Tomohiro Masuda

A splice-site mutation in a retina-specific exon of BBS8 causes nonsyndromic retinitis pigmentosa.

Tissue-specific alternative splicing is an important mechanism for providing spatiotemporal protein diversity. Here we show that an in-frame splice mutation in BBS8, one of the genes involved in pleiotropic Bardet-Biedl syndrome (BBS), is sufficient to cause nonsyndromic retinitis pigmentosa (RP). A genome-wide scan of a consanguineous RP pedigree mapped the trait to a 5.6 Mb region; subsequent systematic sequencing of candidate transcripts identified a homozygous splice-site mutation in a previously unknown BBS8 exon. The allele segregated with the disorder, was absent from controls, was completely invariant across evolution, and was predicted to lead to the elimination of a 10 amino acid sequence from the protein. Subsequent studies showed the exon to be expressed exclusively in the retina and enriched significantly in the photoreceptor layer. Importantly, we found this exon to represent the major BBS8 mRNA species in the mammalian photoreceptor, suggesting that the encoded 10 amino acids play a pivotal role in the function of BBS8 in this organ. Understanding the role of this additional sequence might therefore inform the mechanism of retinal degeneration in patients with syndromic BBS or other related ciliopathies.

BEST1 expression in the retinal pigment epithelium is modulated by OTX family members

A number of genes preferentially expressed in the retinal pigment epithelium (RPE) are associated with retinal degenerative disease. One of these, BEST1, encodes bestrophin-1, a protein that when mutated causes Best macular dystrophy. As a model for RPE gene regulation, we have been studying the mechanisms that control BEST1 expression, and recently demonstrated that members of the MITF-TFE family modulate BEST1 transcription. The human BEST1 upstream region from -154 to +38 bp is sufficient to direct expression in the RPE, and positive-regulatory elements exist between -154 and -104 bp. Here, we show that the -154 to -104 bp region is necessary for RPE expression in transgenic mice and contains a predicted OTX-binding site (Site 1). Since another non-canonical OTX site (Site 2) is located nearby, we tested the function of these sites using BEST1 promoter/luciferase constructs by in vivo electroporation and found that mutation of both sites reduces promoter activity. Three OTX family proteins - OTX1, OTX2 and CRX - bound to both Sites 1 and 2 in vitro, and all of them increased BEST1 promoter activity. Surprisingly, we found that human and bovine RPE expressed not only OTX2 but also CRX, the CRX genomic region in bovine RPE was hypersensitive to DNase I, consistent with active transcription, and that both OTX2 and CRX bound to the BEST1 proximal promoter in vivo. These results demonstrate for the first time CRX expression in the RPE, and suggest that OTX2 and CRX may act as positive modulators of the BEST1 promoter in the RPE.

Dynamic usage of alternative splicing exons during mouse retina development

Alternative processing of pre-mRNA plays an important role in protein diversity and biological function. Previous studies on alternative splicing (AS) often focused on the spatial patterns of protein isoforms across different tissues. Here we studied dynamic usage of AS across time, during murine retina development. Over 7000 exons showed dynamical changes in splicing, with differential splicing events occurring more frequently in early development. The overall splicing patterns for exclusive and inclusive exons show symmetric trends and genes with symmetric splicing patterns that tend to have similar biological functions. Furthermore, we observed that within the retina, retina-enriched genes that are preferentially expressed at the adult stage tend to have more dynamically spliced exons compared to other genes, suggesting that genes maintaining retina homeostasis also play an important role in development via a series of AS events. Interestingly, the transcriptomes of retina-enriched genes largely reflect the retinal developmental process. Finally, we identified a number of candidate cis-regulatory elements for retinal AS by analyzing the relative occurrence of sequence motifs in exons or flanking introns. The occurrence of predicted regulatory elements showed strong correlation with the expression level of known RNA binding proteins, suggesting the high quality of the identified cis-regulatory elements.

The Pex1-G844D mouse: A model for mild human Zellweger spectrum disorder

Zellweger spectrum disorder (ZSD) is a disease continuum that results from inherited defects in PEX genes essential for normal peroxisome assembly. These autosomal recessive disorders impact brain development and also cause postnatal liver, adrenal, and kidney dysfunction, as well as loss of vision and hearing. The hypomorphic PEX1-G843D missense allele, observed in approximately 30% of ZSD patients, is associated with milder clinical and biochemical phenotypes, with some homozygous individuals surviving into early adulthood. Nonetheless, affected children with the PEX1-G843D allele have intellectual disability, failure to thrive, and significant sensory deficits. To enhance our ability to test candidate therapies that improve human PEX1-G843D function, we created the novel Pex1-G844D knock-in mouse model that represents the murine equivalent of the common human mutation. We show that Pex1-G844D homozygous mice recapitulate many classic features of mild ZSD cases, including growth retardation and fatty livers with cholestasis. In addition, electrophysiology, histology, and gene expression studies provide evidence that these animals develop a retinopathy similar to that observed in human patients, with evidence of cone photoreceptor cell death. Similar to skin fibroblasts obtained from ZSD patients with a PEX1-G843D allele, we demonstrate that murine cells homozygous for the Pex1-G844D allele respond to chaperone-like compounds, which normalizes peroxisomal β-oxidation. Thus, the Pex1-G844D mouse provides a powerful model system for testing candidate therapies that address the most common genetic cause of ZSD. In addition, this murine model will enhance studies focused on mechanisms of pathogenesis.

Transcription Factor SOX9 Plays a Key Role in the Regulation of Visual Cycle Gene Expression in the Retinal Pigment Epithelium

Background: The visual cycle is an enzymatic cascade that regenerates the visual chromophore. Results: Visual cycle gene expression is regulated by SOX9 in combination with OTX2 or LHX2 and can be modulated by common microRNAs. Conclusion: A core transcriptional network involving SOX9 regulates visual cycle genes. Significance: Understanding visual cycle gene regulation may have implications for treating retinal degenerative diseases. The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible regulation of visual cycle genes by common miRNAs.

Use of laser capture microdissection for analysis of retinal mRNA/miRNA expression and DNA methylation.

Laser capture microdissection (LCM) is a useful method to isolate specific cells or cell layers of interest from heterogeneous tissues, such as the retina. The collected cells can be used for DNA, RNA, or protein analysis. We have applied LCM technology to isolate cells from the outer nuclear, inner nuclear, and ganglion cell layers of the retina for mRNA and microRNA (miRNA) expression and epigenetic (DNA methylation) analysis. Here, we describe the methods we have employed for sample preparation, LCM-based isolation of retinal layers, RNA/DNA extraction, RNA quality check, microRNA analysis by quantitative PCR, and DNA methylation analysis by bisulfite sequencing.

Gene regulation: it matters who you hang out with.

How broadly expressed transcription factors contribute to tissue-specific gene expression is not well understood. Andzelm, Cherry et al. (2015) demonstrate that myocyte enhancer factor 2D (MEF2D) binds and activates retina-specific regulatory regions by cooperative interaction with the tissue-specific transcription factor CRX.