Abstract
The retinoid X receptor beta gene (Rxrb) is located just upstream of the alpha2(XI) collagen chain gene (Col11a2) in a head-to-tail manner. However, the domain structures of these genes are unknown. Col11a2 is specifically expressed in cartilage. In the present study, we found Rxrb expression in various tissues with low expression in the cartilage. Col11a2 1st intron enhancer directed cartilage specific expression when linked to the heterologous promoter in transgenic mice. These results suggest the presence of enhancer-blocking elements that insulate Rxrb promoter from the Col11a2 enhancer. So far, most of insulators examined in vertebrates contain a binding site for CTCF. We found two possible CTCF-binding sites: one (11P) in the intergenic region between Rxrb and Col11a2 by electrophoretic mobility shift assays, and the other in the 4th intron of RXRB by data base search. To examine the function of these elements, we prepared bacterial artificial chromosome (BAC) transgene constructs containing a 142-kb genomic DNA insert with RXRB and COL11A2 sequences in the middle. Mutation of 11P significantly decreased the RXRB promoter activity in muscular cells and significantly increased expression levels of RXRB in chondrosarcoma cells. In transgenic mouse assays, the wild-type BAC transgene partly recapitulated endogenous Rxrb expression patterns. A 507-bp deletion mutation including 11P enhanced the cartilage-specific activity of the RXRB promoter in BAC transgenic mice. Chromatin immunoprecipitation analysis showed that CTCF was associated with RX4, but not with 11P. Our results showed that the intergenic sequence including 11P insulates Rxrb promoter from Col11a2 enhancer, possibly associating with unknown factors that recognize a motif similar to CTCF.
Highlights
Cartilage is a highly specialized tissue that serves as the template for skeletal development and lines the joint surface
We examined whether the intergenic sequence and CCCTC-binding factor (CTCF)-binding site affected the activities of the Rxrb promoter by using bacterial artificial chromosome (BAC) transgene constructs that cover the entire Rxrb and Col11a2 genes
RXRB and COL11A2 are located in the major histocompatibility complex region of chromosome 6p in humans
Summary
Retinoid X receptor  gene; BAC, bacterial artificial chromosome; BORIS, Brother of the Regulator of Imprinted Sites; ChIP, chromatin immunoprecipitation; Col11a2, ␣2(XI) collagen chain gene (Col11a2); CTCF, CCCTC-binding factor; EMSA, electrophoretic mobility shift assay; RCS, rat chondrosarcoma; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; Nf, neurofilament; RXR, retinoid X receptor; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AD, adenovirus. The enhancer blocking activity of insulators is associated with a binding site for the CCCTC-binding factor (CTCF), which recognizes long and diverse nucleotide sequences. Despite the proximity of Rxrb and Col11a2, the dissimilarities in their expression patterns and functions suggest the existence of an intergenic boundary. To investigate this possibility, we first clarified differences in the expression patterns of the two genes and examined whether the cartilage-specific enhancer of Col11a2 could affect transcriptional activities of heterologous promoters. We examined whether the intergenic sequence and CTCF-binding site affected the activities of the Rxrb promoter by using bacterial artificial chromosome (BAC) transgene constructs that cover the entire Rxrb and Col11a2 genes
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