Cloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A “self-cleaving” peptides have garnered increased interest for their polycistronic nature, small size and high “cleavage” efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.

Rationale Generation of induced cardiac myocytes (iCMs) directly from fibroblasts offers great opportunities for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate of fibroblasts to fully reprogrammed iCMs, which could in part be attributed to unbalanced expression of reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) using the current gene delivery approach. Objective We aimed to establish a system to express distinct ratios of G, M, T proteins in fibroblasts and determine the effect of G, M, T stoichiometry on iCM reprogramming. Methods and Results We took advantage of the inherent feature of the polycistronic system and generated all possible combinations of G, M, T with identical 2A sequences in a single transgene. We demonstrated that each splicing order of G, M, T gave rise to distinct G, M, T protein expression levels. Combinations that resulted in higher protein level of Mef2c with lower levels of Gata4 and Tbx5 significantly enhanced reprogramming efficiency compared to separate G, M, T transduction. Importantly, after further optimization, the MGT vector resulted in more than 10-fold increase in the number of mature beating iCMs loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiac myocyte markers. Conclusion Our results demonstrate that stoichiometry of G, M, T protein expression influences the efficiency and quality of iCM reprogramming. The established optimal G, M, T expression condition will provide a valuable platform for future iCM studies.

SUMMARY Direct reprogramming of induced cardiomyocytes (iCMs) suffers from low efficiency and requires extensive epigenetic repatterning, although the underlying mechanisms are largely unknown. To address these issues, we screened for epigenetic regulators of iCM reprogramming and found that reducing levels of the polycomb complex gene Bmi1 significantly enhanced induction of beating iCMs from neonatal and adult mouse fibroblasts. The inhibitory role of Bmi1 in iCM reprogramming is mediated through direct interactions with regulatory regions of cardiogenic genes, rather than regulation of cell proliferation. Reduced Bmi1 expression corresponded with increased levels of the active histone mark H3K4me3 and reduced levels of repressive H2AK199ub at cardiogenic loci, and de-repression of cardiogenic gene expression during iCM conversion. Furthermore, Bmi1 deletion could substitute for Gata4 during iCM reprogramming. Thus, Bmi1 acts as a critical epigenetic barrier to iCM production. Bypassing this barrier simplifies iCM generation and increases yield, potentially streamlining iCM production for therapeutic purposes.

Summary Direct lineage conversion offers a new strategy for tissue regeneration and disease modeling. Despite recent success in directly reprogramming fibroblasts into various cell types, the precise changes that occur as fibroblasts progressively convert to target cell fates remain unclear. The inherent heterogeneity and asynchronous nature of the reprogramming process renders it difficult to study using bulk genomic techniques. Here, to overcome this limitation, we applied single-cell RNA-seq to analyze global transcriptome changes at early stages of induced cardiomyocyte (iCM) reprogramming1–4. Using unsupervised dimensionality reduction and clustering algorithms, we identified molecularly distinct subpopulations of cells along reprogramming. We also constructed routes of iCM formation, and delineated the relationship between cell proliferation and iCM induction. Further analysis of global gene expression changes during reprogramming revealed an unexpected down-regulation of factors involved in mRNA processing and splicing. Detailed functional analysis of the top candidate splicing factor Ptbp1 revealed that it is a critical barrier to the acquisition of CM-specific splicing patterns in fibroblasts. Concomitantly, Ptbp1 depletion promoted cardiac transcriptome acquisition and increased iCM reprogramming efficiency. Additional quantitative analysis of our dataset revealed a strong correlation between the expression of each reprogramming factor and the progress of individual cells through the reprogramming process, and led to the discovery of novel surface markers for enrichment of iCMs. In summary, our single cell transcriptomics approaches enabled us to reconstruct the reprogramming trajectory and to uncover heretofore unrecognized intermediate cell populations, gene pathways and regulators involved in iCM induction.

Direct conversion of fibroblasts into induced cardiomyocytes (iCMs) offers an alternative strategy for cardiac disease modeling and regeneration. During iCM reprogramming, the starting fibroblasts must overcome existing epigenetic barriers to acquire the CM-like chromatin pattern. However, epigenetic dynamics along this reprogramming process have not been studied. Here, we took advantage of our recently generated polycistronic system and determined the dynamics of two critical histone marks, H3K27me3 and H3K4me3, in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. We observed reduced H3K27me3 and increased H3K4me3 at cardiac promoters as early as day 3, paralleled by a rapid significant increase in their mRNA expression. In contrast, H3K27me3 at loci encoding fibroblast marker genes did not increase until day 10 and H3K4me3 progressively decreased along the reprogramming process; these changes were accompanied by a gradual decrease in the mRNA expression of fibroblast marker genes. Further analyses of fibroblast-enriched transcription factors revealed a similarly late deposition of H3K27me3 and decreased mRNA expression of Sox9, Twist1 and Twist2, three important players in epithelial-mesenchymal transition. Our data suggest early rapid activation of the cardiac program and later progressive suppression of fibroblast fate at both epigenetic and transcriptional levels. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that not every single CpG was equally demethylated during early stage of iCM reprogramming. Rather, there are specific CpGs, whose demethylation states correlated tightly with transcription activation, that we propose are the major contributing CpGs. Our work thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci during iCM reprogramming and could provide future genome-wide epigenetic studies with important guidance such as the appropriate time window and loci to be utilized as positive and negative controls.