Zhen Shao

An Erythroid Enhancer of BCL11A Subject to Genetic Variation Determines Fetal Hemoglobin Level

BCL11A Variants Recent chromatin mapping data have suggested that trait-associated variants often mark regulatory DNA. However, there has been little rigorous experimental investigation of regulatory variation. Bauer et al. (p. 253; see the Perspective by Hardison and Blobel) performed an in-depth study of the BCL11A fetal hemoglobin-associated locus. The trait-associated variants revealed a chromatin signature that enhanced erythroid development. The enhancer was required for erythroid expression of BCL11A and thus for globin gene expression. Fine-mapping reveals a promising therapeutic target for genome engineering in the β-hemoglobinopathies. [Also see Perspective by Hardison and Blobel] Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage–specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the β-hemoglobinopathies.

Correction of Sickle Cell Disease in Adult Mice by Interference with Fetal Hemoglobin Silencing

Manipulation of a transcriptional repressor promotes expression of protective fetal globin genes. Persistence of human fetal hemoglobin (HbF, α2γ2) in adults lessens the severity of sickle cell disease (SCD) and the β-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of γ-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.

MAnorm: a robust model for quantitative comparison of ChIP-Seq data sets

ChIP-Seq is widely used to characterize genome-wide binding patterns of transcription factors and other chromatin-associated proteins. Although comparison of ChIP-Seq data sets is critical for understanding cell type-dependent and cell state-specific binding, and thus the study of cell-specific gene regulation, few quantitative approaches have been developed. Here, we present a simple and effective method, MAnorm, for quantitative comparison of ChIP-Seq data sets describing transcription factor binding sites and epigenetic modifications. The quantitative binding differences inferred by MAnorm showed strong correlation with both the changes in expression of target genes and the binding of cell type-specific regulators.

Combinatorial Assembly of Developmental Stage-Specific Enhancers Controls Gene Expression Programs during Human Erythropoiesis

Gene-distal enhancers are critical for tissue-specific gene expression, but their genomic determinants within a specific lineage at different stages of development are unknown. Here we profile chromatin state maps, transcription factor occupancy, and gene expression profiles during human erythroid development at fetal and adult stages. Comparative analyses of human erythropoiesis identify developmental stage-specific enhancers as primary determinants of stage-specific gene expression programs. We find that erythroid master regulators GATA1 and TAL1 act cooperatively within active enhancers but confer little predictive value for stage specificity. Instead, a set of stage-specific coregulators collaborates with master regulators and contributes to differential gene expression. We further identify and validate IRF2, IRF6, and MYB as effectors of an adult-stage expression program. Thus, the combinatorial assembly of lineage-specific master regulators and transcriptional coregulators within developmental stage-specific enhancers determines gene expression programs and temporal regulation of transcriptional networks in a mammalian genome.

Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation

Here, we describe that lysine-specific demethylase 1 (Lsd1/KDM1a), which demethylates histone H3 on Lys4 or Lys9 (H3K4/K9), is an indispensible epigenetic governor of hematopoietic differentiation. Integrative genomic analysis, combining global occupancy of Lsd1, genome-wide analysis of its substrates H3K4 monomethylation and dimethylation, and gene expression profiling, reveals that Lsd1 represses hematopoietic stem and progenitor cell (HSPC) gene expression programs during hematopoietic differentiation. We found that Lsd1 acts at transcription start sites, as well as enhancer regions. Loss of Lsd1 was associated with increased H3K4me1 and H3K4me2 methylation on HSPC genes and gene derepression. Failure to fully silence HSPC genes compromised differentiation of hematopoietic stem cells as well as mature blood cell lineages. Collectively, our data indicate that Lsd1-mediated concurrent repression of enhancer and promoter activity of stem and progenitor cell genes is a pivotal epigenetic mechanism required for proper hematopoietic maturation. DOI: http://dx.doi.org/10.7554/eLife.00633.001

ETV1 directs androgen metabolism and confers aggressive prostate cancer in targeted mice and patients.

Distinguishing aggressive from indolent disease and developing effective therapy for advanced disease are the major challenges in prostate cancer research. Chromosomal rearrangements involving ETS transcription factors, such as ERG and ETV1, occur frequently in prostate cancer. How they contribute to tumorigenesis and whether they play similar or distinct in vivo roles remain elusive. Here we show that in mice with ERG or ETV1 targeted to the endogenous Tmprss2 locus, either factor cooperated with loss of a single copy of Pten, leading to localized cancer, but only ETV1 appeared to support development of invasive adenocarcinoma under the background of full Pten loss. Mechanistic studies demonstrated that ERG and ETV1 control a common transcriptional network but largely in an opposing fashion. In particular, while ERG negatively regulates the androgen receptor (AR) transcriptional program, ETV1 cooperates with AR signaling by favoring activation of the AR transcriptional program. Furthermore, we found that ETV1 expression, but not that of ERG, promotes autonomous testosterone production. Last, we confirmed the association of an ETV1 expression signature with aggressive disease and poorer outcome in patient data. The distinct biology of ETV1-associated prostate cancer suggests that this disease class may require new therapies directed to underlying programs controlled by ETV1.

Dynamic Control of Enhancer Repertoires Drives Lineage and Stage-Specific Transcription during Hematopoiesis

Enhancers are the primary determinants of cell identity, but the regulatory components controlling enhancer turnover during lineage commitment remain largely unknown. Here we compare the enhancer landscape, transcriptional factor occupancy, and transcriptomic changes in human fetal and adult hematopoietic stem/progenitor cells and committed erythroid progenitors. We find that enhancers are modulated pervasively and direct lineage- and stage-specific transcription. GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. Examination of lineage-specific enhancers identifies transcription factors and their combinatorial patterns in enhancer turnover. Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription. Thus, genome-wide enhancer profiling coupled with in situ enhancer editing provide critical insights into the functional complexity of enhancers during development.

Distinct and Combinatorial Functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in Mouse Embryonic Stem Cell Identity

Self-renewal and pluripotency of embryonic stem cells (ESCs) are established by multiple regulatory pathways operating at several levels. The roles of histone demethylases (HDMs) in these programs are incompletely defined. We conducted a functional RNAi screen for HDMs and identified five potential HDMs essential for mouse ESC identity. In-depth analyses demonstrate that the closely related HDMs Jmjd2b and Jmjd2c are necessary for self-renewal of ESCs and induced pluripotent stem cell generation. Genome-wide occupancy studies reveal that Jmjd2b unique, Jmjd2c unique, and Jmjd2b-Jmjd2c common target sites belong to functionally separable Core, Polycomb repressive complex (PRC), and Myc regulatory modules, respectively. Jmjd2b and Nanog act through an interconnected regulatory loop, whereas Jmjd2c assists PRC2 in transcriptional repression. Thus, two HDMs of the same subclass exhibit distinct and combinatorial functions in control of the ESC state. Such complexity of HDM function reveals an aspect of multilayered transcriptional control.

The mitochondrial respiratory chain is essential for haematopoietic stem cell function

Adult and fetal haematopoietic stem cells (HSCs) display a glycolytic phenotype, which is required for maintenance of stemness; however, whether mitochondrial respiration is required to maintain HSC function is not known. Here we report that loss of the mitochondrial complex III subunit Rieske iron-sulfur protein (RISP) in fetal mouse HSCs allows them to proliferate but impairs their differentiation, resulting in anaemia and prenatal death. RISP-null fetal HSCs displayed impaired respiration resulting in a decreased NAD+/NADH ratio. RISP-null fetal HSCs and progenitors exhibited an increase in both DNA and histone methylation associated with increases in 2-hydroxyglutarate (2HG), a metabolite known to inhibit DNA and histone demethylases. RISP inactivation in adult HSCs also impaired respiration resulting in loss of quiescence concomitant with severe pancytopenia and lethality. Thus, respiration is dispensable for adult or fetal HSC proliferation, but essential for fetal HSC differentiation and maintenance of adult HSC quiescence.

Prediction of Polycomb target genes in mouse embryonic stem cells

Polycomb group (PcG) proteins are important epigenetic regulators, yet the underlying targeting mechanism in mammals is still poorly understood. We have developed a computational approach to predict genome-wide PcG target genes in mouse embryonic stem cells. We use TF binding and motif information as predictors and apply the Bayesian Additive Regression Trees (BART) model for classification. Our model has good prediction accuracy. The performance can be mainly explained by five TF features (Zf5, Tcfcp2l1, Ctcf, E2f1, Myc). Our analysis of H3K27me3 and gene expression data suggests that genomic sequence is highly correlated with the overall PcG target plasticity. We have also compared the PcG target sequence signatures between mouse and Drosophila and found that they are strikingly different. Our predictions may be useful for de novo search for Polycomb response elements (PRE) in mammals.