Publications by authors named "David Lara-Astiaso"

22 Publications

  • Page 1 of 1

Characterization of freshly isolated bone marrow mesenchymal stromal cells from healthy donors and patients with multiple myeloma: transcriptional modulation of the microenvironment.

Haematologica 2020 09 1;105(9):e470-473. Epub 2020 Sep 1.

Clinica Universidad de Navarra, Pamplona, Spain; Instituto de Investigación Sanitaria de Navarra (IDISNA), Pamplona, Spain; Centro de Investigacion Medica Aplicada (CIMA), Pamplona, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain.

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http://dx.doi.org/10.3324/haematol.2019.235135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556476PMC
September 2020

Single-Cell RNA Sequencing Analysis Reveals a Crucial Role for CTHRC1 (Collagen Triple Helix Repeat Containing 1) Cardiac Fibroblasts After Myocardial Infarction.

Circulation 2020 Nov 25;142(19):1831-1847. Epub 2020 Sep 25.

Program of Regenerative Medicine (A.R.-V., S.C.H., P.G.-O., E.I., G.A., G.M., B.P., F.P.), Program of Hemato-Oncology, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain.

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 as a novel regulator of the healing scar process and a target for future translational studies.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.044557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730974PMC
November 2020

Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma.

Genome Res 2020 Sep 20;30(9):1217-1227. Epub 2020 Aug 20.

Centro de Investigación Biomédica en Red de Cáncer, CIBERONC, 28029 Madrid, Spain.

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 (), a major regulator of cellular redox status and, in addition, identified 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.
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http://dx.doi.org/10.1101/gr.265520.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545147PMC
September 2020

Deep MRD profiling defines outcome and unveils different modes of treatment resistance in standard- and high-risk myeloma.

Blood 2021 Jan;137(1):49-60

Clinica Universidad de Navarra, Centro de Investigación Medica Aplicada (CIMA), Instituto de Investigación Sanitaria de Navarra (IDISNA), Centro de Investigación Biomédica en Red-Cáncer (CIBER-ONC) CB16/12/00369, Pamplona, Spain.

Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival (PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of ∼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species-mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.
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http://dx.doi.org/10.1182/blood.2020006731DOI Listing
January 2021

Immunogenomic identification and characterization of granulocytic myeloid-derived suppressor cells in multiple myeloma.

Blood 2020 07;136(2):199-209

Clinica Universidad de Navarra, Centro de Investigacion Medica Aplicada (CIMA), Instituto de Investigacion Sanitaria de Navarra (IDISNA), CIBER-ONC numbers CB16/12/00369, CB16/12/00489, Pamplona, Spain.

Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14-CD15+CD33+HLADR- cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P < .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC-related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM.
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http://dx.doi.org/10.1182/blood.2019004537DOI Listing
July 2020

Functional and transcriptomic analysis of extracellular vesicles identifies calprotectin as a new prognostic marker in peripheral arterial disease (PAD).

J Extracell Vesicles 2020 19;9(1):1729646. Epub 2020 Feb 19.

Laboratory of Atherothrombosis, Program of Cardiovascular Diseases, Cima Universidad de Navarra, Pamplona, Spain.

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 ( < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years,  < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification ( < 0.01). Furthermore, S100A9 was also expressed in femoral plaque derived EVs and tissues. In summary, we found that PAD patients release EVs, mainly of platelet origin, highly positive for AnnexinV and rich in transcripts related to platelet biology and immune responses. Amputation risk prediction improved with calprotectin and was significantly higher when combined with hs-CRP. Our results suggest that EVs can be a promising component of liquid biopsy to identify the molecular signature of PAD patients.
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http://dx.doi.org/10.1080/20013078.2020.1729646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048174PMC
February 2020

Genome-wide analysis of the H3K27me3 epigenome and transcriptome in Brassica rapa.

Gigascience 2019 12;8(12)

Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus de Montegancedo, 28223, Pozuelo de Alarcón (Madrid), Spain.

Background: Genome-wide maps of histone modifications have been obtained for several plant species. However, most studies focus on model systems and do not enforce FAIR data management principles. Here we study the H3K27me3 epigenome and associated transcriptome of Brassica rapa, an important vegetable cultivated worldwide.

Findings: We performed H3K27me3 chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis by 3'-end RNA sequencing from B. rapa leaves and inflorescences. To analyze these data we developed a Reproducible Epigenomic Analysis pipeline using Galaxy and Jupyter, packaged into Docker images to facilitate transparency and reuse. We found that H3K27me3 covers roughly one-third of all B. rapa protein-coding genes and its presence correlates with low transcript levels. The comparative analysis between leaves and inflorescences suggested that the expression of various floral regulatory genes during development depends on H3K27me3. To demonstrate the importance of H3K27me3 for B. rapa development, we characterized a mutant line deficient in the H3K27 methyltransferase activity. We found that braA.clf mutant plants presented pleiotropic alterations, e.g., curly leaves due to increased expression and reduced H3K27me3 levels at AGAMOUS-like loci.

Conclusions: We characterized the epigenetic mark H3K27me3 at genome-wide levels and provide genetic evidence for its relevance in B. rapa development. Our work reveals the epigenomic landscape of H3K27me3 in B. rapa and provides novel genomics datasets and bioinformatics analytical resources. We anticipate that this work will lead the way to further epigenomic studies in the complex genome of Brassica crops.
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http://dx.doi.org/10.1093/gigascience/giz147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892454PMC
December 2019

CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I.

Nat Commun 2018 12 21;9(1):5454. Epub 2018 Dec 21.

Regenerative Medicine Program, Center for Applied Medical Research (CIMA), University of Navarra, IdiSNA, Pamplona, 31008, Spain.

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.
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http://dx.doi.org/10.1038/s41467-018-07827-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303323PMC
December 2018

Inhibitor of Differentiation-1 Sustains Mutant -Driven Progression, Maintenance, and Metastasis of Lung Adenocarcinoma via Regulation of a FOSL1 Network.

Cancer Res 2019 02 18;79(3):625-638. Epub 2018 Dec 18.

Department of Oncology, Clínica Universidad de Navarra, Pamplona, Spain.

Because of the refractory nature of mutant lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant 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 LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of was a marker of poor survival in patients harboring mutant, but not wild-type . Abrogation of Id1 induced G-M arrest and apoptosis in mutant LUAD cells. , loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the 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 signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring as a critical gene in mutant 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 -mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1479DOI Listing
February 2019

Early metazoan cell type diversity and the evolution of multicellular gene regulation.

Nat Ecol Evol 2018 07 25;2(7):1176-1188. Epub 2018 Jun 25.

Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot, Israel.

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.
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http://dx.doi.org/10.1038/s41559-018-0575-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040636PMC
July 2018

Diverse continuum of CD4 T-cell states is determined by hierarchical additive integration of cytokine signals.

Proc Natl Acad Sci U S A 2017 08 17;114(31):E6447-E6456. Epub 2017 Jul 17.

Department of Immunology, Weizmann Institute of Science, 76100 Rehovot, Israel

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.
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http://dx.doi.org/10.1073/pnas.1615590114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547583PMC
August 2017

Genomic Characterization of Murine Monocytes Reveals C/EBPβ Transcription Factor Dependence of Ly6C Cells.

Immunity 2017 05;46(5):849-862.e7

Department of Immunology, Weizmann Institute of Science, 76100 Rehovot, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.immuni.2017.04.018DOI Listing
May 2017

Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq.

Cell 2016 Dec;167(7):1883-1896.e15

Department of Immunology, Weizmann Institute, Rehovot 76100, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2016.11.039DOI Listing
December 2016

The Spectrum and Regulatory Landscape of Intestinal Innate Lymphoid Cells Are Shaped by the Microbiome.

Cell 2016 Aug 18;166(5):1231-1246.e13. Epub 2016 Aug 18.

Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2016.07.043DOI Listing
August 2016

Co-ChIP enables genome-wide mapping of histone mark co-occurrence at single-molecule resolution.

Nat Biotechnol 2016 09 25;34(9):953-61. Epub 2016 Jul 25.

The Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

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.
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http://dx.doi.org/10.1038/nbt.3652DOI Listing
September 2016

Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors.

Cell 2015 Dec 25;163(7):1663-77. Epub 2015 Nov 25.

Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2015.11.013DOI Listing
December 2015

Immunogenetics. Chromatin state dynamics during blood formation.

Science 2014 Aug 7;345(6199):943-9. Epub 2014 Aug 7.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Chromatin modifications are crucial for development, yet little is known about their dynamics during differentiation. Hematopoiesis provides a well-defined model to study chromatin state dynamics; however, technical limitations impede profiling of homogeneous differentiation intermediates. We developed a high-sensitivity indexing-first chromatin immunoprecipitation approach to profile the dynamics of four chromatin modifications across 16 stages of hematopoietic differentiation. We identify 48,415 enhancer regions and characterize their dynamics. We find that lineage commitment involves de novo establishment of 17,035 lineage-specific enhancers. These enhancer repertoire expansions foreshadow transcriptional programs in differentiated cells. Combining our enhancer catalog with gene expression profiles, we elucidate the transcription factor network controlling chromatin dynamics and lineage specification in hematopoiesis. Together, our results provide a comprehensive model of chromatin dynamics during development.
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http://dx.doi.org/10.1126/science.1256271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4412442PMC
August 2014

Deterministic direct reprogramming of somatic cells to pluripotency.

Nature 2013 Oct 18;502(7469):65-70. Epub 2013 Sep 18.

The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.
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http://dx.doi.org/10.1038/nature12587DOI Listing
October 2013

Complement anaphylatoxins C3a and C5a induce a failing regenerative program in cardiac resident cells. Evidence of a role for cardiac resident stem cells other than cardiomyocyte renewal.

Springerplus 2012 Dec 12;1(1):63. Epub 2012 Dec 12.

Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, 28029 Spain.

Cardiac healing, which follows myocardial infarction, is a complex process guided by intricate interactions among different components. Some resident cell populations with a potential role in cardiac healing have already been described in cardiac tissues. These non-cardiomyocyte cell subsets, globally described as cardiac pluripotent/progenitor cells (CPCs), are able to differentiate into all three major cardiac cell lineages (endothelial, smooth muscle and cardiomyocyte cells) in experimental settings. Nevertheless, physiological cardiac healing results in a fibrous scar, which remains to be fully modelled experimentally. Since a role for complement anaphylatoxins (C3a and C5a) has been described in several regeneration/repair processes, we examined the effects that C3a and C5a exert on a defined population of CPCs. We found that C3a and C5a are able to enhance CPC migration and proliferation. In vitro studies showed that this effect is linked to activation of telomerase mRNA and partial preservation of telomere length, in an NFκB-dependent manner. In addition, anaphylatoxin signalling modulates the CPC phenotype, increasing myofibroblast differentiation and reducing endothelial and cardiac gene expression. These findings may denote that C3a and C5a are able to maintain/increase the cardiac stem cell pool within the heart, whilst simultaneously facilitating and modulating resident cell differentiation. We found that this modulation was directed towards scar forming cells, which increased fibroblast/myofibroblast generation and suggests that both these anaphylatoxins could play a relevant role in the damage-coupled activation of resident cells, and regulation of the cardiac healing process after injury.
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http://dx.doi.org/10.1186/2193-1801-1-63DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592996PMC
December 2012

High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states.

Nat Protoc 2013 Mar 21;8(3):539-54. Epub 2013 Feb 21.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Dynamic protein binding to DNA elements regulates genome function and cell fate. Although methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research, they are laborious and costly, thereby limiting progress. Here we present a protocol for mapping in vivo protein-DNA interactions using a high-throughput chromatin immunoprecipitation (HT-ChIP) approach. By using paramagnetic beads, we streamline the entire ChIP and indexed library construction process: sample transfer and loss is minimized and the need for manually labor-intensive procedures such as washes, gel extraction and DNA precipitation is eliminated. All of this allows for fully automated, cost effective and highly sensitive 96-well ChIP sequencing (ChIP-seq). Sample preparation takes 3 d from cultured cells to pooled libraries. Compared with previous methods, HT-ChIP is more suitable for large-scale in vivo studies, specifically those measuring the dynamics of a large number of different chromatin modifications/transcription factors or multiple perturbations.
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http://dx.doi.org/10.1038/nprot.2013.023DOI Listing
March 2013