Diane E. Borst

Site-specific DNA hypomethylation permits expression of the IRBP gene.

Interphotoreceptor retinoid binding protein (IRBP), a putative component of the visual cycle, is expressed selectively in the retina and pineal gland. This study examined whether site-specific DNA hypomethylation plays a role in this expression regulation. Southern blotting of HpaII and MspI digests of DNA from various bovine and murine tissues (whole brain, retina, pineal gland, superior colliculus, cortex, thymus, habenular nucleus, cornea, liver, tail, and kidney) revealed that specific CpG dinucleotides in the IRBP gene promoter are hypomethylated in DNA from retinal photoreceptor cells and pineal gland compared to DNA from other tissues. These sites are methylated in DNA from non-photoreceptor retinal cells. Exogenous methylation of these sites diminished DNA:protein binding in electrophoretic mobility shift assays. HpaII methylation of chloramphenicol acetyltransferase reporter constructs suppressed IRBP but not SV40 promoter activity in transiently transfected primary cultures of embryonic chick retinal cells. These data indicate that specific cytosines in the bovine and murine IRBP promoters are unmethylated in photoreceptive cells but methylated in other tissues. This differential DNA methylation may modulate IRBP gene expression since exogenous methylation of the murine sites suppresses reporter gene transcription, apparently by inhibiting DNA:protein binding events.

N‐RAP expression during mouse heart development

N‐RAP gene expression and N‐RAP localization were studied during mouse heart development using semiquantitative reverse transcriptase‐polymerase chain reaction and immunofluorescence. N‐RAP mRNA was detected at embryonic day (E) 10.5, significantly increased from E10.5 to E16.5, and remained essentially constant from E16.5 until 21 days after birth. In E9.5–10.5 heart tissue, N‐RAP protein was primarily associated with developing premyofibril structures containing α‐actinin, as well as with the Z‐lines and M‐lines of more‐mature myofibrils. In contrast, N‐cadherin was concentrated in patches at the periphery of the cardiomyocytes. N‐RAP labeling markedly increased between E10.5 and E16.5; almost all of the up‐regulated N‐RAP was associated with intercalated disk structures, and the proportion of mature sarcomeres containing N‐RAP decreased. In adult hearts, specific N‐RAP staining was only observed at the intercalated disks and was not found in the sarcomeres. The results are consistent with N‐RAP functioning as a catalytic scaffolding molecule, with low levels of the scaffold being sufficient to repetitively catalyze key steps in myofibril assembly. Developmental Dynamics 233:201–212, 2005.

Expression and alternative splicing of N-RAP during mouse skeletal muscle development.

N-RAP alternative splicing and protein localization were studied in developing skeletal muscle tissue from pre- and postnatal mice and in fusing primary myotubes in culture. Messages encoding N-RAP-s and N-RAP-c, the predominant isoforms of N-RAP detected in adult skeletal muscle and heart, respectively, were present in a 5:1 ratio in skeletal muscle isolated from E16.5 embryos. N-RAP-s mRNA levels increased three-fold over the first 3 weeks of postnatal development, while N-RAP-c mRNA levels remained low. N-RAP alternative splicing during myotube differentiation in culture was similar to the pattern observed in embryonic and neonatal muscle, with N-RAP-s expression increasing and N-RAP-c mRNA levels remaining low. In both developing skeletal muscle and cultured myotubes, N-RAP protein was primarily associated with developing myofibrillar structures containing alpha-actinin, but was not present in mature myofibrils. The results establish that N-RAP-s is the predominant spliced form of N-RAP present throughout skeletal muscle development.

Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells

PURPOSE To determine whether antisense oligonucleotides (AODNs) targeted against CRX, a photoreceptor-specific trans-acting factor, suppress CRX expression and interphotoreceptor retinoid binding protein (IRBP) promoter activity. METHODS Cultures of human retinoblastoma cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing a mouse IRBP promoter and AODNs directed against CRX. RT-PCR using primers specific to CRX, OTX2, GAPDH, or RNase H was conducted on total RNA isolated from retinoblastoma cells at various times following transfection with AODNs. RESULTS Transfection of retinoblastoma cells with IRBP promoter CAT constructs alone produced high activity. Co-transfection with AODNs suppressed IRBP promoter activity in a concentration-dependent manner, with half-maximal effect produced at about 2 nM AODN concentration. Transfection with CAT constructs containing an SV40 promoter produced high activity that was unaffected by co-transfection with AODNs. RT-PCR products were obtained for all target sequences. CRX RT-PCR product from cells transfected with AODNs was greatly diminished following transfection with an AODN whereas control transcripts, including that of OTX2, were relatively unaffected. CONCLUSIONS The CRX-specific AODNs specifically and potently suppressed CRX expression and IRBP promoter activity, as measured by RT-PCR and transient transfection assays, respectively. Little or no effect was seen on controls. These data suggest that endogenous CRX is required for IRBP promoter activity in retinoblastoma cells.

Region specific mitochondrial gene expression in the human retina.

The human mitochondrial genome has not been previously known to differentially express specific mRNA transcripts. Results of northern analysis, using total RNA from two different retinal regions, demonstrate that there is differential expression of five mitochondrial genes. There is a correlation of regional expression of one of these differentially expressed genes with the gene responsible for the majority of cases of foveo-macular mitochondropathy. These findings suggest that there is selective control over specific mitochondrial messenger steady state levels.