Functional dissection of the mouse tyrosinase locus control region identifies a new putative boundary activity

Abstract

Locus control regions (LCRs) are complex high-order chromatin structures harbouring several regulatory elements, including enhancers and boundaries. We have analysed the mouse tyrosinase LCR functions, in vitro, in cell lines and, in vivo, in transgenic mice and flies. The LCR-core (2.1 kb), located at -15 kb and carrying a previously described tissue-specific DNase I hypersensitive site, operates as a transcriptional enhancer that efficiently transactivates heterologous promoters in a cell-specific orientation-independent manner. Furthermore, we have investigated the boundary activity of these sequences in transgenic animals and cells. In mice, the LCR fragment (3.7 kb) rescued a weakly expressed reference construct that displays position effects. In Drosophila, the LCR fragment and its core insulated the expression of a white minigene reporter construct from chromosomal position effects. In cells, sequences located 5' from the LCR-core displayed putative boundary activities. We have obtained genomic sequences surrounding the LCR fragment and found a LINE1 repeated element at 5'. In B16 melanoma and L929 fibroblast mouse cells, this element was found heavily methylated, supporting the existence of putative boundary elements that could prevent the spreading of condensed chromatin from the LINE1 sequences into the LCR fragment, experimentally shown to be in an open chromatin structure.

Zebrafish enhancer detection (ZED) vector: A new tool to facilitate transgenesis and the functional analysis of cis‐regulatory regions in zebrafish

“…The PCR fragment was cloned in TOPO vector (Invitrogen, pCR®8/GW/TOPO® TA cloning KIT) and transferred to the insulator test vector by in vitro recombination (Invitrogen, Gateway LR clonase II Enzyme mix). The GAB insulator element was excised as a 0.57-kb HindIII/EcoRV DNA fragment from plasmid pGAB (A. Garcia-Diaz and L. Montoliu, unpublished), carrying a fusion of the G and AB boundary elements previously described within the locus control region (LCR) of the mouse tyrosinase gene (Giraldo et al, 2003). The fragment was cloned blunt into the KpnI site of the pminiTol2-Z48-CARGFP plasmid.…”

“…To introduce the positive control to this vector, we first excised the Cardiac ActinDsRed-polyA cassette from the ISCarDsR2/Pax6GFP3 vector (gift from H. Ogino and R. Grainger) and cloned into the XbaI/BamHI sites of pBluescript KSϩ vector. In this plasmid, we then introduced the GAB insulator from the pGAB vector (Giraldo et al, 2003) into the XhoI/KpnI sites. We then amplified the GAB insulatorCardiac Actin-DsRed-polyA fragment with the following primers: 5ЈttaattaaT-GAAGTTCCTATACTTTCTAGAGA-ATAGGAACTTCCTCACTATAGGGC-GAATTGG-3Ј and 5ЈttaattaaGAAGT-TCCTATTCTCTAGAAAGTATAGG-AACTTCACGCAATTAACCCTCAC-TAAAGG-3Ј.…”

A mobile insulator system to detect and disrupt cis-regulatory landscapes in vertebrates

“…Insulators are common CREs present in all vertebrate genomes, and although the ED's insulator is a chimera of chicken (Chung et al 1993) and mouse (Giraldo et al 2003) insulators, it is functional in zebrafish, evidencing that these elements function similarly in many different vertebrate genomes. As it has been shown that Tol2 is also effective in mouse transgenesis (Sumiyama et al 2010), we injected the ED vector along with the Tol2 mRNA in mouse embryos at the 1-cell stage as reported (Sumiyama et al 2010).…”

“…The ED insulator is composed of two sequences, the chicken HS4 insulator (Chung et al 1993) and a sequence from the LCR located upstream of the mouse Tyr gene (Giraldo et al 2003). By using an insulator assay in zebrafish, we show that the two sequences, which independently have insulator activity (Bessa et al 2009), are much more effective insulators when linked together.…”

“…All of them contain a GFP transgene whose expression is controlled by a gata2a minimal promoter (Bessa et al 2009), a fusion of the GAB insulator from the mouse tyrosinase gene (Giraldo et al 2003), and the 59HS4 insulator from the chicken beta-globin gene (Chung et al 1993;Recillas-Targa et al 2002) and the mini-Tol2 backbone (Urasaki et al 2006). IMP16 and IMP17 contain in the RFP enhancer trap, the meis1 minimal promoter driving the DsRed2 reporter gene.…”