COMPARATIVE GENE ANALYSIS OF CRABP-II GENE UPREGULATED BY ZIC1 USING AFFYMETRIX GENECHIP MICROARRAY
Abstract
Zic1 is a highly conserved zinc finger transcription factor that plays a very early developmental role in establishing different regions of the future nervous system. Studies from several model organ- isms suggest important roles for zic1 gene in patterning the neural plate, in formation of the neural crest, and in cerebellar development (Kuo et al., 1998; Li et al., 2006).
Retinoids are natural or synthetic derivatives of vitamin A (retinol). There is increasing evidence that retinoids regulate cell differentiation and pattern formation during vertebrate embryogenesis and the neoplastic cells development (Kochhar, 1967). Vitamin A (retinol) is essential for vision, reproduction, normal embryonic development, and the regulation of the growth and differentiation of diverse cell types in adult tissues. In addition, there is a strong evidence for beneficial retinoid responses in preventing or treating clinical tumors based on preclinical, epidemiologi- cal, and clinical findings (Nason-Burchenal and Dmitrovsky, 1999).
The best known biologically active retinoid, alltrans retinoic acid (RA), has long been known to be teratogenic in humans (Kochhar, 1967) and reported to cause severe birth defects when adminis- tered to pregnant women (Lammer et al., 1985). The spectrum of birth defects caused by RA includes cleft palate, specific central nervous system (CNS) defects and congenital heart and limb defects, and is conserved in rodents, chickens and frogs suggesting the exis- tence of a well-defined class of RA- sensitive events during vertebrate embryo- genesis (Sive et al., 1990).
CRABPs (cellular retinoic acid binding proteins) belong to members of a superfamily of lipid-binding proteins that are thought to act by maintaining tolerable concentrations of intracellular RA, as modulators of RA catabolism and as intra- cellular transporters for RA from the cyto- plasm to the nuclear receptor (Mansfield et al., 1998). Based on their significance roles in binding lipids, CRABPs are thought to be candidate genes for meat
quality-related traits in domestic animals. The mapping of the porcine CRABPI and CRABP-II genes is one step towards fur- ther investigation on their possible roles in meat quality traits (Lee et al., 2006).
Interestingly, the gene for CRABP- II is located on human chromosome 1q21-23, which is a region that has been linked with disorders such as familial combined hyperlipidemia (FCHL), type 2 diabetes mellitus, and partial lipodystrophy, all of which are characterized by dyslipidemia (Salazar et al., 2007).
CRABP-II has been proved to be upregulated by RA during neural differ- entiation of Xenopus laevis (Ho et al.,
1994). It was also proved to be a direct target of human embryonal carcinoma through the use of cDNA-based microar- ray (Freemantle et al., 2002). There is growing evidence suggesting that CRABP- II may be regulated by factors other than RA. CRABP-II is directly regulated by estrogen in the rat uterus through an estrogen receptor (ER) binding site in the promoter region of CRABP-II (Li et al., 2003). CRABP-II is overexpressed in a wide variety of cancers. In breast cancer cell lines, estrogen induces CRABP-II expression, possibly through retinoic acid receptor In addition, overexpression of CRABP-II has been observed in several breast cancer cell lines and primary tumors (Lu et al., 2005). From analysis of the published microarray data, CRABP-II was found to be expressed in these cell lines, irrespective of the estrogen status. A recent study identified both MycN and CRABP-II overexpression in Wilms tumors (Li et al., 2005), and expression of MycN correlates with CRABP-II levels in these tumors. CRABP-II expression is upregulated in parathyroid adenomas in correlation with tumor suppressor gene HRPT2 downregulation (Zebracka et al., 2007).
To understand the molecular mechanisms through which Zic1 acts, it is necessary to identify genes that are di- rectly regulated by Zic1 and to determine their individual roles. CRABP-II proved to be upregulated by Zic1 from our previous study (Cornish et al., 2009, under review). CRABP-II seemed to have a great impor- tance since it was found to be over expressed during several cancer diseases. Considerable conservation on the levels of gene structure and amino acid sequence was observed between amphibian and mammalian CRABP-II family genes (Ho et al., 1994). Therefore, the objective of this study is to determine the degree of functional relatedness present among CRABP-II proteins of phylogenetically distant organisms. In addition, we describe the expression patterns using real-time PCR and in situ hybridization in addition to the results of comparative gene analysis for CRABP-II gene versions isolated from different organisms.
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