Interferon α selectively affects expression of the human myeloid cell nuclear differentiation antigen in late stage cells in the monocytic but not the granulocytic lineage

The human myeloid cell nuclear differentiation antigen (MNDA) is expressed constitutively in cells of the myeloid lineage, appearing in myeloblast cells in some cases of acute myeloid leukemia and consistently being detected in promyelocyte stage cells as well as in all later stage cells including peripheral blood monocytes and granulocytes. The human myeloid leukemia cell lines, HL-60, U937, and THP-1, express similar levels of immunochemically detectable MNDA. Although, the level of MNDA mRNA in primary monocytes is very low it was up-regulated at 6 h following the addition of interferon α. The effect of interferon α on the MNDA mRNA is also observed in the cell lines HL-60, U937, and THP-1. The MNDA mRNA level in primary granulocytes was unaffected by addition of interferon α and other agents including interferon γ, endotoxin, poly (I) · poly (C), and FMLP. The MNDA mRNA level in the myeloid cell lines was also unaffected by the latter four agents. Induction of differentiation in the myeloid cell lines with phorbol ester induces monocyte differentiation which was accompanied by a decrease in MNDA mRNA level. This reduced level of mRNA could then be elevated with subsequent interferon α treatment. The effects of phorbol ester on MNDA mRNA appeared to be associated with induced differentiation since inhibiting cell proliferation did not alter the level of MNDA mRNA and cell cycle variation in MNDA mRNA levels were not observed. The ability of interferon α to up-regulate MNDA mRNA in phorbol ester treated myeloid cell lines is consistent with the observations made in primary monocytes. Granulocyte differentiation induced by retinoic acid treatment of HL-60 cells did not alter the MNDA mRNA level which was also unchanged following subsequent treatment with interferon α. The lack of interferon α effects on retinoic acid treated HL-60 cells is consistent with its inability to influence MNDA mRNA level in primary granulocytes.     

Cloning and expression of the human myeloid cell nuclear differentiation antigen: regulation by interferon alpha.

The human myeloid cell nuclear differentiation antigen (MNDA) is a protein of 406 amino acids that is expressed specifically in granulocytes, monocytes and earlier stage cells of these lineages. Degenerate oligonucleotides that could encode regions of MNDA amino acid sequence were used to amplify the MNDA cDNA sequence using the polymerase chain reaction. The amplified cDNA product was sequenced to confirm that it encoded the MNDA protein. It was then used as a probe to isolate five clones from a human bone marrow lambda gt10 cDNA library. A clone containing a 1,672 base pair cDNA insert was sequenced and found to encode the entire MNDA open reading frame, as well as 5' and 3' untranslated regions. The primary structure of the MNDA contains extensive regions of sequence similarity with the protein products of the interferon-inducible genes: 204 and interferon regulatory factor 2. In addition, a 12-base sequence matching the interferon-stimulated response element consensus sequence [GAAAN(N)GAAA] is located in the 5' untranslated region of the MNDA cDNA. The 1.8 kb MNDA mRNA was detected only in cells that express the antigen and the level of MNDA mRNA was elevated in cells treated with either recombinant or natural interferon alpha. The MNDA mRNA was not induced by interferon alpha in cells that do not exhibit a constitutive level of the MNDA mRNA. The MNDA contains sequence motifs found in gene regulatory proteins. The expression and the primary structure of the MNDA indicates that it plays a role in the granulocyte/monocyte cell-specific response to interferon.     

Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2α in Dp71 promoter activity

In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E‐115 cell line. We demonstrated that Dp71 expression is up‐regulated in response to cAMP‐mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5′‐flanking 159‐bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA‐less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2α bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over‐expression of Sp1 and AP2α, as well as knock‐down experiments on Sp1 and AP2α gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2α, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2α binding, which ultimately releases the promoter from repression.     

人细胞周期相关激酶启动子的克隆和初步分析

克隆人的细胞周期相关激酶(CCRK)基因启动子,分析与其表达有关的转录调控因子。通过巢式PCR方法,从人的基因组总DNA中分离出CCRK基因5′端非翻译区大小为1072bp的片段。这段片段的3′端起始点在第一个外显子里第一个翻译密码子ATG( 1)上游128bp处。以1072bp为模板,对启动子进行5′端删除分析,分别扩增951bp(-1072／-128)、564bp(-692／-128)、313bp(-441／-128)、127bp(-239／-128)的片段,与pGL3-Basic荧光素酶报告载体重组构建,人工加上克隆位点,5′端带有KpnI、3′端带有XhoI位点。瞬时转染恶性神经胶质瘤细胞株U373,通过双荧光素酶活性进行分析。对分离出的1072bp的片段进行测序,结果与GenBank(Accession AF035013)的序列比较,同源序列达到98％。双荧光素酶活性分析564bp片段有最强的活性,313bp片段为有活性的最小片段。本研究为进一步研究分析CCRK的核心启动子和与其相关的转录因子奠定基础。     

脂肪细胞分化相关转录因子的结构和功能

关于脂肪细胞分化调控的研究主要集中在转录因子的作用上。目前了解得比较清楚的分化转录因子有多种,其中CAAT增强子结合蛋白家族（C/EBPs）中的C／EBPα和过氧化物酶体增殖物激活受体家族（PPARs）中的PPARy是转录调控中起主要作用的两种因子。两者有各自的结构特点和功能,但在脂肪细胞中它们之间相互协同促进细胞的分化成熟。本文主要就C／EBPs和PPARs家族中主要成员的结构和功能及相互作用进行综述。