Sp family of transcription factors is involved in iron-induced collagen alpha1(I) gene expression

The purpose of this study was to identify the cis-acting elements and the trans-acting factors involved in the iron-induced expression of the collagen alpha1(I) (COL1aI) gene. Rat hepatic stellate cells were cultured in the presence of 50 microM ferric chloride, 50 microM ascorbic acid, and 250 microM citric acid (Fe/AA/CA), and the effects on collagen gene expression and the binding of nuclear proteins to the COL1aI promoter were measured. The Fe/AA/CA treatment induced a time- and dose-dependent increase in the cellular levels of COL1aI mRNA that was abrogate by pretreating cells with cycloheximide, antioxidants, and inhibitors of aldehyde-protein adduct formation. Transient transfection experiments showed that Fe/AA/CA exerted its effect through regulatory elements located between -220 and -110 bp of the COL1aI promoter. Gel retardation assays showed that Fe/AA/CA increased the binding of nuclear proteins to two elements located between -161 and -110 bp of the COL1aI promoter. These bindings were blocked by unlabeled consensus Sp1 oligonucleotide and supershifted with Sp1 and Sp3 antibodies. Finally, Fe/AA/CA increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Treatment with Fe/AA/CA stimulates COL1aI gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between -161 and -110 bp of the COL1aI promoter.     

儿童孤独症相关基因NRXN1β启动子功能分析

Neurexins是神经特异性突触蛋白,Neurexin1β结构的异常与孤独症密切相关。为分析孤独症相关基因NRXN1β最小启动子和调节基因转录的功能元件,本文构建了含NRXN1β基因上游调控区不同区域的荧光素酶报告基因质粒,转染HEK293细胞后,利用检测双荧光素酶报告基因的转录活性以确定NRXN1β基因最小启动子区,进而筛选出相应的显著增强或抑制报告基因活性的功能区;同时,为鉴定顺式作用元件,利用基因定点突变技术对基因功能区内和临近DNA序列进行连续的碱基突变;最后,采用转录因子预测工具对启动子功能区内的转录调控元件进行分析。结果首次发现NRXN1β最小启动子区位于-88~+156 bp,-88~-73 bp和+156~+149 bp可增强启动子活性,+229~+419 bp可抑制启动子活性,且-84~-63 bp为能够显著性增强启动子活性的顺式作用元件,该区域可能存在DBP(Albumin D-site-binding protein,DBP)和ABF1(Autonomously replicating sequence-binding factor 1,ABF1)两个转录因子结合位点。     

Pi 1 binding sites are negative regulators of bcl-2 expression in pre-B cells.

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.     

Sp1 and Sp3 transcription factors mediate malondialdehyde-induced collagen alpha 1(I) gene expression in cultured hepatic stellate cells

Malondialdehyde, the end product of lipid peroxidation, has been shown to stimulate collagen alpha1(I) (Col1a1) gene expression. However, mechanisms of this effect are unclear. The purpose of this study was to clarify these mechanisms. Rat hepatic stellate cells were cultured in the presence of 200 microm malondialdehyde, and the effects on collagen gene expression and the binding of nuclear proteins to the col1a1 promoter were analyzed. Malondialdehyde treatment induced an increase in the cellular levels of col1a1 mRNA that was abrogated by pretreating cells with cycloheximide, p-hydroxymercuribenzoate, pyridoxal 5'-phosphate, and mithramycin. Transient transfections showed that malondialdehyde exerted its effect through regulatory elements located between -220 and -110 bp of the col1a1 promoter. Gel retardation assays demonstrated that malondialdehyde increased the binding of nuclear proteins to two elements located between -161 and -110 bp of the col1a1 promoter. These bindings were supershifted with Sp1 and Sp3 antibodies. Finally, malondialdehyde increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Our data indicated that treatment of hepatic stellate cells with malondialdehyde stimulated col1a1 gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between -161 and -110 bp of the col1a1 promoter.     

The homeodomain protein, Pit-1/GHF-1, is capable of binding to and activating cell-specific elements of both the growth hormone and prolactin gene promoters

Studies were conducted to determine whether the trans-acting protein Pit-1/GHF-1 can bind to and activate promoter elements in both the GH and PRL genes that are necessary for cell-specific expression. Four pituitary cell lines that differentially express the endogenous GH and PRL genes were examined for their ability to activate GH and PRL promoter constructs containing sequences necessary for cell-specific expression (CSEs). Plasmids containing one CSE, -96 PRL and -104 GH, were similarly expressed in each of the four cell lines. Of the plasmids containing two CSEs, -173 PRL was always activated to a greater extent than -145 GH, with this relative activation being stronger in GC and GH1 cells than in 235-1 and GH4C1 cells. Protein-DNA binding assays were used to show that the GH and PRL CSEs specifically bound two highly abundant nuclear proteins (31 and 33 kDa). The two proteins were present at similar levels in all four pituitary cell lines and were recognized by a Pit-1/GHF-1 antibody. In contrast, HeLa and Rat2 cells did not activate transfected GH or PRL plasmids and did not contain nuclear proteins that specifically bound to the GH and PRL CSEs. However, cotransfection of these cells with the expression vector RSV-Pit-1/GHF-1 resulted in the activation of -173 PRL and -145 GH (PRL greater than GH). HeLa cells transfected with RSV-Pit-1/GHF-1 also contained 31- and 33-kDa nuclear proteins that bound to the GH and PRL CSEs. These results show that Pit-1/GHF-1 is present at levels in pituitary cell lines that are sufficient to activate the minimal elements in both the GH and PRL promoters necessary for cell-specific expression of these genes.     

Transport of the IgE receptor alpha-chain is controlled by a multicomponent intracellular retention signal

The human high affinity IgE receptor (FcepsilonRI) is a central component of the allergic response and is expressed as either a trimeric alphagamma2 or tetrameric alphabetagamma2 complex. It has been previously described that the cytoplasmic domain (CD) of the alpha-chain carries a dilysine motif at positions -3/-7 from the C terminus that functions in intracellular retention prior to assembly with other FcepsilonRI subunits. In this report we have further explored the role of the -3/-7 dilysine signal in controlling steady-state alpha-chain transport by mutational analysis and found little surface expression of a -3/-7 dialanine alpha-chain mutant but significant Golgi localization. We compared the transport properties of a series of alpha-chain cytoplasmic domain truncation mutants and observed that truncation mutants lacking 23 or more C-terminal residues showed a dramatic increase in steady-state transport suggesting a role for the membrane-proximal CD sequence in alpha-chain retention. By performing alanine-scanning mutagenesis we identified a dilysine sequence (Lys(212)-Lys(216)) proximal to the transmembrane domain (TMD) that is important for both alpha-chain cell-surface expression and intracellular stability. Furthermore, co-mutation of the Lys(212)-Lys(216) residues with the -3/-7 dilysine signal produced a dramatic increase in alpha-chain surface expression that was further increased by co-mutation of the lone charged residue (Asp(192)) in the TMD thereby defining three regions that function to regulate alpha-chain transport and in a highly synergistic manner.