Abstract Body: Congenital heart defects (CHDs) are highly enriched in Down syndrome (trisomy 21), yet the mechanisms linking gene dosage to aberrant cardiac development remain incompletely defined. In our recent work, we identified the nucleosome-binding protein HMGN1 as a dosage-sensitive regulator that shifts atrioventricular canal cardiomyocytes (AVCMs) toward a ventricular cardiomyocyte-like state, while allele-specific reduction of HMGN1 in trisomic cells restored AVCM gene expression programs. Removal of one allele of Hmgn1 in the trisomic mouse model resulted in similar gene program shifts, and rescue of valvuloseptal defects, providing definitive in vivo evidence regarding the necessity of Hmgn1 duplication for the cardiac defects in the DS model. Here, we investigated the mechanisms underlying HMGN1 function using single-molecule chromatin footprinting (SAMOSA) integrated with HMGN1 occupancy, single-cell chromatin accessibility, and transcriptomic data. We restricted analysis to regulatory regions that are accessible in AVCM chromatin states. Within chromatin regions accessible in the AVC state, subnucleosome state classification revealed a significant enrichment of “breathing”, or partially disassembled nucleosomes in trisomic cells, suggesting a more permissive state for transcriptional activation. This chromatin change was recapitulated with CRISPR activation of just HMGN1 in disomic cells, and was rescued by CRISPR interference of HMGN1 in trisomic cells. Motifs for MEF2C, a transcriptional regulator of ventricular cardiomyocyte gene expression, were enriched in dynamically opening AVCM chromatin regions, as was MEF2C expression in trisomic AVCMs. These results support a model in which HMGN1 dosage promotes nucleosome destabilization at key regulatory elements, facilitating MEF2-driven transcriptional reprogramming in AVC cardiomyocytes. We propose that an HMGN1-dependent, MEF2-driven regulatory axis underlies AVC cardiomyocyte mis-specification and septal defects, offering a chromatin-based contribution to the cardiac phenotype observed in trisomy 21, possibly in conjunction with other genes such as Dyrk1a.