bims-crepig 2021-08-08 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

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

Dissecting OCT4 defines the role of nucleosome binding in pluripotency.
CLAMP and Zelda function together to promote Drosophila zygotic genome activation.
ZMYND8-regulated IRF8 transcription axis is an acute myeloid leukemia dependency.
The role of epigenetic modifications, long-range contacts, enhancers and topologically associating domains in the regulation of glioma grade-specific genes.
Systematic assessment of gene co-regulation within chromatin domains determines differentially active domains across human cancers.
Identification of the transcription factor MAZ as a regulator of erythropoiesis.
LKB1 inactivation modulates chromatin accessibility to drive metastatic progression.
miR-766-5p targets super-enhancers by downregulating CBP and BRD4.
K-mer Content Changes with Node Degree in Promoter-Enhancer Network of Mouse ES Cells.
m6Am-seq reveals the dynamic m6Am methylation in the human transcriptome.
In situ Chromatin Interaction Analysis Using Paired-End Tag Sequencing.
Investigating crosstalk between H3K27 acetylation and H3K4 trimethylation in CRISPR/dCas-based epigenome editing and gene activation.
De novo DNA methylation controls neuronal maturation during adult hippocampal neurogenesis.
Cryo-EM structure of the nucleosome core particle containing Giardia lamblia histones.
osr1 couples intermediate mesoderm cell fate with temporal dynamics of vessel progenitor cell differentiation.
PAX9 determines epigenetic state transition and cell fate in cancer.
Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin.
Diverse heterochromatin-associated proteins repress distinct classes of genes and repetitive elements.
EBNA2 driven enhancer switching at the CIITA-DEXI locus suppresses HLA class II gene expression during EBV infection of B-lymphocytes.
H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.
Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma.
Differential H4K16ac levels ensure a balance between quiescence and activation in hematopoietic stem cells.
SUMOylated non-canonical polycomb PRC1.6 complex as a prerequisite for recruitment of transcription factor RBPJ.
Sc-compReg enables the comparison of gene regulatory networks between conditions using single-cell data.
E2F1 and epigenetic modifiers orchestrate breast cancer progression by regulating oxygen-dependent ESRP1 expression.
Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network.
Regulation Network of Colorectal-Cancer-Specific Enhancers in the Progression of Colorectal Cancer.
The oncogenic transcription factor FUS-CHOP can undergo nuclear liquid-liquid phase separation.
FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes.
Genome-wide and sister chromatid-resolved profiling of protein occupancy in replicated chromatin with ChOR-seq and SCAR-seq.
Enhancer decommissioning imposes an epigenetic barrier to sensory hair cell regeneration.
The Δ40p53 isoform inhibits p53-dependent eRNA transcription and enables regulation by signal-specific transcription factors during p53 activation.
HERON: A Novel Tool Enables Identification of Long, Weakly Enriched Genomic Domains in ChIP-seq Data.
Repurposing RNA sequencing for discovery of RNA modifications in clinical cohorts.
A dual role of YAP in driving TGFβ-mediated endothelial-to-mesenchymal transition.
The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage.

Nat Cell Biol. 2021 Aug 05.

Dissecting OCT4 defines the role of nucleosome binding in pluripotency.

Gareth A Roberts, Burak Ozkan, Ivana Gachulincová, Michael R O'Dwyer, Elisa Hall-Ponsele, Manoj Saxena, Philip J Robinson, Abdenour Soufi.

Pioneer transcription factors such as OCT4 can target silent genes embedded in nucleosome-dense regions. How nucleosome interaction enables transcription factors to target chromatin and determine cell identity remains elusive. Here, we systematically dissect OCT4 to show that nucleosome binding is encoded within the DNA-binding domain and yet can be uncoupled from free-DNA binding. Furthermore, accelerating the binding kinetics of OCT4 to DNA enhances nucleosome binding. In cells, uncoupling nucleosome binding diminishes the ability of OCT4 to individually access closed chromatin, while more dynamic nucleosome binding results in expansive genome scanning within closed chromatin. However, both uncoupling and enhancing nucleosome binding are detrimental to inducing pluripotency from differentiated cells. Remarkably, stable interactions between OCT4 and nucleosomes are continuously required for maintaining the accessibility of pluripotency enhancers in stem cells. Our findings reveal how the affinity and residence time of OCT4-nucleosome complexes modulate chromatin accessibility during cell fate changes and maintenance.

DOI: https://doi.org/10.1038/s41556-021-00727-5
Elife. 2021 Aug 03. pii: e69937. [Epub ahead of print]10

CLAMP and Zelda function together to promote Drosophila zygotic genome activation.

Jingyue Duan, Leila Rieder, Megan M Colonnetta, Annie Huang, Mary Mckenney, Scott Watters, Girish Deshpande, William Jordan, Nicolas Fawzi, Erica Larschan.

  During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, we hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as CLAMP. Here, we demonstrate that Drosophila embryonic development requires maternal CLAMP to: 1) activate zygotic transcription; 2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; 3) enhance chromatin accessibility and facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, CLAMP functions as a pioneer factor which plays a targeted yet essential role in ZGA.

Keywords:  D. melanogaster; genetics; genomics

DOI:  https://doi.org/10.7554/eLife.69937
Mol Cell. 2021 Jul 28. pii: S1097-2765(21)00587-6. [Epub ahead of print]

ZMYND8-regulated IRF8 transcription axis is an acute myeloid leukemia dependency.

Zhendong Cao, Krista A Budinich, Hua Huang, Diqiu Ren, Bin Lu, Zhen Zhang, Qingzhou Chen, Yeqiao Zhou, Yu-Han Huang, Fatemeh Alikarami, Molly C Kingsley, Alexandra K Lenard, Aoi Wakabayashi, Eugene Khandros, Will Bailis, Jun Qi, Martin P Carroll, Gerd A Blobel, Robert B Faryabi, Kathrin M Bernt, Shelley L Berger, Junwei Shi.

  The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML.

Keywords:  IRF8; MEF2D; ZMYND8; acute myeloid leukemia; epigenetics; transcriptional addiction

DOI:  https://doi.org/10.1016/j.molcel.2021.07.018
Sci Rep. 2021 Aug 02. 11(1): 15668

The role of epigenetic modifications, long-range contacts, enhancers and topologically associating domains in the regulation of glioma grade-specific genes.

Ilona E Grabowicz, Bartek Wilczyński, Bożena Kamińska, Adria-Jaume Roura, Bartosz Wojtaś, Michał J Dąbrowski.

Genome-wide studies have uncovered specific genetic alterations, transcriptomic patterns and epigenetic profiles associated with different glioma types. We have recently created a unique atlas encompassing genome-wide profiles of open chromatin, histone H3K27ac and H3Kme3 modifications, DNA methylation and transcriptomes of 33 glioma samples of different grades. Here, we intersected genome-wide atlas data with topologically associating domains (TADs) and demonstrated that the chromatin organization and epigenetic landscape of enhancers have a strong impact on genes differentially expressed in WHO low grade versus high grade gliomas. We identified TADs enriched in glioma grade-specific genes and/or epigenetic marks. We found the set of transcription factors, including REST, E2F1 and NFKB1, that are most likely to regulate gene expression in multiple TADs, containing specific glioma-related genes. Moreover, many genes associated with the cell-matrix adhesion Gene Ontology group, in particular 14 PROTOCADHERINs, were found to be regulated by long-range contacts with enhancers. Presented results demonstrate the existence of epigenetic differences associated with chromatin organization driving differential gene expression in gliomas of different malignancy.

DOI: https://doi.org/10.1038/s41598-021-95009-3
Genome Biol. 2021 Aug 03. 22(1): 218

Systematic assessment of gene co-regulation within chromatin domains determines differentially active domains across human cancers.

Marie Zufferey, Yuanlong Liu, Daniele Tavernari, Marco Mina, Giovanni Ciriello.

   BACKGROUND: Spatial interactions and insulation of chromatin regions are associated with transcriptional regulation. Domains of frequent chromatin contacts are proposed as functional units, favoring and delimiting gene regulatory interactions. However, contrasting evidence supports the association between chromatin domains and transcription.
RESULT: Here, we assess gene co-regulation in chromatin domains across multiple human cancers, which exhibit great transcriptional heterogeneity. Across all datasets, gene co-regulation is observed only within a small yet significant number of chromatin domains. We design an algorithmic approach to identify differentially active domains (DADo) between two conditions and show that these provide complementary information to differentially expressed genes. Domains comprising co-regulated genes are enriched in the less active B sub-compartments and for genes with similar function. Notably, differential activation of chromatin domains is not associated with major changes of domain boundaries, but rather with changes of sub-compartments and intra-domain contacts.
CONCLUSION: Overall, gene co-regulation is observed only in a minority of chromatin domains, whose systematic identification will help unravel the relationship between chromatin structure and transcription.

Keywords:  Chromatin compartment domains; Gene co-regulation; Hi-C

DOI:  https://doi.org/10.1186/s13059-021-02436-6
Blood Adv. 2021 Aug 10. 5(15): 3002-3015

Identification of the transcription factor MAZ as a regulator of erythropoiesis.

Darya Deen, Falk Butter, Deborah E Daniels, Ivan Ferrer-Vicens, Daniel C J Ferguson, Michelle L Holland, Vasiliki Samara, Jacqueline A Sloane-Stanley, Helena Ayyub, Matthias Mann, Jan Frayne, David Garrick, Douglas Vernimmen.

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.

DOI: https://doi.org/10.1182/bloodadvances.2021004609
Nat Cell Biol. 2021 Aug 02.

LKB1 inactivation modulates chromatin accessibility to drive metastatic progression.

Sarah E Pierce, Jeffrey M Granja, M Ryan Corces, Jennifer J Brady, Min K Tsai, Aubrey B Pierce, Rui Tang, Pauline Chu, David M Feldser, Howard Y Chang, Michael C Bassik, William J Greenleaf, Monte M Winslow.

Metastasis is the leading cause of cancer-related deaths and enables cancer cells to compromise organ function by expanding in secondary sites. Since primary tumours and metastases often share the same constellation of driver mutations, the mechanisms that drive their distinct phenotypes are unclear. Here we show that inactivation of the frequently mutated tumour suppressor gene LKB1 (encoding liver kinase B1) has evolving effects throughout the progression of lung cancer, which leads to the differential epigenetic re-programming of early-stage primary tumours compared with late-stage metastases. By integrating genome-scale CRISPR-Cas9 screening with bulk and single-cell multi-omic analyses, we unexpectedly identify LKB1 as a master regulator of chromatin accessibility in lung adenocarcinoma primary tumours. Using an in vivo model of metastatic progression, we further show that loss of LKB1 activates the early endoderm transcription factor SOX17 in metastases and a metastatic-like sub-population of cancer cells within primary tumours. The expression of SOX17 is necessary and sufficient to drive a second wave of epigenetic changes in LKB1-deficient cells that enhances metastatic ability. Overall, our study demonstrates how the downstream effects of an individual driver mutation can change throughout cancer development, with implications for stage-specific therapeutic resistance mechanisms and the gene regulatory underpinnings of metastatic evolution.

DOI: https://doi.org/10.1038/s41556-021-00728-4
Cancer Res. 2021 Aug 05. pii: canres.0649.2021. [Epub ahead of print]

miR-766-5p targets super-enhancers by downregulating CBP and BRD4.

Yasuyuki Gen, Tomoki Muramatsu, Jun Inoue, Johji Inazawa.

Super-enhancers (SE) are clusters of transcription enhancers that drive gene expression. SEs are typically characterized by high levels of acetylation of histone H3 lysine 27 (H3K27ac), which is catalyzed by the histone lysine acetyltransferase CREB binding protein (CBP). Cancer cells frequently acquire tumor-specific SEs at key oncogenes, such as MYC, which induce several hallmarks of cancer. BRD4 is recruited to SEs and consequently functions as an epigenetic reader to promote transcription of SE-marked genes in cancer cells. miRNAs can be potent candidates for nucleic acid therapeutics for cancer. We previously identified miR-766-5p as a miRNA that downregulated MYC expression and inhibited cancer cell growth in vitro. In this study, we show that miR-766-5p directly targets CBP and BRD4. Concurrent suppression of CBP and BRD4 cooperatively downregulated MYC expression in cancer cells but not in normal cells. Chromatin immunoprecipitation analysis revealed that miR-766-5p reduced levels of H3K27ac at MYC SEs via CBP suppression. Moreover, miR-766-5p suppressed expression of a BRD4-NUT fusion protein that drives NUT midline carcinoma (NMC). In vivo administration of miR-766-5p suppressed tumor growth in two xenograft models. Collectively, these data suggest that targeting SEs using miR-766-5p-based therapeutics may serve as an effective strategy for the treatment of MYC-driven cancers.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-0649
Int J Mol Sci. 2021 Jul 28. pii: 8067. [Epub ahead of print]22(15):

K-mer Content Changes with Node Degree in Promoter-Enhancer Network of Mouse ES Cells.

Kinga Szyman, Bartek Wilczyński, Michał Dąbrowski.

  Maps of Hi-C contacts between promoters and enhancers can be analyzed as networks, with cis-regulatory regions as nodes and their interactions as edges. We checked if in the published promoter-enhancer network of mouse embryonic stem (ES) cells the differences in the node type (promoter or enhancer) and the node degree (number of regions interacting with a given promoter or enhancer) are reflected by sequence composition or sequence similarity of the interacting nodes. We used counts of all k-mers (k = 4) to analyze the sequence composition and the Euclidean distance between the k-mer count vectors (k-mer distance) as the measure of sequence (dis)similarity. The results we obtained with 4-mers are interpretable in terms of dinucleotides. Promoters are GC-rich as compared to enhancers, which is known. Enhancers are enriched in scaffold/matrix attachment regions (S/MARs) patterns and depleted of CpGs. Furthermore, we show that promoters are more similar to their interacting enhancers than vice-versa. Most notably, in both promoters and enhancers, the GC content and the CpG count increase with the node degree. As a consequence, enhancers of higher node degree become more similar to promoters, whereas higher degree promoters become less similar to enhancers. We confirmed the key results also for human keratinocytes.

Keywords:  4-mer; CpG; Hi-C; S/MAR; dinucleotide; embryonic stem cell

DOI:  https://doi.org/10.3390/ijms22158067
Nat Commun. 2021 Aug 06. 12(1): 4778

m6Am-seq reveals the dynamic m6Am methylation in the human transcriptome.

Hanxiao Sun, Kai Li, Xiaoting Zhang, Jun'e Liu, Meiling Zhang, Haowei Meng, Chengqi Yi.

N6,2'-O-dimethyladenosine (m6Am), a terminal modification adjacent to the mRNA cap, is a newly discovered reversible RNA modification. Yet, a specific and sensitive tool to directly map transcriptome-wide m6Am is lacking. Here, we report m6Am-seq, based on selective in vitro demethylation and RNA immunoprecipitation. m6Am-seq directly distinguishes m6Am and 5'-UTR N6-methyladenosine (m6A) and enables the identification of m6Am at single-base resolution and 5'-UTR m6A in the human transcriptome. Using m6Am-seq, we also find that m6Am and 5'-UTR m6A respond dynamically to stimuli, and identify key functional methylation sites that may facilitate cellular stress response. Collectively, m6Am-seq reveals the high-confidence m6Am and 5'-UTR m6A methylome and provides a robust tool for functional studies of the two epitranscriptomic marks.

DOI: https://doi.org/10.1038/s41467-021-25105-5
Curr Protoc. 2021 Aug;1(8): e174

In situ Chromatin Interaction Analysis Using Paired-End Tag Sequencing.

Ping Wang, Yuliang Feng, Kun Zhu, Haoxi Chai, Ya-Ting Chang, Xiaofei Yang, Xiyuan Liu, Chen Shen, Eva Gaga, Byoungkoo Lee, Minji Kim, Xiaoan Ruan, Yijun Ruan.

  Chromatin Interaction Analysis Using Paired-End Tag Sequencing (ChIA-PET) is an established method to map protein-mediated chromatin interactions. A limitation, however, is that it requires a hundred million cells per experiment, which hampers its broad application in biomedical research, particularly in studies in which it is impractical to obtain a large number of cells from rare samples. To reduce the required input cell number while retaining high data quality, we developed an in situ ChIA-PET protocol, which requires as few as 1 million cells. Here, we describe detailed step-by-step procedures for performing in situ ChIA-PET from cultured cells, including both an experimental protocol for sample preparation and data generation and a computational protocol for data processing and visualization using the ChIA-PIPE pipeline. As the protocol significantly simplifies the experimental procedure, reduces ligation noise, and decreases the required input of cells compared to previous versions of ChIA-PET protocols, it can be applied to generate high-resolution chromatin contact maps mediated by various protein factors for a wide range of human and mouse primary cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and data generation Support Protocol: Bridge linker preparation Basic Protocol 2: Data processing and visualization.

Keywords:  ChIA-PET; chromatin interaction; in situ ChIA-PET

DOI:  https://doi.org/10.1002/cpz1.174
Sci Rep. 2021 Aug 05. 11(1): 15912

Investigating crosstalk between H3K27 acetylation and H3K4 trimethylation in CRISPR/dCas-based epigenome editing and gene activation.

Weiye Zhao, Ying Xu, Yufan Wang, Dan Gao, Jasmine King, Yajie Xu, Fu-Sen Liang.

Epigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions. While histone PTMs have been correlated with certain gene expression status, the causalities remain elusive. Histone H3 Lysine 27 acetylation (H3K27ac) and histone H3 Lysine 4 trimethylation (H3K4me3) are both associated with active genes, and located at active promoters and enhancers or around transcriptional start sites (TSSs). Although crosstalk between histone lysine acetylation and H3K4me3 has been reported, relationships between specific epigenetic marks during transcriptional activation remain largely unclear. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-based epigenome editing methods, we discovered that the ectopic introduction of H3K27ac in the promoter region lead to H3K4me3 enrichment around TSS and transcriptional activation, while H3K4me3 installation at the promoter cannot induce H3K27ac increase and failed to activate gene expression. Blocking the reading of H3K27ac by BRD proteins using inhibitor JQ1 abolished H3K27ac-induced H3K4me3 installation and downstream gene activation. Furthermore, we uncovered that BRD2, not BRD4, mediated H3K4me3 installation and gene activation upon H3K27ac writing. Our studies revealed the relationships between H3K27ac and H3K4me3 in gene activation process and demonstrated the application of CRISPR/dCas-based epigenome editing methods in elucidating the crosstalk between epigenetic mechanisms.

DOI: https://doi.org/10.1038/s41598-021-95398-5
EMBO J. 2021 Aug 02. e107100

De novo DNA methylation controls neuronal maturation during adult hippocampal neurogenesis.

Sara Zocher, Rupert W Overall, Gabriel Berdugo-Vega, Nicole Rund, Anne Karasinsky, Vijay S Adusumilli, Christina Steinhauer, Sina Scheibenstock, Kristian Händler, Joachim L Schultze, Federico Calegari, Gerd Kempermann.

  Adult neurogenesis enables the life-long addition of functional neurons to the hippocampus and is regulated by both cell-intrinsic molecular programs and behavioral activity. De novo DNA methylation is crucial for embryonic brain development, but its role during adult hippocampal neurogenesis has remained unknown. Here, we show that de novo DNA methylation is critical for maturation and functional integration of adult-born neurons in the mouse hippocampus. Bisulfite sequencing revealed that de novo DNA methyltransferases target neuronal enhancers and gene bodies during adult hippocampal neural stem cell differentiation, to establish neuronal methylomes and facilitate transcriptional up-regulation of neuronal genes. Inducible deletion of both de novo DNA methyltransferases Dnmt3a and Dnmt3b in adult neural stem cells did not affect proliferation or fate specification, but specifically impaired dendritic outgrowth and synaptogenesis of newborn neurons, thereby hampering their functional maturation. Consequently, abolishing de novo DNA methylation modulated activation patterns in the hippocampal circuitry and caused specific deficits in hippocampus-dependent learning and memory. Our results demonstrate that proper establishment of neuronal methylomes during adult neurogenesis is fundamental for hippocampal function.

Keywords:  DNA methylation; Dnmt3a; adult neurogenesis; hippocampus; neuron maturation

DOI:  https://doi.org/10.15252/embj.2020107100
Nucleic Acids Res. 2021 Aug 05. pii: gkab644. [Epub ahead of print]

Cryo-EM structure of the nucleosome core particle containing Giardia lamblia histones.

Shoko Sato, Yoshimasa Takizawa, Fumika Hoshikawa, Mariko Dacher, Hiroki Tanaka, Hiroaki Tachiwana, Tomoya Kujirai, Yukari Iikura, Cheng-Han Ho, Naruhiko Adachi, Indu Patwal, Andrew Flaus, Hitoshi Kurumizaka.

Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A-H2B and DNA association with the G. lamblia H3-H4 were weaker than those for human H3-H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability.

DOI: https://doi.org/10.1093/nar/gkab644
Development. 2021 Aug 02. pii: dev.198408. [Epub ahead of print]

osr1 couples intermediate mesoderm cell fate with temporal dynamics of vessel progenitor cell differentiation.

Elliot A Perens, Jessyka T Diaz, Agathe Quesnel, Amjad Askary, J Gage Crump, Deborah Yelon.

  Transcriptional regulatory networks refine gene expression boundaries to define the dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that establish the boundary between the IM and neighboring vessel progenitors are poorly understood. Here, we delineate roles for the zinc finger transcription factor Osr1 in kidney and vessel progenitor development. Zebrafish osr1 mutants display decreased IM formation and premature emergence of lateral vessel progenitors (LVPs). These phenotypes contrast with the increased IM and absent LVPs observed with loss of the bHLH transcription factor Hand2, and loss of hand2 partially suppresses osr1 mutant phenotypes. hand2 and osr1 are expressed together in the posterior mesoderm, but osr1 expression decreases dramatically prior to LVP emergence. Overexpressing osr1 during this timeframe inhibits LVP development while enhancing IM formation and can rescue the osr1 mutant phenotype. Together, our data demonstrate that osr1 modulates the extent of IM formation and the temporal dynamics of LVP development, suggesting that a balance between levels of osr1 and hand2 expression is essential to demarcate the kidney and vessel progenitor territories.

Keywords:  Intermediate mesoderm; Kidney; Vessel progenitors; Zebrafish; hand2

DOI:  https://doi.org/10.1242/dev.198408
Cancer Res. 2021 Aug 02. pii: canres.CAN-21-1114-A.2021. [Epub ahead of print]

PAX9 determines epigenetic state transition and cell fate in cancer.

Zibo Zhao, Aileen P Szczepanski, Natsumi Tsuboyama, Hiam Abdala-Valencia, Young Ah Goo, Benjamin D Singer, Elizabeth T Bartom, Feng Yue, Lu Wang.

Abnormalities in genetic and epigenetic modifications can lead to drastic changes in gene expression profiles that are associated with various cancer types. Small cell lung cancer (SCLC) is an aggressive and deadly form of lung cancer with limited effective therapies currently available. By utilizing a genome-wide CRISPR-Cas9 dropout screen in SCLC cells, we identified paired box protein 9 (PAX9) as an essential factor that is overexpressed in human malignant SCLC tumor samples and is transcriptionally driven by the BAP1/ASXL3/BRD4 epigenetic axis. Genome-wide studies revealed that PAX9 occupies distal enhancer elements and represses gene expression by restricting enhancer activity. In multiple SCLC cell lines, genetic depletion of PAX9 led to significant induction of a primed-active enhancer transition, resulting in increased expression of a large number of neural differentiation and tumor-suppressive genes. Mechanistically, PAX9 interacted and co-functioned with the nucleosome remodeling and deacetylase (NuRD) complex at enhancers to repress nearby gene expression, which was reversed by pharmacological HDAC inhibition. Overall, this study provides mechanistic insight into the oncogenic function of the PAX9/NuRD complex epigenetic axis in human SCLC and suggests that re-activation of primed enhancers may have potential therapeutic efficacy in treating SCLC expressing high levels of PAX9.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-1114
Nat Genet. 2021 Aug;53(8): 1177-1186

Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin.

Phillip A Doerfler, Ruopeng Feng, Yichao Li, Lance E Palmer, Shaina N Porter, Henry W Bell, Merlin Crossley, Shondra M Pruett-Miller, Yong Cheng, Mitchell J Weiss.

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.

DOI: https://doi.org/10.1038/s41588-021-00904-0
Nat Cell Biol. 2021 Aug 05.

Diverse heterochromatin-associated proteins repress distinct classes of genes and repetitive elements.

Ryan L McCarthy, Kelsey E Kaeding, Samuel H Keller, Yu Zhong, Liqin Xu, Antony Hsieh, Yong Hou, Greg Donahue, Justin S Becker, Oscar Alberto, Bomyi Lim, Kenneth S Zaret.

Heterochromatin, typically marked by histone H3 trimethylation at lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously, we identified 172 proteins that are embedded in sonication-resistant heterochromatin (srHC) harbouring H3K9me3. Here, we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many common genes and repeat elements. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that the enhancer of rudimentary homologue (ERH) is conserved from Schizosaccharomyces pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.

DOI: https://doi.org/10.1038/s41556-021-00725-7
PLoS Pathog. 2021 Aug 05. 17(8): e1009834

EBNA2 driven enhancer switching at the CIITA-DEXI locus suppresses HLA class II gene expression during EBV infection of B-lymphocytes.

Chenhe Su, Fang Lu, Samantha S Soldan, R Jason Lamontagne, Hsin-Yao Tang, Giorgia Napoletani, Paul J Farrell, Italo Tempera, Andrew V Kossenkov, Paul M Lieberman.

Viruses suppress immune recognition through diverse mechanisms. Epstein-Barr Virus (EBV) establishes latent infection in memory B-lymphocytes and B-cell malignancies where it impacts B-cell immune function. We show here that EBV primary infection of naïve B-cells results in a robust down-regulation of HLA genes. We found that the viral encoded transcriptional regulatory factor EBNA2 bound to multiple regulatory regions in the HLA locus. Conditional expression of EBNA2 correlated with the down regulation of HLA class II transcription. EBNA2 down-regulation of HLA transcription was found to be dependent on CIITA, the major transcriptional activator of HLA class II gene transcription. We identified a major EBNA2 binding site downstream of the CIITA gene and upstream of DEXI, a dexamethasone inducible gene that is oriented head-to-head with CIITA gene transcripts. CRISPR/Cas9 deletion of the EBNA2 site upstream of DEXI attenuated CIITA transcriptional repression. EBNA2 caused an increase in DEXI transcription and a graded change in histone modifications with activation mark H3K27ac near the DEXI locus, and a loss of activation marks at the CIITA locus. A prominent CTCF binding site between CIITA and DEXI enhancers was mutated and further diminished the effects of EBNA2 on CIITA. Analysis of HiC data indicate that DEXI and CIITA enhancers are situated in different chromosome topological associated domains (TADs). These findings suggest that EBNA2 down regulates HLA-II genes through the down regulation of CIITA, and that this down regulation is an indirect consequence of EBNA2 enhancer formation at a neighboring TAD. We propose that enhancer competition between these neighboring chromosome domains represents a novel mechanism for gene regulation demonstrated by EBNA2.

DOI: https://doi.org/10.1371/journal.ppat.1009834
EMBO Rep. 2021 Aug 04. 22(8): e52462

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Xuanzhao Jiang, Jiayu Wen, Elizabeth Paver, Yu-Huan Wu, Gege Sun, Amanda Bullman, Jane E Dahlstrom, David J Tremethick, Tatiana A Soboleva.

  Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

Keywords:  H2A.B; RNA polymerase I and II; chromatin; histone variants; transcription

DOI:  https://doi.org/10.15252/embr.202152462
Nature. 2021 Aug 04.

Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma.

Davide G Berta, Heli Kuisma, Niko Välimäki, Maritta Räisänen, Maija Jäntti, Annukka Pasanen, Auli Karhu, Jaana Kaukomaa, Aurora Taira, Tatiana Cajuso, Sanna Nieminen, Rosa-Maria Penttinen, Saija Ahonen, Rainer Lehtonen, Miika Mehine, Pia Vahteristo, Jyrki Jalkanen, Biswajyoti Sahu, Janne Ravantti, Netta Mäkinen, Kristiina Rajamäki, Kimmo Palin, Jussi Taipale, Oskari Heikinheimo, Ralf Bützow, Eevi Kaasinen, Lauri A Aaltonen.

One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility1, and are a common cause of hysterectomy2. They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA23. Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex4, and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice5. Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.

DOI: https://doi.org/10.1038/s41586-021-03747-1
Sci Adv. 2021 Aug;pii: eabi5987. [Epub ahead of print]7(32):

Differential H4K16ac levels ensure a balance between quiescence and activation in hematopoietic stem cells.

Cecilia Pessoa Rodrigues, Asifa Akhtar.

Hematopoietic stem cells (HSCs) are able to reconstitute the bone marrow while retaining their self-renewal property. Individual HSCs demonstrate heterogeneity in their repopulating capacities. Here, we found that the levels of the histone acetyltransferase MOF (males absent on the first) and its target modification histone H4 lysine 16 acetylation are heterogeneous among HSCs and influence their proliferation capacities. The increased proliferative capacities of MOF-depleted cells are linked to their expression of CD93. The CD93+ HSC subpopulation simultaneously shows transcriptional features of quiescent HSCs and functional features of active HSCs. CD93+ HSCs were expanded and exhibited an enhanced proliferative advantage in Mof +/- animals reminiscent of a premalignant state. Accordingly, low MOF and high CD93 levels correlate with poor survival and increased proliferation capacity in leukemia. Collectively, our study indicates H4K16ac as an important determinant for HSC heterogeneity, which is linked to the onset of monocytic disorders.

DOI: https://doi.org/10.1126/sciadv.abi5987
Epigenetics Chromatin. 2021 Jul 31. 14(1): 38

SUMOylated non-canonical polycomb PRC1.6 complex as a prerequisite for recruitment of transcription factor RBPJ.

Małgorzata Sotomska, Robert Liefke, Francesca Ferrante, Heiko Schwederski, Franz Oswald, Tilman Borggrefe.

   BACKGROUND: Notch signaling controls cell fate decisions in many contexts during development and adult stem cell homeostasis and, when dysregulated, leads to carcinogenesis. The central transcription factor RBPJ assembles the Notch coactivator complex in the presence of Notch signaling, and represses Notch target gene expression in its absence.
RESULTS: We identified L3MBTL2 and additional members of the non-canonical polycomb repressive PRC1.6 complex in DNA-bound RBPJ associated complexes and demonstrate that L3MBTL2 directly interacts with RBPJ. Depletion of RBPJ does not affect occupancy of PRC1.6 components at Notch target genes. Conversely, absence of L3MBTL2 reduces RBPJ occupancy at enhancers of Notch target genes. Since L3MBTL2 and additional members of the PRC1.6 are known to be SUMOylated, we investigated whether RBPJ uses SUMO-moieties as contact points. Indeed, we found that RBPJ binds to SUMO2/3 and that this interaction depends on a defined SUMO-interaction motif. Furthermore, we show that pharmacological inhibition of SUMOylation reduces RBPJ occupancy at Notch target genes.
CONCLUSIONS: We propose that the PRC1.6 complex and its conjugated SUMO-modifications provide a favorable environment for binding of RBPJ to Notch target genes.

Keywords:  Epigenetics; Notch signaling; Polycomb repressive complex; RBPJ; Sumoylation

DOI:  https://doi.org/10.1186/s13072-021-00412-9
Nat Commun. 2021 Aug 06. 12(1): 4763

Sc-compReg enables the comparison of gene regulatory networks between conditions using single-cell data.

Zhana Duren, Wenhui Sophia Lu, Joseph G Arthur, Preyas Shah, Jingxue Xin, Francesca Meschi, Miranda Lin Li, Corey M Nemec, Yifeng Yin, Wing Hung Wong.

The comparison of gene regulatory networks between diseased versus healthy individuals or between two different treatments is an important scientific problem. Here, we propose sc-compReg as a method for the comparative analysis of gene expression regulatory networks between two conditions using single cell gene expression (scRNA-seq) and single cell chromatin accessibility data (scATAC-seq). Our software, sc-compReg, can be used as a stand-alone package that provides joint clustering and embedding of the cells from both scRNA-seq and scATAC-seq, and the construction of differential regulatory networks across two conditions. We apply the method to compare the gene regulatory networks of an individual with chronic lymphocytic leukemia (CLL) versus a healthy control. The analysis reveals a tumor-specific B cell subpopulation in the CLL patient and identifies TOX2 as a potential regulator of this subpopulation.

DOI: https://doi.org/10.1038/s41467-021-25089-2
Oncogenesis. 2021 Aug 06. 10(8): 58

E2F1 and epigenetic modifiers orchestrate breast cancer progression by regulating oxygen-dependent ESRP1 expression.

Cheemala Ashok, Neha Ahuja, Subhashis Natua, Jharna Mishra, Atul Samaiya, Sanjeev Shukla.

Epithelial splicing regulatory protein 1 (ESRP1) is an RNA binding protein that governs the alternative splicing events related to epithelial phenotypes. ESRP1 contributes significantly at different stages of cancer progression. ESRP1 expression is substantially elevated in carcinoma in situ compared to the normal epithelium, whereas it is drastically ablated in cancer cells within hypoxic niches, which promotes epithelial to mesenchymal transition (EMT). Although a considerable body of research sought to understand the EMT-associated ESRP1 downregulation, the regulatory mechanisms underlying ESRP1 upregulation in primary tumors remained largely uncharted. This study seeks to unveil the regulatory mechanisms that spatiotemporally fine-tune the ESRP1 expression during breast carcinogenesis. Our results reveal that an elevated expression of transcription factor E2F1 and increased CpG hydroxymethylation of the E2F1 binding motif conjointly induce ESRP1 expression in breast carcinoma. However, E2F1 fails to upregulate ESRP1 despite its abundance in oxygen-deprived breast cancer cells. Mechanistically, impelled by the hypoxia-driven reduction of tet methylcytosine dioxygenase 3 (TET3) activity, CpG sites across the E2F1 binding motif lose the hydroxymethylation marks while gaining the de novo methyltransferase-elicited methylation marks. These two oxygen-sensitive epigenetic events work in concert to repel E2F1 from the ESRP1 promoter, thereby diminishing ESRP1 expression under hypoxia. Furthermore, E2F1 skews the cancer spliceome by upregulating splicing factor SRSF7 in hypoxic breast cancer cells. Our findings provide previously unreported mechanistic insights into the plastic nature of ESRP1 expression and insinuate important implications in therapeutics targeting breast cancer progression.

DOI: https://doi.org/10.1038/s41389-021-00347-6
Mol Metab. 2021 Aug 02. pii: S2212-8778(21)00160-5. [Epub ahead of print] 101313

Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network.

Valérie Schreiber, Reuben Mercier, Sara Jiménez, Tao Ye, Emmanuel García-Sánchez, Annabelle Klein, Aline Meunier, Sabitri Ghimire, Catherine Birck, Bernard Jost, Kristian Honnens de Lichtenberg, Christian Honoré, Palle Serup, Gérard Gradwohl.

   OBJECTIVE: Mice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin secreting beta cells, causing diabetes. In human, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function and downstream genetic programs regulated directly by NEUROG3 remain elusive. Therefore, we mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (iPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets.
METHODS: We generated a novel hiPSC line (NEUROG3-HA-P2A-Venus), where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs and their NEUROG3-dependence. In addition, we searched whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage.
RESULTS: CUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1263 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements frequently overlapping within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA or RFX transcription factors. We found that 22% of the genes downregulated in NEUROG3-/- PEPs, and 10% of genes enriched in NEUROG3-Venus positive endocrine cells are bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 binds to 138 transcription factor genes, some with important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself, as well as many others of unknown islet function. Unexpectedly, we uncovered that NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8 and PCSK1). Thus, analysis of NEUROG3 occupancy suggests that the transient expression of NEUROG3 not only promotes islet destiny in uncommitted pancreatic progenitors but could also initiate endocrine programs essential for beta-cell function. Lastly, we identified eight T2DM risk SNPs within NEUROG3 bound regions.
CONCLUSION: Mapping of NEUROG3 genome occupancy in PEPs uncovered an unexpectedly broad, direct control of the endocrine genes, raising novel hypotheses on how this master regulator controls islet and beta cell differentiation.

Keywords:  CUT&RUN; NEUROG3; SNPs; T2DM; iPSC; islet progenitors

DOI:  https://doi.org/10.1016/j.molmet.2021.101313
Int J Mol Sci. 2021 Aug 03. pii: 8337. [Epub ahead of print]22(15):

Regulation Network of Colorectal-Cancer-Specific Enhancers in the Progression of Colorectal Cancer.

Bohan Chen, Yiping Ma, Jinfang Bi, Wenbin Wang, Anshun He, Guangsong Su, Zhongfang Zhao, Jiandang Shi, Lei Zhang.

  Enhancers regulate multiple genes via higher-order chromatin structures, and they further affect cancer progression. Epigenetic changes in cancer cells activate several cancer-specific enhancers that are silenced in normal cells. These cancer-specific enhancers are potential therapeutic targets of cancer. However, the functions and regulation networks of colorectal-cancer-specific enhancers are still unknown. In this study, we profile colorectal-cancer-specific enhancers and reveal their regulation network through the analysis of HiChIP data that were derived from a colorectal cancer cell line and Hi-C and RNA-seq data that were derived from tissue samples by in silico analysis and in vitro experiments. Enhancer-promoter loops in colorectal cancer cells containing colorectal-cancer-specific enhancers are involved in more than 50% of the topological associated domains (TADs) changed in colorectal cancer cells compared to normal colon cells. In addition, colorectal-cancer-specific enhancers interact with 152 genes that are significantly and highly expressed in colorectal cancer cells. These colorectal-cancer-specific enhancer target genes include ITGB4, RECQL4, MSLN, and GDF15. We propose that the regulation network of colorectal-cancer-specific enhancers plays an important role in the progression of colorectal cancer.

Keywords:  Hi-C; HiChIP; TAD; colorectal-cancer-specific enhancer; long-range interaction

DOI:  https://doi.org/10.3390/ijms22158337
J Cell Sci. 2021 Aug 06. pii: jcs.258578. [Epub ahead of print]

The oncogenic transcription factor FUS-CHOP can undergo nuclear liquid-liquid phase separation.

Izzy Owen, Debra Yee, Hala Wyne, Theodora Myrto Perdikari, Victoria Johnson, Jeremy Smyth, Robert Kortum, Nicolas L Fawzi, Frank Shewmaker.

  Myxoid liposarcoma is caused by a chromosomal translocation resulting in a fusion protein comprised of the N-terminus of FUS (fused in sarcoma) and the full-length transcription factor CHOP (CCAAT/Enhancer Binding Protein Homologous Protein). FUS functions in RNA metabolism and CHOP is a stress-induced transcription factor. The FUS-CHOP fusion protein causes unique gene expression and oncogenic transformation. Though it is clear the FUS segment is required for oncogenic transformation, the mechanism of FUS-CHOP-induced transcriptional activation is unknown. Recently, some transcription factors and super enhancers were proposed to undergo liquid-liquid phase separation and form membraneless compartments that recruit transcription machinery to gene promoters. Since phase separation of FUS depends on its N-terminus, transcriptional activation by FUS-CHOP could result from the N-terminus driving nuclear phase transitions. Here, we characterized FUS-CHOP in cells and in vitro, and observed novel phase-separating properties relative to unmodified CHOP. Our data indicate FUS-CHOP forms phase-separated condensates that colocalize with BRD4, a marker of super enhancer condensates. We provide evidence that the FUS-CHOP phase transition is a novel oncogenic mechanism and potential therapeutic target for myxoid liposarcoma.

Keywords:  CHOP; FUS; Liquid-liquid Phase Separation; Oncogenic Fusion Protein; Transcriptional Activation

DOI:  https://doi.org/10.1242/jcs.258578
Cells. 2021 Jul 12. pii: 1757. [Epub ahead of print]10(7):

FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes.

Miriam Pagin, Mattias Pernebrink, Mattia Pitasi, Federica Malighetti, Chew-Yee Ngan, Sergio Ottolenghi, Giulio Pavesi, Claudio Cantù, Silvia K Nicolis.

  The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC's long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a "mixed" neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the "neuronal" genes are bound by both SOX2 and AP1.

Keywords:  CUT&RUN; Fos; Socs3; Sox2; chromatin; gliogenesis; neural stem cells; neurogenesis; transcription factors

DOI:  https://doi.org/10.3390/cells10071757
Nat Protoc. 2021 Aug 06.

Genome-wide and sister chromatid-resolved profiling of protein occupancy in replicated chromatin with ChOR-seq and SCAR-seq.

Nataliya Petryk, Nazaret Reverón-Gómez, Cristina González-Aguilera, Maria Dalby, Robin Andersson, Anja Groth.

Elucidating the mechanisms underlying chromatin maintenance upon genome replication is critical for the understanding of how gene expression programs and cell identity are preserved across cell divisions. Here, we describe two recently developed techniques, chromatin occupancy after replication (ChOR)-seq and sister chromatids after replication (SCAR)-seq, that profile chromatin occupancy on newly replicated DNA in mammalian cells in 5 d of bench work. Both techniques share a common strategy that includes pulse labeling of newly synthesized DNA and chromatin immunoprecipitation (ChIP), followed by purification and high-throughput sequencing. Whereas ChOR-seq quantitatively profiles the post-replicative abundance of histone modifications and chromatin-associated proteins, SCAR-seq distinguishes chromatin occupancy between nascent sister chromatids. Together, these two complementary techniques have unraveled key mechanisms controlling the inheritance of modified histones during replication and revealed locus-specific dynamics of histone modifications across the cell cycle. Here, we provide the experimental protocols and bioinformatic pipelines for these methods.

DOI: https://doi.org/10.1038/s41596-021-00585-3
Dev Cell. 2021 Jul 27. pii: S1534-5807(21)00559-1. [Epub ahead of print]

Enhancer decommissioning imposes an epigenetic barrier to sensory hair cell regeneration.

Litao Tao, Haoze V Yu, Juan Llamas, Talon Trecek, Xizi Wang, Zlatka Stojanova, Andrew K Groves, Neil Segil.

  Adult mammalian tissues such as heart, brain, retina, and the sensory structures of the inner ear do not effectively regenerate, although a latent capacity for regeneration exists at embryonic and perinatal times. We explored the epigenetic basis for this latent regenerative potential in the mouse inner ear and its rapid loss during maturation. In perinatal supporting cells, whose fate is maintained by Notch-mediated lateral inhibition, the hair cell enhancer network is epigenetically primed (H3K4me1) but silenced (active H3K27 de-acetylation and trimethylation). Blocking Notch signaling during the perinatal period of plasticity rapidly eliminates epigenetic silencing and allows supporting cells to transdifferentiate into hair cells. Importantly, H3K4me1 priming of the hair cell enhancers in supporting cells is removed during the first post-natal week, coinciding with the loss of transdifferentiation potential. We hypothesize that enhancer decommissioning during cochlear maturation contributes to the failure of hair cell regeneration in the mature organ of Corti.

Keywords:  ATOH1 targetome; H3K4me1; enhancer decommissioning; epigenetics; inner ear; maturation; regeneration; sensory hair cell; transdifferentiation potential

DOI:  https://doi.org/10.1016/j.devcel.2021.07.003
PLoS Biol. 2021 Aug 05. 19(8): e3001364

The Δ40p53 isoform inhibits p53-dependent eRNA transcription and enables regulation by signal-specific transcription factors during p53 activation.

Cecilia B Levandowski, Taylor Jones, Margaret Gruca, Sivapriya Ramamoorthy, Robin D Dowell, Dylan J Taatjes.

The naturally occurring Δ40p53 isoform heterotetramerizes with wild-type p53 (WTp53) to regulate development, aging, and stress responses. How Δ40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with a well-tested strategy that expressed Δ40p53:WTp53 as a single transcript, ensuring a 2:2 tetramer stoichiometry. Human MCF10A cell lines expressing Δ40p53:WTp53, WTp53, or WTp53:WTp53 (as controls) from the native TP53 locus were examined with transcriptomics (precision nuclear run-on sequencing [PRO-seq] and RNA sequencing [RNA-seq]), metabolomics, and other methods. Δ40p53:WTp53 was transcriptionally active, and, although phenotypically similar to WTp53 under normal conditions, it failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via Δ40p53:WTp53-dependent inhibition of enhancer RNA (eRNA) transcription and subsequent failure to induce mRNA biogenesis, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil [5FU]) also showed Δ40p53:WTp53-specific changes in mRNA induction; however, other transcription factors (TFs; e.g., E2F2) could then drive the response, yielding similar outcomes vs. WTp53. Our results establish that Δ40p53 tempers WTp53 function to enable compensatory responses by other stimulus-specific TFs. Such modulation of WTp53 activity may be an essential physiological function for Δ40p53. Moreover, Δ40p53:WTp53 functional distinctions uncovered herein suggest an eRNA requirement for mRNA biogenesis and that human p53 evolved as a tetramer to support eRNA transcription.

DOI: https://doi.org/10.1371/journal.pbio.3001364
Int J Mol Sci. 2021 Jul 29. pii: 8123. [Epub ahead of print]22(15):

HERON: A Novel Tool Enables Identification of Long, Weakly Enriched Genomic Domains in ChIP-seq Data.

Anna Macioszek, Bartek Wilczynski.

  The explosive development of next-generation sequencing-based technologies has allowed us to take an unprecedented look at many molecular signatures of the non-coding genome. In particular, the ChIP-seq (Chromatin ImmunoPrecipitation followed by sequencing) technique is now very commonly used to assess the proteins associated with different non-coding DNA regions genome-wide. While the analysis of such data related to transcription factor binding is relatively straightforward, many modified histone variants, such as H3K27me3, are very important for the process of gene regulation but are very difficult to interpret. We propose a novel method, called HERON (HiddEn MaRkov mOdel based peak calliNg), for genome-wide data analysis that is able to detect DNA regions enriched for a certain feature, even in difficult settings of weakly enriched long DNA domains. We demonstrate the performance of our method both on simulated and experimental data.

Keywords:  ChIP-seq; histone methylation; peak calling

DOI:  https://doi.org/10.3390/ijms22158123
Sci Adv. 2021 Aug;pii: eabd2605. [Epub ahead of print]7(32):

Repurposing RNA sequencing for discovery of RNA modifications in clinical cohorts.

Kar-Tong Tan, Ling-Wen Ding, Chan-Shuo Wu, Daniel G Tenen, Henry Yang.

The study of RNA modifications in large clinical cohorts can reveal relationships between the epitranscriptome and human diseases, although this is especially challenging. We developed ModTect (https://github.com/ktan8/ModTect), a statistical framework to identify RNA modifications de novo by standard RNA-sequencing with deletion and mis-incorporation signals. We show that ModTect can identify both known (N 1-methyladenosine) and previously unknown types of mRNA modifications (N 2,N 2-dimethylguanosine) at nucleotide-resolution. Applying ModTect to 11,371 patient samples and 934 cell lines across 33 cancer types, we show that the epitranscriptome was dysregulated in patients across multiple cancer types and was additionally associated with cancer progression and survival outcomes. Some types of RNA modification were also more disrupted than others in patients with cancer. Moreover, RNA modifications contribute to multiple types of RNA-DNA sequence differences, which unexpectedly escape detection by Sanger sequencing. ModTect can thus be used to discover associations between RNA modifications and clinical outcomes in patient cohorts.

DOI: https://doi.org/10.1126/sciadv.abd2605
J Cell Sci. 2021 Aug 01. pii: jcs251371. [Epub ahead of print]134(15):

A dual role of YAP in driving TGFβ-mediated endothelial-to-mesenchymal transition.

Cecilia Savorani, Matteo Malinverno, Roberta Seccia, Claudio Maderna, Monica Giannotta, Linda Terreran, Eleonora Mastrapasqua, Stefano Campaner, Elisabetta Dejana, Costanza Giampietro.

  Endothelial-to-mesenchymal transition (EndMT) is the biological process through which endothelial cells transdifferentiate into mesenchymal cells. During embryo development, EndMT regulates endocardial cushion formation via TGFβ/BMP signaling. In adults, EndMT is mainly activated during pathological conditions. Hence, it is necessary to characterize molecular regulators cooperating with TGFβ signaling in driving EndMT, to identify potential novel therapeutic targets to treat these pathologies. Here, we studied YAP, a transcriptional co-regulator involved in several biological processes, including epithelial-to-mesenchymal transition (EMT). As EndMT is the endothelial-specific form of EMT, and YAP (herein referring to YAP1) and TGFβ signaling cross-talk in other contexts, we hypothesized that YAP contributes to EndMT by modulating TGFβ signaling. We demonstrate that YAP is required to trigger TGFβ-induced EndMT response, specifically contributing to SMAD3-driven EndMT early gene transcription. We provide novel evidence that YAP acts as SMAD3 transcriptional co-factor and prevents GSK3β-mediated SMAD3 phosphorylation, thus protecting SMAD3 from degradation. YAP is therefore emerging as a possible candidate target to inhibit pathological TGFβ-induced EndMT at early stages.

Keywords:  Endothelial-to-mesenchymal transition; SMAD3; TGFβ pathway; YAP

DOI:  https://doi.org/10.1242/jcs.251371
Proc Natl Acad Sci U S A. 2021 Aug 10. pii: e2021998118. [Epub ahead of print]118(32):

The chromatin-binding domain of Ki-67 together with p53 protects human chromosomes from mitotic damage.

Osama Garwain, Xiaoming Sun, Divya Ramalingam Iyer, Rui Li, Lihua Julie Zhu, Paul D Kaufman.

  Vertebrate mammals express a protein called Ki-67 which is most widely known as a clinically useful marker of highly proliferative cells. Previous studies of human cells indicated that acute depletion of Ki-67 can elicit a delay at the G1/S boundary of the cell cycle, dependent on induction of the checkpoint protein p21. Consistent with those observations, we show here that acute Ki-67 depletion causes hallmarks of DNA damage, and the damage occurs even in the absence of checkpoint signaling. This damage is not observed in cells traversing S phase but is instead robustly detected in mitotic cells. The C-terminal chromatin-binding domain of Ki-67 is necessary and sufficient to protect cells from this damage. We also observe synergistic effects when Ki-67 and p53 are simultaneously depleted, resulting in increased levels of chromosome bridges at anaphase, followed by the appearance of micronuclei. Therefore, these studies identify the C terminus of Ki-67 as an important module for genome stability.

Keywords:  DNA damage; chromatin; mitosis

DOI:  https://doi.org/10.1073/pnas.2021998118