Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis

The Krüppel-like factor 1 (KLF1) and KLF2 positively regulate embryonic β-globin expression and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1(-/-) KLF2(-/-) double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1(-/-), and KLF1(-/-) KLF2(-/-) mice. Among these, the gene for Myc (c-Myc) emerged as a central node in the most significant gene network. The expression of the Myc gene is synergistically regulated by KLF1 and KLF2, and both factors bind the Myc promoters. To characterize the role of Myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia, analogous to KLF1(-/-) KLF2(-/-) embryos. In the absence of Myc, circulating erythroid cells do not show the normal increase in α- and β-like globin gene expression but, interestingly, have accelerated erythroid cell maturation between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate Myc to control the primitive erythropoietic program.     

KLFs对珠蛋白基因表达和红系分化的调控作用

Krüppel样因子(Krüppel-like factors, KLFs)是一组与真核基因转录调控密切相关的锌指蛋白.KLFs高度保守的羧基末端含3个串联的Cys2His2型锌指结构,用于结合GC盒和CACCC盒等DNA序列. 红细胞中特异表达的珠蛋白基因和许多红系调控因子中都含有CACCC盒.已有研究发现,多个KLFs通过结合CACCC盒参与调控珠蛋白基因表达和红系分化,例如,KLF1通过结合β-珠蛋白启动子和位点控制区(locus control region, LCR),促进β-珠蛋白的表达、γ-向β-珠蛋白基因的转换和红系分化;KLF2、KLF11和KLF13分别促进ε-和γ-珠蛋白基因的表达;KLF4促进α-和γ-珠蛋白基因的表达;KLF3和KLF8则抑制ε-和γ-珠蛋白基因的表达. 本文综述了KLFs调控珠蛋白基因表达和红系分化的研究进展.     

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.     

Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions

Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions.