ABSTRACT
Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast, early hematopoietic, and vascular development. We have previously shown that an upstream enhancer, Eng -8, together with the promoter region, mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements, we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8, +7+9 enhancers in both blood and endothelial cells. By contrast Pu.1, an Ets factor specific to the blood lineage, and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence.
Subject(s)
Antigens, CD/genetics , Blood/metabolism , Endothelium/metabolism , GATA Transcription Factors/physiology , Hemangioblasts/physiology , Proto-Oncogene Protein c-ets-1/physiology , Receptors, Cell Surface/genetics , Animals , Antigens, CD/metabolism , Cell Differentiation/genetics , Cells, Cultured , Embryo, Mammalian , Embryonic Development/genetics , Endoglin , GATA Transcription Factors/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Hemangioblasts/metabolism , Hematopoietic System/metabolism , Humans , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Protein c-ets-1/metabolism , Receptors, Cell Surface/metabolismABSTRACT
Conservation of the vertebrate body plan has been attributed to the evolutionary stability of gene-regulatory networks (GRNs). We describe a regulatory circuit made up of Gata2, Fli1, and Scl/Tal1 and their enhancers, Gata2-3, Fli1+12, and Scl+19, that operates during specification of hematopoiesis in the mouse embryo. We show that the Fli1+12 enhancer, like the Gata2-3 and Scl+19 enhancers, targets hematopoietic stem cells (HSCs) and relies on a combination of Ets, Gata, and E-Box motifs. We show that the Gata2-3 enhancer also uses a similar cluster of motifs and that Gata2, Fli1, and Scl are expressed in embryonic day-11.5 dorsal aorta where HSCs originate and in fetal liver where they multiply. The three HSC enhancers in these tissues and in ES cell-derived hemangioblast equivalents are bound by each of these transcription factors (TFs) and form a fully connected triad that constitutes a previously undescribed example of both this network motif in mammalian development and a GRN kernel operating during the specification of a mammalian stem cell.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , GATA2 Transcription Factor/metabolism , Hematopoiesis/physiology , Hematopoietic Stem Cells/physiology , Proto-Oncogene Protein c-fli-1/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/genetics , Binding Sites , Blood Vessels/embryology , Blood Vessels/physiology , Embryo, Mammalian/physiology , Enhancer Elements, Genetic , GATA2 Transcription Factor/chemistry , GATA2 Transcription Factor/genetics , Gene Expression Regulation , Mice , Proto-Oncogene Protein c-fli-1/chemistry , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , T-Cell Acute Lymphocytic Leukemia Protein 1ABSTRACT
Transcription factors are key regulators of hematopoietic stem cells (HSCs), yet the molecular mechanisms that control their expression are largely unknown. Previously, we demonstrated that expression of Scl/Tal1, a transcription factor required for the specification of HSCs, is controlled by Ets and GATA factors. Here we characterize the molecular mechanisms controlling expression of Lyl1, a paralog of Scl also required for HSC function. Two closely spaced promoters directed expression to hematopoietic progenitor, megakaryocytic, and endothelial cells in transgenic mice. Conserved binding sites required for promoter activity were bound in vivo by GATA-2 and the Ets factors Fli1, Elf1, Erg, and PU.1. However, despite coregulation of Scl and Lyl1 by the same Ets and GATA factors, Scl expression was initiated prior to Lyl1 in embryonic stem (ES) cell differentiation assays. Moreover, ectopic expression of Scl but not Lyl1 rescued hematopoietic differentiation in Scl-/- ES cells, thus providing a molecular explanation for the vastly different phenotypes of Scl-/- and Lyl1-/- mouse embryos. Furthermore, coregulation of Scl and Lyl1 later during development may explain the mild phenotype of Scl-/- adult HSCs.
