The COMPASS protein family catalyzes histone H3 Lys 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and ...bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells. We found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, DNA demethylation on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4 trimethylation underlies transcriptional regulation on MLL2/COMPASS targets.
SPOROS: A pipeline to analyze DISE/6mer seed toxicity Bartom, Elizabeth T; Kocherginsky, Masha; Paudel, Bidur ...
PLOS computational biology/PLoS computational biology,
03/2022, Letnik:
18, Številka:
3
Journal Article
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microRNAs (miRNAs) are (18-22nt long) noncoding short (s)RNAs that suppress gene expression by targeting the 3' untranslated region of target mRNAs. This occurs through the seed sequence located in ...position 2-7/8 of the miRNA guide strand, once it is loaded into the RNA induced silencing complex (RISC). G-rich 6mer seed sequences can kill cells by targeting C-rich 6mer seed matches located in genes that are critical for cell survival. This results in induction of Death Induced by Survival gene Elimination (DISE), through a mechanism we have called 6mer seed toxicity. miRNAs are often quantified in cells by aligning the reads from small (sm)RNA sequencing to the genome. However, the analysis of any smRNA Seq data set for predicted 6mer seed toxicity requires an alternative workflow, solely based on the exact position 2-7 of any short (s)RNA that can enter the RISC. Therefore, we developed SPOROS, a semi-automated pipeline that produces multiple useful outputs to predict and compare 6mer seed toxicity of cellular sRNAs, regardless of their nature, between different samples. We provide two examples to illustrate the capabilities of SPOROS: Example one involves the analysis of RISC-bound sRNAs in a cancer cell line (either wild-type or two mutant lines unable to produce most miRNAs). Example two is based on a publicly available smRNA Seq data set from postmortem brains (either from normal or Alzheimer's patients). Our methods (found at https://github.com/ebartom/SPOROS and at Code Ocean: https://doi.org/10.24433/CO.1732496.v1) are designed to be used to analyze a variety of smRNA Seq data in various normal and disease settings.
Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise. A heterozygous point mutation of histone H3 occurs in more ...than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M). Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.
Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila ...melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.
Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar ...macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.
During B lymphopoiesis, recombination of the locus encoding the immunoglobulin κ-chain complex (Igk) requires expression of the precursor to the B cell antigen receptor (pre-BCR) and escape from ...signaling via the interleukin 7 receptor (IL-7R). By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that a STAT5 tetramer bound the Igk intronic enhancer (E(κi)), which led to recruitment of the histone methyltransferase Ezh2. Ezh2 marked trimethylation of histone H3 at Lys27 (H3K27me3) throughout the κ-chain joining region (J(κ)) to the κ-chain constant region (C(κ)). In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R-STAT5 signaling, and the transcription factor E2A bound E(κi), which resulted in acquisition of H3K4me1 and acetylated histone H4 (H4Ac). Genome-wide analyses showed a STAT5 tetrameric binding motif associated with transcriptional repression. Our data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression.
Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from ...the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.
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•UBE2O acts downstream of the interleukin-1 pathway to regulate MLL/COMPASS stability•Stabilizing wild-type MLL protein inhibits MLL leukemia cell proliferation•UBE2O and IRAK inhibition alters a common set of MLL chimera target genes•Targeting the IL-1 pathway is a potential therapeutic strategy for MLL leukemia
Stabilizing wild-type MLL proteins is a potential therapeutic approach for leukemia resulting from MLL translocations.
Post-translational modification of ribosomal subunit proteins (RPs) is emerging as an important means of regulating gene expression. Recently, regulatory ubiquitination of small RPs RPS10 and RPS20 ...by the ubiquitin ligase ZNF598 was found to function in ribosome sensing and stalling on internally polyadenylated mRNAs during ribosome quality control (RQC). Here, we reveal that ZNF598 and RPS10 negatively regulate interferon-stimulated gene (ISG) expression in primary cells, depletion of which induced ISG expression and a broad antiviral state. However, cell lines lacking interferon responses revealed that ZNF598 E3 ligase activity and ubiquitination of RPS20, but not RPS10, were specifically required for poxvirus replication and synthesis of poxvirus proteins whose encoding mRNAs contain unusual 5′ poly(A) leaders. Our findings reveal distinct functions for ZNF598 and its downstream RPS targets, one that negatively regulates ISG expression and infection by a range of viruses while the other is positively exploited by poxviruses.
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•ZNF598 negatively regulates interferon-stimulated gene (ISG) expression•ZNF598 specifically functions in poxvirus infection of cell lines lacking ISG responses•Poxvirus protein synthesis requires ZNF598 ubiquitin ligase activity•Poxvirus replication requires ubiquitination of the ZNF598 substrate, RPS20
In addition to repressing poly(A) readthrough during ribosome quality control, DiGiuseppe et al. report that ZNF598 and RPS10 negatively regulate interferon-stimulated gene expression (ISG). However, in cell lines lacking ISG responses, ZNF598 ubiquitin ligase activity and RPS20 ubiquitination are specifically required by poxviruses, which produce unusual mRNAs with 5′ poly(A) leaders.
Glioblastoma (GBM) is the most aggressive primary brain tumor in adults and is associated with a median overall survival (mOS) of 16-21 months. Our previous work found a negative association between ...advanced aging and the survival benefit after treatment with immunotherapy in an experimental brain tumor model. Given the recent phase III clinical success of immunotherapy in patients with many types of cancer, but not for patients with GBM, we hypothesize that aging enhances immunosuppression in the brain and contributes to the lack of efficacy for immunotherapy to improve mOS in patients with malignant glioma. Herein, we compare epidemiological data for the incidence and mortality of patients with central nervous system (CNS) cancers, in addition to immune-related gene expression in the normal human brain, as well as peripheral blood immunological changes across the adult lifespan.
Data were extracted from the National Cancer Institute's surveillance, epidemiology, and end results (SEER)-, the Broad Institute's Genotype Tissue Expression project (GTEx)-, and the University of California San Francisco's 10k Immunomes-databases and analyzed for associations with aging.
The proportion of elderly individuals, defined as ≥65 years of age, has predominantly increased for more than 100 years in the United States. Over time, the rise in elderly United States citizens has correlated with an increased incidence and mortality rate associated with primary brain and other CNS cancer. With advanced aging, human mRNA expression for factors associated with immunoregulation including immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO) and programmed death-ligand 1 (PD-L1), as well as the dendritic cell surface marker, CD11c, increase in the brain of normal human subjects, coincident with increased circulating immunosuppressive Tregs and decreased cytolytic CD8
T cells in the peripheral blood. Strikingly, these changes are maximally pronounced in the 60-69 year old group; consistent with the median age of a diagnosis for GBM.
These data demonstrate a significant association between normal human aging and increased immunosuppression in the circulation and CNS; particularly late in life. Our data raise several hypotheses including that, aging: (i) progressively suppresses normal immunosurveillance and thereby contributes to GBM cell initiation and/or outgrowth; (ii) decreases immunotherapeutic efficacy against malignant glioma.
Age-associated bone marrow changes include myeloid skewing and mutations that lead to clonal hematopoiesis. Molecular mechanisms for these events are ill defined, but decreased expression of ...Irf8/Icsbp (interferon regulatory factor 8/interferon consensus sequence binding protein) in aging hematopoietic stem cells may contribute. Irf8 functions as a leukemia suppressor for chronic myeloid leukemia, and young Irf8−/− mice have neutrophilia with progression to acute myeloid leukemia (AML) with aging. Irf8 is also required to terminate emergency granulopoiesis during the innate immune response, suggesting this may be the physiologic counterpart to leukemia suppression by this transcription factor. Identifying Irf8 effectors may define mediators of both events and thus contributors to age-related bone marrow disorders. In this study, we identified RASSF5 (encoding Nore1) as an Irf8 target gene and investigated the role of Nore1 in hematopoiesis. We found Irf8 activates RASSF5 transcription and increases Nore1a expression during emergency granulopoiesis. Similar to Irf8−/− mice, we found that young Rassf5−/− mice had increased neutrophils and progressed to AML with aging. We identified enhanced DNA damage, excess clonal hematopoiesis, and a distinct mutation profile in hematopoietic stem cells from aging Rassf5−/− mice compared with wildtype. We found sustained emergency granulopoiesis in Rassf5−/− mice, with repeated episodes accelerating AML, also similar to Irf8−/− mice. Identifying Nore1a downstream from Irf8 defines a pathway involved in leukemia suppression and the innate immune response and suggests a novel molecular mechanism contributing to age-related clonal myeloid disorders.
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