Characterization and purification of a protein binding to the cis-acting element for brain-specific exon 1 of the mouse aromatase gene

The functional differences between male and female brains commit to the existence of androgen that the testis secretes during the perinatal period. Androgen exerts its action on the brain after conversion to estrogen by brain aromatase. The aromatase appears in some neural nuclei such as in the hypothalamus and amygdala, and has been indicated to be involved in the expression of sexuality by the results of neurobehavioral analyses involving aromatase-knockout mice. We analyzed the brain-specific promoter in order to clarify the control mechanism for the expression of brain aromatase, which is deeply concerned in the sexual differentiation of the brain. The 202 bp upstream region of brain-specific exon 1 contains at least three kinds of cis-acting elements, Arom-A, -Aβ and -B. In particular, the binding activities as to the Aβ sequence show a tissue-specific pattern. Gel shift analysis revealed that the Aβ binding factor recognizes the TTGGCCCCT sequence. Aβ binding activity is detectable at the perinatal stage, but is undetectable at the adult stage in the brain. Furthermore, a protein which binds to the Aβ sequence was purified from the fetal mouse brain. The molecular mass of the Aβ binding protein was estimated to be 49 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene

The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS.     

AUF1-like protein binds specifically to DAS cis-acting element that regulates mouse alpha-fetoprotein gene expression

Alpha-fetoprotein (AFP) is one of the major serum proteins in the early life of mammals. We have previously identified a novel cis-acting element designated as DAS at the 5'-flanking region of the AFP gene and demonstrated that the DAS sequence can be specifically recognized by nuclear protein DAP-II in AFP-producing hepatoma cells and retinoic acid (RA)-induced AFP-producing F9 cells. In this study, we used DNA affinity chromatography to purify the DAP-II proteins from the nuclear extracts (NE) of RA-treated F9 cells. The purified DAP-II complex mainly contained five proteins, with molecular weights of 45, 42, 32, 30, and 20 kDa, respectively. The identification of these proteins was determined by MALDI-TOF mass spectrometric analysis and a database search. These proteins were found to belong to the AUF1 RNA-binding protein family. Protein (30 kDa), one of five proteins in an isolated DAP-II complex, was matched with amino acid sequence highly similar to muAUF1-3. The expression of this protein is inducible by RA, and the pattern of the protein expression is the same as DAP-II proteins in F9 cells after treatment with RA during differentiation. Our results suggest that the 30-kDa protein is a novel isoform of AUF1 family and is the main component of the DAP-II complex that binds to the DAS sequence.     

A cis-acting element and associated binding factor required for CNS expression of the Drosophila melanogaster dopa decarboxylase gene.

A cis-acting sequence from the Drosophila melanogaster dopa decarboxylase (Ddc) gene is selectively required for Ddc expression in the central nervous system. We analyze several parameters influencing the function of the sequence element and describe a factor which interacts with it and mediates CNS expression of Ddc. The element, element I, can function in vivo when included on a synthetic oligonucleotide inserted near its normal location, or closer to the RNA startpoint. It displays partial activity when inverted. Two different 2-bp mutations in element I abolish its ability to stimulate neuronal Ddc expression in the CNS. A factor present in embryonic nuclear extracts specifically protects element I in DNase I footprinting assays. The binding affinity of this factor is reduced by each alteration of element I that inhibits neuronal expression, indicating a role in mediating CNS expression of Ddc. Element I alone has no detectable activity when placed adjacent to a heterologous promoter, although 2.2 kb of 5' Ddc sequences direct correct cell-specific expression of a heterologous promoter.     

Characterization of a cis-acting element involved in cell-specific expression of the zebrafish brain aromatase gene

The cytochrome P450 Aromatase is the key enzyme catalyzing the conversion of androgens into estrogens. In zebrafish, the brain aromatase is encoded by cyp19b. Expression of cyp19b is restricted to radial glial cells bordering forebrain ventricles and is strongly stimulated by estrogens during development. At the promoter level, we have previously shown that an estrogen responsive element (ERE) is required for induction by estrogens. Here, we investigated the role of ERE flanking regions in the control of cell-specific expression. First, we show that a 20 bp length motif, named G x RE (glial x responsive element), acts in synergy with the ERE to mediate the estrogenic induction specifically in glial cells. Second, we demonstrate that, in vitro, this sequence binds factors exclusively present in glial or neuro-glial cells and is able to confer a glial specificity to an artificial estrogen-dependent gene. Taken together, these results contribute to the understanding of the molecular mechanisms allowing cyp19b regulation by estrogens and allowed to identify a promoter sequence involved in the strong estrogen inducibility of cyp19b which is specific for glial cells. The exceptional aromatase activity measured in the brain of teleost fish could rely on such mechanisms.     

Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora

In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtained from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17kDa protein matched with the regulatory beta-subunit of calcineurin (Ca(2+)-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression.     

Identification of cis-acting DNA elements involved in the regulation of angiotensinogen gene expression.

Angiotensinogen is the precursor molecule of one of the most potent vasoactive substances, angiotensin-II. Angiotensinogen is normally synthesized in the liver and secreted into the plasma where it is converted into angiotensin-II by the combined proteolytic action of renin and angiotensin converting enzyme. Angiotensinogen levels in the plasma are modulated by a number of pathological and physiological factors. In order to understand the regulation of angiotensinogen gene expression, we have constructed an expression vector in which 688 bp of the 5'-flanking region of the rat angiotensinogen gene were attached to the chloramphenicol acetyl transferase (CAT) coding sequence. We have also obtained 5'-sequential deletion mutants from the rat angiotensinogen promoter attached to the CAT gene, and have identified multiple cis-acting DNA sequences involved in the regulation of angiotensinogen gene expression by transient transfection of these recombinant DNA molecules in human hepatoma cell lines, Hep3B, and HepG2.     

Conservation between animals and plants of the cis-acting element involved in the unfolded protein response

Using Arabidopsis thaliana, we identified the cis-element involved in the plant unfolded protein response (UPR). In transgenic plants, tunicamycin stimulated expression of a reporter gene under the control of the BiP promoter and promoter analysis identified a 24 bp sequence crucial to this induction. When fused with a minimal promoter, a hexamer of this sequence was sufficient for induction of a reporter gene in protoplasts treated with tunicamycin or dithiothreitol. Induction rate equivalent to original promoter was observed when the assay was conducted in transgenic plants. This 24 bp sequence contained two elements also responsible for the UPR in animals. Either of these elements was sufficient for the plant UPR, indicating conservation between animals and plants of cis-elements involved in the UPR.     

Androgen regulated expression of a spermine binding protein gene in mouse ventral prostate.

A full length cDNA (MP25) encoding the major mouse prostatic secretory glycoprotein (p25), whose expression is androgen dependent, has been cloned and characterised. Steady-state levels of mRNA are decreased approximately 100-fold after 3 days castration but are restored progressively over 4 days with testosterone treatment. The secreted glycoprotein appears to be a spermine binding protein since the nucleotide and predicted amino acid sequence of MP25 shares extensive homology with a spermine binding protein (SBP) found in rat ventral prostate. Genomic clones indicate that there is a single gene for SBP which consists of 4 exons, the first of which is only 11bp in length. The second exon encodes the signal peptide, the third contains a portion of the spermine binding protein unique to the mouse and the largest exon encodes the bulk of the secreted protein.     

固醇调节元件结合蛋白1及其靶基因网络

固醇调节元件结合蛋白1(Sterol regulatory element-binding protein 1, SREBP-1)是重要的核转录因子之一, 能调控内源性胆固醇、脂肪酸、甘油三酯和磷脂合成所需酶的表达, 以维持血脂动态平衡。研究表明, SREBP-1及其靶基因网络的异常可引起胰岛素抵抗、Ⅱ型糖尿病、心功能紊乱、血管并发症和肝脂肪变等一系列代谢性疾病。近年高通量组学技术的发展极大扩展了对SREBP-1靶基因及其转录调控模式的了解。文章对SREBP-1蛋白结构、活化过程、DNA结合位点及其调控的靶基因等方面的研究进展进行了综述, 并着重介绍了基于组学数据的转录调控网络的构建, 这将有助于更好的认识SREBP-1在脂类代谢中的作用, 为深入探讨脂质代谢性疾病的治疗提供新线索。     

Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.

Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.