Abstract
Purpose :
Transcriptome analyses of photoreceptors have identified numerous genes with alternative promoter usage. Importantly, many of these alternate promoters are regulated by NRL and CRX, suggesting a crucial role in photoreceptors. However, how alternative promoter usage confers photoreceptor-specific regulation and function to widely expressed genes is not well-characterized. In the present work, we have explored the regulation and function of Hcls1 (hematopoietic cell-specific Lyn substrate 1), implicated in actin remodeling and receptor tyrosine kinase (RTK) signaling in immune cells but with unknown function in the retina.
Methods :
RNA-seq analyses of mouse retina and flow-sorted photoreceptors identified genes with putative alternative promoter usage in photoreceptors. ATAC-seq, epigenetic marks (H3K4me3, H3K27ac) as well as NRL and CRX ChIP-seq data analyses predicted new transcription start sites. 5’-RACE was used to identify novel Hcls1 photoreceptor-specific transcripts. Expression of these transcripts was examined by in situ hybridization. Electrophoretic mobility shift assays (EMSA) were used to validate the NRL and CRX binding sites.
Results :
We identified by 5’-RACE analysis a novel photoreceptor specific isoform of Hcls1 (Hcls1r), generated by alternative promoter usage. The expression of Hcls1r is concordant with photoreceptor morphogenesis and maturation trajectory. The protein encoded by this new transcript does not include the N-terminal domain involved in actin remodeling but retains signaling elements associated with receptor tyrosine kinase (RTK) signaling, which drives essential cellular processes in response to extracellular cues. In situ hybridization analysis confirmed the enrichment of this novel Hcls1 transcript in the photoreceptor layer. EMSA demonstrated the binding of Nrl and Crx to conserved Hcls1 intronic sequences, close to the transcription start site of the alternate Hcls1r transcript and contained open chromatin regions.
Conclusions :
We propose that Hcls1r is a part of the regulatory network associated with photoreceptor function. Specifically, we predict an important function of Hcls1r in RTK signaling to modulate responses to the retinal microenvironment in photoreceptors and maintenance of homeostasis. We hypothesize that alternative promoter usage contributes to highly specialized structural and functional complexity of retinal photoreceptors.
This is a 2021 ARVO Annual Meeting abstract.