Single cell studies have transformed our understanding of cellular heterogeneity in disease but the need for fresh starting material can be an obstacle, especially in the context of international multicenter studies and archived tissue. We developed a protocol to obtain high-quality cells and nuclei from dissected human skeletal muscle archived in the preservative Allprotect® Tissue Reagent. After fluorescent imaging microscopy confirmed intact nuclei, we performed four protocol variations that compared sequencing metrics between cells and nuclei enriched by either filtering or flow cytometry sorting. Cells and nuclei (either sorted or filtered) produced statistically identical transcriptional profiles and recapitulated 8 cell types present in skeletal muscle. Flow cytometry sorting successfully enriched for higher-quality cells and nuclei but resulted in an overall decrease in input material. Our protocol provides an important resource for obtaining high-quality single cell genomic material from archived tissue and to streamline global collaborative efforts. Optimized methods and protocols for single cell and single nucleus RNA sequencing of preserved skeletal muscle provide an important resource for obtaining high-quality single cell genomic material from archived tissue.

Changes in gene expression drive differentiation along distinct cell lineages, and these shifts in gene expression are associated with alterations in chromatin accessibility and modifications reflecting activation or repression. We used deep sequencing of polyA+ RNA to map the transcriptomes of the megakaryocyte-erythroid progenitor (MEP) and cells of its two daughter lineages, erythroblasts (ERY) and megakaryocytes (MEG) in mice to reveal insights into differentiation. Transcriptome comparisons revealed that MEPs already expressed much of the MEG program while continuing to express genes associated with parallel myeloid lineages. By contrast, ERY underwent an extensive program of gene induction along with repression of pan-hematopoietic and MEG genes. Maps of transcription factor (TF) occupancy also indicated distinct modes of regulation for the MEG and ERY programs, with MEG genes preferentially occupied by hematopoietic TFs in multipotent progenitors and continued occupancy post-commitment, in contrast to erythroid genes that were primarily occupied in committed ERY. Previous work had indicated a surprising discordance in the clustering of MEP with other hematopoietic cell types by RNA-seq versus chromatin states. We combined the differential expression data with chromatin accessibility across blood cell types to identify trends that contribute to this discordance. Specifically, candidate cis-regulatory elements (cCREs) in some ERY-specific genes were precociously actuated in the bipotential cell populations, and some other genes were expressed in both the MEP population and MEG but their cCREs have less chromatin accessibility in MEP. This discordance in cell type clustering by different modalities of functional genomics may reflect the different contributions of subpopulations in the MEP to the different modalities measured.

Single cell genomics analysis often utilizes freshly collected or flash frozen tissue, which is not always available or whose collection is not feasible in research and clinical contexts. Similarly, often the only tissue available on research participants and patients is archived or biobanked. This protocol includes four methods using a combination of flow cytometry and confocal microscopy to extract high quality single cell RNA sequencing data (scRNA-Seq data) from tissues preserved in the nucleic acid stabilizing fixative AllProtect‱ Tissue Reagent.

Inducible pluripotent stem cells (iPSCs) derived from patient samples have significantly enhanced our ability to model neurological diseases. Comparative studies of dopaminergic (DA) neurons differentiated from iPSCs derived from siblings with Gaucher disease discordant for parkinsonism provides a valuable avenue to explore genetic modifiers contributing to GBA1 -associated parkinsonism in disease-relevant cells. However, such studies are often complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich dopaminergic (DA) neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene following a T2A self-cleavage peptide, placing its expression under the control of the TH promoter. This allows for TH+ DA neuron enrichment through geneticin selection. This method enabled us to generate comparable, high-purity DA neuron cultures from iPSC lines derived from three sisters that we followed for over a decade: one sibling is a healthy individual, and the other two have Gaucher disease (GD) with GBA1 genotype N370S/c.203delC+R257X (p.N409S/c.203delC+p.R296X). Notably, the younger sister with GD later developed Parkinson disease (PD). A comprehensive analysis of these high-purity DA neurons revealed that although GD DA neurons exhibited decreased levels of glucocerebrosidase (GCase), there was no substantial difference in GCase protein levels or lipid substrate accumulation between DA neurons from the GD and GD/PD sisters, suggesting that the PD discordance is related to of other genetic modifiers.

Single cell studies have transformed our understanding of cellular heterogeneity in disease but the need for fresh starting material can be an obstacle, especially in the context of international multicenter studies and archived tissue. We developed a protocol to obtain high-quality cells and nuclei from dissected human skeletal muscle archived in the preservative Allprotect® Tissue Reagent (ATR). After fluorescent imaging microscopy confirmed intact nuclei, we performed four protocol variations that compared sequencing metrics between cells and nuclei enriched by either filtering or flow cytometry sorting. Cells and nuclei (either sorted or filtered) produced statistically identical transcriptional profiles and recapitulated 8 cell types present in skeletal muscle. Flow cytometry sorting successfully enriched for higher-quality cells and nuclei but resulted in an overall decrease in input material. Our protocol provides an important resource for obtaining high-quality single cell genomic material from archived tissue and to streamline global collaborative efforts.

Metabolic dependences highlight a cell's reliance on specific pathways to meet its bioenergetic needs, with these pathways being interrogated using chemical inhibitors to assess their significance. While surrogate markers of bioenergetics (e.g., oxygen consumption) have yielded important insights, we asked whether metabolic dependences could be defined using ATP as a biomarker. To address this gap, we developed Mitochondrial/Energy Flow Cytometry (MitE-Flo), a method that evaluates the contributions of glycolysis, fatty acid oxidation (FAO), and oxidative phosphorylation (OXPHOS) to cellular ATP content. In models of mitochondrial disease due to complex I or complex IV deficiency, we identified impaired OXPHOS with a compensatory shift to glycolysis. To define the utility of ATP monitoring in immunometabolism research, we analyzed previously inaccessible cell populations: light zone (LZ) and dark zone (DZ) germinal center (GC) B cells. Highly proliferative DZ B cells exhibited elevated ATP levels and a preference for FAO and OXPHOS over glycolysis, with uniform increased activity across ETC complexes. In contrast, less proliferative LZ B cells showed lower ATP levels and an equal reliance on glycolysis and OXPHOS. Using ATP as a biomarker to define metabolic dependences provides valuable insights into disease states and elusive immune cell subtypes, thereby enhancing the metabolism research toolkit.

Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

Spinal bulbar muscular atrophy (SBMA), the first identified CAG-repeat expansion disorder, is an X-linked neuromuscular disorder involving CAG-repeat-expansion mutations in the androgen receptor (AR) gene. We utilized CRISPR-Cas9 gene editing to engineer novel isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control, and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing FLAG-tagged wildtype and mutant AR, respectively. Adapting a small-molecule cocktail-directed approach, we differentiate the isogenic hiPSC models into motor neuron-like cells with a highly enriched population to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in disease motor neurons. We demonstrate that ligand-free mutant AR causes drastic mitochondrial dysfunction in neurites of differentiated disease motor neurons due to gain-of-function mechanisms, and such cytotoxicity can be amplified upon ligand (androgens) treatment. We further show that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and multiple other mitochondrial impairments. These findings counter the established notion that androgens are requisite for mutant AR-induced cytotoxicity in SBMA, reveal a compelling mechanistic link between ligand-free mutant AR, F-ATP synthase, and mitochondrial dysfunction, and provide innovative insights into motor neuron-specific therapeutic interventions for SBMA.

Middle meningeal artery embolization (MMAE) is an effective minimally invasive option for chronic subdural hematoma (cSDH). Dexamethasone and statins have been reported to improve the resolution of cSDH and reduce its recurrence. However, only 1 study has investigated the role of statins in patients treated with MMAE, and there is no such study on dexamethasone. Thus, we used the TriNetX research network to determine whether adding dexamethasone or statin along with primary MMAE is associated with a benefit in outcomes.We queried all primary MMAE cases for cSDH between January 1st, 2012, and July 1st, 2022, in the TriNetX research network. We included patients greater than or equal to 18 years old and separated them regarding statin and dexamethasone use around the time of MMAE. Outcomes were evaluated within 6 months and 3 years after MMAE, and analyses were performed before and after propensity score matching.The study included 372 patients with chronic subdural hematoma who underwent MMAE and were on dexamethasone, 339 not on dexamethasone, 391 on statins, and 278 not on statins. After propensity score matching, the dexamethasone cohorts included 250 patients each and only headache remained more prevalent in the dexamethasone cohort at both 6 months (21.2% vs. 10.0%, P = 0.001) and 3 years (23.6% vs. 12.4%, P = 0.001). After propensity score matching, the statin cohorts included 150 patients each and no differences in outcomes were found at both 6 months and 3 months after MMAE.Patients treated with primary MMAE and were on dexamethasone or statins had no differences in mortality and functional/provider dependence compared to those who were not on dexamethasone or statins. Patients on dexamethasone had a higher prevalence of headaches.

BACKGROUND:Epigenetic modification of chromatin plays a pivotal role in regulating gene expression during cell differentiation. The scale and complexity of epigenetic data pose significant challenges for biologists to identify the regulatory events controlling cell differentiation. RESULTS:To reduce the complexity, we developed a package, called Snapshot, for clustering and visualizing candidate cis-regulatory elements (cCREs) based on their epigenetic signals during cell differentiation. This package first introduces a binarized indexing strategy for clustering the cCREs. It then provides a series of easily interpretable figures for visualizing the signal and epigenetic state patterns of the cCREs clusters during the cell differentiation. It can also use different hierarchies of cell types to highlight the epigenetic history specific to any particular cell lineage. We demonstrate the utility of Snapshot using data from a consortium project for ValIdated Systematic IntegratiON (VISION) of epigenomic data in hematopoiesis. CONCLUSION:The package Snapshot can identify all distinct clusters of genomic locations with unique epigenetic signal patterns during cell differentiation. It outperforms other methods in terms of interpreting and reproducing the identified cCREs clusters. The package of Snapshot is available at GitHub: https://github.com/guanjue/Snapshot .

Trisomy 8 mosaicism (T8M) has been associated with a Behçet’s-like inflammatory disease, but immunologic features and treatment responses are not well-characterized. Here, we characterize 20 individuals with constitutional T8M and inflammatory disease. Most participants had congenital dysmorphologies and developmental delay. Two developed hematologic malignancies, two had bleeding diathesis due to platelet dense granule deficiency, and most had macrocytosis. The majority had recurrent fever and severe oral ulcerations, while nearly half had genital ulcers or rash. Colchicine, apremilast, and IL-1 and TNFa inhibitors were effective therapies. With ddPCR on sorted cell populations, we found that cells from the myeloid lineage (monocytes, neutrophils, megakaryocytes, erythroid progenitors) had a significantly higher percentage of trisomy 8 mosaicism compared to those from the lymphoid lineage (T and B cells) in both the peripheral blood and bone marrow, suggesting that trisomy 8 is tolerated to a greater degree by myeloid cells. Furthermore, we found that participants with T8M had more classical monocytes and had upregulation of genes associated with activated neutrophils and monocytes in their whole blood compared to healthy controls. With single cell RNAseq, we identifed which cells were trisomy 8 and disomy based on chromosome 8 gene expression and found that trisomy 8 monocytes had distinct transcriptional signatures and alteration of innate immune genes. Our findings suggest that patients with T8M are prone to a distinct autoinflammatory disease which we propose calling trisomy 8 associated autoinflammatory disease (TRIAD) and have complications due to dysregulation of cells arising from the myeloid lineage. This work was supported by the Intramural Research Program of NIAID, NHGRI, and NHLBI, NIH.

T follicular helper (Tfh) cells provide signals to initiate and maintain the germinal center (GC) reaction and are crucial for the generation of robust, long-lived antibody responses, but how the GC microenvironment affects Tfh cells is not well understood. Here we develop an in vivo T cell-intrinsic CRISPR-knockout screen to evaluate Tfh and Th1 cells in an acute viral infection model to identify regulators of Tfh cells in their physiological setting. Using a screen of druggable-targets, alongside genetic, transcriptomic and cellular analyses, we identify a function of HIF-1α in suppressing mTORC1-mediated and Myc-related pathways, and provide evidence that VHL-mediated degradation of HIF-1α is required for Tfh development; an expanded in vivo CRISPR screen reveals multiple components of these pathways that regulate Tfh versus Th1 cells, including signaling molecules, cell-cycle regulators, nutrient transporters, metabolic enzymes and autophagy mediators. Collectively, our data serve as a resource for studying Tfh versus Th1 decisions, and implicate the VHL-HIF-1α axis in fine-tuning Tfh generation.

Chromatin immunoprecipitation followed by massively parallel, high throughput sequencing (ChIP-seq) is the method of choice for genome-wide identification of DNA segments bound by specific transcription factors or in chromatin with particular histone modifications. However, the quality of ChIP-seq datasets varies widely, with a substantial fraction being of intermediate to poor quality. Thus, it is important to discern and control the factors that contribute to variation in ChIP-seq. In this study, we focused on sonication, a user-controlled variable, to produce sheared chromatin. We systematically varied the amount of shearing of fixed chromatin from a mouse erythroid cell line, carefully measuring the distribution of resultant fragment lengths prior to ChIP-seq. This systematic study was complemented with a retrospective analysis of additional experiments. We found that the level of sonication had a pronounced impact on the quality of ChIP-seq signals. Over-sonication consistently reduced quality, while the impact of under-sonication differed among transcription factors, with no impact on sites bound by CTCF but frequently leading to the loss of sites occupied by TAL1 or bound by POL2. The bound sites not observed in low-quality datasets were inferred to be a mix of both direct and indirect binding. We leveraged these findings to produce a set of CTCF ChIP-seq datasets in rare, primary hematopoietic progenitor cells. Our observation that the amount of chromatin sonication is a key variable in success of ChIP-seq experiments indicates that monitoring the level of sonication can improve ChIP-seq quality and reproducibility and facilitate ChIP-seq in rare cell types.

Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell tran-scriptome profiling of 135,929 CD34(+) lineage(-) hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.

The hierarchical model of hematopoiesis posits that hematopoietic stem and progenitor cells produce common myeloid progenitors (CMP). CMP can become granulocyte/monocyte progenitors (GMP) or bipotential megakaryocyte/erythroid progenitors (MEP). MEP can produce megakaryocytic (Mk) or erythroid (Ery) cells. However, we and others have shown that early mouse and human progenitor populations express many Mk genes (Heuston, Epig. Chrom., 2016), while single cell studies have identified lineage-specific colony forming cells in progenitor populations thought to be multipotent (Psaila, Genome Biol., 2016). To identify the earliest mouse Ery and Mk cells, we performed single cell RNASeq on 10000 stem and progenitor cells (Lin-Sca1+Kit+), 12000 CMP (Lin-Sca1-Kit+CD16/32-CD34+), 6000 MEP (Lin-Sca1-Kit+CD16/32-CD34-) and 8000 GMP (Lin-Sca1-Kit+CD16/32+CD34+). TSNE analysis of expression in the 4 populations identified 33 clusters, which were correlated to biological functions using gene set enrichment analysis. In LSK, no cells with an Ery RNA profile were found, while 56% of cells co-expressed Mk-associated (e.g., Meis1, Fli1) and lymphoid genes. In CMP, 12% of the cells co-expressed Ery (e.g., Gata1, Fog1) and Mk (e.g., Pf4, Cd41) genes, while 23% had an Mk-specific profile (e.g., Fli1, Cd41) enriched for platelet biology processes (p< 3E-18). Unlike traditional models, over 94% of MEP had Ery RNA profiles enriched for ribosome synthesis and heme-biology processes (p< 4E-10). To establish developmental relationships, we performed pseudotime analysis using the Monocle and Scanpy software packages. These programs model differentiation by mapping similar transcriptomes together. Map nodes indicate lineage commitment points and cells further from a node are more differentiated. Combined analysis of LSK, CMP, and MEP generated a model with a single node and 2 trajectories. LSK with Mk and lymphoid RNA profiles diverged at the node, as did 14% of CMP. 31% of CMP with an Mk RNA profile were downstream of the node. Further downstream were cells with mixed Ery/Mk profiles, and furthest from the node were MEP with Ery profiles. A separate pseudotime analysis of CMP only 2 trajectories: one with decreasing Mk- and increasing Ery RNA profiles, and a second with an early Mk endomitotic RNA profile. Pseudotime analysis of MEP only identified a linear trajectory: cells at one end expressed early Ery RNA profiles, and cells at the other end had RNA profiles similar to those of burst-forming unit-erythroid (BFU-E). We generated a predictive set of RNAs for each TSNE cluster. We used index-sorting with 11 markers (Kit, Sca1, CD34, CD16/32, CD36, CD41, CD48, CD123, CD150, CD9, Flk2) to isolate single cells for custom high-throughput multiplex qPCR. This allowed confirmation of cell frequency within TSNE clusters while identifying surface markers for prospective isolation of cell subsets. We focused on 2 populations: CMP-E, which had an Ery RNA profile (10% of clustered CMP and 12% of CMP in the qPCR assay), and CMP-MkE, which had Mk and Ery RNA profiles (12% of clustered CMP and 13% of CMP in the qPCR assay). We prospectively isolated CMP-E and CMP-MkE to compare RNASeq profiles, ATACSeq profiles, and colony forming ability against those of bulk CMP, Ery, and Mk. In CMP-E, 54% of RNAs were expressed in both CMP and ERY, while 41% were expressed only in CMP (p < 6E-72). In contrast, 41% of CMP-E ATACSeq peaks were present in CMP and ERY, while 57% of CMP-E peaks were present only in CMP (p < 1E-3). We conclude that in CMP-E, the RNASeq profile is more erythroid than the ATACSeq profile. In CMP-MkE, 89% of RNAs were expressed in both CMP and Mk, while 7% were expressed only in CMP (p < 8E-190). Likewise, 88% of CMP-MkE ATACSeq peaks were present in both CMP and Mk, while 3% were present only in CMP (p < 1E-3). We conclude that in CMP-MkE, the RNASeq and ATACSeq profiles are equivalent. In soft agar assays, 21% of CMP-E and 3% of CMP-MkE colonies contained BFU-E, compared to 9% of control colonies. We conclude that the CMP-E and CMP-MkE populations are skewed towards the ERY and MK lineages, but are not erythro-megakaryocyte restricted. Our data support a model in which there are two megakaryocyte precursor populations and no erythroid populations in LSK. A third megakaryocyte population in CMP gives rise to erythroid cells. Finally, our data show that transcriptional changes precede chromatin accessibility changes in the earliest erythroid cells. Disclosures No relevant conflicts of interest to declare.

Progenitor-like CD8(+) T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8(+) Tcells responding to acute and chronic viral infections, we found that progenitor-like CD8(+) T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8(+) T cells and was required for the programming of progenitor-like CD8(+) T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.

Background: Enhancers and promoters are cis-acting regulatory elements associated with lineage-specific gene expression. Previous studies showed that different categories of active regulatory elements are in regions of open chromatin, and each category is associated with a specific subset of post-translationally marked histones. These regulatory elements are systematically activated and repressed to promote commitment of hematopoietic stem cells along separate differentiation paths, including the closely related erythrocyte (ERY) and megakaryocyte (MK) lineages. However, the order in which these decisions are made remains unclear. Results: To characterize the order of cell fate decisions during hematopoiesis, we collected primary cells from mouse bone marrow and isolated 10 hematopoietic populations to generate transcriptomes and genome-wide maps of chromatin accessibility and histone H3 acetylated at lysine 27 binding (H3K27ac). Principle component analysis of transcriptional and open chromatin profiles demonstrated that cells of the megakaryocyte lineage group closely with multipotent progenitor populations, whereas erythroid cells form a separate group distinct from other populations. Using H3K27ac and open chromatin profiles, we showed that 89% of immature MK (iMK)-specific active regulatory regions are present in the most primitive hematopoietic cells, 46% of which contain active enhancer marks. These candidate active enhancers are enriched for transcription factor binding site motifs for megakaryopoiesis-essential proteins, including ERG and ETS1. In comparison, only 64% of ERY-specific active regulatory regions are present in the most primitive hematopoietic cells, 20% of which containing active enhancer marks. These regions were not enriched for any transcription factor consensus sequences. Incorporation of genome-wide DNA methylation identified significant levels of de novo methylation in iMK, but not ERY. Conclusions: Our results demonstrate that megakaryopoietic profiles are established early in hematopoiesis and are present in the majority of the hematopoietic progenitor population. However, megakaryopoiesis does not constitute a "default" differentiation pathway, as extensive de novo DNA methylation accompanies megakaryopoietic commitment. In contrast, erythropoietic profiles are not established until a later stage of hematopoiesis, and require more dramatic changes to the transcriptional and epigenetic programs. These data provide important insights into lineage commitment and can contribute to ongoing studies related to diseases associated with differentiation defects.

The hierarchical model of hematopoiesis posits that hematopoietic stem cells (HSC) give rise to myeloid progenitors (CMP), that can become further restricted to bipotential granulocyte/monocyte progenitors (GMP) or megakaryocyte/erythroid progenitors (MEP). We and others have shown that this model may not accurately depict hematopoiesis. Recent studies have shown that shown that populations of mouse hematopoietic stem and progenitor cells (LSK) have a strong megakaryocyte (Mk) transcriptional profile (Heuston, 2018, Epig. & Chrom.), and single cell studies have identified lineage committed cells in progenitor populations thought to be multipotent. For example, we recently reported that human MEP contain 3 populations: erythroid (Ery) primed, Mk primed, and bipotential (Psaila, 2016; Gen. Bio.).