Andrew J. Woo

A Myc Network Accounts for Similarities between Embryonic Stem and Cancer Cell Transcription Programs

c-Myc (Myc) is an important transcriptional regulator in embryonic stem (ES) cells, somatic cell reprogramming, and cancer. Here, we identify a Myc-centered regulatory network in ES cells by combining protein-protein and protein-DNA interaction studies and show that Myc interacts with the NuA4 complex, a regulator of ES cell identity. In combination with regulatory network information, we define three ES cell modules (Core, Polycomb, and Myc) and show that the modules are functionally separable, illustrating that the overall ES cell transcription program is composed of distinct units. With these modules as an analytical tool, we have reassessed the hypothesis linking an ES cell signature with cancer or cancer stem cells. We find that the Myc module, independent of the Core module, is active in various cancers and predicts cancer outcome. The apparent similarity of cancer and ES cell signatures reflects, in large part, the pervasive nature of Myc regulatory networks.

Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

ABSTRACT The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

Identification of ZBP-89 as a Novel GATA-1-Associated Transcription Factor Involved in Megakaryocytic and Erythroid Development

ABSTRACT A complete understanding of the transcriptional regulation of developmental lineages requires that all relevant factors be identified. Here, we have taken a proteomic approach to identify novel proteins associated with GATA-1, a lineage-restricted zinc finger transcription factor required for terminal erythroid and megakaryocytic maturation. We identify the Krüppel-type zinc finger transcription factor ZBP-89 as being a component of multiprotein complexes involving GATA-1 and its essential cofactor Friend of GATA-1 (FOG-1). Using chromatin immunoprecipitation assays, we show that GATA-1 and ZBP-89 cooccupy cis-regulatory elements of certain erythroid and megakaryocyte-specific genes, including an enhancer of the GATA-1 gene itself. Loss-of-function studies in zebrafish and mice demonstrate an in vivo requirement for ZBP-89 in megakaryopoiesis and definitive erythropoiesis but not primitive erythropoiesis, phenocopying aspects of FOG-1- and GATA-1-deficient animals. These findings identify ZBP-89 as being a novel transcription factor involved in erythroid and megakaryocytic development and suggest that it serves a cooperative function with GATA-1 and/or FOG-1 in a developmental stage-specific manner.

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.

A Proteomics Approach for the Identification of DNA Binding Activities Observed in the Electrophoretic Mobility Shift Assay

Transcription factors lie at the center of gene regulation, and their identification is crucial to the understanding of transcription and gene expression. Traditionally, the isolation and identification of transcription factors has been a long and laborious task. We present here a novel method for the identification of DNA-binding proteins seen in electrophoretic mobility shift assay (EMSA) using the power of two-dimensional electrophoresis coupled with mass spectrometry. By coupling SDS-PAGE and isoelectric focusing to EMSA, the molecular mass and pI of a protein complex seen in EMSA were estimated. Candidate proteins were then identified on a two-dimensional array at the predetermined pI and molecular mass coordinates and identified by mass spectrometry. We show here the successful isolation of a functionally relevant transcription factor and validate the identity through EMSA supershift analysis.

A Src family kinase–Shp2 axis controls RUNX1 activity in megakaryocyte and T-lymphocyte differentiation

Hematopoietic development occurs in complex microenvironments and is influenced by key signaling events. Yet how these pathways communicate with master hematopoietic transcription factors to coordinate differentiation remains incompletely understood. The transcription factor RUNX1 plays essential roles in definitive hematopoietic stem cell (HSC) ontogeny, HSC maintenance, megakaryocyte (Mk) maturation, and lymphocyte differentiation. It is also the most frequent target of genetic alterations in human leukemia. Here, we report that RUNX1 is phosphorylated by Src family kinases (SFKs) and that this occurs on multiple tyrosine residues located within its negative regulatory DNA-binding and autoinhibitory domains. Retroviral transduction, chemical inhibitor, and genetic studies demonstrate a negative regulatory role of tyrosine phosphorylation on RUNX1 activity in Mk and CD8 T-cell differentiation. We also demonstrate that the nonreceptor tyrosine phosphatase Shp2 binds directly to RUNX1 and contributes to its dephosphorylation. Last, we show that RUNX1 tyrosine phosphorylation correlates with reduced GATA1 and enhanced SWI/SNF interactions. These findings link SFK and Shp2 signaling pathways to the regulation of RUNX1 activity in hematopoiesis via control of RUNX1 multiprotein complex assembly.

Surfactant Protein–C Chromatin-Bound Green Fluorescence Protein Reporter Mice Reveal Heterogeneity of Surfactant Protein C–Expressing Lung Cells

The regeneration of alveolar epithelial cells is a critical aspect of alveolar reorganization after lung injury. Although alveolar Type II (AT2) cells have been described as progenitor cells for alveolar epithelia, more remains to be understood about how their progenitor cell properties are regulated. A nuclear, chromatin-bound green fluorescence protein reporter (H2B-GFP) was driven from the murine surfactant protein-C (SPC) promoter to generate SPC H2B-GFP transgenic mice. The SPC H2B-GFP allele allowed the FACS-based enrichment and gene expression profiling of AT2 cells. Approximately 97% of AT2 cells were GFP-labeled on Postnatal Day 1, and the percentage of GFP-labeled AT2 cells decreased to approximately 63% at Postnatal Week 8. Isolated young adult SPC H2B-GFP(+) cells displayed proliferation, differentiation, and self-renewal capacity in the presence of lung fibroblasts in a Matrigel-based three-dimensional culture system. Heterogeneity within the GFP(+) population was revealed, because cells with distinct alveolar and bronchiolar gene expression arose in three-dimensional cultures. CD74, a surface marker highly enriched on GFP(+) cells, was identified as a positive selection marker, providing 3-fold enrichment for AT2 cells. In vivo, GFP expression was induced within other epithelial cell types during maturation of the distal lung. The utility of the SPC H2B-GFP murine model for the identification of AT2 cells was greatest in early postnatal lungs and more limited with age, when some discordance between SPC and GFP expression was observed. In adult mice, this allele may allow for the enrichment and future characterization of other SPC-expressing alveolar and bronchiolar cells, including putative stem/progenitor cell populations.

Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation

About 10% of Down syndrome (DS) infants are born with a transient myeloproliferative disorder (DS-TMD) that spontaneously resolves within the first few months of life. About 20%-30% of these infants subsequently develop acute megakaryoblastic leukemia (DS-AMKL). Somatic mutations leading to the exclusive production of a short GATA1 isoform (GATA1s) occur in all cases of DS-TMD and DS-AMKL. Mice engineered to exclusively produce GATA1s have marked megakaryocytic progenitor (MkP) hyperproliferation during early fetal liver (FL) hematopoiesis, but not during postnatal BM hematopoiesis, mirroring the spontaneous resolution of DS-TMD. The mechanisms that underlie these developmental stage-specific effects are incompletely understood. Here, we report a striking upregulation of type I IFN-responsive gene expression in prospectively isolated mouse BM- versus FL-derived MkPs. Exogenous IFN-α markedly reduced the hyperproliferation FL-derived MkPs of GATA1s mice in vitro. Conversely, deletion of the α/β IFN receptor 1 (Ifnar1) gene or injection of neutralizing IFN-α/β antibodies increased the proliferation of BM-derived MkPs of GATA1s mice beyond the initial postnatal period. We also found that these differences existed in human FL versus BM megakaryocytes and that primary DS-TMD cells expressed type I IFN-responsive genes. We propose that increased type I IFN signaling contributes to the developmental stage-specific effects of GATA1s and possibly the spontaneous resolution of DS-TMD.

CpG island-mediated global gene regulatory modes in mouse embryonic stem cells

Both transcriptional and epigenetic regulations are fundamental for the control of eukaryotic gene expression. Here we perform a compendium analysis of >200 large sequencing data sets to elucidate the regulatory logic of global gene expression programs in mouse embryonic stem (ES) cells. We define four major classes of DNA-binding proteins (Core, PRC, MYC and CTCF) based on their target co-occupancy, and discover reciprocal regulation between the MYC and PRC classes for the activity of nearly all genes under the control of the CpG island (CGI)-containing promoters. This CGI-dependent regulatory mode explains the functional segregation between CGI-containing and CGI-less genes during early development. By defining active enhancers based on the co-occupancy of the Core class, we further demonstrate their additive roles in CGI-containing gene expression and cell type-specific roles in CGI-less gene expression. Altogether, our analyses provide novel insights into previously unknown CGI-dependent global gene regulatory modes.

Role of ZBP-89 in human globin gene regulation and erythroid differentiation

The molecular mechanisms underlying erythroid-specific gene regulation remain incompletely understood. Closely spaced binding sites for GATA, NF-E2/maf, and CACCC interacting transcription factors play functionally important roles in globin and other erythroid-specific gene expression. We and others recently identified the CACCC-binding transcription factor ZBP-89 as a novel GATA-1 and NF-E2/mafK interacting partner. Here, we examined the role of ZBP-89 in human globin gene regulation and erythroid maturation using a primary CD34(+) cell ex vivo differentiation system. We show that ZBP-89 protein levels rise dramatically during human erythroid differentiation and that ZBP-89 occupies key cis-regulatory elements within the globin and other erythroid gene loci. ZBP-89 binding correlates strongly with RNA Pol II occupancy, active histone marks, and high-level gene expression. ZBP-89 physically associates with the histone acetyltransferases p300 and Gcn5/Trrap, and occupies common sites with Gcn5 within the human globin loci. Lentiviral short hairpin RNAs knockdown of ZBP-89 results in reduced Gcn5 occupancy, decreased acetylated histone 3 levels, lower globin and erythroid-specific gene expression, and impaired erythroid maturation. Addition of the histone deacetylase inhibitor valproic acid partially reverses the reduced globin gene expression. These findings reveal an activating role for ZBP-89 in human globin gene regulation and erythroid differentiation.