Homologous recombination in human embryonic stem cells: a tool for advancing cell therapy and understanding and treating human disease.

Human embryonic stem cells (hESCs) hold great promise for ushering in an era of novel cell therapies to treat a wide range of rare and common diseases, yet they also provide an unprecedented opportunity for basic research to yield clinical benefit. HESCs can be used to better understand human development, to model human diseases, to understand the contribution of specific mutations to the pathogenesis of disease, and to develop human cell-based screening systems to identify novel therapeutic agents and evaluate potential toxicity of therapeutic agents under development. Such basic research will benefit greatly from efficient methods to perform targeted gene modification, an area of hESC investigation that is currently in its infancy. Moreover, the reality of hESC-based cellular therapies will require improved methods for generating the specific cells of interest, and reporter cell lines generated through targeted gene modifications are expected to play an important role in developing optimal cell-specific differentiation protocols. Herein, we review the current status of homologous recombination in hESCs, a gene targeting technique that is sure to continue to improve, and to play an important role in realizing the maximal human benefit from hESCs.

Characterization of megakaryocyte GATA1-interacting proteins: the corepressor ETO2 and GATA1 interact to regulate terminal megakaryocyte maturation.

The transcription factor GATA1 coordinates timely activation and repression of megakaryocyte gene expression. Loss of GATA1 function results in excessive megakaryocyte proliferation and disordered terminal platelet maturation, leading to thrombocytopenia and leukemia in patients. The mechanisms by which GATA1 does this are unclear. We have used in vivo biotinylated GATA1 to isolate megakaryocyte GATA1-partner proteins. Here, several independent approaches show that GATA1 interacts with several proteins in the megakaryocyte cell line L8057 and in primary megakaryocytes. They include FOG1, the NURD complex, the pentameric complex containing SCL/TAL-1, the zinc-finger regulators GFI1B and ZFP143, and the corepressor ETO2. Knockdown of ETO2 expression promotes megakaryocyte differentiation and enhances expression of select genes expressed in terminal megakaryocyte maturation, eg, platelet factor 4 (Pf4). ETO2-dependent direct repression of the Pf4 proximal promoter is mediated by GATA-binding sites and an E-Box motif. Consistent with this, endogenous ETO2, GATA1, and the SCL pentameric complex all specifically bind the promoter in vivo. Finally, as ETO2 expression is restricted to immature megakaryocytes, these data suggest that ETO2 directly represses inappropriate early expression of a subset of terminally expressed megakaryocyte genes by binding to GATA1 and SCL.

ETO-2 associates with SCL in erythroid cells and megakaryocytes and provides repressor functions in erythropoiesis.

Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

Differences in the chromatin structure and cis-element organization of the human and mouse GATA1 loci: implications for cis-element identification.

Cis-element identification is a prerequisite to understand transcriptional regulation of gene loci. From analysis of a limited number of conserved gene loci, sequence comparison has proved a robust and efficient way to locate cis-elements. Human and mouse GATA1 genes encode a critical hematopoietic transcription factor conserved in expression and function. Proper control of GATA1 transcription is critical in regulating myeloid lineage specification and maturation. Here, we compared sequence and systematically mapped position of DNase I hypersensitive sites, acetylation status of histone H3/H4, and in vivo binding of transcription factors over approximately 120 kilobases flanking the human GATA1 gene and the corresponding region in mice. Despite lying in approximately 10 megabase (Mb) conserved syntenic segment, the chromatin structures of the 2 homologous loci are strikingly different. The 2 previously unidentified hematopoietic cis-elements, one in each species, are not conserved in position and sequence and have enhancer activity in erythroid cells. In vivo, they both bind the transcription factors GATA1, SCL, LMO2, and Ldb1. More broadly, there are both species- and regulatory element-specific patterns of transcription factor binding. These findings suggest that some cis-elements regulating human and mouse GATA1 genes differ. More generally, mouse human sequence comparison may fail to identify all cis-elements.