Trapping and Characterization of Novel Retinoid Response Elements

Abstract

Retinoids, such as retinoic acid (RA), play a critical role in normal vertebrate development and physiology. However, embryonic exposure to excess retinoids also causes severe malformations. Retinoids bind RA receptors and retinoid X receptors, thus activating a plethora of genes. Separating the genes induced directly by retinoid-bound receptors from those induced subsequently by other transcription factors is difficult. The loose consensus defining known RA responsive elements (RAREs) further complicates this effort. We developed a yeast-based system to trap functional RAREs in the mouse genome. Several of the clones contain RAREs near RA-induced genes. Mammalian reporter gene analyses and EMSAs showed that these are bona fide RAREs. This functional genomics approach should identify RA-regulated genes that initiate critical signaling cascades in cells.

A Protein Associated with Toll-Like Receptor 4 (PRAT4A) Regulates Cell Surface Expression of TLR4

“…Up-regulation or down-regulation of PRAT4A may influence LPS responses. PRAT4A was previously shown to be induced by all-trans retinoic acid (29). It is important to study induction and reduction of PRAT4A mRNA upon a variety of stimulations.…”

“…The mature peptide has a canonical N-glycosylation site and six cysteine residues. PRAT4A is highly conserved among a variety of species, including cow, chick, pig, catfish, sea squirt, nematode, zebra fish, and fruit fly (29). We examined tissue distribution of mRNA for mouse PRAT4A.…”

Utilization of DR1 as true RARE in regulating the Ssm, a novel retinoic acid-target gene in the mouse testis

“…The pDSS yeast reporter vector encodes b-gal under the control of a minimal cyc1 promoter. A genomic DNA library was constructed to include 1-2 kb genomic DNA upstream of cyc1 promoter (Glozak et al 2003). Thus, induction of b-gal reporter in the presence of ligand-activated PPARd/RXRa would indicate that the genomic DNA piece contains putative DR1s and serves as an enhancer for induction of the b-gal reporter gene.…”

“…Mouse genomic DNA fragments of 1-2 kb were cloned into the pDSS plasmid, which contains the yeast cytochrome c1 (cyc1) minimal promotor and the b-gal reporter gene (Glozak et al 2003). Full-length cDNA encoding mouse PPARd and RXRa were cloned into yeast expression vectors p423ADH and p424ADH (American Type Culture Collection (ATCC), Manassas, VA, USA) respectively (Mumberg et al 1995).…”

“…In an attempt to find target genes that are directly bound and regulated by PPARd/ RXR, we employed a yeast-based system to trap functional DR1s in the mouse genome in the presence of PPARd and RXRa ligands (carbaprostacyclin (cPGI) and 9-cis-RA respectively). This method was previously used to trap functional RAR-responsive elements under RA-enriched environment (Glozak et al 2003). During screening, we identified a putative target genomic region containing two DR1s that promiscuously interacts with RXR homodimer and RAR/RXR heterodimer, as well as PPAR/RXR heterodimer in vitro.…”