A Genetic Disorder Reveals a Hematopoietic Stem Cell Regulatory Network Co-Opted in Leukemia

The molecular regulation of human hematopoietic stem cell (HSC) self-renewal and maintenance is of substantial interest, but limitations in experimental systems and interspecies variation have constrained our knowledge of this process. To better discern in vivo HSC function in humans, we have studied a rare genetic disorder due to MECOM haploinsufficiency that is characterized by neonatal aplastic anemia with an early-onset absence of HSCs in vivo. To establish a faithful model of MECOM haploinsufficiency, we performed CRISPR/Cas9 editing of MECOM in human hematopoietic stem and progenitor cells (HSPCs) and achieved predominantly heterozygous editing in phenotypic long-term (LT)-HSCs, as substantiated through both bulk and single cell assessments. Following MECOM editing, HSPCs showed a significant reduction in the number of phenotypic LT-HSCs as well as multipotential progenitor colonies in vitro, and had impaired engraftment following xenotransplantation into immunodeficient and Kit mutant mice. Next, we sought to use this model of MECOM haploinsufficiency to define the regulatory networks driven by MECOM that are critical for HSC maintenance. We therefore performed single-cell RNA sequencing on several thousand phenotypic LT-HSCs following MECOM editing and identified a list of 724 subtly but significantly differentially expressed genes compared to controls, including 322 genes that are downregulated after MECOM perturbation. Given the profound phenotypic effects associated with loss of MECOM and dysregulation of these gene sets, we sought to identify other cooperating factors that control the MECOM dependent gene network which underlies HSC self-renewal. To do so, we used integrative genomic approaches involving accessible chromatin and gene expression correlations, as well as long range chromatin interaction data across human hematopoiesis, to comprehensively define associations between genes and putative regulatory elements. Inspection of the nominated cis-regulatory elements controlling genes impacted by MECOM perturbation revealed significant enrichment for motifs and chromatin occupancy by several key cooperating transcription factors, including RUNX1, FLI1, and GATA2. In addition to the identification of cooperating transcription factors, we also discovered a strong binding motif enrichment for, and chromatin occupancy by, CTCF. CTCF is crucial to enable differentiation of LT-HSCs, and we found that MECOM regulated genes frequently had associated cis-regulatory elements that were occupied by CTCF. Moreover, these occupied cis-elements became more highly enriched for binding during hematopoietic differentiation. Chromatin conformation analysis revealed that the MECOM regulated genes with cis-elements bound by CTCF underwent chromatin reorganization and became more highly looped as LT-HSCs underwent differentiation, suggesting opposing functions of MECOM and CTCF in the regulation of the MECOM gene network. In light of these findings, we performed tandem perturbation of MECOM and CTCF and demonstrated rescue of LT-HSC loss with the dual perturbation, thereby illuminating a key role for MECOM in constraining CTCF-dependent chromatin reorganization that occurs as HSCs undergo differentiation. Finally, based on the observation that elevated MECOM expression is associated with high-risk myeloid malignancies, we investigated the role of the MECOM-regulated HSC gene network in acute myeloid leukemias (AML). Across three independent AML datasets, we found that the MECOM regulated gene network had an independent and strong prognostic prediction ability that enabled risk stratification beyond currently used approaches, including a variety of molecular criteria and the previously described LSC17 signature. To validate these correlative observations, we performed CRISPR/Cas9 editing of MECOM in the MUTZ-3 AML cell line that is characterized by MECOM overexpression. We found that MECOM editing results in a loss of CD34 + leukemia progenitors and that the same transcriptional network that we identified in LT-HSCs is similarly altered upon MECOM perturbation in these AML cells. Collectively, we use the study of a rare experiment of nature due to MECOM haploinsufficiency resulting in neonatal aplastic anemia to illuminate a gene regulatory network necessary for HSC self-renewal and maintenance that is co-opted in high-risk forms of AML. Disclosures Regev: Genentech: Current Employment; Celsius Therapeutics: Current equity holder in publicly-traded company, Other: Co-founder; Immunitas: Current equity holder in publicly-traded company; ThermoFisher Scientific: Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Neogene Therapeutics: Membership on an entity's Board of Directors or advisory committees; Asimov: Membership on an entity's Board of Directors or advisory committees. Sankaran: Forma: Consultancy; Ensoma: Consultancy; Novartis: Consultancy; Cellarity: Consultancy; Branch Biosciences: Consultancy.

Sexual Dimorphism in Mouse Meiosis

Meiosis is a highly conserved and essential process in gametogenesis in sexually reproducing organisms. However, there are substantial sex-specific differences within individual species with respect to meiosis-related chromatin reorganization, recombination, and tolerance for meiotic defects. A wide range of murine models have been developed over the past two decades to study the complex regulatory processes governing mammalian meiosis. The present review article thus provides a comprehensive overview of the knockout mice that have been employed to study meiosis, with a particular focus on gene- and gametogenesis-related sexual dimorphism observed in these model animals. In so doing, we aim to provide a firm foundation for the future study of sex-specific differences in meiosis at the molecular level.

DNA METHYLATION IN EMBRYOS AND SEEDLINGS OF PLANTS

A complex system of genetic and epigenetic networks controls growth and development of plants. DNA methylation is one of the epigenetic mechanisms involved in suppression of transposon activity, chromatin reorganization, genomic imprinting, and regulation of gene expression. Modulation of the degree of genome methylation is observed during the implementation of morphogenetic development programs and in response to external influences. The change in the degree and pattern of methylation at the embryo-seedling stage in this mini-review is considered. The issues of molecular mechanisms of methylation and its role in the processes of embryo formation, germination and seedling development are discussed.

Locus-specific induction of gene expression from heterochromatin loci during cellular senescence

Cellular senescence is a fate-determined state, accompanied by reorganization of heterochromatin. While lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3K9me3. The fate of these heterochromatic genes during the chromatin reorganization accompanying senescence is unclear. Here we show a small number of lineage-inappropriate genes are derepressed in senescent cells from H3K9me3 regions that gain open chromatin marks. DNA FISH experiments reveal that these gene loci, which are tightly condensed at the nuclear periphery in proliferative cells, are physically decompacted during senescence. Among these gene loci, NLRP3 is predominantly expressed in immune cells, such as macrophages, where it resides within an open topologically associated domain (TAD). In contrast, NLRP3 is derepressed in senescent fibroblasts, potentially due to the local disruption of the H3K9me3-rich TAD that contains it. The role of NLRP3 has been implicated in the amplification of inflammatory cytokine signalling in senescence and aging, underscoring the functional relevance of gene induction from ‘permissive’ H3K9me3 regions in senescent cells.

Rapid nucleus-scale reorganization of chromatin in neurons enables transcriptional adaptation for memory consolidation

The interphase nucleus is functionally organized in active and repressed territories defining the transcriptional status of the cell. However, it remains poorly understood how the nuclear architecture of neurons adapts in response to behaviorally relevant stimuli that trigger fast alterations in gene expression patterns. Imaging of fluorescently tagged nucleosomes revealed that pharmacological manipulation of neuronal activity in vitro and auditory cued fear conditioning in vivo induce nucleus-scale restructuring of chromatin within minutes. Furthermore, the acquisition of auditory fear memory is impaired after infusion of a drug into auditory cortex which blocks chromatin reorganization in vitro. We propose that active chromatin movements at the nucleus scale act together with local gene-specific modifications to enable transcriptional adaptations at fast time scales. Introducing a transgenic mouse line for photolabeling of histones, we extend the realm of systems available for imaging of chromatin dynamics to living animals.