A Far Upstream, Cell Type-specific Enhancer of the Mouse Thrombospondin 3 Gene Is Located within Intron 6 of the Adjacent Metaxin Gene

Abstract

Thrombospondin 3 (TSP3) is a secreted, pentameric glycoprotein whose regulation of expression and function are not well understood. Mouse Thbs3 is located just downstream from the divergently transcribed metaxin gene (Mtx), which encodes an outer mitochondrial membrane import protein. Although Thbs3 and Mtx share a common promoter region, previous studies showed that Mtx is regulated by proximal elements that had little effect on Thbs3 expression. In this study, transient transfection of rat chondrosarcoma cells and NIH-3T3 fibroblasts demonstrated that Thbs3 is regulated in a cell type-specific manner by a position-and orientationindependent far upstream enhancer located within intron 6 of Mtx. Despite its greater proximity to the transcription start site of Mtx, the Thbs3 enhancer did not have a significant effect on Mtx expression. Two DNAprotein complexes, which were both required for activity, were identified when nuclear extracts were assayed with a probe containing the enhancer sequence. The protein in one of these complexes was identified as Sp1, while the other DNA-protein complex remains uncharacterized. A 6-kilobase pair promoter containing the enhancer was able to direct specific expression of the E. coli lacZ gene in transgenic mice, whereas a 2-kilobase pair promoter that lacked the enhancer was inactive. Thus, despite their close proximity, the genes of the Mtx/Thbs3 gene cluster are regulated independently.The thrombospondins (TSPs) 1 comprise a family of five secreted matrix glycoproteins that have a characteristic modular structure consisting of a distinct amino-terminal globular domain, three (TSP1 and 2) or four (TSP3 to -5) type II (epidermal growth factor-like) repeats, seven type III (Ca 2ϩ -binding) repeats, and a homologous carboxyl-terminal globular domain (for reviews, see Refs. 1 and 2). In addition to these common motifs, TSP1 and TSP2 contain a cysteine-rich ␣1(I) procollagen homology domain and three type I (TSP or properdin) repeats, separating the amino-terminal domain and the type II repeats. Unlike TSP1 and TSP2, which are trimers, TSP3 to TSP5/COMP (cartilage oligomeric matrix protein) are pentamers (3-5). Specific domains in TSP1 and TSP2 have been shown to interact with different components of the extracellular matrix, such as types I and V collagen, fibronectin, and laminin, and amino acid sequences within these domains have been implicated in binding to cell surface receptors, including several integrins, CD36, lipoprotein receptor-related protein, and integrin-associated protein (6 -9).

Disclosing hidden transcripts: Mouse natural sense–antisense transcripts tend to be poly(A) negative and nuclear localized

“…We now know that a long ncRNA is expressed from this region. Interestingly, deletion of this region markedly reduced the expression of the Thrombospondin3 gene, but not of the Metaxin1 gene (Collins et al 1998). It is thus possible that the long ncRNA is involved in the regulation of Thrombospondin3 gene expression, although more detailed functional analysis of this ncRNA is needed.…”

“…figure. The positions of the initial and nested primers for the 5Ј RACE analysis are indicated as black arrows above the G430028I15 gene position. The GC boxes identified in the intron of Metaxin1 genes are denoted "G." The square-boxed G is the one to which Sp1 actually bound (Collins et al 1998). The transcriptional start sites identified by 5Ј RACE analysis are indicated with asterisks and bent arrows.…”

“…There are four putative GC-boxes (Sp1-binding sites) in this region (marked as "G" in Fig. 5F), and two of these (square-boxed G) have been proven to bind to Sp1 (Collins et al 1998). We now know that a long ncRNA is expressed from this region.…”

Invoking the power of thrombospondins: Regulation of thrombospondins expression

“…Bornstein's group reported on the promoter of THBS3 as well. Interaction between THBS3 gene and adjacent metaxin gene was revealed (104), and the active SP1-binding elements were identified in the common regulatory region of the two genes (105). Analysis of the transcription initiation sites revealed alternative transcripts (106).…”

Mice with a Disruption of the Thrombospondin 3 Gene Differ in Geometric and Biomechanical Properties of Bone and Have Accelerated Development of the Femoral Head

“…The latter two genes are transcribed divergently and share a common promoter region of about 1 kb (5,9,32). However, despite this genomic arrangement, and the presence of an enhancer of TSP3 in intron 6 of the metaxin gene (10), no metabolic interactions have been identified among any of the products of these four genes.…”