye, Bernardo Reina-San-Martin, Matthieu Stierle, Laszlo Tora, Didier Devys.

  SAGA (Spt-Ada-Gcn5 acetyltransferase) and ATAC (Ada-two-A-containing) are two related coactivator complexes, sharing the same histone acetyltransferase (HAT) subunit. The HAT activities of SAGA and ATAC are required for metazoan development, but the role of these complexes in RNA polymerase II transcription is less understood. To determine whether SAGA and ATAC have redundant or specific functions, we compare the effects of HAT inactivation in each complex with that of inactivation of either SAGA or ATAC core subunits in mouse embryonic stem cells (ESCs). We show that core subunits of SAGA or ATAC are required for complex assembly and mouse ESC growth and self-renewal. Surprisingly, depletion of HAT module subunits causes a global decrease in histone H3K9 acetylation, but does not result in significant phenotypic or transcriptional defects. Thus, our results indicate that SAGA and ATAC are differentially required for self-renewal of mouse ESCs by regulating transcription through different pathways in a HAT-independent manner.

Keywords:  4sU labeling; ATAC; HAT-independent function; Pol II transcription; SAGA; coactivator complexes; histone acetyltransferase; mouse embryonic stem cells; newly synthesized RNA

DOI:  https://doi.org/10.1016/j.celrep.2021.109598
Development. 2021 Apr 01. pii: dev197319. [Epub ahead of print]148(7):

A lineage-specific requirement for YY1 Polycomb Group protein function in early T cell development.

Anna L F V Assumpção, Guoping Fu, Deependra K Singh, Zhanping Lu, Ashley M Kuehnl, Rene Welch, Irene M Ong, Renren Wen, Xuan Pan.

  Yin Yang 1 (YY1) is a ubiquitous transcription factor and mammalian Polycomb Group protein (PcG) with important functions for regulating lymphocyte development and stem cell self-renewal. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that result in histone modifications. Many questions remain unanswered regarding how cell- and tissue-specificity is achieved by PcG proteins. Here, we demonstrate that a conditional knockout of Yy1 in the hematopoietic system results in an early T cell developmental blockage at the double negative (DN) 1 stage with reduced Notch1 signaling. There is a lineage-specific requirement for YY1 PcG function. YY1 PcG domain is required for T and B cell development but not necessary for myeloid cells. YY1 functions in early T cell development are multicomponent and involve both PcG-dependent and -independent regulations. Although YY1 promotes early T cell survival through its PcG function, its function to promote the DN1-to-DN2 transition and Notch1 expression and signaling is independent of its PcG function. Our results reveal how a ubiquitously expressed PcG protein mediates lineage-specific and context-specific functions to control early T cell development.

Keywords:  Hematopoiesis; Mouse; Notch1; Polycomb; T cell; YY1

DOI:  https://doi.org/10.1242/dev.197319
Oncotarget. 2021 Aug 17. 12(17): 1651-1662

Diverse transcriptional regulation and functional effects revealed by CRISPR/Cas9-directed epigenetic editing.

Miguel Vizoso, Jacco van Rheenen.

  DNA methylation is an epigenetic process that controls DNA accessibility and serves as a transcriptomic switch when deposited at regulatory regions. The adequate functioning of this process is indispensable for tissue homeostasis and cell fate determination. Conversely, altered DNA methylation patterns result in abnormal gene transcription profiles that contribute to tumor initiation and progression. However, whether the consequence of DNA methylation on gene expression and cell fate is uniform regardless of the cell type or state could so far not been tested due to the lack of technologies to target DNA methylation in-situ. Here, we have taken advantage of CRISPR/dCas9 technology adapted for epigenetic editing through site-specific targeting of DNA methylation to characterize the transcriptional changes of the candidate gene and the functional effects on cell fate in different tumor settings. As a proof-of-concept, we were able to induce de-novo site-specific methylation of the gene promoter of IGFBP2 up to 90% with long-term and bona-fide inheritance by daughter cells. Strikingly, this modification led to opposing expression profiles of the target gene in different cancer cell models and affected the expression of mesenchymal genes CDH1, VIM1, TGFB1 and apoptotic marker BCL2. Moreover, methylation-induced changes in expression profiles was also accompanied by a phenotypic switch in cell migration and cell morphology. We conclude that in different cell types the consequence of DNA methylation on gene expression and cell fate can be completely different.

Keywords:  CRISPR/Cas9-based system; IGFBP2; epithelial-to-mesenchymal transition; targeted DNA methylation

DOI:  https://doi.org/10.18632/oncotarget.28037
Proc Natl Acad Sci U S A. 2021 Aug 31. pii: e2025948118. [Epub ahead of print]118(35):

METTL14 facilitates global genome repair and suppresses skin tumorigenesis.

Zizhao Yang, Seungwon Yang, Yan-Hong Cui, Jiangbo Wei, Palak Shah, Gayoung Park, Xiaolong Cui, Chuan He, Yu-Ying He.

  Global genome repair (GGR), a subpathway of nucleotide excision repair, corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here, we show that METTL14 (methyltransferase-like 14), a critical component of the N6-methyladenosine (m6A) RNA methyltransferase complex, promotes GGR through regulating m6A mRNA methylation-mediated DDB2 translation and suppresses ultraviolet B (UVB) radiation-induced skin tumorigenesis. UVB irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy. METTL14 knockdown decreases GGR and DDB2 abundance. Conversely, overexpression of wild-type METTL14 but not its enzymatically inactive mutant increases GGR and DDB2 abundance. METTL14 knockdown decreases m6A methylation and translation of the DDB2 transcripts. Adding DDB2 reverses the GGR repair defect in METTL14 knockdown cells, indicating that METTL14 facilitates GGR through regulating DDB2 m6A methylation and translation. Similarly, knockdown of YTHDF1, an m6A reader promoting translation of m6A-modified transcripts, decreases DDB2 protein levels. Both METTL14 and YTHDF1 bind to the DDB2 transcript. In mice, skin-specific heterozygous METTL14 deletion increases UVB-induced skin tumorigenesis. Furthermore, METTL14 as well as DDB2 is down-regulated in human and mouse skin tumors and by chronic UVB irradiation in mouse skin, and METTL14 level is associated with the DDB2 level, suggesting a tumor-suppressive role of METTL14 in UVB-associated skin tumorigenesis in association with DDB2 regulation. Taken together, these findings demonstrate that METTL14 is a target for selective autophagy and acts as a critical epitranscriptomic mechanism to regulate GGR and suppress UVB-induced skin tumorigenesis.

Keywords:  METTL14; UVB; m6A RNA methylation; nucleotide excision repair; ultraviolet radiation

DOI:  https://doi.org/10.1073/pnas.2025948118
Cell Rep. 2021 Aug 24. pii: S2211-1247(21)00957-8. [Epub ahead of print]36(8): 109526

Epigenomic and transcriptional profiling identifies impaired glyoxylate detoxification in NAFLD as a risk factor for hyperoxaluria.

Kathrin Gianmoena, Nina Gasparoni, Adelina Jashari, Philipp Gabrys, Katharina Grgas, Ahmed Ghallab, Karl Nordström, Gilles Gasparoni, Jörg Reinders, Karolina Edlund, Patricio Godoy, Alexander Schriewer, Heiko Hayen, Christian A Hudert, Georg Damm, Daniel Seehofer, Thomas S Weiss, Peter Boor, Hans-Joachim Anders, Manga Motrapu, Peter Jansen, Tobias S Schiergens, Maren Falk-Paulsen, Philip Rosenstiel, Clivia Lisowski, Eduardo Salido, Rosemarie Marchan, Jörn Walter, Jan G Hengstler, Cristina Cadenas.

  Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release-a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients.

Keywords:  AGXT; DNA methylation; HAO1; LDHA; chromatin accessibility; gene expression; glucagon; glycolate; glyoxylate; hydroxyproline; oxalate

DOI:  https://doi.org/10.1016/j.celrep.2021.109526
Development. 2021 Aug 23. pii: dev.199493. [Epub ahead of print]

Lineage-specific control of convergent differentiation by a Forkhead repressor.

Karolina Mizeracka, Julia M Rogers, Jonathan D Rumley, Shai Shaham, Martha L Bulyk, John I Murray, Maxwell G Heiman.

  During convergent differentiation, multiple developmental lineages produce a highly similar or identical cell type. However, few molecular players that drive convergent differentiation are known. Here, we show that the C. elegans Forkhead transcription factor UNC-130 is required in only one of three convergent lineages that produce the same glial cell type. UNC-130 acts transiently as a repressor in progenitors and newly-born terminal cells to allow the proper specification of cells related by lineage rather than by cell type or function. Specification defects correlate with UNC-130:DNA binding, and UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3. We propose that, in contrast to terminal selectors that activate cell-type specific transcriptional programs in terminally differentiating cells, UNC-130 acts early and specifically in one convergent lineage to produce a cell type that also arises from molecularly distinct progenitors in other lineages.

Keywords:  C. elegans; Cell lineage; Convergent differentiation; FoxD3; Glia

DOI:  https://doi.org/10.1242/dev.199493
Proc Natl Acad Sci U S A. 2021 Aug 31. pii: e2109517118. [Epub ahead of print]118(35):

Bromodomain containing 9 (BRD9) regulates macrophage inflammatory responses by potentiating glucocorticoid receptor activity.

Liu Wang, Tae Gyu Oh, Jason Magida, Gabriela Estepa, S M Bukola Obayomi, Ling-Wa Chong, Jovylyn Gatchalian, Ruth T Yu, Annette R Atkins, Diana Hargreaves, Michael Downes, Zong Wei, Ronald M Evans.

  In macrophages, homeostatic and immune signals induce distinct sets of transcriptional responses, defining cellular identity and functional states. The activity of lineage-specific and signal-induced transcription factors are regulated by chromatin accessibility and other epigenetic modulators. Glucocorticoids are potent antiinflammatory drugs; however, the mechanisms by which they selectively attenuate inflammatory genes are not yet understood. Acting through the glucocorticoid receptor (GR), glucocorticoids directly repress inflammatory responses at transcriptional and epigenetic levels in macrophages. A major unanswered question relates to the sequence of events that result in the formation of repressive regions. In this study, we identify bromodomain containing 9 (BRD9), a component of SWI/SNF chromatin remodeling complex, as a modulator of glucocorticoid responses in macrophages. Inhibition, degradation, or genetic depletion of BRD9 in bone marrow-derived macrophages significantly attenuated their responses to both liposaccharides and interferon inflammatory stimuli. Notably, BRD9-regulated genes extensively overlap with those regulated by the synthetic glucocorticoid dexamethasone. Pharmacologic inhibition of BRD9 potentiated the antiinflammatory responses of dexamethasone, while the genetic deletion of BRD9 in macrophages reduced high-fat diet-induced adipose inflammation. Mechanistically, BRD9 colocalized at a subset of GR genomic binding sites, and depletion of BRD9 enhanced GR occupancy primarily at inflammatory-related genes to potentiate GR-induced repression. Collectively, these findings establish BRD9 as a genomic antagonist of GR at inflammatory-related genes in macrophages, and reveal a potential for BRD9 inhibitors to increase the therapeutic efficacies of glucocorticoids.

Keywords:  bromodomain containing 9 (BRD9); inflammation; macrophages

DOI:  https://doi.org/10.1073/pnas.2109517118
Proc Natl Acad Sci U S A. 2021 Aug 31. pii: e2102895118. [Epub ahead of print]118(35):

Identification of a KLF5-dependent program and drug development for skeletal muscle atrophy.

Lin Liu, Hiroyuki Koike, Takehito Ono, Shinichiro Hayashi, Fujimi Kudo, Atsushi Kaneda, Hiroyuki Kagechika, Ichiro Manabe, Tomoki Nakashima, Yumiko Oishi.

  Skeletal muscle atrophy is caused by various conditions, including aging, disuse related to a sedentary lifestyle and lack of physical activity, and cachexia. Our insufficient understanding of the molecular mechanism underlying muscle atrophy limits the targets for the development of effective pharmacologic treatments and preventions. Here, we identified Krüppel-like factor 5 (KLF5), a zinc-finger transcription factor, as a key mediator of the early muscle atrophy program. KLF5 was up-regulated in atrophying myotubes as an early response to dexamethasone or simulated microgravity in vitro. Skeletal muscle-selective deletion of Klf5 significantly attenuated muscle atrophy induced by mechanical unloading in mice. Transcriptome- and genome-wide chromatin accessibility analyses revealed that KLF5 regulates atrophy-related programs, including metabolic changes and E3-ubiquitin ligase-mediated proteolysis, in coordination with Foxo1. The synthetic retinoic acid receptor agonist Am80, a KLF5 inhibitor, suppressed both dexamethasone- and microgravity-induced muscle atrophy in vitro and oral Am80 ameliorated disuse- and dexamethasone-induced atrophy in mice. Moreover, in three independent sets of transcriptomic data from human skeletal muscle, KLF5 expression significantly increased with age and the presence of sarcopenia and correlated positively with the expression of the atrophy-related ubiquitin ligase genes FBXO32 and TRIM63 These findings demonstrate that KLF5 is a key transcriptional regulator mediating muscle atrophy and that pharmacological intervention with Am80 is a potentially preventive treatment.

Keywords:  KLF; RAR ligand; muscle atrophy

DOI:  https://doi.org/10.1073/pnas.2102895118