The intronic Ig heavy chain (IgH) enhancer, which consists of the core enhancer f lanked by 5 and 3 matrix attachment regions, has been implicated in control of IgH locus recombination and transcription. To elucidate the regulatory functions of the core enhancer and its associated matrix attachment regions in the endogenous IgH locus, we have introduced targeted deletions of these elements, both individually and in combination, into an IgH a͞b -heterozygous embryonic stem cell line. These embryonic stem cells were used to generate chimeric mice by recombination activating gene-2 (Rag-2)-deficient blastocyst complementation, and the effects of the introduced mutations were assayed in mutant B cells. We find that the core enhancer is necessary and sufficient to promote normal variable (V), diversity (D), and joining (J) segment recombination in developing B lineage cells and IgH locus transcription in mature B cells. Surprisingly, the 5 and 3 matrix attachment regions were dispensable for these processes. Multiple enhancer elements have been identified within the IgH locus, including the intronic enhancer (E) between J H and C (3, 4) and a series of enhancers (collectively referred to as the 3Ј IgH regulatory region) that lie downstream of C␣ (reviewed in ref. 5). Extensive transfection and transgenic studies delineated the E sequences based on ability to direct lymphoid-specific expression (3, 4, 6-8; reviewed in refs. 9-11). Studies of cell lines with spontaneous E region deletions suggested this enhancer was necessary for IgH expression in precursor-B cells (12, 13), but dispensable for expression in terminally differentiated B cell lines (14-17).Transfection assays defined a small 220-bp core element (hereafter referred as cE) within E, which is necessary and sufficient for transcriptional stimulation; cE contains multiple binding sites for both ubiquitous and cell-specific factors with negative and positive activity (reviewed in ref. 18). Biochemical assays further identified two AT-rich nuclear matrix attachment regions (MARs) flanking cE (19). MARs are generally defined by the ability to bind to the nuclear matrix, which is a rather poorly defined protein fraction containing factors important for regulation of gene expression in addition to structural scaffold components (reviewed in refs. 20-25). Despite a strictly biochemical definition, several functions for MARs have been proposed (reviewed in refs. 20, 23-27). For example, MARs have been implicated in defining physical boundaries between genes (27, 28). In addition, MARs often are found in close association with active elements such as enhancers (19, 27, 28), promoters (29, 30), and putative replication origins (31, 32), potentially serving to anchor these elements to specific nuclear matrix sites. MARs have also been described as regions susceptible to histone H1 displacement and, therefore, chromatin ''opening'' by way of the interaction of minor groove binding proteins like HMG-I͞Y (33).The E-associated MARs initially were implicated as a...

GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.

The intronic IgH enhancer E(mu), which consists of the core enhancer (cE(mu) flanked by 5' and 3' matrix attachment regions (MAR), has been implicated in the control of IgH locus recombination and transcription. Both cE(mu) and the MAR are required to enhance transcription of an IgH transgene. To elucidate the regulatory functions of cE(mu) versus its associated MAR in IgH class switch recombination (CSR), we have assayed ES cell lines which have targeted deletions of these elements, both individually and in combination, by the Rag-2-deficient blastocyst complementation method. Mutant B cells from chimeric mice were activated in culture and the influence of the mutations on CSR was assessed by analysis of B cell hybridomas. We find that the cE(mu) is necessary and sufficient for providing the functions of E(mu) required for efficient CSR at the IgH locus. However, the 5' and 3' MAR sequences, as well as the known I(mu) transcription start sites and the bulk of I(mu) coding sequences, were dispensable for the process.

The zinc finger transcription factor GATA-1 is essential for both primitive (embryonic) and definitive (adult) erythropoiesis. To define the roles of GATA-1 in the production and differentiation of primitive and definitive erythrocytes, we established GATA-1-null embryonic stem cell lines in which GATA-1 was able to be conditionally expressed by using the tetracycline conditional gene expression system. The cells were subjected to hematopoietic differentiation by coculturing on OP9 stroma cells. We expressed GATA-1 in the course of primitive and definitive erythropoiesis and analyzed the ability of GATA-1 to rescue the defective erythropoiesis caused by the GATA-1 null mutation. Our results show that GATA-1 functions in the proliferation and maturation of erythrocytes in a distinctive manner. The early-stage expression of GATA-1 during both primitive and definitive erythropoiesis was sufficient to promote the proliferation of red blood cells. In contrast, the late-stage expression of GATA-1 was indispensable to the terminal differentiation of primitive and definitive erythrocytes. Thus, GATA-1 affects the proliferation and differentiation of erythrocytes by different mechanisms. IntroductionHematopoiesis is the sequential proliferation and differentiation process that produces more than 8 distinct, mature blood cells. The process is tightly controlled by various lineage-specific transcription factors. 1 Among them, GATA transcription factors are one of the most well-studied transcription factors. The GATA protein is in the zinc finger transcription factor family and is categorized by recognizing the consensus motif WGATAR in a conserved multifunctional domain consisting of 2 C4-type zinc fingers. [2][3][4][5][6] The GATA family includes GATA-1, GATA-2, 5,7-9 and GATA-3, 10 which are essential hematopoietic factors, and GATA-4, GATA-5, and GATA-6, which regulate heart, lung, and gut cell development. 11,12 GATA-1, the founding member of the GATA family, has been mapped to the X chromosome, and the GATA-1 protein has been detected in erythroid, megakaryocytic, eosinophilic, and mast cell lines within the hematopoietic system. [13][14][15][16][17][18][19] From a developmental point of view, erythropoiesis consists of 2 waves of red blood cell production, primitive and definitive erythropoiesis. 20 The differences between primitive and definitive erythropoiesis are attributable to not only the emerging time but also to the origin, morphology, and globin gene expression. Primitive erythropoiesis, the first wave of erythroid production, generates nucleated, primitive erythrocytes (EryPs) that express embryonic hemoglobin at the blood island in the embryonic yolk sac. A recent work reported that murine primitive erythroblasts also enucleated and continued to circulate through late gestation and even into the postnatal period, indicating that the primitive erythropoiesis in mammals shared many processes with its definitive counterpart. 21 Definitive erythropoiesis, the second wave of erythropoiesis, commences in the fe...