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In FXS, an expanded CGG repeat tract in the FMR1 gene induces epigenetic silencing during embryogenesis. FMR1 silencing can be reversed with 5-aza-deoxyctidine (5-aza-dC), a nonspecific epigenetic reactivator; however, continuous administration of 5-aza-dC is problematic due to its toxicity. We describe an approach to restore FMR1 expression in FXS neurons by transient treatment with 5-aza-dC, followed by treatment with 2HE-5NMe, which binds the CGG repeat expansion in the FMR1 mRNA and could block the resilencing of the FMR1 gene after withdrawal of 5-aza-dC. Methods: This study uses immunofluorescence and fluorescent in situ hybridization (FISH) to measure whether FMR1 expression is maintained in FXS post-mitotic neurons treated with 2HE-5NMe. Genome-wide profiling of histone marks was used to monitor epigenetic changes and drug selectivity in response to 5-aza-dC followed by 2HE-5NMe treatment. Changes to dendritic morphology were visualized using confocal microscopy. Results: In this study, we find that 2HE-5Nme maintains FMR1 in a reactivated state after reactivation using 5-aza-dC in post-mitotic neurons. FMR1 reactivation in neurons results in the re-expression of FMRP and reversal of FXS-associated dendritic spine defects. Conclusions: These results demonstrate that an RNA-binding small molecule can achieve gene-specific epigenetic control and provide an approach for the restoration of FMRP in FXS neurons.<\/jats:p>","DOI":"10.3390\/genes16030278","type":"journal-article","created":{"date-parts":[[2025,2,25]],"date-time":"2025-02-25T12:01:17Z","timestamp":1740484877000},"page":"278","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Sustained Epigenetic Reactivation in Fragile X Neurons with an RNA-Binding Small Molecule"],"prefix":"10.3390","volume":"16","author":[{"given":"Christina W.","family":"Kam","sequence":"first","affiliation":[{"name":"Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6926-4843","authenticated-orcid":false,"given":"Jason G.","family":"Dumelie","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gabriele","family":"Ciceri","sequence":"additional","affiliation":[{"name":"The Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA"},{"name":"Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1839-631X","authenticated-orcid":false,"given":"Wang-Yong","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Physics, University of Tennessee at Chattanooga, Chattanooga, TN 37403, USA"},{"name":"Department of Chemistry, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew D.","family":"Disney","sequence":"additional","affiliation":[{"name":"Department of Chemistry, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA"},{"name":"Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, FL 33458, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lorenz","family":"Studer","sequence":"additional","affiliation":[{"name":"The Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA"},{"name":"Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3615-6958","authenticated-orcid":false,"given":"Samie R.","family":"Jaffrey","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1097","DOI":"10.1126\/science.252.5009.1097","article-title":"Instability of a 550-Base Pair DNA Segment and Abnormal Methylation in Fragile X Syndrome","volume":"252","author":"Rousseau","year":"1991","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1016\/0092-8674(91)90397-H","article-title":"Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome","volume":"65","author":"Verkerk","year":"1991","journal-title":"Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1086\/342931","article-title":"Histone modifications depict an aberrantly heterochromatinized FMR1 gene in fragile x syndrome","volume":"71","author":"Coffee","year":"2002","journal-title":"Am. 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