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Despite significant clinical interest in the molecular level characterization of GATA1 mutations, a comprehensive understanding of their impact on DNA binding is limited. Efforts to conduct detailed studies on full\u2010length recombinant GATA1 have faced significant technical challenges, while alternative approaches are limited by low throughput or qualitative nature. Here, we introduce a native holdup (nHU) assay designed to systematically quantify DNA\u2010protein interactions and is suitable for studying the impact of transcription factor mutations on DNA binding affinity. First, using the erythroid\u2010specific\n                    <jats:italic>ATP2B4<\/jats:italic>\n                    promoter as a model, we demonstrate that nHU can capture sequence\u2010specific interactions and detect even subtle differences in DNA binding affinities. Then, we quantitatively characterize the impact of pathological mutations on DNA binding affinities in the context of full\u2010length human GATA1. Our findings reveal that the GATA1s isoform, lacking the N\u2010terminal transactivation domain (N\u2010TAD), binds to DNA with increased affinity, while the R307C mutation reduces binding to the\n                    <jats:italic>ATP2B4<\/jats:italic>\n                    erythroid promoter. In harmony with these observations, GATA1s exhibits increased functional activity, while the R307C mutation results in decreased activity. This study demonstrates the power of the nHU assay for studying DNA interactions of transcription factor variants and providing insight into the molecular mechanism of related diseases.\n                  <\/jats:p>","DOI":"10.1111\/febs.70483","type":"journal-article","created":{"date-parts":[[2026,3,17]],"date-time":"2026-03-17T11:51:13Z","timestamp":1773748273000},"page":"5149-5169","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Quantitative analysis of\n                    <scp>DNA<\/scp>\n                    \u2010\n                    <scp>GATA1<\/scp>\n                    binding alterations linked to hematopoietic disorders"],"prefix":"10.1111","volume":"293","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5912-699X","authenticated-orcid":false,"given":"Boglarka","family":"Zambo","sequence":"first","affiliation":[{"name":"\u00c9quipe Labellis\u00e9e Ligue contre le cancer Institut de G\u00e9n\u00e9tique 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FischerS FreyerL LallemandY DardenneP&Gomez PerdigueroE(2025)Ontogeny drives stage\u2010specific effects of a Gata1 mutation.bioRxiv2025.01.15.633164.","DOI":"10.1101\/2025.01.15.633164"},{"key":"e_1_2_10_57_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.95397"}],"container-title":["The FEBS 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