In vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation

Abstract

Between birth and adulthood cardiomyocytes (CMs) undergo dramatic changes in size, ultrastructure, metabolism, and gene expression, in a process collectively referred to as CM maturation. The transcriptional network that coordinates CM maturation is poorly understood, creating a bottleneck for cardiac regenerative medicine. Forward genetic screens are a powerful, unbiased method to gain novel insights into transcriptional networks, yet this approach has rarely been used in vivo in mammals because of high resource demands. Here we utilized somatic mutagenesis to perform the first reported in vivo CRISPR genetic screen within a mammalian heart. We discovered and validated several novel transcriptional regulators of CM maturation. Among them were RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicated that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

Transcriptomic entropy benchmarks stem cell-derived cardiomyocyte maturation against endogenous tissue at single cell level

“…Our results suggest that increased focus should be placed on trying to understand regulators of perinatal maturation in vivo, and determining discrepancies in activity of these regulators in vitro. In particular, we anticipate that entropy score can serve as a readout for mosaic-based screening systems for identifying perinatal CM maturation regulators [56]. Whether similar mechanisms underlie maturation arrest in other PSC-derived cells remains a question for further studies.…”

Maturing heart muscle cells: Mechanisms and transcriptomic insights

“…Currently, the best systems to study in vivo pathways of maturation rely on mouse models of post-natal heart development amenable to genetic modification. Using an MYH7-YFP mouse line as a reporter of maturation in a CRISPR/Cas9 knockout screen, Vandusen et al found RNF20/40 to be key epigenetic regulators required for maturation [ 85 ]. Knockdown of estrogen-related receptor (ERR) isoforms ERRα and ERRβ postnatally showed that these regulators are critical for CM maturation [ 86 ].…”

Maturation of Stem Cell-Derived Cardiomyocytes: Foe in Translation Medicine

“…Mechanistically, pIC treatment regulates early Notch signaling and increases the epigenetic activating modification H3K9ac in cardiac myofilament genes promoter regions. In a recent study, in vivo CRISPR screening identifies RNF20/40 ( 123 ), which monoubiqui-tinates H2B at lysine 120 by exerting E3 ligase activity. RNF20/40 is essential for CMs maturation, which is proven to regulate metabolism during CM maturation directly.…”