Missense mutations in CRX homeodomain cause dominant retinopathies through two distinct mechanisms

Reviewer #1 (Public Review):

The manuscript by Zheng et al. examined the disease-causing mechanisms of two missense mutations within the homeodomain (HD) of CRX protein. Both mutations were found in humans and can produce severe dominant retinopathy. The authors investigated the two CRX HD mutants via in vitro DNA-binding assay (Spec-seq), in vivo chromatin-binding assay (ChIP-seq), in vivo expression assay of downstream target genes (RNA-seq), and retinal histological and functional assays. They concluded that p.E80A increased the transactivation activity of CRX and resulted in precocious photoreceptor differentiation, whereas p.K88N significantly changed the binding specificity of CRX and led to defects in photoreceptor differentiation and maintenance. The authors performed a significant amount of analyses. The claims are sufficiently supported by the data. The results not only uncovered the underlying disease-causing mechanisms, but also can significantly improve our understanding of the interaction between HD-TF and DNA during development.

Minor concerns:
1. The E80A, K88N and R90W (previously reported by the same group) mutations are located very close to each other in the homeodomain (Figure 1A), but had distinct effects on the activity of CRX. Has the structure of the homeodomain (of CRX) been resolved? If so, could the authors discuss this phenomenon (mutations close to each other but have distinct effects) based on the HD-DNA structure? In addition, has this phenomenon been observed in other homeodomain TFs?
2. The authors should briefly summarize the effects/disease-causing-mechanisms of all the reported CRX mutations in the discussion part. The readers can then have a better overview of the topic.
3. CRX can also function as a pioneer factor (reported by the same group). Would these HD mutations distinctively affect chromatin accessibility (which then leads to ectopic binding on the genome)?
4. The discussion part can be shortened and simplified.

Reviewer #2 (Public Review):

Zheng et al., investigated the molecular and functional mechanisms of two homeodomain missense mutations causing human retinal photoreceptor degeneration diseases in photoreceptor development regulated by the CRX transcription factor. They analyzed the E80A mutation associated with dominant cone-rod dystrophy (CRD) and the K88N mutation associated with dominant Leber Congenital Amaurosis (LCA). The authors found that E80A CRX binds to the same target DNA sites as WT CRX, but the binding specificity of K88N CRX is altered from that of WT in an in vitro assay. They generated Crx(E80A) and Crx(K88N) KI mice and performed ChIP assay and observed that K88N CRX binds to novel genomic regions from the WT-binding sites, while E80A binds to the WT sites. In addition, using the KI mice, they found that E80A and K88N differently affect the expression of Crx target genes. This study is well executed with proper and solid methodologies, and the manuscript is clearly written. This study gives us the insights how single missense CRX mutations lead to different types of human retinal photoreceptor degeneration diseases.

While the study has strengths in principle, it has a couple of weaknesses. One is how well E80A KI mice function as a pathological model of dominant CRD, in which cones are mainly first affected, is not clearly shown in this study. More data investigating how cones are affected by performing histological, molecular, and physiological analyses will be helpful and useful. For example, in the Discussion, the authors describe that E80A associates with S-cone opsin promoter results is "data now shown". This data must be presented for the readers. In addition, more molecular insights as to how E80A affects cones will strengthen this study. Another point is that it will be very valuable if the authors could show how E80A and K88N differently affect the 3D structure of the CRX homeodomain. Even a simulation model would be valuable.

Author Response

Reviewer #1 (Public Review):

The manuscript by Zheng et al. examined the disease-causing mechanisms of two missense mutations within the homeodomain (HD) of CRX protein. Both mutations were found in humans and can produce severe dominant retinopathy. The authors investigated the two CRX HD mutants via in vitro DNA-binding assay (Spec-seq), in vivo chromatin-binding assay (ChIP-seq), in vivo expression assay of downstream target genes (RNA-seq), and retinal histological and functional assays. They concluded that p.E80A increased the transactivation activity of CRX and resulted in precocious photoreceptor differentiation, whereas p.K88N significantly changed the binding specificity of CRX and led to defects in photoreceptor differentiation and maintenance. The authors performed a significant amount of analyses. The claims are sufficiently supported by the data. The results not only uncovered the underlying disease-causing mechanisms, but also can significantly improve our understanding of the interaction between HD-TF and DNA during development.

Thank you for summarizing the key findings and strengths of our manuscript.

Minor concerns:

1. The E80A, K88N and R90W (previously reported by the same group) mutations are located very close to each other in the homeodomain (Figure 1A), but had distinct effects on the activity of CRX. Has the structure of the homeodomain (of CRX) been resolved? If so, could the authors discuss this phenomenon (mutations close to each other but have distinct effects) based on the HD-DNA structure?

In paragraphs 2, 4, 5 of the discussion section, we have added explanations on how each mutation could affect CRX HD-DNA interactions differently based on published structural studies. And we further explain how these biochemical changes relate to the molecular perturbations and cellular phenotypes seen in vivo.

In addition, has this phenomenon been observed in other homeodomain TFs?

Disease associated missense mutations at residues HD50 (K88) and HD52 (R90) have also been reported in other HD TFs implicated in CNS development (see discussion paragraph 7). Distinctively, different substitutions at CRX E80 residue have been reported in multiple CoRD cases, suggesting its essential role in HD-DNA-mediated regulation during retinal development. These new points are now included in the discussion section.

1. The authors should briefly summarize the effects/disease-causing-mechanisms of all the reported CRX mutations in the discussion part. The readers can then have a better overview of the topic.

We have added a concise summary of previously proposed CRX mutation classification scheme, all characterized Crx mutant mouse models and their pathogenic mechanisms. Please see paragraph 9 in the discussion section.

1. CRX can also function as a pioneer factor (reported by the same group). Would these HD mutations distinctively affect chromatin accessibility (which then leads to ectopic binding on the genome)?

Prior evidence has demonstrated that regulatory regions for many photoreceptor genes failed to stay accessible upon loss of CRX in the Crx-/- model (PMID: 30068366). It is unclear with the existing data whether CRX could initiate the chromatin remodeling (true pioneering function) of these regions, or it simply maintains the accessibility once these regions became accessible. Future studies comparing epigenomic landscape changes in mutant Crx KI models at various ages can be informative, particularly for the CRX K88N ectopic binding events. Determining how the CRX K88N mutant protein alters chromatin landscape important for photoreceptor fate and/or differentiation during development would shed light on the nature of these ectopic binding events.

1. The discussion part can be shortened and simplified.

We have re-written the discussion section to make it concise and to incorporate discussions on mutant CRX HD structures. Please see the revised manuscript.

Reviewer #2 (Public Review):

Zheng et al., investigated the molecular and functional mechanisms of two homeodomain missense mutations causing human retinal photoreceptor degeneration diseases in photoreceptor development regulated by the CRX transcription factor. They analyzed the E80A mutation associated with dominant cone-rod dystrophy (CRD) and the K88N mutation associated with dominant Leber Congenital Amaurosis (LCA). The authors found that E80A CRX binds to the same target DNA sites as WT CRX, but the binding specificity of K88N CRX is altered from that of WT in an in vitro assay. They generated Crx(E80A) and Crx(K88N) KI mice and performed ChIP assay and observed that K88N CRX binds to novel genomic regions from the WT-binding sites, while E80A binds to the WT sites. In addition, using the KI mice, they found that E80A and K88N differently affect the expression of Crx target genes. This study is well executed with proper and solid methodologies, and the manuscript is clearly written. This study gives us the insights how single missense CRX mutations lead to different types of human retinal photoreceptor degeneration diseases.

We greatly appreciate the reviewer’s summary and positive comments.

While the study has strengths in principle, it has a couple of weaknesses. One is how well E80A KI mice function as a pathological model of dominant CRD, in which cones are mainly first affected, is not clearly shown in this study. More data investigating how cones are affected by performing histological, molecular, and physiological analyses will be helpful and useful. For example, in the Discussion, the authors describe that E80A associates with S-cone opsin promoter results is "data now shown". This data must be presented for the readers. In addition, more molecular insights as to how E80A affects cones will strengthen this study.

The mouse retina is rod dominant and contains only a small number of cones (3% of all photoreceptors) that are born prenatally. This poses technical challenges to appropriately assess cone-specific changes during disease initiation/progression. We are in the process of developing cellular/molecular tools to investigate how cones are being affected in Crx E80A KI model, but this is beyond the scope of the current study.

At the same time, we have added a supplemental panel showing that, based on P0 retinal immunostaining of the early cone marker RXRγ, cones were initially born, and fate specified in CrxE80A retinas (see Figure S7A). Since the E80A protein also hyper-activated S-cone opsin promoter-luciferase (Sop-luc) reporter in HEK293 cells (see Figure S7B), we predict that CRX E80A affects cone photoreceptor differentiation in a similar manner as rod photoreceptors. Furthermore, the cone transcriptional program might be more prone to perturbations by abnormal CRX activities. These possibilities require future investigations. For this manuscript, we have included all these points in the discussion section.

Another point is that it will be very valuable if the authors could show how E80A and K88N differently affect the 3D structure of the CRX homeodomain. Even a simulation model would be valuable.

Please see our answer to Point 1 of Reviewer #1. In short, we have added in the discussion section our explanations on how each mutation could affect CRX HD-DNA interactions differently based on structural studies. We further explain how these biochemical changes relate to the molecular perturbations and cellular phenotypes seen in vivo. Additionally, since TF-DNA interactions are diverse and dynamic across binding sites with different sequence features and genomic environments, future studies that systematically and quantitatively evaluate CRX transcriptional activity at different regulatory sequences would be important.