Spi-B can functionally replace PU.1 in myeloid but not lymphoid development

Mature macrophages, neutrophils and lymphoid cells do not develop in PU.1(-/-) mice. In contrast, mice lacking the highly related protein Spi-B generate all hematopoietic lineages but display a B-cell receptor signaling defect. These distinct phenotypes could result from functional differences between PU.1 and Spi-B or their unique temporal and tissue-specific expression (PU.1: myeloid and B cells; Spi-B: B cells only). To address this question, we introduced the Spi-B cDNA into the murine PU.1 locus by homologous recombination. In the absence of PU.1, Spi-B rescued macrophage and granulocyte development when assayed by in vitro differentiation of embryonic stem cells. Adherent, CD11b(+)/F4/80(+) cells capable of phagocytosis were detected in PU.1(Spi-B/Spi-B) embryoid bodies, and myeloid colonies were present in hematopoietic progenitor assays. Despite its ability to rescue myeloid differentiation, Spi-B did not rescue lymphoid development in a RAG-2(-/-) complementation assay. These results demonstrate an important difference between PU.1 and Spi-B. Careful comparison of these Ets factors will delineate important functional domains of PU.1 involved in lymphocyte lineage commitment and/or maturation.     

Systematic RNAi studies on the role of Sp/KLF factors in globin gene expression and erythroid differentiation

Sp/KLF family of factors regulates gene expression by binding to the CACCC/GC/GT boxes in the DNA through their highly conserved three zinc finger domains. To investigate the role of this family of factors in erythroid differentiation and globin gene expression, we first measured the expression levels of selected Sp/KLF factors in primary cells of fetal and adult stages of erythroid development. This quantitative analysis revealed that their expression levels vary significantly in cells of either stages of the erythroid development. Significant difference in their expression levels was observed between fetal and adult erythroid cells for some Sp/KLF factors. Functional studies using RNA interference revealed that the silencing of Sp1 and KLF8 resulted in elevated level of gamma globin expression in K562 cells. In addition, K562 cells become visibly red after Sp1 knockdown. Benzidine staining revealed significant hemoglobinization of these cells, indicating erythroid differentiation. Moreover, the expression of PU.1, ETS1 and Notch1 is significantly down-regulated in the cells that underwent erythroid differentiation following Sp1 knockdown. Overexpression of PU.1 or ETS1 efficiently blocked the erythroid differentiation caused by Sp1 knockdown in K562 cells. The expression of c-Kit, however, was significantly up-regulated. These data indicate that Sp1 may play an important role in erythroid differentiation.     

KLF1和KLF9对K562细胞红系分化的协同调控作用

Krüppel样因子(Krüppel-like factors,KLFs)是锌指蛋白超家族的一个亚家族,参与细胞内的多种生理、病理过程,该家族成员在红细胞分化发育过程中发挥非常重要的作用,但是家族成员间对红系分化的协同调控作用还鲜有报道。本课题组前期研究发现,KIF家族成员KLF1和KLF9在已分化的红系细胞中的表达水平显著高于造血干细胞。为进一步探讨二者在红系分化中是否存在协同作用,本研究在K562细胞中分别过表达/敲低表达KLF1和KLF9,检测二者表达的相关性,发现KLF1和KLF9的基因表达呈现正相关,且二者共表达可以显著促进K562细胞红系分化,特异地增强β-珠蛋白的表达。通过对KLF1、KLF9单独和共同过表达、敲低表达的K562细胞转录组数据的分析发现二者可能通过PI3K-Akt和FoxO通路协同调控红系分化,FOS、TF、IL8是协同调控的候选靶基因。本研究结果为后续深入研究KLF1和KLF9协同调控红系分化的分子机制奠定了基础。