David Lara-Astiaso

Deterministic direct reprogramming of somatic cells to pluripotency

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.

Chromatin state dynamics during blood formation

Opening and closing blood enhancers As cells develop and differentiate into different types, the shape and accessibility of their DNA can change. Lara-Astiaso et al. studied this phenomenon in blood. They developed a technique that examines a relatively small number of cells to identify the changes that affect DNA during blood development. They found that the DNA of noncoding regions, called enhancers, is set in an open position when cells are undifferentiated and able to take on a variety of roles and gradually closes as cells mature into their final forms. Science, this issue p. 943 A chromatin precipitation technique identifies changes during the differentiation of blood cells. 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.

Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors

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.

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

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.

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

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.

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

Summary 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/EBP&bgr; and C/EBP&bgr;‐deficient mice lacked Ly6C− monocytes. Mechanistically, C/EBP&bgr; bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor. Graphical Abstract Figure. No Caption available. HighlightsSteady‐state Ly6C+ and Ly6C− monocytes are homogeneous populationsLy6Cint monocytes comprise a heterogeneous population expressing MHCIIC/EBP&bgr; regulates monocyte differentiation from Ly6C+ into Ly6C− cellsExpression of the monocyte survival factor Nr4a1 is regulated by C/EBP&bgr; &NA; Monocytes are circulating, short‐lived blood cells. Here, Mildner et al. (2017) use transcriptome and epigenome profiling to study murine monocyte identities and subset interrelations. They highlight the critical role of C/EBP&bgr; in monocyte conversion and reveal that while Ly6C+ and Ly6C− monocytes are homogeneous in steady state, Ly6Cint cells display heterogeneity.

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

Significance Understanding the logic by which cells respond to complex signal combinations is challenging. We used CD4+ T cells as a model system to study signal integration by systematically mapping their differentiation in response to a large number of cytokine combinations. We find that, in response to varied cytokine mixtures, cells coexpress lineage-specifying proteins at diverse levels, such that the cell population spans a continuum of intermediate states between canonical cell phenotypes. Mathematical modeling explains these results using hierarchical summation of cytokine inputs and correctly predicts population response to new input conditions. These findings suggest that complex cellular responses can be effectively described using relatively simple hierarchical summation rules, providing a framework for prediction of cellular responses to signal combinations. 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.

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

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.Analysis of cell-type-specific transcription in non-bilaterian animals provides insight into the evolution of the gene regulatory networks that underlie metazoan cell types.