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A large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

ABSTRACT: Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased 'fate bias'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.

SUBMITTER: Emmerich K

PROVIDER: S-EPMC11361637 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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A large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

Emmerich Kevin K Hageter John J Hoang Thanh T Lyu Pin P Sharrock Abigail V AV Ceisel Anneliese A Thierer James J Chunawala Zeeshaan Z Nimmagadda Saumya S Palazzo Isabella I Matthews Frazer F Zhang Liyun L White David T DT Rodriguez Catalina C Graziano Gianna G Marcos Patrick P May Adam A Mulligan Tim T Reibman Barak B Saxena Meera T MT Ackerley David F DF Qian Jiang J Blackshaw Seth S Horstick Eric E Mumm Jeff S JS

Development (Cambridge, England) 20240812 15

Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration ...[more]

PMID: 39007397

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Project description:BackgroundDespite advances in B7 homolog 3 protein (B7-H3) based immunotherapy, the development of drug resistance remains a major clinical concern. The heterogeneity and emerging loss of B7-H3 expression are the main causes of drug resistance and treatment failure in targeted therapies, which reveals an urgent need to elucidate the mechanism underlying the regulation of B7-H3 expression. In this study, we identified and explored the crucial role of the transcription factor SPT20 homolog (SP20H) in B7-H3 expression and tumor progression.MethodsHere, we performed CRISPR/Cas9-based genome scale loss-of-function screening to identify regulators of B7-H3 in human ovarian cancer cells. Signaling pathways altered by SP20H knockout were revealed by RNA sequencing. The regulatory role and mechanism of SP20H in B7-H3 expression were validated using loss-of-function and gain-of-function assays in vitro. The effects of inhibiting SP20H on tumor growth and efficacy of anti-B7-H3 treatment were evaluated in tumor-bearing mice.ResultsWe identified SUPT20H (SP20H) as negative and eIF4E as positive regulators of B7-H3 expression in various cancer cells. Furthermore, we provided evidence that either SP20H loss or TNF-α stimulation in tumor cells constitutively activates p38 MAPK-eIF4E signaling, thereby upregulating B7-H3 expression. Loss of SP20H upregulated B7-H3 expression both in vitro and in vivo. Additionally, deletion of SP20H significantly suppressed tumor growth and increased immune cells infiltration in tumor microenvironment. More importantly, antibody-drug conjugates targeting B7-H3 exhibited superior antitumor performance against SP20H-deficient tumors relative to control groups.ConclusionsActivation of p38 MAPK-eIF4E signaling serves as a key event in the transcription initiation and B7-H3 protein expression in tumor cells. Genetically targeting SP20H upregulates target antigen expression and sensitizes tumors to anti-B7-H3 treatment. Collectively, our findings provide new insight into the mechanisms underlying B7-H3 expression and introduce a potential synergistic target for existing antibody-based targeted therapy against B7-H3.

Dataset Information

A large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

Publications

A large-scale CRISPR screen reveals context-specific genetic regulation of retinal ganglion cell regeneration.

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