Cardiac morphogenesis requires complex and well-orchestrated transcriptional programs. PRDI-BF1 and RIZ homology (PR) domain-containing 16 (PRDM16) is a member of the conserved PRDM family that function as transcriptional regulators and methyltransferases in diverse cell types. PRDM16 is expressed in both murine and human cardiomyocytes (CMs). Mutations in PRDM16 are associated with congenital heart disease (CHD), highlighting its importance for cardiac development. Furthermore, a previous study has reported that global Prdm16-deficient mice (gKO) die perinatally and display heart abnormalities. However, little is known as to the specific role of PRDM16 in CMs, or molecular mechanisms by which loss of PRDM16 results in CHD. Further, requirements for the DNA-binding function(s) and/or histone methyltransferase (HMT) activity of PRDM16 in CMs are yet to be determined. To examine the role of PRDM16 in CMs, we generated a Prdm16 CM-specific knockout (cKO) mouse model. Our preliminary data revealed that Prdm16 cKO mice die before postnatal day 7, suggesting that the primary cause of lethality in gKO mice is due to loss of PRDM16 in CMs. Prdm16 cKO mice exhibited dramatic left ventricular dilation, first observed at embryonic day (E)15.5. Taken together, the foregoing observations suggest that PRDM16 plays a critical role in developing CMs. To determine the target genes of Prdm16 in developing CMs, we performed RNA- and ChIP-sequencing analysis of ventricular tissue isolated from Prdm16 cKO and control hearts at E13.5. Results demonstrated significant alterations in gene expression, with 69.3% of dysregulated genes having PRDM16 binding peaks, suggesting that PRDM16 plays a critical role in the transcriptional program of developing CMs. Accordingly, our hypothesis is that PRDM16 plays an essential role in CMs by exhibiting unique functional activities that shape key events in transcriptional regulation of cardiac morphogenesis, and mutation to abolish its DNA-binding or HMT activity will impair specific aspects of PRDM16 function to lead to cardiac developmental defects. To study the specific roles of PRDM16 DNA-binding and HMT activity in developing CMs, we have generated two novel mouse models, in which the DNA-binding or HMT activity has been abolished, respectively, by mutating functionally critical amino acid(s). In the ?DB? mutant, a critical ?DNA-binding? Arginine (R1000) is mutated to Glutamine (Q), resulting in the loss of DNA-binding activity of PRDM16. In the ?HMT? mutant, the Tyrosine (Y113) and Valine (V115) in the PR domain have been mutated to Phenylalanine (F) and Glycine (G), respectively, resulting in loss of HMT activity.
Our specific aims are to (1) determine the role and mechanisms by which PRDM16 is required in cardiac development by histological, physiological, biochemical, and molecular analyses of Prdm16 cKO mice; and (2) determine specific roles of DNA-binding and/or HMT activities of PRDM16 in developing CMs by analyzing ?DB? and ?HMT? mutant mice, in which either the DNA-binding or HMT activity has been abolished.
Mutations in PRDM16, a transcriptional regulator and histone methyltransferase, are associated with congenital heart disease (CHD), highlighting its importance for cardiac development. However, little is known as to the specific role of PRDM16 in cardiomyocytes, or molecular mechanisms by which loss of PRDM16 results in CHD. Proposed studies are aimed at understanding the function of PRDM16 in developing cardiomyocytes at the molecular, cellular, and physiological levels, and deciphering the requirements for the DNA-binding function(s) and/or histone methyltransferase activity of PRDM16 in cardiomyocytes.
