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Background: Cardiac fibroblasts (CFs) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Because of the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is lacking. The purpose of this study was to define CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate CF function. Methods: Collagen1α1–GFP (green fluorescent protein)–positive CFs were characterized after myocardial infarction (MI) by single-cell and bulk RNA sequencing, assay for transposase-accessible chromatin sequencing, and functional assays. Swine and patient samples were studied using bulk RNA sequencing. Results: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear profibrotic signature, express high levels of Cthrc1 (collagen triple helix repeat containing 1), and localize into the scar. Noncanonical transforming growth factor–β signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Absence of CTHRC1 results in pronounced lethality attributable to ventricular rupture. A population of CFs with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. Conclusions: We report CF heterogeneity and their dynamics during the course of MI and redefine the CFs that respond to cardiac injury and participate in myocardial remodeling. Our study identifies CTHRC1 as a novel regulator of the healing scar process and a target for future translational studies.

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G2–M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD. Significance: These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.

Peripheral arterial disease (PAD) is associated with a high risk of cardiovascular events and death and is postulated to be a critical socioeconomic cost in the future. Extracellular vesicles (EVs) have emerged as potential candidates for new biomarker discovery related to their protein and nucleic acid cargo. In search of new prognostic and therapeutic targets in PAD, we determined the prothrombotic activity, the cellular origin and the transcriptomic profile of circulating EVs. This prospective study included control and PAD patients. Coagulation time (Procoag-PPL kit), EVs cellular origin and phosphatidylserine exposure were determined by flow cytometry in platelet-free plasma (n = 45 PAD). Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n = 12/group). The serum concentration of the differentially expressed gene S100A9, in serum calprotectin (S100A8/A9), was validated by ELISA in control (n = 100) and PAD patients (n = 317). S100A9 was also determined in EVs and tissues of human atherosclerotic plaques (n = 3). Circulating EVs of PAD patients were mainly of platelet origin, predominantly Annexin V positive and were associated with the procoagulant activity of platelet-free plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients (p

A hallmark of metazoan evolution is the emergence of genomic mechanisms that implement cell-type-specific functions. However, the evolution of metazoan cell types and their underlying gene regulatory programmes remains largely uncharacterized. Here, we use whole-organism single-cell RNA sequencing to map cell-type-specific transcription in Porifera (sponges), Ctenophora (comb jellies) and Placozoa species. We describe the repertoires of cell types in these non-bilaterian animals, uncovering diverse instances of previously unknown molecular signatures, such as multiple types of peptidergic cells in Placozoa. Analysis of the regulatory programmes of these cell types reveals variable levels of complexity. In placozoans and poriferans, sequence motifs in the promoters are predictive of cell-type-specific programmes. By contrast, the generation of a higher diversity of cell types in ctenophores is associated with lower specificity of promoter sequences and the existence of distal regulatory elements. Our findings demonstrate that metazoan cell types can be defined by networks of transcription factors and proximal promoters, and indicate that further genome regulatory complexity may be required for more diverse cell type repertoires.

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (TXN), a major regulator of cellular redox status and, in addition, identified PRDM5 as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype. This research was funded by the European Union's Seventh Framework Programme through the Blueprint Consortium (grant agreement 282510), Fundació La Marató de TV3, Instituto de Salud Carlos III (ISCIII) PI14/01867, PI16/02024, and PI17/00701, TRASCAN (EPICA), MINECO Explora (RTHALMY), Departamento de Salud del Gobierno de Navarra 40/2016, Gilead Fellowship Program (GLD16/00142), Multiple Myeloma Research Foundation Networks of excellence, the International Myeloma Foundation (Brian van Novis) and the Qatar National Research Fund award 7-916-3-237. Furthermore, the authors acknowledge the support of the Generalitat de Catalunya Suport Grups de Recerca AGAUR 2017-SGR-736 and 2017-SGR-1142, TRASCAN-iMMunocelland European Research Council starting grant (MYELOMANEXT), CIBERONC (CB16/12/00489, CB16/12/00369 and CB16/12/00225), co-financed with FEDER funds, the Accelerator award CRUK/AIRC/AECC joint funder-partnership, as well as NCI R01 CA180475 and a MMRF collaborative grant. R.O. was supported by a FPU Fellowship of the Spanish Government, M.K. by an AOI grant of the Spanish Association Against Cancer and N.R. by the Acció instrumental d'incorporació de scientífics i tecnòlegs PERIS 2016 from the Generalitat de Catalunya. This work was partially developed at the Centro Esther Koplowitz (CEK, Barcelona, Spain). We particularly acknowledge the patients for their participation and the Biobank of the University of Navarra for its collaboration.

Histone modifications play an important role in chromatin organization and transcriptional regulation, but despite the large amount of genome-wide histone modification data collected in different cells and tissues, little is known about co-occurrence of modifications on the same nucleosome. Here we present a genome-wide quantitative method for combinatorial indexed chromatin immunoprecipitation (co-ChIP) to characterize co-occurrence of histone modifications on nucleosomes. Using co-ChIP, we study the genome-wide co-occurrence of 14 chromatin marks (70 pairwise combinations), and find previously undescribed co-occurrence patterns, including the co-occurrence of H3K9me1 and H3K27ac in super-enhancers. Finally, we apply co-ChIP to measure the distribution of the bivalent H3K4me3-H3K27me3 domains in two distinct mouse embryonic stem cell (mESC) states and in four adult tissues. We observe dynamic changes in 5,786 regions and discover both loss and de novo gain of bivalency in key tissue-specific regulatory genes, suggesting a functional role for bivalent domains during different stages of development. These results show that co-ChIP can reveal the complex interactions between histone modifications.

CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1−/− mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites., Substrate reduction therapies (SRT) are a promising therapeutic approach for monogenic inherited metabolic diseases. Here the authors evaluate the therapeutic potential of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I and demonstrate its safety and efficacy.

Background: Deep understanding of the complexity and diversity of the tumor immune microenvironment (TIME) and its influence on response to therapy is needed to improve the ability to predict, monitor and guide immunotherapeutic responsiveness. Among different cell types in the MM-TIME, granulocytic MDSCs (G-MDSCs) have a prominent role in promoting tumor growth and inducing immune suppression; however, their identification and monitoring is imprecise because the phenotypic profile of MDSCs in the MM-TIME is not well-established. Aim: To provide the detailed phenotypic profile of G-MDSCs based on the immune suppressive potential, gene regulatory network and clinical significance of distinct granulocytic subsets in the MM-TIME. Methods: First, we used multidimensional flow cytometry (MFC) to evaluate the preestablished phenotype of G-MDSCs in bone marrow (BM) samples from controls (n=4) and MM patients (n=5). We then used principal component analysis (PCA) to unbiasedly identify different granulocytic subsets in the MM-TIME, and FACS for in vitro experiments to determine their immune suppressive potential (n=9) and for RNAseq to analyze the molecular profile of G-MDSCs in MM (n=5) vs controls (n=5). Subsequently, the clinical significance of the different granulocytic subsets was investigated by comparing their numbers at diagnosis, in MM patients (n=124) achieving MRD-negativity vs MRD-positivity after treatment with VRD induction (x6) followed by autologous transplant and VRD consolidation (x2) (GEM2012MENOS65 clinical trial). Results: In humans, G-MDSCs have been defined as a unique cluster displaying a CD11b-, CD14-, CD15+, CD33+ and HLADR- phenotype, comprising 1% of total BM nucleated cells in healthy individuals and approximately 25% in MM patients. However, we found that the percentage of cells with a CD11b-CD14-CD15+CD33+HLADR- phenotype was similar in the BM of controls and MM patients (median of 8% in both, P>.99). Since these cells were not expanded in MM and represented only 24% of total neutrophils, we next used MFC and PCA to unbiasedly identify other cell clusters within neutrophils. Accordingly, 3 major subsets were identified in neutrophils from controls and MM patients, based on homogeneous CD14-CD15+CD33+HLADR- expression but differential reactivity against CD11b, CD13 and CD16: CD11b-CD13lo/-CD16- (19% and 24%), CD11b+CD13lo/-CD16- (46% and 47%) and CD11b+CD13+CD16+ (35% and 29%). Afterwards, we used FACSorting to deplete or isolate individually, each of the 3 neutrophil subsets from the BM MM-TIME and determine its immune suppressive potential in 2 functional assays: 1) the proliferation rate of autologous T cells in presence of CD3/CD28 stimulatory beads and, 2) the cytotoxic potential of autologous T-cells against MM cells using a BCMAxCD3 bispecific antibody. Interestingly, we noted a significant decrease in T cell proliferation when these were stimulated in the presence of CD11b+CD13+CD16+ neutrophils (0.5-fold, p =.03) but not the CD11b-CD13lo/-CD16- and CD11b+CD13lo/-CD16- subsets. In addition, we noted that the cytotoxic potential of T cells engaged by the BCMAxCD3 bispecific antibody significantly increased with the depletion of CD11b+CD13lo/-CD16- and CD11b+CD13+CD16+ subsets (3-fold and 4-fold, respectively; p ≤.04) but not CD11b-CD13lo/-CD16- neutrophils. Furthermore, RNAseq of the 3 subsets in controls and MM patients revealed that genes related with the IL-4, IL-10 and IL-13 immunosuppressive pathways were specifically upregulated in the CD11b+CD13+CD16+ subset. Finally, based on the surrogacy between the achievement of MRD-negativity and prolonged survival, we compared the distribution of the 3 granulocytic subsets in the BM-TIME at diagnosis and observed that patients reaching MRD-negativity (n=56) displayed significantly lower percentages of total neutrophils (46% vs 52%, p =.002), particularly of the CD11b+CD13lo/-CD16- (11% vs 15%, p =.003) and CD11b+CD13+CD16+ (31% vs 35%, p =.07) subsets vs MRD-positive cases (n=68). Conclusions: We have determined the correlation between the phenotypic, molecular and immunosuppressive potential of unique granulocytic subsets. Thus, we have identified optimal markers for monitoring G-MDCSs in patients with MM (ie. CD11b, CD13, CD16) and unveiled that, in contrast to previous findings, the more mature granulocytes are the only stages with immunosuppressive potential. Disclosures Puig: Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria. Martinez Lopez:Celgene: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau. Oriol:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rios:Amgen, Celgene, Janssen, and Takeda: Consultancy. Rosinol:Janssen, Celgene, Amgen, Takeda: Honoraria. Mateos:Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bladé:Celgene: Honoraria; Janssen: Honoraria; Amgen: Honoraria. San-Miguel:Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria; Sanofi: Honoraria; Roche: Honoraria.

During cell differentiation, progenitor cells integrate signals from their environment that guide their development into specialized phenotypes. The ways by which cells respond to complex signal combinations remain difficult to analyze and model. To gain additional insight into signal integration, we systematically mapped the response of CD4+ T cells to a large number of input cytokine combinations that drive their differentiation. We find that, in response to varied input combinations, cells differentiate into a continuum of cell fates as opposed to a limited number of discrete phenotypes. Input cytokines hierarchically influence the cell population, with TGFβ being most dominant followed by IL-6 and IL-4. Mathematical modeling explains these results using additive signal integration within hierarchical groups of input cytokine combinations and correctly predicts cell population response to new input conditions. These findings suggest that complex cellular responses can be effectively described using a segmented linear approach, providing a framework for prediction of cellular responses to new cytokine combinations and doses, with implications to fine-tuned immunotherapies.

In multicellular organisms, dedicated regulatory circuits control cell type diversity and responses. The crosstalk and redundancies within these circuits and substantial cellular heterogeneity pose a major research challenge. Here, we present CRISP-seq, an integrated method for massively parallel single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-pooled screens. We show that profiling the genomic perturbation and transcriptome in the same cell enables us to simultaneously elucidate the function of multiple factors and their interactions. We applied CRISP-seq to probe regulatory circuits of innate immunity. By sampling tens of thousands of perturbed cells in vitro and in mice, we identified interactions and redundancies between developmental and signaling-dependent factors. These include opposing effects of Cebpb and Irf8 in regulating the monocyte/macrophage versus dendritic cell lineages and differential functions for Rela and Stat1/2 in monocyte versus dendritic cell responses to pathogens. This study establishes CRISP-seq as a broadly applicable, comprehensive, and unbiased approach for elucidating mammalian regulatory circuits.

Innate lymphoid cells (ILCs) are critical modulators of mucosal immunity, inflammation, and tissue homeostasis, but their full spectrum of cellular states and regulatory landscapes remains elusive. Here, we combine genome-wide RNA-seq, ChIP-seq, and ATAC-seq to compare the transcriptional and epigenetic identity of small intestinal ILCs, identifying thousands of distinct gene profiles and regulatory elements. Single-cell RNA-seq and flow and mass cytometry analyses reveal compartmentalization of cytokine expression and metabolic activity within the three classical ILC subtypes and highlight transcriptional states beyond the current canonical classification. In addition, using antibiotic intervention and germ-free mice, we characterize the effect of the microbiome on the ILC regulatory landscape and determine the response of ILCs to microbial colonization at the single-cell level. Together, our work characterizes the spectrum of transcriptional identities of small intestinal ILCs and describes how ILCs differentially integrate signals from the microbial microenvironment to generate phenotypic and functional plasticity.

Background: Despite significant improvements in the treatment of MM, the outcome of patients with HR cytogenetics remains poor despite similar complete remission (CR) rates as compared to SR cases. Relapses among patients in CR are attributed to the persistence of MRD, but knowledge about the impact of MRD in patients with SR and HR cytogenetics, treated with modern therapies and monitored with next-generation techniques, is limited. Similarly, there is virtually no data about in vivo mechanisms of resistance in SR and HR MM; however, since MRD represents those very few cells that are resistant to treatment, it could be hypothesized that profiling MRD cells may shed light into the mechanisms of resistance in both SR and HR patients. Aim: To determine the clinical impact of MRD in MM patients with SR vs HR cytogenetics, and to identify transcriptional mechanisms determining MRD resistance by investigating the transcriptome of MRD cells in both patient subgroups. Methods: This study was conducted in a series of 390 patients enrolled in the PETHEMA/GEM2012 trial (6 induction cycles with VRD followed by ASCT and 2 courses of consolidation with VRD). FISH was analyzed on CD138 purified PCs at diagnosis. MRD was predefined to be prospectively assessed following induction, transplant and consolidation, using next-generation flow (NGF) according to EuroFlow. In 40 patients [28 with SR and 12 with HR cytogenetics: i.e., t(4;14), t(14;16) and/or del(17p)], diagnostic and MRD tumor cells persisting after VRD-induction were isolated by FACS according to patient-specific aberrant phenotypes. Due to the small number of sorted MRD cells (median of 25,600) we used a 3' end RNAseq method optimized for generating libraries from low-input starting material (MARSeq). Differential expression analyses were performed with DESeq2 R package. Results: At the latest time-point in which MRD was assessed, MRD-positive rates progressively increased (p =.006) from SR patients (148/300, 49%) to cases with t(4;14) (24/42, 57%) and del(17p) (29/38, 76%). Furthermore, MRD levels were significantly superior in patients with del(17p) compared to SR FISH (0.02% vs 0.006%, p =.009), while MRD levels in patients with t(4;14) (0.004%) were similar to those in SR MM. Only 10 patients had a t(14;16) and 4 were MRD-positive. Among patients achieving MRD-negativity (.05). Conversely, 3-year PFS rates for MRD-positive patients decreased from those having SR FISH to those with t(4;14) and del(17p) (59%, 46% and 24%, respectively), with statistically significant differences between the first and the latest subgroups (p Since clearance of MRD notably lowered the risk of relapse and persistence of MRD significantly shortened the PFS in each cytogenetic group (p ≤.001), we investigated the unique features of MRD cells persisting after VRD-induction by comparing their transcriptome to that of patient-matched tumor cells at diagnosis (n=40). Accordingly, MRD cells showed 763 genes significantly deregulated (Padj Conclusions: This is one of the largest studies integrating patients' cytogenetics and MRD status. Our results, based on intensive treatment and MRD monitoring using NGF, unveil that achieving MRD-negativity may overcome the poor prognosis of HR cytogenetics. By contrast, persistent MRD significantly reduces PFS rates, particularly in patients with del(17p). Interestingly, MRD cells from SR and HR patients may have different transcriptional mechanisms leading to VRD resistance, and further understanding of these could provide knowledge on how to eradicate MRD in both patient subgroups. Disclosures Puig: Takeda: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Garcia-Sanz:Affimed: Research Funding. Martinez-Lopez:BMS: Research Funding; Pfizer: Research Funding; Vivia: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Research Funding. Oriol:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rios:Amgen, Celgene, Janssen, and Takeda: Consultancy. De La Rubia:Ablynx: Consultancy, Other: Member of Advisory Board. Mateos:GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bladé:Janssen: Honoraria. San-Miguel:Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria; Sanofi: Honoraria; Celgene: Honoraria; Roche: Honoraria; Janssen: Honoraria.

Summary Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.

Monocytes are circulating, short-lived mononuclear phagocytes, which in mice and man comprise two main subpopulations. Murine Ly6C+ monocytes display developmental plasticity and are recruited to complement tissue-resident macrophages and dendritic cells on demand. Murine vascular Ly6C- monocytes patrol the endothelium, act as scavengers, and support vessel wall repair. Here we characterized population and single cell transcriptomes, as well as enhancer and promoter landscapes of the murine monocyte compartment. Single cell RNA-seq and transplantation experiments confirmed homeostatic default differentiation of Ly6C+ into Ly6C- monocytes. The main two subsets were homogeneous, but linked by a more heterogeneous differentiation intermediate. We show that monocyte differentiation occurred through de novo enhancer establishment and activation of pre-established (poised) enhancers. Generation of Ly6C- monocytes involved induction of the transcription factor C/EBPβ and C/EBPβ-deficient mice lacked Ly6C- monocytes. Mechanistically, C/EBPβ bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor.