bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2020‒12‒27
nine papers selected by
Connor Rogerson
University of Cambridge, MRC Cancer Unit

  1. Nucleic Acids Res. 2020 Dec 21. pii: gkaa1155. [Epub ahead of print]
      Chromatin immunoprecipitation (IP) followed by sequencing (ChIP-seq) is the gold standard to detect transcription-factor (TF) binding sites in the genome. Its success depends on appropriate controls removing systematic biases. The predominantly used controls, i.e. DNA input, correct for uneven sonication, but not for nonspecific interactions of the IP antibody. Another type of controls, 'mock' IP, corrects for both of the issues, but is not widely used because it is considered susceptible to technical noise. The tradeoff between the two control types has not been investigated systematically. Therefore, we generated comparable DNA input and mock IP experiments. Because mock IPs contain only nonspecific interactions, the sites predicted from them using DNA input indicate the spurious-site abundance. This abundance is highly correlated with the 'genomic activity' (e.g. chromatin openness). In particular, compared to cell lines, complex samples such as whole organisms have more spurious sites-probably because they contain multiple cell types, resulting in more expressed genes and more open chromatin. Consequently, DNA input and mock IP controls performed similarly for cell lines, whereas for complex samples, mock IP substantially reduced the number of spurious sites. However, DNA input is still informative; thus, we developed a simple framework integrating both controls, improving binding site detection.
  2. Nat Commun. 2020 Dec 21. 11(1): 6422
      Wnt/β-catenin signaling is crucial for intestinal carcinogenesis and the maintenance of intestinal cancer stem cells. Here we identify the histone methyltransferase Mll1 as a regulator of Wnt-driven intestinal cancer. Mll1 is highly expressed in Lgr5+ stem cells and human colon carcinomas with increased nuclear β-catenin. High levels of MLL1 are associated with poor survival of colon cancer patients. The genetic ablation of Mll1 in mice prevents Wnt/β-catenin-driven adenoma formation from Lgr5+ intestinal stem cells. Ablation of Mll1 decreases the self-renewal of human colon cancer spheres and halts tumor growth of xenografts. Mll1 controls the expression of stem cell genes including the Wnt/β-catenin target gene Lgr5. Upon the loss of Mll1, histone methylation at the stem cell promoters switches from activating H3K4 tri-methylation to repressive H3K27 tri-methylation, indicating that Mll1 sustains stem cell gene expression by antagonizing gene silencing through polycomb repressive complex 2 (PRC2)-mediated H3K27 tri-methylation. Transcriptome profiling of Wnt-mutated intestinal tumor-initiating cells reveals that Mll1 regulates Gata4/6 transcription factors, known to sustain cancer stemness and to control goblet cell differentiation. Our results demonstrate that Mll1 is an essential epigenetic regulator of Wnt/β-catenin-induced intestinal tumorigenesis and cancer stemness.
  3. Bioinformatics. 2020 Dec 21. pii: btaa1048. [Epub ahead of print]
      MOTIVATION: Whole genome bisulfite sequencing (WGBS), measures DNA methylation at base pair resolution resulting in large bedGraph like coverage files. Current options for processing such files are hindered by discrepancies in file format specification, speed and memory requirements.RESULTS: We developed methrix, an R package, which provides a toolset for systematic analysis of large datasets. Core functionality of the package includes a comprehensive bedGraph or similar tab-separated text file reader - which summarizes methylation calls based on annotated reference indices, infers and collapses strands, and handles uncovered reference CpG sites while facilitating a flexible input file format specification. Additional optimized functions for quality control filtering, sub-setting, and visualization allow user-friendly and effective processing of WGBS results. Easy integration with tools for differentially methylated region (DMR) calling and annotation further eases the analysis of genome-wide methylation data. Overall, methrix enriches established WGBS workflows by bringing together computational efficiency and versatile functionality.
    AVAILABILITY AND IMPLEMENTATION: Methrix is implemented as an R package, made available under MIT license at and can be installed from the Bioconductor repository.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  4. Cell Syst. 2020 Dec 14. pii: S2405-4712(20)30463-4. [Epub ahead of print]
      Transcription factors (TFs) bind regulatory DNA to control gene expression, and mutations to either TFs or DNA can alter binding affinities to rewire regulatory networks and drive phenotypic variation. While studies have profiled energetic effects of DNA mutations extensively, we lack similar information for TF variants. Here, we present STAMMP (simultaneous transcription factor affinity measurements via microfluidic protein arrays), a high-throughput microfluidic platform enabling quantitative characterization of hundreds of TF variants simultaneously. Measured affinities for ∼210 mutants of a model yeast TF (Pho4) interacting with 9 oligonucleotides (>1,800 Kds) reveal that many combinations of mutations to poorly conserved TF residues and nucleotides flanking the core binding site alter but preserve physiological binding, providing a mechanism by which combinations of mutations in cis and trans could modulate TF binding to tune occupancies during evolution. Moreover, biochemical double-mutant cycles across the TF-DNA interface reveal molecular mechanisms driving recognition, linking sequence to function. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.
    Keywords:  DNA specificity; bHLH; basic helix-loop-helix; binding affinity; conformational selection; double-mutant cycle; microfluidics; protein-DNA binding; scanning mutagenesis; transcription factor; transcriptional regulation
  5. Cell Death Dis. 2020 Dec 15. 11(12): 1073
      The major challenge in chemotherapy lies in the gain of therapeutic resistance properties of cancer cells. The relatively small fraction of chemo-resistant cancer cells outgrows and are responsible for tumor relapse, with acquired invasiveness and stemness. We demonstrate that zinc-finger MYND type-8 (ZMYND8), a putative chromatin reader, suppresses stemness, drug resistance, and tumor-promoting genes, which are hallmarks of cancer. Reinstating ZMYND8 suppresses chemotherapeutic drug doxorubicin-induced tumorigenic potential (at a sublethal dose) and drug resistance, thereby resetting the transcriptional program of cells to the epithelial state. The ability of ZMYND8 to chemo-sensitize doxorubicin-treated metastatic breast cancer cells by downregulating tumor-associated genes was further confirmed by transcriptome analysis. Interestingly, we observed that ZMYND8 overexpression in doxorubicin-treated cells stimulated those involved in a good prognosis in breast cancer. Consistently, sensitizing the cancer cells with ZMYND8 followed by doxorubicin treatment led to tumor regression in vivo and revert back the phenotypes associated with drug resistance and stemness. Intriguingly, ZMYND8 modulates the bivalent or poised oncogenes through its association with KDM5C and EZH2, thereby chemo-sensitizing the cells to chemotherapy for better disease-free survival. Collectively, our findings indicate that poised chromatin is instrumental for the acquisition of chemo-resistance by cancer cells and propose ZMYND8 as a suitable epigenetic tool that can re-sensitize the chemo-refractory breast carcinoma.
  6. RNA Biol. 2020 Dec 19. 1-14
      RNA-sequencing (RNA-seq) analysis of gene expression and alternative splicing should be routine and robust but is often a bottleneck for biologists because of different and complex analysis programs and reliance on specialized bioinformatics skills. We have developed the '3D RNA-seq' App, an R shiny App and web-based pipeline for the comprehensive analysis of RNA-seq data from any organism. It represents an easy-to-use, flexible and powerful tool for analysis of both gene and transcript-level gene expression to identify differential gene/transcript expression, differential alternative splicing and differential transcript usage (3D) as well as isoform switching from RNA-seq data. 3D RNA-seq integrates state-of-the-art differential expression analysis tools and adopts best practice for RNA-seq analysis. The program is designed to be run by biologists with minimal bioinformatics experience (or by bioinformaticians) allowing lab scientists to analyse their RNA-seq data. It achieves this by operating through a user-friendly graphical interface which automates the data flow through the programs in the pipeline. The comprehensive analysis performed by 3D RNA-seq is extremely rapid and accurate, can handle complex experimental designs, allows user setting of statistical parameters, visualizes the results through graphics and tables, and generates publication quality figures such as heat-maps, expression profiles and GO enrichment plots. The utility of 3D RNA-seq is illustrated by analysis of data from a time-series of cold-treated Arabidopsis plants and from dexamethasone-treated male and female mouse cortex and hypothalamus data identifying dexamethasone-induced sex- and brain region-specific differential gene expression and alternative splicing.
    Keywords:  Arabidopsis; RNA-seq; dexamethasone; differential alternative splicing; differential gene/transcript expression; differential transcript usage; interactive GUI; mouse; time-series
  7. Nat Microbiol. 2020 Dec 21.
      Streptococcus pneumoniae is a natural colonizer of the human respiratory tract and an opportunistic pathogen. Although epithelial cells are among the first to encounter pneumococci, the cellular processes and contribution of epithelial cells to the host response are poorly understood. Here, we show that a S. pneumoniae serotype 6B ST90 strain, which does not cause disease in a murine infection model, induces a unique NF-κB signature response distinct from an invasive-disease-causing isolate of serotype 4 (TIGR4). This signature is characterized by activation of p65 and requires a histone demethylase KDM6B. We show, molecularly, that the interaction of the 6B strain with epithelial cells leads to chromatin remodelling within the IL-11 promoter in a KDM6B-dependent manner, where KDM6B specifically demethylates histone H3 lysine 27 dimethyl. Remodelling of the IL-11 locus facilitates p65 access to three NF-κB sites that are otherwise inaccessible when stimulated by IL-1β or TIGR4. Finally, we demonstrate through chemical inhibition of KDM6B with GSK-J4 inhibitor and through exogenous addition of IL-11 that the host responses to the 6B ST90 and TIGR4 strains can be interchanged both in vitro and in a murine model of infection in vivo. Our studies therefore reveal how a chromatin modifier governs cellular responses during infection.
  8. Nat Genet. 2020 Dec 21.
      Expression quantitative trait loci (eQTLs) studies provide associations of genetic variants with gene expression but fall short of pinpointing functionally important eQTLs. Here, using H3K27ac HiChIP assays, we mapped eQTLs overlapping active cis-regulatory elements that interact with their target gene promoters (promoter-interacting eQTLs, pieQTLs) in five common immune cell types (Database of Immune Cell Expression, Expression quantitative trait loci and Epigenomics (DICE) cis-interactome project). This approach allowed us to identify functionally important eQTLs and show mechanisms that explain their cell-type restriction. We also devised an approach to eQTL discovery that relies on HiChIP-based promoter interaction maps as a structural framework for deciding which SNPs to test for association with gene expression, and observe ultra-long-distance pieQTLs (>1 megabase away), including several disease-risk variants. We validated the functional role of pieQTLs using reporter assays, CRISPRi, dCas9-tiling guides and Cas9-mediated base-pair editing. In this article we present a method for functional eQTL discovery and provide insights into relevance of noncoding variants for cell-specific gene regulation and for disease association beyond conventional eQTL mapping.
  9. Epigenomics. 2020 Dec 22.
      Aim: To develop a method for estimating cell-specific effects in epigenomic association studies in the presence of cell type heterogeneity. Materials & methods: We utilized Monte Carlo Expectation-Maximization (MCEM) algorithm with Metropolis-Hastings sampler to reconstruct the 'missing' cell-specific methylations and to estimate their associations with phenotypes free of confounding by cell type proportions. Results: Simulations showed reliable performance of the method under various settings including when the cell type is rare. Application to a real dataset recapitulated the directly measured cell-specific methylation pattern in whole blood. Conclusion: This work provides a framework to identify important cell groups and account for cell type composition useful for studying the role of epigenetic changes in human traits and diseases.
    Keywords:  DNA methylation; cell-type-specific effect; deconvolution; epigenome-wide association study