RUNX1 colludes with NOTCH1 to reprogram chromatin in T cell acute lymphoblastic leukemia.

Runt-related transcription factor 1 (RUNX1) is oncogenic in diverse types of leukemia and epithelial cancers where its expression is associated with poor prognosis. Current models suggest that RUNX1 cooperates with other oncogenic factors (e.g., NOTCH1, TAL1) to drive the expression of proto-oncogenes in T cell acute lymphoblastic leukemia (T-ALL) but the molecular mechanisms controlled by RUNX1 and its cooperation with other factors remain unclear. Integrative chromatin and transcriptional analysis following inhibition of RUNX1 and NOTCH1 revealed a surprisingly widespread role of RUNX1 in the establishment of global H3K27ac levels and that RUNX1 is required by NOTCH1 for cooperative transcription activation of key NOTCH1 target genes including MYC, DTX1, HES4, IL7R, and NOTCH3. Super-enhancers were preferentially sensitive to RUNX1 knockdown and RUNX1-dependent super-enhancers were disrupted following the treatment of a pan-BET inhibitor, I-BET151.

SMARCB1 Loss in Poorly Differentiated Chordomas Drives Tumor Progression.

Poorly differentiated (PD) chordoma, a rare, aggressive tumor originating from notochordal tissue, shows loss of SMARCB1 expression, a core component of the Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes. To determine the impact of SMARCB1 re-expression on cell growth and gene expression, two SMARCB1-negative PD chordoma cell lines with an inducible SMARCB1 expression system were generated. After 72 hours of induction of SMARCB1, both SMARCB1-negative PD chordoma cell lines continued to proliferate. This result contrasted with those observed with SMARCB1-negative rhabdoid cell lines in which SMARCB1 re-expression caused the rapid inhibition of growth. We found that the lack of growth inhibition may arise from the loss of CDKN2A (p16INK4A) expression in PD chordoma cell lines. RNA-sequencing of cell lines after SMARCB1 re-expression showed a down-regulation for rRNA and RNA processing as well as metabolic processing and increased expression of genes involved in cell adhesion, cell migration, and development. Taken together, these data establish that SMARCB1 re-expression in PD chordomas alters the repertoire of SWI/SNF complexes, perhaps restoring those associated with cellular differentiation. These novel findings support a model in which SMARCB1 inactivation blocks the conversion of growth-promoting SWI/SNF complexes to differentiation-inducing ones, and they implicate SMARCB1 loss as a late event in tumorigenic progression. Importantly, the absence of growth inhibition after SMARCB1 restoration creates a unique opportunity to identify therapeutic vulnerabilities.

Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents.

The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.

Stress hematopoiesis induces a proliferative advantage in TET2 deficiency.

TET2 loss-of-function mutations are recurrent events in a wide range of hematological malignancies and a physiologic occurrence in blood cells of healthy older adults. It is currently unknown what determines if a person harboring a somatic TET2 mutation will progress to myelodysplastic syndrome or acute myeloid leukemia. Here we develop a zebrafish tet2 mutant through which we show that tet2 loss leads to restricted hematopoietic differentiation combined with a modest upregulation of p53, which is also characteristic of many inherited bone marrow failure syndromes. Uniquely in the context of emergency hematopoiesis by external stimuli, such as infection or cytokine stimulation, lack of tet2 leads hematopoietic stem cells to undergo excessive proliferation, resulting in an accumulation of immature cells, which are poised to become leukemogenic following additional genetic/epigenetic perturbations. This same phenomenon observed in zebrafish extends to human hematopoietic stem cells, identifying TET2 as a critical relay switch in the context of stress hematopoiesis.

Prenatal Adversity Alters the Epigenetic Profile of the Prefrontal Cortex: Sexually Dimorphic Effects of Prenatal Alcohol Exposure and Food-Related Stress.

Prenatal adversity or stress can have long-term consequences on developmental trajectories and health outcomes. Although the biological mechanisms underlying these effects are poorly understood, epigenetic modifications, such as DNA methylation, have the potential to link early-life environments to alterations in physiological systems, with long-term functional implications. We investigated the consequences of two prenatal insults, prenatal alcohol exposure (PAE) and food-related stress, on DNA methylation profiles of the rat brain during early development. As these insults can have sex-specific effects on biological outcomes, we analyzed epigenome-wide DNA methylation patterns in prefrontal cortex, a key brain region involved in cognition, executive function, and behavior, of both males and females. We found sex-dependent and sex-concordant influences of these insults on epigenetic patterns. These alterations occurred in genes and pathways related to brain development and immune function, suggesting that PAE and food-related stress may reprogram neurobiological/physiological systems partly through central epigenetic changes, and may do so in a sex-dependent manner. Such epigenetic changes may reflect the sex-specific effects of prenatal insults on long-term functional and health outcomes and have important implications for understanding possible mechanisms underlying fetal alcohol spectrum disorder and other neurodevelopmental disorders.

MYC-induced human acute myeloid leukemia requires a continuing IL-3/GM-CSF costimulus.

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.

Epiclomal: Probabilistic clustering of sparse single-cell DNA methylation data.

We present Epiclomal, a probabilistic clustering method arising from a hierarchical mixture model to simultaneously cluster sparse single-cell DNA methylation data and impute missing values. Using synthetic and published single-cell CpG datasets, we show that Epiclomal outperforms non-probabilistic methods and can handle the inherent missing data characteristic that dominates single-cell CpG genome sequences. Using newly generated single-cell 5mCpG sequencing data, we show that Epiclomal discovers sub-clonal methylation patterns in aneuploid tumour genomes, thus defining epiclones that can match or transcend copy number-determined clonal lineages and opening up an important form of clonal analysis in cancer. Epiclomal is written in R and Python and is available at https://github.com/shahcompbio/Epiclomal.

The Pathognomonic FOXL2 C134W Mutation Alters DNA-Binding Specificity.

The somatic missense point mutation c.402C>G (p.C134W) in the FOXL2 transcription factor is pathognomonic for adult-type granulosa cell tumors (AGCT) and a diagnostic marker for this tumor type. However, the molecular consequences of this mutation and its contribution to the mechanisms of AGCT pathogenesis remain unclear. To explore these mechanisms, we engineered V5-FOXL2WT- and V5-FOXL2C134W-inducible isogenic cell lines and performed chromatin immunoprecipitation sequencing and transcriptome profiling. FOXL2C134W associated with the majority of the FOXL2 wild-type DNA elements as well as a large collection of unique elements genome wide. This model enabled confirmation of altered DNA-binding specificity for FOXL2C134W and identification of unique targets of FOXL2C134W including SLC35F2, whose expression increased sensitivity to YM155. Our results suggest FOXL2C134W drives AGCT by altering the binding affinity of FOXL2-containing complexes to engage an oncogenic transcriptional program. SIGNIFICANCE: A mechanistic understanding of FOXL2C134W-induced regulatory state alterations drives discovery of a rationally designed therapeutic strategy.

De novo pathogenic DNM1L variant in a patient diagnosed with atypical hereditary sensory and autonomic neuropathy.

BACKGROUND: Profiling the entire genome at base pair resolution in a single test offers novel insights into disease by means of dissection of genetic contributors to phenotypic features. METHODS: We performed genome sequencing for a patient who presented with atypical hereditary sensory and autonomic neuropathy, severe epileptic encephalopathy, global developmental delay, and growth hormone deficiency. RESULTS: Assessment of the variants detected by mapped sequencing reads followed by Sanger confirmation revealed that the proband is a compound heterozygote for rare variants within RETREG1 (FAM134B), a gene associated with a recessive form of hereditary sensory and autonomic neuropathy, but not with epileptic encephalopathy or global developmental delay. Further analysis of the data also revealed a heterozygous missense variant in DNM1L, a gene previously implicated in an autosomal dominant encephalopathy, epilepsy, and global developmental delay and confirmed by Sanger sequencing to be a de novo variant not present in parental genomes. CONCLUSIONS: Our findings emphasize the importance of genome-wide sequencing in patients with a well-characterized genetic disease with atypical presentation. This approach reduces the potential for misdiagnoses.

Autophagy Regulation of Metabolism Is Required for CD8+ T Cell Anti-tumor Immunity.

Autophagy is a cell survival process essential for the regulation of immune responses to infections. However, the role of T cell autophagy in anti-tumor immunity is less clear. Here, we demonstrate a cell-autonomous role for autophagy in the regulation of CD8+ T-cell-mediated control of tumors. Mice deficient for the essential autophagy genes Atg5, Atg14, or Atg16L1 display a dramatic impairment in the growth of syngeneic tumors. Moreover, T cells lacking Atg5 have a profound shift to an effector memory phenotype and produce greater amounts of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α). Mechanistically, Atg5-/- CD8+ T cells exhibit enhanced glucose metabolism that results in alterations in histone methylation, increases in H3K4me3 density, and transcriptional upregulation of both metabolic and effector target genes. Nonetheless, glucose restriction is sufficient to suppress Atg5-dependent increases in effector function. Thus, autophagy-dependent changes in CD8+ T cell metabolism directly regulate anti-tumor immunity.

High-Resolution Single-Cell DNA Methylation Measurements Reveal Epigenetically Distinct Hematopoietic Stem Cell Subpopulations.

Increasing evidence of functional and transcriptional heterogeneity in phenotypically similar cells examined individually has prompted interest in obtaining parallel methylome data. We describe the development and application of such a protocol to index-sorted murine and human hematopoietic cells that are highly enriched in their content of functionally defined stem cells. Utilizing an optimized single-cell bisulfite sequencing protocol, we obtained quantitative DNA methylation measurements of up to 5.7 million CpGs in single hematopoietic cells. In parallel, we developed an analytical strategy (PDclust) to define single-cell DNA methylation states through pairwise comparisons of single-CpG methylation measurements. PDclust revealed that a single-cell epigenetic state can be described by a small (<1%) stochastically sampled fraction of CpGs and that these states are reflective of cell identity and state. Using relationships revealed by PDclust, we derive near complete methylomes for epigenetically distinct subpopulations of hematopoietic cells enriched for functional stem cell content.

Single-cell analysis identifies a CD33+ subset of human cord blood cells with high regenerative potential.

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy.

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

The [PSI +] yeast prion does not wildly affect proteome composition whereas selective pressure exerted on [PSI +] cells can promote aneuploidy.

The yeast Sup35 protein is a subunit of the translation termination factor, and its conversion to the [PSI +] prion state leads to more translational read-through. Although extensive studies have been done on [PSI +], changes at the proteomic level have not been performed exhaustively. We therefore used a SILAC-based quantitative mass spectrometry approach and identified 4187 proteins from both [psi -] and [PSI +] strains. Surprisingly, there was very little difference between the two proteomes under standard growth conditions. We found however that several [PSI +] strains harbored an additional chromosome, such as chromosome I. Albeit, we found no evidence to support that [PSI +] induces chromosomal instability (CIN). Instead we hypothesized that the selective pressure applied during the establishment of [PSI +]-containing strains could lead to a supernumerary chromosome due to the presence of the ade1-14 selective marker for translational read-through. We therefore verified that there was no prevalence of disomy among newly generated [PSI +] strains in absence of strong selection pressure. We also noticed that low amounts of adenine in media could lead to higher levels of mitochondrial DNA in [PSI +] in ade1-14 cells. Our study has important significance for the establishment and manipulation of yeast strains with the Sup35 prion.

Atrophin controls developmental signaling pathways via interactions with Trithorax-like.

Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-pallidoluysian atrophy, a neurodegenerative disease. Drosophila Atrophin (Atro) mutants display many phenotypes, including neurodegeneration, segmentation, patterning and planar polarity defects. Despite Atro's critical role in development and disease, relatively little is known about Atro's binding partners and downstream targets. We present the first genomic analysis of Atro using ChIP-seq against endogenous Atro. ChIP-seq identified 1300 potential direct targets of Atro including engrailed, and components of the Dpp and Notch signaling pathways. We show that Atro regulates Dpp and Notch signaling in larval imaginal discs, at least partially via regulation of thickveins and fringe. In addition, bioinformatics analyses, sequential ChIP and coimmunoprecipitation experiments reveal that Atro interacts with the Drosophila GAGA Factor, Trithorax-like (Trl), and they bind to the same loci simultaneously. Phenotypic analyses of Trl and Atro clones suggest that Atro is required to modulate the transcription activation by Trl in larval imaginal discs. Taken together, these data indicate that Atro is a major Trl cofactor that functions to moderate developmental gene transcription.

Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Active Enhancer States and Associated Transcription Factor Networks.

The normal adult human mammary gland is a continuous bilayered epithelial system. Bipotent and myoepithelial progenitors are prominent and unique components of the outer (basal) layer. The inner (luminal) layer includes both luminal-restricted progenitors and a phenotypically separable fraction that lacks progenitor activity. We now report an epigenomic comparison of these three subsets with one another, with their associated stromal cells, and with three immortalized, non-tumorigenic human mammary cell lines. Each genome-wide analysis contains profiles for six histone marks, methylated DNA, and RNA transcripts. Analysis of these datasets shows that each cell type has unique features, primarily within genomic regulatory regions, and that the cell lines group together. Analyses of the promoter and enhancer profiles place the luminal progenitors in between the basal cells and the non-progenitor luminal subset. Integrative analysis reveals networks of subset-specific transcription factors.

Nucleosome Density ChIP-Seq Identifies Distinct Chromatin Modification Signatures Associated with MNase Accessibility.

Nucleosome position, density, and post-translational modification are widely accepted components of mechanisms regulating DNA transcription but still incompletely understood. We present a modified native ChIP-seq method combined with an analytical framework that allows MNase accessibility to be integrated with histone modification profiles. Application of this methodology to the primitive (CD34+) subset of normal human cord blood cells enabled genomic regions enriched in one versus two nucleosomes marked by histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 trimethylation (H3K27me3) to be associated with their transcriptional and DNA methylation states. From this analysis, we defined four classes of promoter-specific profiles and demonstrated that a majority of bivalent marked promoters are heterogeneously marked at a single-cell level in this primitive cell type. Interestingly, extension of this approach to human embryonic stem cells revealed an altered relationship between chromatin modification state and nucleosome content at promoters, suggesting developmental stage-specific organization of histone methylation states.