Stress Responsive bZIP Transcription Factors ATF4 and BACH1 Cooperate With MAF-Family bZIP Protein NRL to Fine-Tune Rod Photoreceptor Gene Expression

Purpose: Musculoaponeurotic fibrosarcoma (MAF) family basic motif leucine zipper (bZIP) transcription factor neural retina leucine zipper (NRL) determines rod cell fate and controls expression of rod genes in concert with multiple regulatory proteins. Mutations in NRL, its targets, and interacting proteins are associated with retinopathies. Because bZIP heterodimerization expands target sequence selectivity, we set out to identify bZIP protein interactors of NRL. Methods: Interactors were identified by yeast two-hybrid and co-immunoprecipitation, validated by high-resolution microscopy and proximity ligation, and functionally assessed by reporter assays. We used Cleavage Under Targets and Tagmentation (CUT&Tag) to map activating transcription factor 4 (ATF4) and BTB and CNC homology 1 (BACH1) occupancy and single-cell RNA sequencing (scRNA-seq) to assess gene expression changes in response to their knockdown in mouse retina. Results: We identified two bZIP proteins, ATF4 and BACH1, as interactors of NRL. We demonstrate a direct interaction of NRL and ATF4 via leucine zipper domain and validate their co-localization in rod photoreceptors. NRL and BACH1 are also partially colocalized, but their interaction likely requires additional factors. Reporter assays show that ATF4 promotes NRL-mediated transactivation of rhodopsin promoter, whereas BACH1 appears to act as a suppressor. CUT&Tag revealed shared and distinct binding sites for NRL, ATF4, and BACH1 in promoters of rod-expressed genes, including phototransduction genes. scRNA-seq further indicated a concordance of higher ATF4 and NRL expression with upregulation of phototransduction genes in distinct rod subpopulations. Conclusions: We suggest that the NRL-mediated gene regulatory network includes transient and stable but context-dependent protein-protein interactions, which control quantitatively precise gene expression patterns in mature rod photoreceptors. Our findings suggest therapeutic potential for retinopathies involving photoreceptor dysfunction through targeted gene expression modulation.