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The role of DOT1L in regulating the epigenetic signature of cardiomyocytes during heart development

Final Report Summary - THE CARDIAC CODE (The role of DOT1L in regulating the epigenetic signature of cardiomyocytes during heart development)

Cardiovascular diseases are an extremely relevant socio-economical burden: congenital heart disease (CHD) is the most common form of inborn malformation, and adult heart disease is a major cause of morbidity and mortality in developed countries. Congenital and adult heart disease share a common link: regulation of cardiomyocyte (CM) proliferation. Impaired CM proliferation during embryogenesis is a frequent cause of defective cardiogenesis, whereas the reduced regenerative potential of the adult heart after injury can be explained by the fact that adult CMs lack proliferative potential. In fact, after myocardial infarction, dead CMs are replaced by an extensive fibrotic scar. CMs switch from a proliferative state in embryos, to a non-replicative state shortly after birth. Understanding molecular and cellular mechanisms underlying this transition is critical for deciphering molecular origins of cardiac malformations and to develop new therapies for cardiac regeneration in adulthood. Stage and cell-type-specific gene expression programs are established by the coordinated action of transcription factors and epigenetic enzymes. Epigenetic modifications are crucial for stabilizing transcriptional programs, but little is known about the “epigenetic code” of histone methylation during cardiogenesis. Importantly, our previous studies in mouse embryonic stem cell-derived CMs suggested that the histone methyl transferase DOT1L is required for proper CM differentiation. Following up on such in vitro observations, the “Cardiac Code” project was designed to test the hypothesis that DOT1L activity in CMs plays a key role in embryonic heart development, defining a H3K79 dependent epigenetic landscape essential for cardiomyocyte differentiation and cardiomyocyte cell cycle regulation.
To test this hypothesis, the following Specific Aims have been proposed: 1) To investigate effects of early cardiomyocyte-specific deletion of Dot1L on cardiac morphogenesis and cardiomyocyte cell biology in vivo. 2) To identify genes directly regulated, positively or negatively, by DOT1L-mediated H3K79 methylation in vivo.

To achieve these objectives, “the Cardiac Code” project was carried out by the fellow Dr. Paola Cattaneo in the framework of an international collaboration between the laboratory of Prof. Sylvia Evans at the University of California San Diego (UCSD) (San Diego, USA), one of the world’s leading experts on the gene regulatory circuits driving mammalian cardiogenesis, and Prof. Gianluigi Condorelli at the National Research Council, Institute of Genetic and Biomedical Research (CNR-IRGB) (Milan, Italy), expert in epigenetics and molecular cardiology.

During the outgoing phase of the Marie Curie fellowship (conducted at UCSD), Dr. Cattaneo has achieved critical results for completion of the aims initially proposed. Using murine models to study mammalian heart development, Dr. Cattaneo has characterized the dynamics of expression of the Dot1L methyltrasnferase enzyme and the dynamics of H3K79 methylation during distinct phases of cardiogenesis in vivo. These experiments suggested that this enzyme and the modification it promotes might play a critical role in mammalian cardiogenesis. Importantly, Dr. Cattaneo confirmed this hypothesis by generating mutant mice that harbor a cardiac specific deletion of Dot1L. Dr. Cattaneo showed that such mutants display abnormal cardiogenesis and die perinatally due to cardiac enlargement and dysfunction. This finding is extremely relevant because it suggests that abnormal activity of Dot1L during gestation can lead to congenital heart disease. Adding more relevance to the congenital cardiac malformations observed in the mutants, Dr. Cattaneo observed that Dot1L-depleted neonatal cardiomyocytes displayed higher proliferative rates than those observed in control littermates. This is an extremely relevant finding in the context of adult heart regeneration. The major reason why the adult heart is incapable of efficient regeneration after injury is the lack of proliferative activity characteristic of adult cardiomyocytes. The observation that Dot1L mutants have increased proliferation suggests this enzyme might be involved in the mechanism that drives perinatal cardiomyocytes into an unreplicative state.

At UCSD Dr. Cattaneo received extensive training and expanded her skills in cardiac development and organogenesis and learnt all technical approaches associated with the study of these processes. In particular, she became proficient in the complex logistics associated with the generation and maintenance of a colony of transgenic mice and learned the spatiotemporal patterns of expression of all commonly used constitutive and inducible myocardial recombinase lines. Importantly, she acquired expertise in state state-of-the-art approaches for lineage tracing, a sort of knowledge that is crucial for the proper design of strategies for performing gene ablation studies. The impact of her work is documented by original research articles in high-impact peer reviewed journals (JCI 2015, Cell Stem Cell 2017, Circ Res 2017), as well as one review on cellular and molecular responses taking place in the heart after injury (J Mol Cell Cardiol. 2015)

During the returning phase at IRGB-CNR in Italy, using Next generation Sequencing approaches – for both genomic and epigenomic analysis - Dr. Cattaneo has characterized the dynamics of gene expression and methylation profiles changes in Dot1L mutant and control cardiomyocytes. These approaches have very high resolution and allowed to gain information for each single gene locus in the whole genome. Dr. Cattaneo performed RNA-seq and ChIP-seq experiments at two distinct stages of heart development, and was able to identify genes specifically altered in this transition phase from a late gestation stage to a post-natal stage. Genes upregulated during this transition belonged mainly to pathways related to metabolism, with an enrichment in genes associated with oxidative phosphorylation. On the other hand, genes downregulated during this transition were mainly associated with cell cycle. Analysis on Dot1L mutant versus control cardiomyocytes, indicated that the mutant cardiomyocytes were retaining the expression profile of a more immature stage. To add an additional layer of information, bioinformatics analyses integrating transcriptomic (RNA-seq) and epigenomic (ChIP-seq) datasets in control and mutant CMs allowed to distinguish between direct and indirect targets of Dot1L. These results were critical to unravel the mechanism by which absence of Dot1L during cardiac development results in increased proliferation and they suggest that abnormal activity of Dot1L during gestation can lead to congenital heart disease.

In the returning phase Dr. Cattaneo expanded her training in bioinformatics, genomics and epigenomics analyses working together with the bioinformatics team of the host institute. As part of her transfer of knowledge activities and to improve her management and tutorial skills, during the returning phase at IRGB-CNR, Dr. Cattaneo taught technical and conceptual lessons to several of the PhD students and post-doctoral fellows of the Cardiology Department. She guided them in their projects and career path and transferred them the knowledge acquired at UCSD regarding scientific matters such as mouse transgenesis, heart development, regeneration and fibrosis as well as managing matters, such as fellowship application and time managing.

In Conclusion, the “Cardiac Code” project significantly increased our knowledge of mechanisms by which DOT1L- mediated methylation is critical for heart development, and mechanisms underlying CM cell cycle regulation. This knowledge will help in designing regenerative therapies for the heart, and will also yield fundamental insights into how epigenetic mechanisms regulate gene expression in cardiomyocytes. A manuscript describing the latest findings of the “Cardiac Code” project is in preparation and will be submitted in the near future to a major peer-review journal.

The Marie Curie postdoctoral experience had an important impact on the maturation of Dr. Cattaneo as scientist and independent researcher in the areas of cardiac developmental biology. Dr. Cattaneo obtained a tenured position as Researcher-Assistant Professor at the IRGB-CNR and was recently successful in two grant applications that will allow her to start her independent group and recruit PhD students and post-docs to train in the field of transcription regulation in cardiac development and regeneration.