Fine-tuned KDM1A alternative splicing regulates human cardiomyogenesis through an enzymatic-independent mechanism
The histone demethylase KDM1A is a multi-faceted regulator of critical developmental processes, including mesodermal and cardiac tube formation during mice gastrulation. The fine-tuning of KDM1A splicing has been linked to regulating the transcriptional program of excitable cells such as neurons. However, it is unknown whether modulating the expression of KDM1A isoforms is crucial for the specification and maintenance of cell identity of other cell types sensitive to electrical cues such as the cardiomyocytes. Here, we investigated the role of ubKDM1A and KDM1A+2a ubiquitous splice variants during cardiomyogenesis and evaluated their impact on the regulation of cardiac differentiation in vitro. We discovered a temporal modulation of ubKDM1A and KDM1A+2a isoform levels during human and mouse fetal cardiac development. Therefore, we generated human embryonic stem cells (hESCs) exclusively devoid of one or both isoforms and assessed their potential to derive cardiomyocytes. KDM1A depletion severely impaired cardiac differentiation. Conversely, KDM1A+2a-/- hESCs give rise to functional cardiomyocytes, displaying increased beating amplitude and frequency compared to wild-type cells. Transcriptomic profiling revealed that KDM1A-/- cardiomyocytes fail to activate an effective cardiac transcriptional program, while the depletion of KDM1A+2a enhances the expression of key cardiogenic markers. Notably, the impaired cardiac differentiation of KDM1A-/- cells can be rescued by re-expressing ubKDM1A or catalytically deficient ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. These data demonstrate a divergent role of the two KDM1A isoforms that is independent of their enzymatic activity. Through an exhaustive biochemical and genome-wide binding characterization, we excluded that the opposite ubKDM1A- and KDM1A+2a-mediated regulation of cardiac differentiation resides into differential substrate specificity, H3K4 demethylation efficiency, core-partners binding affinity, or alternative genome binding profiles. Our findings prove the existence of a divergent scaffolding role of KDM1A splice variants during hESC differentiation into cardiomyocytes.
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Mass spectrometry: Approximately 2 mg of total hESC total extract were immunoprecipitated in presence of Anti-FLAG M2 affinity gel (Sigma, 2220) overnight at 4°C and eluted with 3XFLAG- Peptide (Sigma, F3290) eluted in Elution Buffer (0.5M Tris-HCl, pH7.5, 1M NaCl) according to manufacturer’s instructions. Immunoprecipitation eluates were denatured in loading buffer and separated by SDS-PAGE to about 1 cm. Proteins in gel bands were destained using 50 mM NH4HCO3 containing 50% ACN (v/v), dehydrated using 100% ACN, and reduced by 10 mM dithiothreitol (DTT) in 100 mM NH4HCO3. Cysteine residues were alkylated using the IOA alkylating agent. Gel bands were subjected to a series of washing, dehydration, and hydration steps and then subjected to re-swelling overnight in gel Trypsin digestion steps. Trypsin digested proteins were isolated through the supernatant collection by extraction buffers 5% acetic acid, 50% ACN, and 0.1% TFA in 75% ACN. Samples were dried, reconstituted, and load on the Q ExactiveTM Mass Spectrometer instrument. The Mascot software was used to identify the peptide mass fingerprint from molecular ion peaks, sequence query, and matched MS/MS ion spectra. The significant threshold was set at < 0.05 and the ion score of the expected cut-off was set at 20, the search was done through the Uniprot database, showing the percentage coverage, the number of significant matches, and the number of peptide sequences.