The non-canonical roles of NIPBL in cardiomyocytes

About This Grant

SUMMARY Cornelia de Lange Syndrome (CdLS) is a genetic disorder encompassing a spectrum of abnormalities in multiple organs. Heterozygous pathogenic mutations of NIPBL gene account for over 60% of CdLS cases. Although a significant portion (>30%) of CdLS patients experience congenital heart disease (CHD) or decreased cardiac function in adulthood, the regulatory role of NIPBL in human cardiac cells remains unknown. Consequently, no specific treatment has been developed to address cardiac phenotypes in CdLS patients. NIPBL is known as a cohesin loader factor that facilitates the interactions between DNA and cohesin complex, which is essential for sister chromatid cohesion and DNA looping during mitosis and cell proliferation. More recent studies have shown that NIPBL mutations lead to a mild, yet universal, remodeling of the transcription of a constellation of genes, possibly through the modification of chromatin architecture and enhancer-promoter interactions. However, double knockout of NIPBL is embryonic lethal, which impedes the study of NIPBL’s role in a clean background. Also, the scarcity of human cardiac tissue has further constrained our understanding of NIPBL in human cardiac cells. To address this knowledge gap, we have generated inducible NIPBL depletion human induced pluripotent stem cell (iPSC) based on a degradation tag (dTAG) system, enabling the investigation of NIPBL’s role in human cardiomyocyte (CM). Our preliminary studies showed that NIPBL-null induced significant changes in a small group of cardiac genes in CMs, rather than the mild changes of a constellation of genes reported in other cell types. Moreover, NIPBL interacts with nuclear proteins at specific DNA loci, such as BRD4 and GATA4, suggesting its function as a transcription regulator in CMs. Our further studies showed the formation of chromatin complex by NIPBL and BRD4 are key to maintaining genome stability and transcription homeostasis in stressed CMs. Thus, we hypothesize that NIPBL, in addition to being a cohesin loader, is a key transcriptional regulator and chromatin stabilizer in CMs, and NIPBL haploinsufficiency contributes to the transcriptional remodeling in CdLS patient heart. In our plan: we will first define the regulatory role of NIPBL in cardiac function and transcription landscape in NIPBL-null iPSC-CMs with functional assays and scRNA-seq (Aim 1). Then, we will examine the role of NIPBL as a transcriptional regulator in CMs through 1) confirm the interactions between NIPBL and transcription factors (TFs) with advanced imaging assay; 2) understand how NIPBL-TF interactions regulate DNA binding landscapes and transcription activity; 3) identify novel binding partners of NIPBL with Co-IP mass spectrometry (Aim 2). Lastly, we will validate the role of NIPBL-BRD4 interaction in maintaining genomic and transcriptional homeostasis and testify the beneficial effect of BRD4 inhibition in stressed CMs and animal heart (Aim 3). Our study will examine the non-canonical role of NIPBL in CMs, potentially revealing novel therapeutic targets for cardiac remodeling induced by NIPBL haploinsufficiency (CdLS) or functional stresses.