Abstract 60: Histone Modification H3k4me2 Controls Vascular Smooth Muscle Cell Lineage Memory during Vascular Injury

Abstract

Smooth muscle cells (SMC) have the ability to undergo reversible phenotypic switch (i.e. dedifferentiation/redifferentiation) characterized by loss and re-expression of SMC marker genes, a process occurring during blood vessel repair and adaptive remodeling. Using SMC lineage-tracing mice, we showed that newly generated SMC derive from pre-existing SMC that transiently de-differentiate, participate in repair, and then redifferentiate following wire-induced femoral injury. However, a critical unresolved question is how dedifferentiated SMC retain their lineage identity. We previously demonstrated that SMC acquire a unique epigenetic signature during differentiation, H3K4me2 on the SMC marker genes, retained in SMC during PDGF-BB-induced phenotypic switch. Our hypothesis is that H3K4me2 on SMC genes biases SMC re-differentiation and functions as a lineage memory mechanism during vascular injury-repair. We demonstrated that H3K4me2 on SMC genes is retained in dedifferentiated and redifferentiated SMC in vivo following vascular injury using our unique ISH-PLA epigenetic assay. We also found that H3K4me2 is present and retained on the SMC regulatory gene Myocardin during PDGF-BB treatment, reinforcing significance of H3K4me2 as a regulator of SMC identity. To directly assess the functional role of H3K4me2 on the SMC repertoire, we generated a fusion protein consisting of the H3K4 demethylase LSD1 coupled to the SRF-CArG binding domain of Myocardin (Myocd-LSD1WT) to perform SMC-specific locus-selective removal of H3K4me2 on the SMC marker genes. We also generated loss-of-function mutant of Myocd-LSD1 to ensure that effects are due to H3K4me2 removal and not steric hindrance. Stable expression of Myocd-LSD1WT in SMC induced locus selective removal of H3K4me2 on SMC CArG genes without impacting non-SMC CArG-dependent genes like c-fos. Myocd-LSD1WT also inhibits expression of the SMC marker genes and abolishes the ability of SMC to redifferentiate in PDGF-BB induced phenotypic switch. None of these effects were observed in Myocd-LSD1Mut SMC. Results are the first to our knowledge to provide direct functional evidence that locus specific epigenetic modifications regulate cell lineage memory during reversible phenotypic transitions.