Characterization of human Collagen XVIII promoter 2: Interaction of Sp1, Sp3 and YY1 with the regulatory region and a SNP that increases transcription in hepatocytes

Abstract

Different levels of Collagen XVIII expression have been associated with several pathological processes such as cancer, liver fibrosis, diabetic retinopathy and Alzheimer's disease. Understanding the transcriptional regulation of Collagen XVIII might elucidate some pathways related to the progression of these diseases. The promoter 2 of COL18A1 gene is poorly understood and is responsible for the transcription of this gene in several adult tissues such as liver, eyes and brain. This study focused upon characterization of cis-regulatory elements interacting with human COL18A1 promoter 2 and identification of SNPs in this region in different ethnic groups. Our results show that there are five conserved regions (I to V) between human and mouse promoter 2 and that the human COL18A1 core promoter is located between nucleotides − 186 and − 21. Sp1 and Sp3 bind to conserved regions I and V, while Sp3 and YY1 interact with region II. We have verified that the SNP at position − 700 (T > G) is embedded in two common haplotypes, which have different frequencies between European and African descendents. The allele − 700G increases transcription and binding for a still unknown transcription factor. SNP − 700 affects Sp3 and YY1 interaction with this region, even though it is not part of these transcription factors' predicted binding sites. Therefore, our results show for the first time that Sp3 and YY1 interact with human COL18A1 promoter 2, and that nucleotide − 700 is part of a binding motif for a still unknown TF that is involved in the expression of this gene in hepatocytes. In addition, we also confirm the involvement of Sp1 in the regulation of this gene.

Introduction

Type XVIII collagen is a member of the MULTIPLEXIN collagen family characterized by polypeptide chains with multiple triple-helical domains separated and flanked by non-triple-helical regions (Oh et al., 1994, Rehn and Pihlajaniemi, 1994). This collagen, a homotrimer molecule made of three α1 chains, is a heparan sulfate proteoglycan constituent of epithelial and endothelial basement membranes of a wide variety of tissues (Saarela et al., 1998a). Human type XVIII collagen, encoded by the COL18A1 gene, has three isoforms, which differ in their N-terminal non-collagenous domain (NC11), namely: NC11-303, NC11-493 and NC11-728. The COL18A1 gene spans 105 kbp and contains 43 exons and 2 putative promoter regions, namely: promoter 1 located 5′ from exon 1, and promoter 2 within intron 2 (Saarela et al., 1998b, Elamaa et al., 2003). The NC11-303 isoform is associated with promoter 1 and the other two isoforms have their transcription regulated by the promoter 2. Variant NC11-728 is expressed in various human fetal tissues (Elamaa et al., 2003) and adult brain and retina (Suzuki et al., 2002), while NC11-493 is highly expressed in liver (Saarela et al., 1998b). Liétard et al. (2000) have shown that the mouse Col18a1 promoter 2 is up regulated by Sp1 and HNF3 and, that it has a silencer-like element that may be regulated by HNF3 and NF1/CTF interactions with this regulatory region, but, so far, data on human COL18A1 regulatory regions are limited to the fact that there are two distinct promoters (Saarela et al., 1998b, Elamaa et al., 2003).

Although COL18A1 function is not completely understood, it is well known that its deficiency causes several ocular problems and neuronal cell migration disorders in patients with Knobloch syndrome (Sertié et al., 2000, Suzuki et al., 2002, Kliemann et al., 2003). Type XVIII collagen expression varies in many pathological conditions such as cancer (Musso et al., 2001), liver fibrosis (Musso et al., 1998), Alzheimer's disease (van Horssen et al., 2002) and diabetic retinopathy (Noma et al., 2002). Also, the 184-aminoacid proteolytic carboxyterminus fragment of Collagen XVIII, called endostatin, has been characterized as a potent endogenous inhibitor of angiogenesis (O'Reilly et al., 1997). Interestingly, there is a wide variation in serum levels of endostatin, mainly originated from NC11-493 proteolysis, in a normal human population (Zorick et al., 2001). The characterization of the regulatory mechanisms underlying the COL18A1 gene expression will help elucidate the variability of Collagen XVIII and endostatin levels both in physical and pathological conditions. Therefore, in the present study we have focused upon characterization of cis-regulatory elements interacting with human COL18A1 promoter 2 and identification of functional SNPs in this region in different ethnic groups.