Dempsey Nancy J, Ojalvo Laureen S, Wu Davina W, Little Jane A
Hematology, Oncology, and Transplantation, Department of Internal Medicine and Masonic Cancer Center, University of Minnesota, in-care-of LCDB, NIH, Bldg 50, Rm 3154, 9000 Rockville Pike, Bethesda, MD, USA.
Blood. 2003 Dec 1;102(12):4214-22. doi: 10.1182/blood-2002-12-3766. Epub 2003 Aug 14.
Short-chain fatty acids (SCFAs) and dimethyl sulfoxide (DMSO) induce adult erythroid differentiation in murine erythroleukemia (MEL) cells, but only SCFAs concurrently up-regulate expression from the endogenous embryonic globin gene epsilony. The epsilony promoter, linked to a reporter gene and stably transfected into MEL cells, was tested during adult erythroid differentiation. Both the epsilony-CACCC site at -114 bp and enhancer sequences (hypersensitive site 2 [HS2]) from the beta-globin locus control region (LCR) were essential to maximal SCFA-mediated induction of expression from these constructs in MEL cells. Gel-shift analyses of binding activity from SCFA-induced MEL cell nuclear extracts showed in vitro binding by specificity proteins 1 and 3 (SP1, SP3) and basic or erythroid Krüppel-like factors (BKLF, EKLF) at the epsilony-CACCC site. In a functional analysis, transient cotransfections in nonerythroid NIH/3T3 cells of SP1, SP3, BKLF, or EKLF and HS2 epsilony promoter-luciferase constructs, with or without coactivators (p300, CREB-binding protein [CBP], or p300/CBP-associated factor [PCAF]) and SCFAs, were performed. SP1, SP3, and EKLF further increased expression from HS2 epsilony promoter constructs following exposure to SCFAs. This effect was variably augmented by coactivators and was diminished in EKLF mutants that were unable to undergo histone/factor-acetyl transferase (H/FAT)-mediated acetylation. In addition, acetylation of SP1 was detectable in NIH/3T3 cells following exposure to SCFAs. In sum, LCR sequence and an embryonic globin gene promoter CACCC site were essential to that promoter's up-regulation during SCFA-mediated induction of adult erythroid differentiation in vitro. Of factors that interact at the CACCC site, SCFA-mediated acetylation is implicated in SP1 and EKLF, and may be a mechanism through which SCFAs induce embryonic/fetal globin gene promoters during adult erythroid differentiation.
短链脂肪酸（SCFAs）和二甲基亚砜（DMSO）可诱导小鼠红白血病（MEL）细胞向成年红细胞分化，但只有SCFAs能同时上调内源性胚胎珠蛋白基因ε的表达。将与报告基因相连并稳定转染至MEL细胞的ε启动子，在成年红细胞分化过程中进行了检测。位于-114 bp处的ε-CACCC位点以及来自β-珠蛋白基因座控制区（LCR）的增强子序列（超敏位点2 [HS2]），对于SCFAs介导的这些构建体在MEL细胞中表达的最大诱导作用至关重要。对SCFA诱导的MEL细胞核提取物的结合活性进行凝胶迁移分析，结果显示特异性蛋白1和3（SP1、SP3）以及碱性或红细胞类Krüppel样因子（BKLF、EKLF）在ε-CACCC位点存在体外结合。在功能分析中，对SP1、SP3、BKLF或EKLF与HS2 ε启动子-荧光素酶构建体进行瞬时共转染，转染至非红细胞NIH/3T3细胞，同时添加或不添加共激活因子（p300、CREB结合蛋白[CBP]或p300/CBP相关因子[PCAF]）以及SCFAs。暴露于SCFAs后，SP1、SP3和EKLF进一步增加了HS2 ε启动子构建体的表达。共激活因子可不同程度地增强这种效应，而在无法进行组蛋白/因子乙酰转移酶（H/FAT）介导的乙酰化的EKLF突变体中，这种效应减弱。此外，暴露于SCFAs后，在NIH/3T3细胞中可检测到SP1的乙酰化。总之，LCR序列和一个胚胎珠蛋白基因启动子CACCC位点对于该启动子在SCFAs介导的体外成年红细胞分化诱导过程中的上调至关重要。在与CACCC位点相互作用的因子中，SCFAs介导的乙酰化与SP1和EKLF有关，可能是SCFAs在成年红细胞分化过程中诱导胚胎/胎儿珠蛋白基因启动子的一种机制。
