Myocardial Reprogramming by HMGN1 Underlies Heart Defects in Trisomy 21 (CutnRun)

Congenital heart defects (CHD) are the most common form of developmental abnormalities, occurring in ~1% of live births, and can arise due to altered dosage of genes essential for cardiogenesis. Aneuploidy accounts for nearly 15% of CHD and the most frequent form involves trisomy of chromosome 21 (Ch21), resulting in Down Syndrome (DS). Here we used single cell RNA-seq, CRISPR-activation with single cell RNA-seq, single cell ATAC-seq and Cut&Tag epigenomic profiling to define the molecular disruptions occuring with Trisomy 21 and upregulation of the Ch21 epigenetic factor HMGN1. These experiments were performed in human pluripotent stem cells (hiPSCs) or in directed differentiation to cardiomyocyte lineages at days 10 and 20. Overall design: Cut and Run samples for HMGN1-Flag. Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) were collected at day 20 for CUT&RUN analysis. Cells were dissociated using 0.25% Trypsin-EDTA (1X) (Gibco, 25200-072) and neutralized with 1% FBS in PBS. For each condition, 150,000 cells per sample were processed according to the manufacturer's instructions using the CUTANA™ CUT&RUN Kit (Epicypher, 14-1048). Cells were first washed and bound to activated Concanavalin A (ConA)–coated magnetic beads in the Bead Activation Buffer. Nuclei were permeabilized with Digitonin-containing buffer, and all buffers were supplemented with cOmplete™ Mini, EDTA-free Protease Inhibitor Cocktail (Millipore Sigma, 11836170001). Samples were incubated with 0.5 µg of H3K4me3 primary positive control antibody (Epicypher 13-0060), 1 µL of IgG control antibody, and 1 µg of Monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich, F1804-200 µG) per sample at 4 °C overnight on a rotating mixer. After antibody binding, samples were washed by cell permeabilization buffer. Protein A-MNase fusion enzyme was added to each sample and incubated at 4 °C for 1 hour with mixing. Targeted chromatin digestion was initiated by adding calcium-containing digestion buffer and incubating at 0 °C for 30 minutes. Reactions were stopped with Stop Buffer supplemented with glycogen and RNase A, followed by incubation at 37 °C for 10 minutes to release digested chromatin fragments. DNA was purified using phenol-chloroform extraction or DNA Clean & Concentrator kit (Zymo Research), followed by SPRIselect bead cleanup (Beckman Coulter, B23317) and 80% ethanol washes. Elution was performed with 0.1X TE buffer, and DNA concentration was measured using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32851). Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645), indexed with i5/i7 dual indices, and amplified by PCR. Library DNA size distribution was assessed using the Agilent Bioanalyzer High Sensitivity DNA kit (Agilent, 5067-4626). Final libraries were pooled and sequenced on an Illumina NextSeq 500/550 using the Mid Output v2 kit (Illumina, 20024904) with the following cycle configuration: Read 1: 75 bp, Index 1: 8 bp, Index 2: 8 bp, Read 2: 75 bp.