Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching.

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT-box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR-1 binding sites for non-steroid nuclear hormone receptors. We show that a complex previously named NF-E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human epsilon-globin promoter leads to substantial reduction in epsilon expression in embryonic erythroid cells, indicating that CP1 activates epsilon expression; in contrast, destruction of the DR-1 elements yields striking epsilon expression in definitive erythropoiesis, indicating that the NF-E3 complex acts as a developmental repressor of the epsilon gene. We also show that NF-E3 is immunologically related to COUP-TF orphan nuclear receptors. One of these, COUP-TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra-embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF-E3/COUP-TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF-E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.     

Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: relevance to the cell-lineage models of globin switching

The cellular control of the switch from embryonic to fetal globin formation in man was investigated with studies of globin expression in erythroid cells of 35- to 56-day-old embryos. Analyses of globins synthesized in vivo and in cultures of erythroid progenitors (burst-forming units, BFUe) showed that cells of the yolk sac (primitive) erythropoiesis, in addition to embryonic chains, produced fetal and adult globins and that cells of the definitive (liver) erythropoiesis, in addition to fetal and adult globins, produce embryonic globins. That embryonic, fetal, and adult globins were coexpressed by cells of the same lineage was documented by analysis of globin chains in single BFUe colonies: all 67 yolk sac-origin BFUe colonies and 42 of 43 liver-origin BFUe colonies synthesized epsilon-, gamma-, and beta-chains. These data showed that during the switch from embryonic to adult globin formation, embryonic and definitive globin chains are coexpressed in the primitive, as well as in the definitive, erythroid cells. Such results are compatible with the postulate that the switch from embryonic to fetal globin synthesis represents a time-dependent change in programs of progenitor cells rather than a change in hemopoietic cell lineages.     

Erythroid overexpression of C/EBPgamma in transgenic mice affects gamma-globin expression and fetal liver erythropoiesis

The CCAAT boxes of the beta-like globin genes interact with three proteins: NF-Y, GATA-1 and NFE-6. We demonstrate that NFE-6 contains C/EBPgamma, and address its role in globin gene regulation by erythroid overexpression of C/EBPgamma, and a dominant-negative form C/EBPgammaDeltaB, in mice. Elevated levels of C/EBPgamma, but not C/EBPgammaDeltaB, increase expression of the (fetal) gamma-globin relative to the (adult) beta-globin gene. Interestingly, fetal liver erythropoiesis is ablated when the C/EBPgamma and C/EBPgammaDeltaB levels are further increased in homozygous transgenics. We suggest that targeted expression of dominant-negative leucine zipper proteins is a generally applicable approach to ablate specific tissues in mice.     

The 5'HS4 core element of the human beta-globin locus control region is required for high-level globin gene expression in definitive but not in primitive erythropoiesis.

To assess the contribution of DNase I-hypersensitive site 4 (HS4) of the beta-globin locus control region (LCR) to overall LCR function we deleted a 280 bp fragment encompassing the core element of 5'HS4 from a 248 kb beta-globin locus yeast artificial chromosome (beta-YAC) and analyzed globin gene expression during development in beta-YAC transgenic mice. Four transgenic lines were established; each contained at least one intact copy of the beta-globin locus. The deletion of the 5'HS4 core element had no effect on globin gene expression during embryonic erythropoiesis. In contrast, deletion of the 5'HS4 core resulted in a significant decrease of gamma and beta-globin gene expression during definitive erythropoiesis in the fetal liver and a decrease of beta-globin gene expression in adult blood. We conclude that the core element of 5'HS4 is required for globin gene expression only in definitive erythropoiesis. Absence of the core element of HS4 may limit the ability of the LCR to provide an open chromatin domain and/or enhance gamma and beta-globin gene expression in the adult erythroid cells.     

A global role for zebrafish klf4 in embryonic erythropoiesis

There are two waves of erythropoiesis, known as primitive and definitive waves in mammals and lower vertebrates including zebrafish. The founding member of the Kruppel-like factor (KLF) family of CACCC-box binding proteins, EKLF/Klf1, is essential for definitive erythropoiesis in mammals but only plays a minor role in primitive erythropoiesis. Morpholino knockdown experiments have shown a role for zebrafish klf4 in primitive erythropoiesis and hatching gland formation. In order to generate a global understanding of how klf4 might influence gene expression and differentiation, we have performed expression profiling of klf4 morphants, and then performed validation of many putative target genes by qRT-PCR and whole mount in situ hybridization. We found a critical role for klf4 in embryonic globin, heme synthesis and hatching gland gene expression. In contrast, there was an increase in expression of definitive hematopoietic specific genes such as larval globin genes, runx1 and c-myb from 24 hpf, suggesting a selective role for klf4 in primitive rather than definitive erythropoiesis. In addition, we show klf4 preferentially binds CACCC box elements in the primitive zebrafish beta-like globin gene promoters. These results have global implications for primitive erythroid gene regulation by KLF-CACCC box interactions.     

Sox9 transactivation and testicular expression of a novel human gene,KIAA0800

The Sry and Sox9 sex-determination factors initiate and promote testis differentiation by gene transactivation through similar promoter elements. However, knowledge is limited concerning what genes are regulated by Sry/Sox9 in the testis. Identification and characterization of Sry/Sox9-regulated genes are critical for understanding sexual differentiation. We now demonstrate that a novel human gene, KIAA0800, is preferentially expressed in the testis and is transactivated by Sox9. The KIAA0800 promoter is repressed by an upstream element involving a polyT track and two Alu repeats. Two specific Sox9-bindings sites have been identified in the KIAA0800 promoter by using DNaseI footprinting assays and gel electrophoretic mobility shift assays. Sox9 transactivation of the KIAA0800 promoter appears to be exerted mainly through the relief of promoter repression. Genes homologous to the human KIAA0800 exist in organisms with differentiated sex tissues including mouse, Drosophila, and C. elegans, but not in unicellular organisms, including yeast and bacteria. Further, our recent sequence analysis shows that KIAA0800 protein is 97% identical between human and mouse. Thus, KIAA0800 is a novel Sox9-activated gene that is evolutionarily conserved and potentially involved in sexual differentiation.     

Klf1 affects DNase II-alpha expression in the central macrophage of a fetal liver erythroblastic island: a non-cell-autonomous role in definitive erythropoiesis

A key regulatory gene in definitive erythropoiesis is the erythroid Kruppel-like factor (Eklf or Klf1). Klf1 knockout (KO) mice die in utero due to severe anemia, while residual circulating red blood cells retain their nuclei. Dnase2a is another critical gene in definitive erythropoiesis. Dnase2a KO mice are also affected by severe anemia and die in utero. DNase II-alpha is expressed in the central macrophage of erythroblastic islands (CMEIs) of murine fetal liver. Its main role is to digest the DNA of the extruded nuclei of red blood cells during maturation. Circulating erythrocytes retain their nuclei in Dnase2a KO mice. Here, we show that Klf1 is expressed in CMEIs and that it binds and activates the promoter of Dnase2a. We further show that Dnase2a is severely downregulated in the Klf1 KO fetal liver. We propose that this downregulation of Dnase2a in the CMEI contributes to the Klf1 KO phenotype by a non-cell-autonomous mechanism.