bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2021–10–31
twenty-one papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit
  1. Increased ACTL6A occupancy within mSWI/SNF chromatin remodelers drives human squamous cell carcinoma.
  2. A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus.
  3. Sequential in cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster.
  4. Transcriptional cooperation of PBX1 and PAX6 in adult neural progenitor cells.
  5. Brd4 is required for chondrocyte differentiation and endochondral ossification.
  6. Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice.
  7. Multilayer omics analysis reveals a non-classical retinoic acid signaling axis that regulates hematopoietic stem cell identity.
  8. Tumor suppressor TET2 safeguards enhancers from aberrant DNA methylation and epigenetic reprogramming in ERα-positive breast cancer cells.
  9. NKX2-1 controls lung cancer progression by inducing DUSP6 to dampen ERK activity.
  10. Comparison of EM-seq and PBAT methylome library methods for low-input DNA.
  11. Chromatin accessibility and gene expression during adipocyte differentiation identify context-dependent effects at cardiometabolic GWAS loci.
  12. Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes.
  13. PIM1 phosphorylation of the androgen receptor and 14-3-3 ζ regulates gene transcription in prostate cancer.
  14. p53 inactivation unmasks histone methylation-independent WDR5 functions that drive self-renewal and differentiation of pluripotent stem cells.
  15. Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior.
  16. Metabolite discovery through global annotation of untargeted metabolomics data.
  17. Chromatin accessibility analysis identifies the transcription factor ETV5 as a suppressor of adipose tissue macrophage activation in obesity.
  18. BRD4 REGULATES TRANSCRIPTION FACTOR ∆Np63αTO DRIVE A CANCER STEM CELL PHENOTYPE IN SQUAMOUS CELL CARCINOMAS.
  19. HES1 protein oscillations are necessary for neural stem cells to exit from quiescence.
  20. A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells.
  21. Endocrine disrupting chemicals differentially alter intranuclear dynamics and transcriptional activation of estrogen receptor-α.
  1. Mol Cell. 2021 Oct 19. pii: S1097-2765(21)00834-0. [Epub ahead of print]
    Increased ACTL6A occupancy within mSWI/SNF chromatin remodelers drives human squamous cell carcinoma.
    Chiung-Ying Chang, Zohar Shipony, Sherry G Lin, Ann Kuo, Xiaochen Xiong, Kyle M Loh, William J Greenleaf, Gerald R Crabtree.
      Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.
    Keywords:  ACTL6A; BAF complex; SWI/SNF; TEAD; YAP; cancer; chromatin remodeling; polycomb; squamous cell carcinoma; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2021.10.005
  2. Dev Cell. 2021 Oct 26. pii: S1534-5807(21)00805-4. [Epub ahead of print]
    A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus.
    Boaz E Aronson, Laurianne Scourzic, Veevek Shah, Emily Swanzey, Andreas Kloetgen, Alexander Polyzos, Abhishek Sinha, Annabel Azziz, Inbal Caspi, Jiexi Li, Bobbie Pelham-Webb, Rachel A Glenn, Thomas Vierbuchen, Hynek Wichterle, Aristotelis Tsirigos, Meelad M Dawlaty, Matthias Stadtfeld, Effie Apostolou.
      Loss of imprinting (LOI) results in severe developmental defects, but the mechanisms preventing LOI remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) in pluripotent stem cells. We demonstrate that the IG-DMR consists of two antagonistic elements: a paternally methylated CpG island that prevents recruitment of TET dioxygenases and a maternally unmethylated non-canonical enhancer that ensures expression of the Gtl2 lncRNA by counteracting de novo DNA methyltransferases. Genetic or epigenetic editing of these elements leads to distinct LOI phenotypes with characteristic alternations of allele-specific gene expression, DNA methylation, and 3D chromatin topology. Although repression of the Gtl2 promoter results in dysregulated imprinting, the stability of LOI phenotypes depends on the IG-DMR, suggesting a functional hierarchy. These findings establish the IG-DMR as a bipartite control element that maintains imprinting by allele-specific restriction of the DNA (de)methylation machinery.
    Keywords:  DNA methylation; Dlk1-Dio3; Dnmt3; IG-DMR; Tet enzymes; bipartite element; enhancer; epigenome editing; genomic imprinting; pluripotent stem cells
    DOI:  https://doi.org/10.1016/j.devcel.2021.10.004
  3. Genes Dev. 2021 Oct 28.
    Sequential in cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster.
    Ana Rita Amândio, Leonardo Beccari, Lucille Lopez-Delisle, Bénédicte Mascrez, Jozsef Zakany, Sandra Gitto, Denis Duboule.
      Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the misregulation of Hoxd genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions, and some doing both, whereas they all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites among paralogous Hox clusters or between various vertebrates.
    Keywords:  3D chromatin; CRISPR–Cas9; Hox genes; enhancer selection; limb development
    DOI:  https://doi.org/10.1101/gad.348934.121
  4. Sci Rep. 2021 Oct 25. 11(1): 21013
    Transcriptional cooperation of PBX1 and PAX6 in adult neural progenitor cells.
    Ann-Christin Hau, Elise Mommaerts, Vera Laub, Tamara Müller, Gunnar Dittmar, Dorothea Schulte.
      PAX6 is a highly conserved transcription factor and key regulator of several neurogenic processes, including the continuous generation of dopaminergic/GABAergic interneurons in the adult ventricular-subventricular (V-SVZ) neurogenic system in mice. Here we report that PAX6 cooperates with the TALE-homeodomain transcription factor PBX1 in this context. Chromatin-immunoprecipitation showed that PBX1 and PAX6 co-occupy shared genomic binding sites in adult V-SVZ stem- and progenitor cell cultures and mouse embryonic stem cells, while depletion of Pbx1 revealed that association of PAX6 with these sites requires the presence of PBX1. Expression profiling together with viral overexpression or knockdown of Pax6 or Pbx1 identified novel PBX1-PAX6 co-regulated genes, including several transcription factors. Computational modeling of genome wide expression identified novel cross-regulatory networks among these very transcription factors. Taken together, the results presented here highlight the intimate link that exists between PAX6 and TALE-HD family proteins and contribute novel insights into how the orchestrated activity of transcription factors shapes adult V-SVZ neurogenesis.
    DOI:  https://doi.org/10.1038/s41598-021-99968-5
  5. Bone. 2021 Oct 23. pii: S8756-3282(21)00400-2. [Epub ahead of print] 116234
    Brd4 is required for chondrocyte differentiation and endochondral ossification.
    Christopher R Paradise, M Lizeth Galvan, Oksana Pichurin, Sofia Jerez, Eva Kubrova, S Sharare Dehghani, Margarita E Carrasco, Roman Thaler, A Noelle Larson, Andre J van Wijnen, Amel Dudakovic.
      Differentiation of multi-potent mesenchymal stromal cells (MSCs) is directed by the activities of lineage-specific transcription factors and co-factors. A subset of these proteins controls the accessibility of chromatin by recruiting histone acetyl transferases or deacetylases that regulate acetylation of the N-termini of H3 and H4 histone proteins. Bromodomain (BRD) proteins recognize these acetylation marks and recruit the RNA pol II containing transcriptional machinery. Our previous studies have shown that Brd4 is required for osteoblast differentiation in vitro. Here, we investigated the role of Brd4 on endochondral ossification in C57BL/6 mice and chondrogenic differentiation in cell culture models. Conditional loss of Brd4 in the mesenchyme (Brd4 cKO, Brd4fl/fl: Prrx1-Cre) yields smaller mice that exhibit alteration in endochondral ossification. Importantly, abnormal growth plate morphology and delayed long bone formation is observed in juvenile Brd4 cKO mice. One week old Brd4 cKO mice have reduced proliferative and hypertrophic zones within the physis and exhibit a delay in the formation of the secondary ossification center. At the cellular level, Brd4 function is required for chondrogenic differentiation and maturation of both ATDC5 cells and immature mouse articular chondrocytes. Mechanistically, Brd4 loss suppresses Sox9 levels and reduces expression of Sox9 and Runx2 responsive endochondral genes (e.g., Col2a1, Acan, Mmp13 and Sp7/Osx). Collectively, our results indicate that Brd4 is a key epigenetic regulator required for normal chondrogenesis and endochondral ossification.
    Keywords:  Brd4; Epigenetics; Genetic animal model; Growth plate; Histone; Limb patterning
    DOI:  https://doi.org/10.1016/j.bone.2021.116234
  6. Nat Commun. 2021 Oct 27. 12(1): 6212
    Histone H4 lysine 16 acetylation controls central carbon metabolism and diet-induced obesity in mice.
    Cecilia Pessoa Rodrigues, Aindrila Chatterjee, Meike Wiese, Thomas Stehle, Witold Szymanski, Maria Shvedunova, Asifa Akhtar.
      Noncommunicable diseases (NCDs) account for over 70% of deaths world-wide. Previous work has linked NCDs such as type 2 diabetes (T2D) to disruption of chromatin regulators. However, the exact molecular origins of these chronic conditions remain elusive. Here, we identify the H4 lysine 16 acetyltransferase MOF as a critical regulator of central carbon metabolism. High-throughput metabolomics unveil a systemic amino acid and carbohydrate imbalance in Mof deficient mice, manifesting in T2D predisposition. Oral glucose tolerance testing (OGTT) reveals defects in glucose assimilation and insulin secretion in these animals. Furthermore, Mof deficient mice are resistant to diet-induced fat gain due to defects in glucose uptake in adipose tissue. MOF-mediated H4K16ac deposition controls expression of the master regulator of glucose metabolism, Pparg and the entire downstream transcriptional network. Glucose uptake and lipid storage can be reconstituted in MOF-depleted adipocytes in vitro by ectopic Glut4 expression, PPARγ agonist thiazolidinedione (TZD) treatment or SIRT1 inhibition. Hence, chronic imbalance in H4K16ac promotes a destabilisation of metabolism triggering the development of a metabolic disorder, and its maintenance provides an unprecedented regulatory epigenetic mechanism controlling diet-induced obesity.
    DOI:  https://doi.org/10.1038/s41467-021-26277-w
  7. Cell Stem Cell. 2021 Oct 20. pii: S1934-5909(21)00414-8. [Epub ahead of print]
    Multilayer omics analysis reveals a non-classical retinoic acid signaling axis that regulates hematopoietic stem cell identity.
    Katharina Schönberger, Nadine Obier, Mari Carmen Romero-Mulero, Pierre Cauchy, Julian Mess, Polina V Pavlovich, Yu Wei Zhang, Michael Mitterer, Jasmin Rettkowski, Maria-Eleni Lalioti, Karin Jäcklein, Jonathan D Curtis, Betty Féret, Pia Sommerkamp, Claudia Morganti, Keisuke Ito, Norbert B Ghyselinck, Eirini Trompouki, Joerg M Buescher, Erika L Pearce, Nina Cabezas-Wallscheid.
      Hematopoietic stem cells (HSCs) rely on complex regulatory networks to preserve stemness. Due to the scarcity of HSCs, technical challenges have limited our insights into the interplay between metabolites, transcription, and the epigenome. In this study, we generated low-input metabolomics, transcriptomics, chromatin accessibility, and chromatin immunoprecipitation data , revealing distinct metabolic hubs that are enriched in HSCs and their downstream multipotent progenitors. Mechanistically, we uncover a non-classical retinoic acid (RA) signaling axis that regulates HSC function. We show that HSCs rely on Cyp26b1, an enzyme conventionally considered to limit RA effects in the cell. In contrast to the traditional view, we demonstrate that Cyp26b1 is indispensable for production of the active metabolite 4-oxo-RA. Further, RA receptor beta (Rarb) is required for complete transmission of 4-oxo-RA-mediated signaling to maintain stem cells. Our findings emphasize that a single metabolite controls stem cell fate by instructing epigenetic and transcriptional attributes.
    Keywords:  4-oxo-RA; Cyp26b1; Rarb; at-RA; epigenetics; hematopoietic stem cells; metabolites; self-renewal; vitamin A
    DOI:  https://doi.org/10.1016/j.stem.2021.10.002
  8. Epigenetics. 2021 Oct 24.
    Tumor suppressor TET2 safeguards enhancers from aberrant DNA methylation and epigenetic reprogramming in ERα-positive breast cancer cells.
    Ruitu Lyu, Xuguo Zhu, Yinghui Shen, Lijun Xiong, Lu Liu, Hang Liu, Feizhen Wu, Christian Argueta, Li Tan.
      Aberrant DNA methylation is an epigenetic hallmark of malignant tumors. The DNA methylation level is regulated by not only DNA methyltransferases (DNMTs) but also Ten-Eleven Translocation (TET) family proteins. However, the exact role of TET genes in breast cancer remains controversial. Here, we uncover that the ERα-positive breast cancer patients with high TET2 mRNA expression had better overall survival rates. Consistently, knockout of TET2 promotes the tumorigenesis of ERα-positive MCF7 breast cancer cells. Mechanistically, TET2 loss leads to aberrant DNA methylation (gain of 5mC) at a large proportion of enhancers, accompanied by significant reduction in H3K4me1 and H3K27ac enrichment. By analyzing the epigenetically reprogrammed enhancers, we identify estrogen responsive element (ERE) as one of the enriched motifs of transcriptional factors. Importantly, TET2 loss impairs 17beta-estradiol (E2)-induced transcription of the epigenetically reprogrammed EREs-associated genes through attenuating the binding of ERα. Taken together, these findings shed light on our understanding of the epigenetic mechanisms underlying the enhancer reprogramming during breast cancer pathogenesis.
    Keywords:  Breast cancer; ERα; TET2; enhancer
    DOI:  https://doi.org/10.1080/15592294.2021.1997405
  9. Oncogene. 2021 Oct 23.
    NKX2-1 controls lung cancer progression by inducing DUSP6 to dampen ERK activity.
    Kelley Ingram, Shiela C Samson, Rediet Zewdu, Rebecca G Zitnay, Eric L Snyder, Michelle C Mendoza.
      The RAS→RAF→MEK→ERK pathway is hyperactivated in the majority of human lung adenocarcinoma (LUAD). However, the initial activating mutations induce homeostatic feedback mechanisms that limit ERK activity. How ERK activation reaches the tumor-promoting levels that overcome the feedback and drive malignant progression is unclear. We show here that the lung lineage transcription factor NKX2-1 suppresses ERK activity. In human tissue samples and cell lines, xenografts, and genetic mouse models, NKX2-1 induces the ERK phosphatase DUSP6, which inactivates ERK. In tumor cells from late-stage LUAD with silenced NKX2-1, re-introduction of NKX2-1 induces DUSP6 and inhibits tumor growth and metastasis. We show that DUSP6 is necessary for NKX2-1-mediated inhibition of tumor progression in vivo and that DUSP6 expression is sufficient to inhibit RAS-driven LUAD. Our results indicate that NKX2-1 silencing, and thereby DUSP6 downregulation, is a mechanism by which early LUAD can unleash ERK hyperactivation for tumor progression.
    DOI:  https://doi.org/10.1038/s41388-021-02076-x
  10. Epigenetics. 2021 Oct 28.
    Comparison of EM-seq and PBAT methylome library methods for low-input DNA.
    Yanan Han, Galina Yurevna Zheleznyakova, Yanara Marincevic-Zuniga, Majid Pahlevan Kakhki, Amanda Raine, Maria Needhamsen, Maja Jagodic.
      DNA methylation is the most studied epigenetic mark involved in regulation of gene expression. For low input samples, a limited number of methods for quantifying DNA methylation genome-wide has been evaluated. Here, we compared a series of input DNA amounts (1-10ng) from two methylome library preparation protocols, enzymatic methyl-seq (EM-seq) and post-bisulfite adaptor tagging (PBAT) adapted from single-cell PBAT. EM-seq takes advantage of enzymatic activity while PBAT relies on conventional bisulfite conversion for detection of DNA methylation. We found that both methods accurately quantified DNA methylation genome-wide. They produced expected distribution patterns around genomic features, high C-T transition efficiency at non-CpG sites and high correlation between input amounts. However, EM-seq performed better in regard to library and sequencing quality, i.e. EM-seq produced larger insert sizes, higher alignment rates and higher library complexity with lower duplication rate compared to PBAT. Moreover, EM-seq demonstrated higher CpG coverage, better CpG site overlap and higher consistency between input series. In summary, our data suggests that EM-seq overall performed better than PBAT in whole genome methylation quantification of low input samples.
    Keywords:  EM-seq; Low input DNA; PBAT; methylome
    DOI:  https://doi.org/10.1080/15592294.2021.1997406
  11. PLoS Genet. 2021 Oct 26. 17(10): e1009865
    Chromatin accessibility and gene expression during adipocyte differentiation identify context-dependent effects at cardiometabolic GWAS loci.
    Hannah J Perrin, Kevin W Currin, Swarooparani Vadlamudi, Gautam K Pandey, Kenneth K Ng, Martin Wabitsch, Markku Laakso, Michael I Love, Karen L Mohlke.
      Chromatin accessibility and gene expression in relevant cell contexts can guide identification of regulatory elements and mechanisms at genome-wide association study (GWAS) loci. To identify regulatory elements that display differential activity across adipocyte differentiation, we performed ATAC-seq and RNA-seq in a human cell model of preadipocytes and adipocytes at days 4 and 14 of differentiation. For comparison, we created a consensus map of ATAC-seq peaks in 11 human subcutaneous adipose tissue samples. We identified 58,387 context-dependent chromatin accessibility peaks and 3,090 context-dependent genes between all timepoint comparisons (log2 fold change>1, FDR<5%) with 15,919 adipocyte- and 18,244 preadipocyte-dependent peaks. Adipocyte-dependent peaks showed increased overlap (60.1%) with Roadmap Epigenomics adipocyte nuclei enhancers compared to preadipocyte-dependent peaks (11.5%). We linked context-dependent peaks to genes based on adipocyte promoter capture Hi-C data, overlap with adipose eQTL variants, and context-dependent gene expression. Of 16,167 context-dependent peaks linked to a gene, 5,145 were linked by two or more strategies to 1,670 genes. Among GWAS loci for cardiometabolic traits, adipocyte-dependent peaks, but not preadipocyte-dependent peaks, showed significant enrichment (LD score regression P<0.005) for waist-to-hip ratio and modest enrichment (P < 0.05) for HDL-cholesterol. We identified 659 peaks linked to 503 genes by two or more approaches and overlapping a GWAS signal, suggesting a regulatory mechanism at these loci. To identify variants that may alter chromatin accessibility between timepoints, we identified 582 variants in 454 context-dependent peaks that demonstrated allelic imbalance in accessibility (FDR<5%), of which 55 peaks also overlapped GWAS variants. At one GWAS locus for palmitoleic acid, rs603424 was located in an adipocyte-dependent peak linked to SCD and exhibited allelic differences in transcriptional activity in adipocytes (P = 0.003) but not preadipocytes (P = 0.09). These results demonstrate that context-dependent peaks and genes can guide discovery of regulatory variants at GWAS loci and aid identification of regulatory mechanisms.
    DOI:  https://doi.org/10.1371/journal.pgen.1009865
  12. Nat Commun. 2021 Oct 26. 12(1): 6184
    Precise measurements of chromatin diffusion dynamics by modeling using Gaussian processes.
    Guilherme M Oliveira, Attila Oravecz, Dominique Kobi, Manon Maroquenne, Kerstin Bystricky, Tom Sexton, Nacho Molina.
      The spatiotemporal organization of chromatin influences many nuclear processes: from chromosome segregation to transcriptional regulation. To get a deeper understanding of these processes, it is essential to go beyond static viewpoints of chromosome structures, to accurately characterize chromatin's diffusion properties. We present GP-FBM: a computational framework based on Gaussian processes and fractional Brownian motion to extract diffusion properties from stochastic trajectories of labeled chromatin loci. GP-FBM uses higher-order temporal correlations present in the data, therefore, outperforming existing methods. Furthermore, GP-FBM allows to interpolate incomplete trajectories and account for substrate movement when two or more particles are present. Using our method, we show that average chromatin diffusion properties are surprisingly similar in interphase and mitosis in mouse embryonic stem cells. We observe surprising heterogeneity in local chromatin dynamics, correlating with potential regulatory activity. We also present GP-Tool, a user-friendly graphical interface to facilitate usage of GP-FBM by the research community.
    DOI:  https://doi.org/10.1038/s41467-021-26466-7
  13. Commun Biol. 2021 Oct 25. 4(1): 1221
    PIM1 phosphorylation of the androgen receptor and 14-3-3 ζ regulates gene transcription in prostate cancer.
    Sophie E Ruff, Nikita Vasilyev, Evgeny Nudler, Susan K Logan, Michael J Garabedian.
      PIM1 is a serine/threonine kinase over-expressed in prostate cancer. We have previously shown that PIM1 phosphorylates the androgen receptor (AR), the primary therapeutic target in prostate cancer, at serine 213 (pS213), which alters expression of select AR target genes. Therefore, we sought to investigate the mechanism whereby PIM1 phosphorylation of AR alters its transcriptional activity. We previously identified the AR co-activator, 14-3-3 ζ, as an endogenous PIM1 substrate in LNCaP cells. Here, we show that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, and that they extensively occupy the same sites on chromatin in an AR-dependent manner. Their occupancy at a number of genes involved in cell migration and invasion results in a PIM1-dependent increase in the expression of these genes. We also use rapid immunoprecipitation and mass spectrometry of endogenous proteins on chromatin (RIME), to find that select AR co-regulators, such as hnRNPK and TRIM28, interact with both AR and 14-3-3 ζ in PIM1 over-expressing cells. We conclude that PIM1 phosphorylation of AR and 14-3-3 ζ coordinates their interaction, which in turn recruits additional co-regulatory proteins to alter AR transcriptional activity.
    DOI:  https://doi.org/10.1038/s42003-021-02723-9
  14. Stem Cell Reports. 2021 Oct 21. pii: S2213-6711(21)00515-4. [Epub ahead of print]
    p53 inactivation unmasks histone methylation-independent WDR5 functions that drive self-renewal and differentiation of pluripotent stem cells.
    Qiang Li, Yuanhao Huang, Jing Xu, Fengbiao Mao, Bo Zhou, Lichao Sun, Brian W Basinski, Michael Aksu, Jie Liu, Yali Dou, Rajesh C Rao.
      p53 alterations occur during culture of pluripotent stem cells (PSCs), but the significance of these events on epigenetic control of PSC fate determination remains poorly understood. Wdr5 deletion in p53-null (DKO) mouse ESCs (mESCs) leads to impaired self-renewal, defective retinal neuroectoderm differentiation, and de-repression of germ cell/meiosis (GCM)-specific genes. Re-introduction of a WDR5 mutant with defective H3K4 methylation activity into DKO ESCs restored self-renewal and suppressed GCM gene expression but failed to induce retinal neuroectoderm differentiation. Mechanistically, mutant WDR5 targets chromatin that is largely devoid of H3K4me3 and regulates gene expression in p53-null mESCs. Furthermore, MAX and WDR5 co-target lineage-specifying chromatin and regulate chromatin accessibility of GCM-related genes. Importantly, MAX and WDR5 are core subunits of a non-canonical polycomb repressor complex 1 responsible for gene silencing. This function, together with canonical, pro-transcriptional WDR5-dependent MLL complex H3K4 methyltransferase activity, highlight how WDR5 mediates crosstalk between transcription and repression during mESC fate choice.
    Keywords:  MAX; WDR5; chromatin; embryonic stem cells; germ cell; meiosis; neuroectoderm; p53; retina; stem cells
    DOI:  https://doi.org/10.1016/j.stemcr.2021.10.002
  15. Genes Dev. 2021 Oct 28.
    Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior.
    Kamila Naxerova, Bruno Di Stefano, Jessica L Makofske, Emma V Watson, Marit A de Kort, Timothy D Martin, Mohammed Dezfulian, Dominik Ricken, Eric C Wooten, Mitzi I Kuroda, Konrad Hochedlinger, Stephen J Elledge.
      Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.
    Keywords:  genetic screening; germ layer formation; human embryonic stem cells
    DOI:  https://doi.org/10.1101/gad.349048.121
  16. Nat Methods. 2021 Oct 28.
    Metabolite discovery through global annotation of untargeted metabolomics data.
    Li Chen, Wenyun Lu, Lin Wang, Xi Xing, Ziyang Chen, Xin Teng, Xianfeng Zeng, Antonio D Muscarella, Yihui Shen, Alexis Cowan, Melanie R McReynolds, Brandon J Kennedy, Ashley M Lato, Shawn R Campagna, Mona Singh, Joshua D Rabinowitz.
      Liquid chromatography-high-resolution mass spectrometry (LC-MS)-based metabolomics aims to identify and quantify all metabolites, but most LC-MS peaks remain unidentified. Here we present a global network optimization approach, NetID, to annotate untargeted LC-MS metabolomics data. The approach aims to generate, for all experimentally observed ion peaks, annotations that match the measured masses, retention times and (when available) tandem mass spectrometry fragmentation patterns. Peaks are connected based on mass differences reflecting adduction, fragmentation, isotopes, or feasible biochemical transformations. Global optimization generates a single network linking most observed ion peaks, enhances peak assignment accuracy, and produces chemically informative peak-peak relationships, including for peaks lacking tandem mass spectrometry spectra. Applying this approach to yeast and mouse data, we identified five previously unrecognized metabolites (thiamine derivatives and N-glucosyl-taurine). Isotope tracer studies indicate active flux through these metabolites. Thus, NetID applies existing metabolomic knowledge and global optimization to substantially improve annotation coverage and accuracy in untargeted metabolomics datasets, facilitating metabolite discovery.
    DOI:  https://doi.org/10.1038/s41592-021-01303-3
  17. Cell Death Dis. 2021 Oct 29. 12(11): 1023
    Chromatin accessibility analysis identifies the transcription factor ETV5 as a suppressor of adipose tissue macrophage activation in obesity.
    Ren-Dong Hu, Wen Zhang, Liang Li, Zu-Qi Zuo, Min Ma, Jin-Fen Ma, Ting-Ting Yin, Cai-Yue Gao, Shu-Han Yang, Zhi-Bin Zhao, Zi-Jun Li, Gui-Bin Qiao, Zhe-Xiong Lian, Kun Qu.
      Activation of adipose tissue macrophages (ATMs) contributes to chronic inflammation and insulin resistance in obesity. However, the transcriptional regulatory machinery involved in ATM activation during the development of obesity is not fully understood. Here, we profiled the chromatin accessibility of blood monocytes and ATMs from obese and lean mice using assay for transposase-accessible chromatin sequencing (ATAC-seq). We found that monocytes and ATMs from obese and lean mice exhibited distinct chromatin accessibility status. There are distinct regulatory elements that are specifically associated with monocyte or ATM activation in obesity. We also discovered several transcription factors that may regulate monocyte and ATM activation in obese mice, specifically a predicted transcription factor named ETS translocation variant 5 (ETV5). The expression of ETV5 was significantly decreased in ATMs from obese mice and its downregulation was mediated by palmitate stimulation. The decrease in ETV5 expression resulted in macrophage activation. Our results also indicate that ETV5 suppresses endoplasmic reticulum (ER) stress and Il6 expression in macrophages. Our work delineates the changes in chromatin accessibility in monocytes and ATMs during obesity, and identifies ETV5 as a critical transcription factor suppressing ATM activation, suggesting its potential use as a therapeutic target in obesity-related chronic inflammation.
    DOI:  https://doi.org/10.1038/s41419-021-04308-0
  18. Cancer Res. 2021 Oct 25. pii: canres.0707.2021. [Epub ahead of print]
    BRD4 REGULATES TRANSCRIPTION FACTOR ∆Np63αTO DRIVE A CANCER STEM CELL PHENOTYPE IN SQUAMOUS CELL CARCINOMAS.
    Matthew L Fisher, Seamus Balinth, Yon Hwangbo, Caizhi Wu, Carlos Ballon, John E Wilkinson, Gary L Goldberg, Alea A Mills.
      Bromodomain containing protein 4 (BRD4) plays a critical role in controlling the expression of genes involved in development and cancer. Inactivation of BRD4 inhibits cancer growth, making it a promising anticancer drug target. The cancer stem cell population is a key driver of recurrence and metastasis in cancer patients. Here we show that cancer stem-like cells can be enriched from squamous cell carcinomas, and that these cells display an aggressive phenotype with enhanced stem cell marker expression, migration, invasion, and tumor growth. BRD4 was highly elevated in this aggressive subpopulation of cells, and its function is critical for these cancer stem cell-like properties. Moreover, BRD4 regulated ∆Np63α, a key transcription factor that is essential for epithelial stem cell function that is often overexpressed in cancers. BRD4 regulated an EZH2/STAT3 complex that led to increased ∆Np63α-mediated transcription. Targeting BRD4 in human squamous cell carcinoma reduces ∆Np63α, leading to inhibition of spheroid formation, migration, invasion and tumor growth. These studies identify a novel BRD4-regulated signaling network in a subpopulation of cancer stem-like cells elucidating a possible avenue for effective therapeutic intervention.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-0707
  19. iScience. 2021 Oct 22. 24(10): 103198
    HES1 protein oscillations are necessary for neural stem cells to exit from quiescence.
    Elli Marinopoulou, Veronica Biga, Nitin Sabherwal, Anzy Miller, Jayni Desai, Antony D Adamson, Nancy Papalopulu.
      Quiescence is a dynamic process of reversible cell cycle arrest. High-level persistent expression of the HES1 transcriptional repressor, which oscillates with an ultradian periodicity in proliferative neural stem cells (NSCs), is thought to mediate quiescence. However, it is not known whether this is due to a change in levels or dynamics. Here, we induce quiescence in embryonic NSCs with BMP4, which does not increase HES1 level, and we find that HES1 continues to oscillate. To assess the role of HES1 dynamics, we express persistent HES1 under a moderate strength promoter, which overrides the endogenous oscillations while maintaining the total HES1 level within physiological range. We find that persistent HES1 does not affect proliferation or entry into quiescence; however, exit from quiescence is impeded. Thus, oscillatory expression of HES1 is specifically required for NSCs to exit quiescence, a finding of potential importance for controlling reactivation of stem cells in tissue regeneration and cancer.
    Keywords:  cell biology; molecular biology; stem cells research
    DOI:  https://doi.org/10.1016/j.isci.2021.103198
  20. Br J Cancer. 2021 Oct 27.
    A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells.
    Takashi Takeda, Yuhki Yokoyama, Hidekazu Takahashi, Daisuke Okuzaki, Kaho Asai, Hiroaki Itakura, Norikatsu Miyoshi, Shogo Kobayashi, Mamoru Uemura, Toshitsugu Fujita, Hiroo Ueno, Masaki Mori, Yuichiro Doki, Hodaka Fujii, Hidetoshi Eguchi, Hirofumi Yamamoto.
       BACKGROUND: KLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC).
    METHODS: In vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter.
    RESULTS: We revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples.
    CONCLUSIONS: Our data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.
    DOI:  https://doi.org/10.1038/s41416-021-01579-4
  21. iScience. 2021 Nov 19. 24(11): 103227
    Endocrine disrupting chemicals differentially alter intranuclear dynamics and transcriptional activation of estrogen receptor-α.
    Michael J Bolt, Pankaj Singh, Caroline E Obkirchner, Reid T Powell, Maureen G Mancini, Adam T Szafran, Fabio Stossi, Michael A Mancini.
      Transcription is a highly regulated sequence of stochastic processes utilizing many regulators, including nuclear receptors (NR) that respond to stimuli. Endocrine disrupting chemicals (EDCs) in the environment can compete with natural ligands for nuclear receptors to alter transcription. As nuclear dynamics can be tightly linked to transcription, it is important to determine how EDCs affect NR mobility. We use an EPA-assembled set of 45 estrogen receptor-α (ERα) ligands and EDCs in our engineered PRL-Array model to characterize their effect upon transcription using fluorescence in situ hybridization and fluorescence recovery after photobleaching (FRAP). We identified 36 compounds that target ERα-GFP to a transcriptionally active, visible locus. Using a novel method for multi-region FRAP analysis we find a strong negative correlation between ERα mobility and inverse agonists. Our findings indicate that ERα mobility is not solely tied to transcription but affected highly by the chemical class binding the receptor.
    Keywords:  Biological sciences; Biophysical Chemistry; Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2021.103227
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