bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2021‒03‒21
thirty-two papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit


  1. Epigenetics Chromatin. 2021 Mar 19. 14(1): 14
      Epigenetic marks do not change the sequence of DNA but affect gene expression in a cell-type specific manner by altering the activities of regulatory elements. Development of new molecular biology assays, sequencing technologies, and computational approaches enables us to profile the human epigenome in three-dimensional structure genome-wide. Here we describe various molecular biology techniques and bioinformatic tools that have been developed to measure the activities of regulatory elements and their chromatin interactions. Moreover, we list currently available three-dimensional epigenomic data sets that are generated in various human cell types and tissues to assist in the design and analysis of research projects.
    Keywords:  Analysis tools; Chromatin interactions; Databases; Epigenomics; Regulatory elements
    DOI:  https://doi.org/10.1186/s13072-021-00390-y
  2. Nat Commun. 2021 Mar 19. 12(1): 1749
      Sonic hedgehog medulloblastoma encompasses a clinically and molecularly diverse group of cancers of the developing central nervous system. Here, we use unbiased sequencing of the transcriptome across a large cohort of 250 tumors to reveal differences among molecular subtypes of the disease, and demonstrate the previously unappreciated importance of non-coding RNA transcripts. We identify alterations within the cAMP dependent pathway (GNAS, PRKAR1A) which converge on GLI2 activity and show that 18% of tumors have a genetic event that directly targets the abundance and/or stability of MYCN. Furthermore, we discover an extensive network of fusions in focally amplified regions encompassing GLI2, and several loss-of-function fusions in tumor suppressor genes PTCH1, SUFU and NCOR1. Molecular convergence on a subset of genes by nucleotide variants, copy number aberrations, and gene fusions highlight the key roles of specific pathways in the pathogenesis of Sonic hedgehog medulloblastoma and open up opportunities for therapeutic intervention.
    DOI:  https://doi.org/10.1038/s41467-021-21883-0
  3. Bioinformatics. 2021 Mar 15. pii: btab173. [Epub ahead of print]
      SUMMARY: Identification of functional transcriptional regulators associated with chromatin in-teractions is an important problem in studies of 3-dimensional genome organization and gene regulation. Direct inference of TR binding has been limited by the resolu-tion of Hi-C data. Here, we present BART3D, a computational method for inferring TRs associated with genome-wide differential chromatin interactions by comparing Hi-C maps from two states, leveraging public ChIP-seq data for human and mouse. We demonstrate that BART3D can detect relevant TRs from dynamic Hi-C profiles with TR perturbation or cell differentiation. BART3D can be a useful tool in 3D genome data analysis and functional genomics research.AVAILABILITY AND IMPLEMENTATION: BART3D is implemented in Python and the source code is available at https://github.com/zanglab/bart3d.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btab173
  4. Mol Cell. 2021 Mar 09. pii: S1097-2765(21)00142-8. [Epub ahead of print]
      During self-renewal, cell-type-defining features are drastically perturbed in mitosis and must be faithfully reestablished upon G1 entry, a process that remains largely elusive. Here, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. We captured distinct waves of transcriptional reactivation with rapid induction of stem cell genes and transient activation of lineage-specific genes. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed partial coordination with transcriptional resetting. Globally, rapid transcriptional and architectural resetting associated with mitotic retention of H3K27 acetylation, supporting a bookmarking function. Indeed, mitotic depletion of H3K27ac impaired the early reactivation of bookmarked, stem-cell-associated genes. However, 3D chromatin reorganization remained largely unaffected, suggesting that these processes are driven by distinct forces upon mitotic exit. This study uncovers principles and mediators of PSC molecular resetting during self-renewal.
    Keywords:  3D chromatin organization; H3K27ac; Hi-C; PRO-seq; bookmarking; cell identity; enhancer-promoter interaction; mitosis; pluripotent stem cells; transcription reactivation
    DOI:  https://doi.org/10.1016/j.molcel.2021.02.032
  5. BMC Res Notes. 2021 Mar 19. 14(1): 104
      OBJECTIVE: To address the challenge of computational identification of cell type-specific regulatory elements on a genome-wide scale.RESULTS: We propose SeqEnhDL, a deep learning framework for classifying cell type-specific enhancers based on sequence features. DNA sequences of "strong enhancer" chromatin states in nine cell types from the ENCODE project were retrieved to build and test enhancer classifiers. For any DNA sequence, positional k-mer (k = 5, 7, 9 and 11) fold changes relative to randomly selected non-coding sequences across each nucleotide position were used as features for deep learning models. Three deep learning models were implemented, including multi-layer perceptron (MLP), Convolutional Neural Network (CNN) and Recurrent Neural Network (RNN). All models in SeqEnhDL outperform state-of-the-art enhancer classifiers (including gkm-SVM and DanQ) in distinguishing cell type-specific enhancers from randomly selected non-coding sequences. Moreover, SeqEnhDL can directly discriminate enhancers from different cell types, which has not been achieved by other enhancer classifiers. Our analysis suggests that both enhancers and their tissue-specificity can be accurately identified based on their sequence features. SeqEnhDL is publicly available at https://github.com/wyp1125/SeqEnhDL .
    Keywords:  Cell type; Classification; DNA sequence; Deep learning; Enhancer
    DOI:  https://doi.org/10.1186/s13104-021-05518-7
  6. Nat Commun. 2021 Mar 19. 12(1): 1781
      Prostate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.
    DOI:  https://doi.org/10.1038/s41467-021-21867-0
  7. Cell Rep. 2021 Mar 16. pii: S2211-1247(21)00153-4. [Epub ahead of print]34(11): 108839
      Naive CD8+ T cell activation results in an autonomous program of cellular proliferation and differentiation. However, the mechanisms that underpin this process are unclear. Here, we profile genome-wide changes in chromatin accessibility, gene transcription, and the deposition of a key chromatin modification (H3K27me3) early after naive CD8+ T cell activation. Rapid upregulation of the histone demethylase KDM6B prior to the first cell division is required for initiating H3K27me3 removal at genes essential for subsequent T cell differentiation and proliferation. Inhibition of KDM6B-dependent H3K27me3 demethylation limits the magnitude of an effective primary virus-specific CD8+ T cell response and the formation of memory CD8+ T cell populations. Accordingly, we define the early spatiotemporal events underpinning early lineage-specific chromatin reprogramming that are necessary for autonomous CD8+ T cell proliferation and differentiation.
    Keywords:  CD8(+) T cell; T cell activation; T cell memory; chromatin; histone demethylase; virus immunity
    DOI:  https://doi.org/10.1016/j.celrep.2021.108839
  8. Mol Biol Evol. 2021 Mar 15. pii: msab075. [Epub ahead of print]
      Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming require efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, ePOU can induce pluripotency with Sox2 alone and in the absence of Sox2 in three factor - ePOU/Klf4/c-Myc cocktails. Biochemical assays combined with genome-wide analyses show that ePOU acquires a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins are not changed in ePOU. Compared to Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: (1) differentially activates several genes hitherto not implicated in reprogramming, (2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and (3) binds a distinct class of retrotransposons. Collectively, these features enabled ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.
    DOI:  https://doi.org/10.1093/molbev/msab075
  9. Genome Res. 2021 Mar 19.
      Single-cell RNA sequencing (scRNA-seq) technology is poised to replace bulk cell RNA sequencing for many biological and medical applications as it allows users to measure gene expression levels in a cell type-specific manner. However, data produced by scRNA-seq often exhibit batch effects that can be specific to a cell type, to a sample, or to an experiment, which prevent integration or comparisons across multiple experiments. Here, we present Dmatch, a method that leverages an external expression atlas of human primary cells and kernel density matching to align multiple scRNA-seq experiments for downstream biological analysis. Dmatch facilitates alignment of scRNA-seq data sets with cell types that may overlap only partially and thus allows integration of multiple distinct scRNA-seq experiments to extract biological insights. In simulation, Dmatch compares favorably to other alignment methods, both in terms of reducing sample-specific clustering and in terms of avoiding overcorrection. When applied to scRNA-seq data collected from clinical samples in a healthy individual and five autoimmune disease patients, Dmatch enabled cell type-specific differential gene expression comparisons across biopsy sites and disease conditions and uncovered a shared population of pro-inflammatory monocytes across biopsy sites in RA patients. We further show that Dmatch increases the number of eQTLs mapped from population scRNA-seq data. Dmatch is fast, scalable, and improves the utility of scRNA-seq for several important applications. Dmatch is freely available online.
    DOI:  https://doi.org/10.1101/gr.261115.120
  10. Proc Natl Acad Sci U S A. 2021 Mar 23. pii: e2025196118. [Epub ahead of print]118(12):
      Specification of Sox2+ proneurosensory progenitors within otic ectoderm is a prerequisite for the production of sensory cells and neurons for hearing. However, the underlying molecular mechanisms driving this lineage specification remain unknown. Here, we show that the Brg1-based SWI/SNF chromatin-remodeling complex interacts with the neurosensory-specific transcriptional regulators Eya1/Six1 to induce Sox2 expression and promote proneurosensory-lineage specification. Ablation of the ATPase-subunit Brg1 or both Eya1/Six1 results in loss of Sox2 expression and lack of neurosensory identity, leading to abnormal apoptosis within the otic ectoderm. Brg1 binds to two of three distal 3' Sox2 enhancers occupied by Six1, and Brg1-binding to these regions depends on Eya1-Six1 activity. We demonstrate that the activity of these Sox2 enhancers in otic neurosensory cells specifically depends on binding to Six1. Furthermore, genome-wide and transcriptome profiling indicate that Brg1 may suppress apoptotic factor Map3k5 to inhibit apoptosis. Together, our findings reveal an essential role for Brg1, its downstream pathways, and their interactions with Six1/Eya1 in promoting proneurosensory fate induction in the otic ectoderm and subsequent neuronal lineage commitment and survival of otic cells.
    Keywords:  Sox2 enhancers; chromatin remodelers; otic neurosensory lineage; regulation of otic Sox2 expression; transcription factors
    DOI:  https://doi.org/10.1073/pnas.2025196118
  11. Clin Cancer Res. 2021 Mar 17. pii: clincanres.CCR-20-2931-A.2020. [Epub ahead of print]
      PURPOSE: Multiple myeloma is a malignancy of plasma cells. Extensive genetic and transcriptional characterization of myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, relatively little is known about the myeloma epigenome.EXPERIMENTAL DESIGN: CD138+CD38+ myeloma cells were isolated from fresh bone marrow aspirate or the same aspirate after freezing for one to six months. Gene expression and chromatin accessibility were compared between fresh and frozen samples by RNA-seq and ATAC-seq. Chromatin accessible regions were used to identify regulatory RNA expression in over 700 samples from newly diagnosed patients in the MMRF CoMMpass trial (NCT01454297).
    RESULTS: Gene expression and chromatin accessibility of cryopreserved myeloma recapitulated that of freshly isolated samples. ATAC-seq performed on a series of biobanked specimens identified thousands of chromatin accessible regions with hundreds being highly coordinated with gene expression. Over 4,700 of these chromatin accessible regions were transcribed in newly diagnosed myelomas from the CoMMpass trial. Regulatory element activity alone recapitulated myeloma gene expression subtypes, and in particular myeloma subtypes with IGH translocations were defined by transcription of distal regulatory elements. Moreover, enhancer activity predicted oncogene expression implicating gene regulatory mechanisms in aggressive myeloma.
    CONCLUSIONS: These data demonstrate the feasibility of using biobanked specimens for retrospective studies of the myeloma epigenome and illustrate the unique enhancer landscapes of myeloma subtypes that are coupled to gene expression and disease progression.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-20-2931
  12. Nat Chem Biol. 2021 Mar 15.
      The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.
    DOI:  https://doi.org/10.1038/s41589-021-00755-0
  13. Cancer Cell. 2021 Mar 10. pii: S1535-6108(21)00118-5. [Epub ahead of print]
      Many cancers, including pancreatic ductal adenocarcinoma (PDAC), depend on autophagy-mediated scavenging and recycling of intracellular macromolecules, suggesting that autophagy blockade should cause tumor starvation and regression. However, until now autophagy-inhibiting monotherapies have not demonstrated potent anti-cancer activity. We now show that autophagy blockade prompts established PDAC to upregulate and utilize an alternative nutrient procurement pathway: macropinocytosis (MP) that allows tumor cells to extract nutrients from extracellular sources and use them for energy generation. The autophagy to MP switch, which may be evolutionarily conserved and not cancer cell restricted, depends on activation of transcription factor NRF2 by the autophagy adaptor p62/SQSTM1. NRF2 activation by oncogenic mutations, hypoxia, and oxidative stress also results in MP upregulation. Inhibition of MP in autophagy-compromised PDAC elicits dramatic metabolic decline and regression of transplanted and autochthonous tumors, suggesting the therapeutic promise of combining autophagy and MP inhibitors in the clinic.
    Keywords:  NRF2; RAS-driven cancer; autophagy; macropinocytosis; p62/SQSTM1
    DOI:  https://doi.org/10.1016/j.ccell.2021.02.016
  14. Elife. 2021 Mar 16. pii: e62250. [Epub ahead of print]10
      Tissue homeostasis requires long-term lineage fidelity of somatic stem cells. Whether and how age-related changes in somatic stem cells impact the faithful execution of lineage decisions remains largely unknown. Here, we address this question using genome-wide chromatin accessibility and transcriptome analysis as well as single cell RNA-seq to explore stem cell-intrinsic changes in the aging Drosophila intestine. These studies indicate that in stem cells of old flies, promoters of Polycomb (Pc) target genes become differentially accessible, resulting in the increased expression of enteroendocrine (EE) cell specification genes. Consistently, we find age-related changes in the composition of the EE progenitor cell population in aging intestines, as well as a significant increase in the proportion of EE-specified intestinal stem cells (ISCs) and progenitors in aging flies. We further confirm that Pc-mediated chromatin regulation is a critical determinant of EE cell specification in the Drosophila intestine. Pc is required to maintain expression of stem cell genes while ensuring repression of differentiation and specification genes. Our results identify Pc group proteins as central regulators of lineage identity in the intestinal epithelium and highlight the impact of age-related decline in chromatin regulation on tissue homeostasis.
    Keywords:  D. melanogaster; genetics; genomics
    DOI:  https://doi.org/10.7554/eLife.62250
  15. Cancer Discov. 2021 Mar 19. pii: candisc.1052.2020. [Epub ahead of print]
      ZFTA (C11orf95)-a gene of unknown function-partners with a variety of transcriptional co-activators in translocations that drive supratentorial ependymoma, a frequently lethal brain tumor. Understanding the function of ZFTA is key to developing therapies that inhibit these fusion proteins. Here, using a combination of transcriptomics, chromatin immunoprecipitation-sequencing, and proteomics, we interrogated a series of deletion-mutant genes to identify a tri-partite transformation mechanism of ZFTA-containing fusions, including: spontaneous nuclear translocation, extensive chromatin binding, and SWI/SNF, SAGA and NuA4/Tip60 HAT chromatin modifier complex recruitment. Thereby, ZFTA tethers fusion proteins across the genome, modifying chromatin to an active state, and enabling its partner transcriptional co-activators to promote promiscuous expression of a transforming transcriptome. Using mouse models, we validate further those elements of ZFTA-fusion proteins that are critical for transformation-including ZFTA zinc fingers and partner gene transactivation domains-thereby unmasking vulnerabilities for therapeutic targeting.
    DOI:  https://doi.org/10.1158/2159-8290.CD-20-1052
  16. Nat Aging. 2021 Feb;1(2): 165-178
      Organisms respond to mitochondrial stress by activating multiple defense pathways including the mitochondrial unfolded protein response (UPRmt). However, how UPRmt regulators are orchestrated to transcriptionally activate stress responses remains largely unknown. Here we identified CBP-1, the worm ortholog of the mammalian acetyltransferases CBP/p300, as an essential regulator of the UPRmt, as well as mitochondrial stress-induced immune response, reduction of amyloid-β aggregation and lifespan extension in Caenorhabditis elegans. Mechanistically, CBP-1 acts downstream of histone demethylases, JMJD-1.2/JMJD-3.1, and upstream of UPRmt transcription factors including ATFS-1, to systematically induce a broad spectrum of UPRmt genes and execute multiple beneficial functions. In mouse and human populations, transcript levels of CBP/p300 positively correlate with UPRmt transcripts and longevity. Furthermore, CBP/p300 inhibition disrupts, while forced expression of p300 is sufficient to activate, the UPRmt in mammalian cells. These results highlight an evolutionarily conserved mechanism that determines mitochondrial stress response, and promotes health and longevity through CBP/p300.
    DOI:  https://doi.org/10.1038/s43587-020-00025-z
  17. Genome Biol. 2021 Mar 16. 22(1): 85
      BACKGROUND: Histone lactylation, a metabolic stress-related histone modification, plays an important role in the regulation of gene expression during M1 macrophage polarization. However, the role of histone lactylation in tumorigenesis remains unclear.RESULTS: Here, we show histone lactylation is elevated in tumors and is associated with poor prognosis of ocular melanoma. Target correction of aberrant histone lactylation triggers therapeutic efficacy both in vitro and in vivo. Mechanistically, histone lactylation contributes to tumorigenesis by facilitating YTHDF2 expression. Moreover, YTHDF2 recognizes the m6A modified PER1 and TP53 mRNAs and promotes their degradation, which accelerates tumorigenesis of ocular melanoma.
    CONCLUSION: We reveal the oncogenic role of histone lactylation, thereby providing novel therapeutic targets for ocular melanoma therapy. We also bridge histone modifications with RNA modifications, which provides novel understanding of epigenetic regulation in tumorigenesis.
    Keywords:  Histone lactylation; Ocular melanoma; Transcriptional activation; YTHDF2; m6A
    DOI:  https://doi.org/10.1186/s13059-021-02308-z
  18. Nat Genet. 2021 Mar 17.
      Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.
    DOI:  https://doi.org/10.1038/s41588-021-00804-3
  19. Elife. 2021 Mar 15. pii: e66668. [Epub ahead of print]10
      Following fertilization, the genomes of the germ cells are reprogrammed to form the totipotent embryo. Pioneer transcription factors are essential for remodeling the chromatin and driving the initial wave of zygotic gene expression. In Drosophila melanogaster, the pioneer factor Zelda is essential for development through this dramatic period of reprogramming, known as the maternal-to-zygotic transition (MZT). However, it was unknown whether additional pioneer factors were required for this transition. We identified an additional maternally encoded factor required for development through the MZT, GAGA Factor (GAF). GAF is necessary to activate widespread zygotic transcription and to remodel the chromatin accessibility landscape. We demonstrated that Zelda preferentially controls expression of the earliest transcribed genes, while genes expressed during widespread activation are predominantly dependent on GAF. Thus, progression through the MZT requires coordination of multiple pioneer-like factors, and we propose that as development proceeds control is gradually transferred from Zelda to GAF.
    Keywords:  D. melanogaster; chromosomes; gene expression
    DOI:  https://doi.org/10.7554/eLife.66668
  20. Nature. 2021 Mar 17.
      Establishment of the mammalian body plan occurs shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. While methods for in vitro culture of pre- and peri-implantation mouse embryos are routinely utilized1,2, approaches for robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we develop highly conducive ex utero post-implantation mouse embryo culture platforms, that enable appropriate development of embryos before gastrulation (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings, while extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of novel static and rotating bottle culture platforms. Histological, molecular, and single-cell RNA-seq analyses validate that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to introducing a variety of embryonic perturbations and micro-manipulations that can be followed ex utero for up to six days. Establishment of a system to robustly grow normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool to investigate embryogenesis, eliminating the uterine barrier to mechanistically interrogate post-implantation morphogenesis and tissue specification in mammals.
    DOI:  https://doi.org/10.1038/s41586-021-03416-3
  21. Oncogene. 2021 Mar 19.
      The survival rate in lung cancer remains stubbornly low and there is an urgent need for the identification of new therapeutic targets. In the last decade, several members of the SWI/SNF chromatin remodeling complexes have been described altered in different tumor types. Nevertheless, the precise mechanisms of their impact on cancer progression, as well as the application of this knowledge to cancer patient management are largely unknown. In this study, we performed targeted sequencing of a cohort of lung cancer patients on genes involved in chromatin structure. In addition, we studied at the protein level the expression of these genes in cancer samples and performed functional experiments to identify the molecular mechanisms linking alterations of chromatin remodeling genes and tumor development. Remarkably, we found that 20% of lung cancer patients show ARID2 protein loss, partially explained by the presence of ARID2 mutations. In addition, we showed that ARID2 deficiency provokes profound chromatin structural changes altering cell transcriptional programs, which bolsters the proliferative and metastatic potential of the cells both in vitro and in vivo. Moreover, we demonstrated that ARID2 deficiency impairs DNA repair, enhancing the sensitivity of the cells to DNA-damaging agents. Our findings support that ARID2 is a bona fide tumor suppressor gene in lung cancer that may be exploited therapeutically.
    DOI:  https://doi.org/10.1038/s41388-021-01748-y
  22. Front Oncol. 2021 ;11 605025
      FOXP2, a member of forkhead box transcription factor family, was first identified as a language-related gene that played an important role in language learning and facial movement. In addition, FOXP2 was also suggested regulating the progression of cancer cells. In previous studies, we found that FOXA2 inhibited epithelial-mesenchymal transition (EMT) in breast cancer cells. In this study, by identifying FOXA2-interacting proteins from FOXA2-pull-down cell lysates with Mass Spectrometry Analysis, we found that FOXP2 interacted with FOXA2. After confirming the interaction between FOXP2 and FOXA2 through Co-IP and immunofluorescence assays, we showed a correlated expression of FOXP2 and FOXA2 existing in clinical breast cancer samples. The overexpression of FOXP2 attenuated the mesenchymal phenotype whereas the stable knockdown of FOXP2 promoted EMT in breast cancer cells. Even though FOXP2 was believed to act as a transcriptional repressor in most cases, we found that FOXP2 could activate the expression of tumor suppressor PHF2. Meanwhile, we also found that FOXP2 could endogenously bind to the promoter of E-cadherin and activate its transcription. This transcriptional activity of FOXP2 relied on its interaction with FOXA2. Furthermore, the stable knockdown of FOXP2 enhanced the metastatic capacity of breast cancer cells in vivo. Together, the results suggested that FOXP2 could inhibit EMT by activating the transcription of certain genes, such as E-cadherin and PHF2, in concert with FOXA2 in breast cancer cells.
    Keywords:  E-cadherin; FOXA2 transcription factor; FOXP2 transcription factor; PHF2; epithelial-mesenchymal transition of breast cancer
    DOI:  https://doi.org/10.3389/fonc.2021.605025
  23. Nat Commun. 2021 03 17. 12(1): 1714
      Advanced prostate cancer (PCa) often develops bone metastasis, for which therapies are very limited and the underlying mechanisms are poorly understood. We report that bone-borne TGF-β induces the acetylation of transcription factor KLF5 in PCa bone metastases, and acetylated KLF5 (Ac-KLF5) causes osteoclastogenesis and bone metastatic lesions by activating CXCR4, which leads to IL-11 secretion, and stimulating SHH/IL-6 paracrine signaling. While essential for maintaining the mesenchymal phenotype and tumorigenicity, Ac-KLF5 also causes resistance to docetaxel in tumors and bone metastases, which is overcome by targeting CXCR4 with FDA-approved plerixafor. Establishing a mechanism for bone metastasis and chemoresistance in PCa, these findings provide a rationale for treating chemoresistant bone metastasis of PCa with inhibitors of Ac-KLF5/CXCR4 signaling.
    DOI:  https://doi.org/10.1038/s41467-021-21976-w
  24. Elife. 2021 Mar 18. pii: e65857. [Epub ahead of print]10
      Genetic regulation of gene expression underlies variation in disease risk and other complex traits. The effect of expression quantitative trait loci (eQTLs) varies across cell types; however, the complexity of mammalian tissues makes studying cell-type eQTLs highly challenging. We developed a novel approach in the model nematode Caenorhabditis elegans that uses single cell RNA sequencing to map eQTLs at cellular resolution in a single one-pot experiment. We mapped eQTLs across cell types in an extremely large population of genetically distinct C. elegans individuals. We found cell-type-specific trans-eQTL hotspots that affect the expression of core pathways in the relevant cell types. Finally, we found single-cell-specific eQTL effects in the nervous system, including an eQTL with opposite effects in two individual neurons. Our results show that eQTL effects can be specific down to the level of single cells.
    Keywords:  C. elegans; genetics; genomics
    DOI:  https://doi.org/10.7554/eLife.65857
  25. Cancer Sci. 2021 Mar 17.
      Cell identity is known to be controlled by regulatory elements within the genome, such as promoters, enhancers, and insulators. These regulatory elements interact with each other in the nucleus and form tissue-specific chromatin structures. Dysregulation of these elements and their interactions can lead to loss of cell identity and promote the development of diseases such as cancer. Tumor cells acquire aberrantly activated enhancers at oncogenic driver genes through various mechanisms. Small genomic alterations such as mutations, insertions, and amplifications can form aberrant enhancers. Genomic rearrangements at the chromosomal level, including translocations and inversions, are also frequently observed in cancers. These rearrangements can result in the repositioning of enhancers to locations near tumor-type specific oncogenes. Chromatin structural changes caused by genomic or epigenomic changes lead to mis-interaction between enhancers and proto-oncogenes, ultimately contributing to tumorigenesis through activation of oncogenic signals. Additional epigenomic mechanisms can also cause aberrant enhancer activation, including those associated with overexpression of oncogenic transcription factors and the mutation of transcriptional cofactors. Exogenous viral DNA can also lead to enhancer aberrations. Here, we review the mechanisms underlying aberrant oncogene activation through enhancer activation and rewiring, both of which are caused by genomic or epigenomic alterations in non-coding regions.
    Keywords:  Cancer; Enhancer; Enhancer rewiring; Epigenomic aberration; Genomic aberration
    DOI:  https://doi.org/10.1111/cas.14884
  26. Nat Commun. 2021 03 17. 12(1): 1703
      The factors regulating cellular identity are critical for understanding the transition from health to disease and responses to therapies. Recent literature suggests that autophagy compromise may cause opposite effects in different contexts by either activating or inhibiting YAP/TAZ co-transcriptional regulators of the Hippo pathway via unrelated mechanisms. Here, we confirm that autophagy perturbation in different cell types can cause opposite responses in growth-promoting oncogenic YAP/TAZ transcriptional signalling. These apparently contradictory responses can be resolved by a feedback loop where autophagy negatively regulates the levels of α-catenins, LC3-interacting proteins that inhibit YAP/TAZ, which, in turn, positively regulate autophagy. High basal levels of α-catenins enable autophagy induction to positively regulate YAP/TAZ, while low α-catenins cause YAP/TAZ activation upon autophagy inhibition. These data reveal how feedback loops enable post-transcriptional determination of cell identity and how levels of a single intermediary protein can dictate the direction of response to external or internal perturbations.
    DOI:  https://doi.org/10.1038/s41467-021-21882-1
  27. Nucleic Acids Res. 2021 Mar 15. pii: gkab157. [Epub ahead of print]
      The hydrolytic deamination of adenosine-to-inosine (A-to-I) by RNA editing is a widespread post-transcriptional modification catalyzed by the adenosine deaminase acting on RNA (ADAR) family of proteins. ADAR-mediated RNA editing modulates cellular pathways involved in innate immunity, RNA splicing, RNA interference, and protein recoding, and has been investigated as a strategy for therapeutic intervention of genetic disorders. Despite advances in basic and translational research, the mechanisms regulating RNA editing are poorly understood. Though several trans-acting regulators of editing have been shown to modulate ADAR protein expression, previous studies have not identified factors that modulate ADAR catalytic activity. Here, we show that RNA editing increases upon intracellular acidification, and that these effects are predominantly explained by both enhanced ADAR base-flipping and deamination rate at acidic pH. We also show that the extent of RNA editing increases with the reduction in pH associated with conditions of cellular hypoxia.
    DOI:  https://doi.org/10.1093/nar/gkab157
  28. Genome Res. 2021 Mar 19.
      Decoding the cell type-specific transcription factor (TF) binding landscape at single-nucleotide resolution is crucial for understanding the regulatory mechanisms underlying many fundamental biological processes and human diseases. However, limits on time and resources restrict the high-resolution experimental measurements of TF binding profiles of all possible TF-cell type combinations. Previous computational approaches either cannot distinguish the cell context-dependent TF binding profiles across diverse cell types or can only provide a relatively low-resolution prediction. Here we present a novel deep learning approach, Leopard, for predicting TF binding sites at single-nucleotide resolution, achieving the average area under receiver operating characteristic curve (AUROC) of 0.982 and the average area under precision recall curve (AUPRC) of 0.208. Our method substantially outperformed the state-of-the-art methods Anchor and FactorNet, improving the predictive AUPRC by 19% and 27%, respectively, when evaluated at 200-bp resolution. Meanwhile, by leveraging a many-to-many neural network architecture, Leopard features a hundredfold to thousandfold speedup compared with current many-to-one machine learning methods.
    DOI:  https://doi.org/10.1101/gr.269613.120
  29. Bioinformatics. 2021 Mar 15. pii: btab170. [Epub ahead of print]
      SUMMARY: Many experimental approaches have been developed to identify transcription start sites (TSS) from genomic scale data. However, experiment specific biases lead to large numbers of false positive calls. Here, we present our integrative approach iTiSS, which is an accurate and generic TSS caller for any TSS profiling experiment in eukaryotes, and substantially reduces the number of false positives by a joint analysis of several complementary data sets.AVAILABILITY AND IMPLEMENTATION: iTiSS is platform independent and implemented in Java (v1.8) and is freely available at https://www.erhard-lab.de/software and https://github.com/erhard-lab/iTiSS.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. The raw data as well as the scripts to reproduce all analyses in this study are available on Zenodo (https://doi.org/10.5281/zenodo.3860525).
    DOI:  https://doi.org/10.1093/bioinformatics/btab170
  30. Stem Cells. 2021 Mar 19.
      The Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSC). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2), individually or in combination. Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Further, pharmacological inhibition by T-5224 of FOS/JUN AP1 complex binding to its targets decreased cell proliferation and expression of the putative target Suppressor of cytokine signaling 3 (Socs3). Additionally, Fos requirement for efficient long-term proliferation was demonstrated by the reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Socs3 gene is strongly downregulated following Sox2 deletion, and its reexpression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 reexpression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; further, we provide direct evidence for FOS and JUN binding on Socs3 promoter, suggesting direct transcriptional regulation. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Proliferation and maintenance of NSC are essential during normal brain development, and, postnatally, for the maintenance of hippocampal function and memory until advanced age. Little is known about the molecular mechanisms that maintain the critical aspects of NSC biology (quiescence and proliferation) in postnatal age. Our work provides a methodology, transduction of genes deregulated following Sox2 deletion, that allows to test many candidate genes for their ability to sustain NSC proliferation. In principle, this may have interesting implications for identifying targets for pharmacological manipulations.
    Keywords:  AP1 inhibitor T-5224; CRISPR; CUT&RUN; Fos; Neural stem cells (NSCs); Socs3; Sox2; lentiviral vector; self-renewal; transcription factors
    DOI:  https://doi.org/10.1002/stem.3373
  31. Nat Commun. 2021 Mar 19. 12(1): 1738
      Strictly controlled inducible gene expression is crucial when engineering biological systems where even tiny amounts of a protein have a large impact on function or host cell viability. In these cases, leaky protein production must be avoided, but without affecting the achievable range of expression. Here, we demonstrate how the central dogma offers a simple solution to this challenge. By simultaneously regulating transcription and translation, we show how basal expression of an inducible system can be reduced, with little impact on the maximum expression rate. Using this approach, we create several stringent expression systems displaying >1000-fold change in their output after induction and show how multi-level regulation can suppress transcriptional noise and create digital-like switches between 'on' and 'off' states. These tools will aid those working with toxic genes or requiring precise regulation and propagation of cellular signals, plus illustrate the value of more diverse regulatory designs for synthetic biology.
    DOI:  https://doi.org/10.1038/s41467-021-21995-7
  32. Cancer Res. 2021 Mar 19. pii: canres.3445.2020. [Epub ahead of print]
      Epigenetic mechanisms such as aberrant DNA methylation (DNAme) are known to drive esophageal squamous cell carcinoma (ESCC), yet they remain poorly understood. Here we studied tumor-specific DNAme in ESCC cases from nine high-incidence countries of Africa, Asia, and South America. Infinium MethylationEPIC array was performed on 108 tumors and 51 normal tissue adjacent to the tumors (NAT) in the discovery phase, and targeted pyrosequencing was performed on 132 tumors and 36 NAT in the replication phase. Top genes for replication were prioritizied by weighting methylation results using RNA-seq data from TCGA and GTEx and validated by qPCR. Methylome analysis comparing tumor and NAT identified 6,796 differentially methylated positions (DMP) and 866 differential methylated regions (DMR) with a 30% methylation (Δβ) difference. The majority of identified DMPs and DMRs were hypermethylated in tumors, particularly in promoters and gene-body regions of genes involved in transcription activation. The top three prioritized genes for replication, PAX9, SIM2 and THSD4 had similar methylation differences in the discovery and replication sets. These genes were exclusively expressed in normal esophageal tissues in GTEx and downregulated in tumors. The specificity and sensitivity of these DNAme events in discriminating tumors from NAT were assessed. Our study identified novel, robust, and crucial tumor-specific DNAme events in ESCC tumors across several high incidence populations of the world. Methylome changes identified in this study may serve as potential targets for biomarker discovery and warrant further functional characterization.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-3445