Host-derived Interleukin 1α induces an immunosuppressive tumor microenvironment via regulating monocyte-to-macrophage differentiation.

Tumor-associated macrophages exhibit high heterogeneity and contribute to the establishment of an immunosuppressive tumor microenvironment (TME). Although numerous studies have demonstrated that extracellular factors promote macrophage proliferation and polarization, the regulatory mechanisms governing the differentiation process to generate phenotypically, and functionally diverse macrophage subpopulations remain largely unexplored. In this study, we examined the influence of interleukin 1α (IL-1α) on the development of an immunosuppressive TME using orthotopic transplantation murine models of breast cancer. Deletion of host Il1α led to the rejection of inoculated congenic tumors. Single-cell sequencing analysis revealed that CX3CR1+ macrophage cells were the primary sources of IL-1α in the TME. The absence of IL-1α reprogrammed the monocyte-to-macrophage differentiation process within the TME, characterized by a notable decrease in the subset of CX3CR+ ductal-like macrophages and an increase in iNOS-expressing inflammatory cells. Comparative analysis of gene signatures in both human and mouse macrophage subsets suggested that IL-1α deficiency shifted the macrophage polarization from M2 to M1 phenotypes, leading to enhanced cytotoxic T lymphocyte activity in the TME. Importantly, elevated levels of IL-1α in human cancers were associated with worse prognosis following immunotherapy. These findings underscore the pivotal role of IL-1α in shaping an immune-suppressive TME through the regulation of macrophage differentiation and activity, highlighting IL-1α as a potential target for breast cancer treatment.

DNA methylation and histone modifications are essential for regulation of stem cell formation and differentiation in zebrafish development.

The complex processes necessary for embryogenesis require a gene regulatory network that is complex and systematic. Gene expression regulates development and organogenesis, but this process is altered and fine-tuned by epigenetic regulators that facilitate changes in the chromatin landscape. Epigenetic regulation of embryogenesis adjusts the chromatin structure by modifying both DNA through methylation and nucleosomes through posttranslational modifications of histone tails. The zebrafish is a well-characterized model organism that is a quintessential tool for studying developmental biology. With external fertilization, low cost and high fecundity, the zebrafish are an efficient tool for studying early developmental stages. Genetic manipulation can be performed in vivo resulting in quick identification of gene function. Large-scale genome analyses including RNA sequencing, chromatin immunoprecipitation and chromatin structure all are feasible in the zebrafish. In this review, we highlight the key events in zebrafish development where epigenetic regulation plays a critical role from the early stem cell stages through differentiation and organogenesis.

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

Regulation of stress-induced hematopoiesis.

PURPOSE OF REVIEW: The hematopoietic compartment is tasked with the establishment and maintenance of the entire blood program in steady-state and in response to stress. Key to this process are hematopoietic stem cells (HSCs), which possess the unique ability to self-renew and differentiate to replenish blood cells throughout an organism's lifetime. Though tightly regulated, the hematopoietic system is vulnerable to both intrinsic and extrinsic factors that influence hematopoietic stem and progenitor cell (HSPC) fate. Here, we review recent advances in our understanding of hematopoietic regulation under stress conditions such as inflammation, aging, mitochondrial defects, and damage to DNA or endoplasmic reticulum. RECENT FINDINGS: Recent studies have illustrated the vast mechanisms involved in regulating stress-induced hematopoiesis, including cytokine-mediated lineage bias, gene signature changes in aged HSCs associated with chronic inflammation, the impact of clonal hematopoiesis and stress tolerance, characterization of the HSPC response to endoplasmic reticulum stress and of several epigenetic regulators that influence HSPC response to cell cycle stress. SUMMARY: Several key recent findings have deepened our understanding of stress hematopoiesis. These studies will advance our abilities to reduce the impact of stress in disease and aging through clinical interventions to treat stress-related outcomes.

Systemic Toxicity Reported for CDK8/19 Inhibitors CCT251921 and MSC2530818 Is Not Due to Target Inhibition.

CDK8/19 kinases, which mediate transcriptional reprogramming, have become an active target for cancer drug discovery. Several small-molecule CDK8/19 inhibitors showed in vivo efficacy and two have entered clinical trials, with no significant toxicities reported. However, Clarke et al. (eLife 2016; 5; e20722) found severe systemic toxicity associated with two potent CDK8/19 inhibitors, Cmpd3 (CCT251921) and Cmpd4 (MSC2530818), and suggested that their toxicity was due to on-target effects. Here, we compared five CDK8/19 inhibitors: Cmpd3, Cmpd4, Senexin B, 16-didehydro-cortistatin A (dCA) and 15w, in different assays. Only Cmpd4 showed striking toxicity in developing zebrafish. In cell-based assays for CDK8 and CDK19 inhibition, Cmpd3, Cmpd4, dCA and 15w showed similar low-nanomolar potency and efficacy against CDK8 and CDK19, while Senexin B was less potent. Only dCA produced sustained inhibition of CDK8/19-dependent gene expression. While toxicity of different compounds did not correlate with their effects on CDK8 and CDK19, kinome profiling identified several off-target kinases for both Cmpd3 and Cmpd4, which could be responsible for their toxicity. Off-target activities could have been achieved in the study of Clarke et al. due to high in vivo doses of Cmpd3 and Cmpd4, chosen for the ability to inhibit STAT1 S727 phosphorylation in tumor xenografts. We show here that STAT1 S727 phosphorylation is induced by various cytokines and stress stimuli in CDK8/19-independent manner, indicating that it is not a reliable pharmacodynamic marker of CDK8/19 activity. These results illustrate the need for careful off-target analysis and dose selection in the development of CDK8/19 inhibitors.

CAT7 and cat7l Long Non-coding RNAs Tune Polycomb Repressive Complex 1 Function during Human and Zebrafish Development.

The essential functions of polycomb repressive complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs), but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs, which co-precipitate with PRC1 from chromatin and found candidates that impact polycomb group protein (PcG)-regulated gene expression in vivo A novel lncRNA from this screen, CAT7, regulates expression and polycomb group binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain that shares high sequence similarity to a non-syntenic zebrafish analog, cat7l Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA, enhanced by interference of a PRC1 component, and suppressed by interference of a known PRC1 target gene, demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs and that CAT7/cat7l represents convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci.

A network of epigenetic regulators guides developmental haematopoiesis in vivo.

The initiation of cellular programs is orchestrated by key transcription factors and chromatin regulators that activate or inhibit target gene expression. To generate a compendium of chromatin factors that establish the epigenetic code during developmental haematopoiesis, a large-scale reverse genetic screen was conducted targeting orthologues of 425 human chromatin factors in zebrafish. A set of chromatin regulators was identified that target different stages of primitive and definitive blood formation, including factors not previously implicated in haematopoiesis. We identified 15 factors that regulate development of primitive erythroid progenitors and 29 factors that regulate development of definitive haematopoietic stem and progenitor cells. These chromatin factors are associated with SWI/SNF and ISWI chromatin remodelling, SET1 methyltransferase, CBP-p300-HBO1-NuA4 acetyltransferase, HDAC-NuRD deacetylase, and Polycomb repressive complexes. Our work provides a comprehensive view of how specific chromatin factors and their associated complexes play a major role in the establishment of haematopoietic cells in vivo.

The transcriptional landscape of hematopoietic stem cell ontogeny.

Transcriptome analysis of adult hematopoietic stem cells (HSCs) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSCs purified from >2,500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network-biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knockdown in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSCs, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.

CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development.

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells.

Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors.

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFß transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFß and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFß deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

Ikaros directly represses the notch target gene Hes1 in a leukemia T cell line: implications for CD4 regulation.

Ikaros and Notch1, two regulators of gene transcription, are critically important at many stages of T cell development. Deregulation of Ikaros and Notch activities cooperate to promote T cell leukemogenesis, providing evidence that they function in converging pathways in developing T cells. In this report, a mechanism for Ikaros:Notch cooperativity is described, revealing a non-redundant role for Ikaros in regulating expression of the Notch target gene Hes1 in a leukemia T cell line. We provide evidence that Ikaros directly represses Hes1 in concert with the transcriptional repressor, RBP-Jkappa, allowing for cross-talk between Notch and Ikaros that impacts regulation of CD4 expression. Taken together, these data describe a potential mechanism for Ikaros' function during T cell development and define Ikaros as an obligate repressor of Hes1.

Ikaros induces quiescence and T-cell differentiation in a leukemia cell line.

Ikaros is a hematopoietic cell-specific zinc finger DNA binding protein that plays an important role in lymphocyte development. Genetic disruption of Ikaros results in T-cell transformation. Ikaros null mice develop leukemia with 100% penetrance. It has been hypothesized that Ikaros controls gene expression through its association with chromatin remodeling complexes. The development of leukemia in Ikaros null mice suggests that Ikaros has the characteristics of a tumor suppressor gene. In this report, we show that the introduction of Ikaros into an established mouse Ikaros null T leukemia cell line leads to growth arrest at the G0/G1 stage of the cell cycle. This arrest is associated with up-regulation of the cell cycle-dependent kinase inhibitor p27kip1, the induction of expression of T-cell differentiation markers, and a global and specific increase in histone H3 acetylation status. These studies provide strong evidence that Ikaros possesses the properties of a bona fide tumor suppressor gene for the T-cell lineage and offer insight into the mechanism of Ikaros's tumor suppressive activity.