Urman, A. | bioRxiv Science

bioRxiv2025

Active DNA demethylation is upstream of rod-photoreceptor fate determination and required for retinal development

Retinal cell fate specification from multipotent retinal progenitors is governed by dynamic changes in chromatin structure and gene expression. Methylation at cytosines in DNA (5mC) is actively regulated for proper control of gene expression and chromatin architecture. Numerous genes display active DNA demethylation across retinal development; a process that requires oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and is controlled by the ten-eleven translocation methylcytosine dioxygenase (TET) enzymes. Using an allelic series of conditional TET enzyme mutants, we determine that DNA demethylation is required upstream of NRL and NR2E3 expression for the establishment of rod-photoreceptor fate. Using histological, behavioral, transcriptomic, and base-pair resolution DNA methylation analyses, we establish that inhibition of active DNA demethylation results in global changes in gene expression and methylation patterns that prevent photoreceptor precursors from adopting a rod-photoreceptor fate, instead producing a retina in which all photoreceptors specify as cones. Our results establish the TET enzymes and DNA demethylation as critical regulators of retinal development and cell fate specification, elucidating a novel mechanism required for the specification of rod-photoreceptors.

developmental biology↗

bioRxiv2024

Non-CG DNA methylation and MeCP2 stabilize repeated tuning of long genes that distinguish closely related neuron types

The extraordinary diversity of neuron types in the mammalian brain is delineated at the highest resolution by subtle gene expression differences that may require specialized molecular mechanisms to be maintained. Neurons uniquely express the longest genes in the genome and utilize neuron-enriched non-CG DNA methylation (mCA) together with the Rett syndrome protein, MeCP2, to control gene expression, but the function of these unique gene structures and machinery in regulating finely resolved neuron type-specific gene programs has not been explored. Here, we employ epigenomic and spatial transcriptomic analyses to discover a major role for mCA and MeCP2 in maintaining neuron type-specific gene programs at the finest scale of cellular resolution. We uncover differential susceptibility to MeCP2 loss in neuronal populations depending on global mCA levels and dissect methylation patterns and intragenic enhancer repression that drive overlapping and distinct gene regulation between neuron types. Strikingly, we show that mCA and MeCP2 regulate genes that are repeatedly tuned to differentiate neuron types at the highest cellular resolution, including spatially resolved, vision-dependent gene programs in the visual cortex. These repeatedly tuned genes display genomic characteristics, including long length, numerous intragenic enhancers, and enrichment for mCA, that predispose them to regulation by MeCP2. Thus, long gene regulation by the MeCP2 pathway maintains differential gene expression between closely-related neurons to facilitate the exceptional cellular diversity in the complex mammalian brain.

neuroscience↗