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.

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 reprogramming of cardiac fibroblasts (CFs) to induced cardiomyocytes (iCMs) is a newly emerged promising approach for cardiac regeneration, disease modeling, and drug discovery. However, its potential has been drastically limited due to the low reprogramming efficiency and largely unknown underlying molecular mechanisms. We have previously screened and identified epigenetic factors related to histone modification during iCM reprogramming. Here, we used shRNAs targeting an additional battery of epigenetic factors involved in chromatin remodeling and RNA splicing factors to further identify inhibitors and facilitators of direct cardiac reprogramming. Knockdown of RNA splicing factors Sf3a1 or Sf3b1 significantly reduced the percentage and total number of cardiac marker positive iCMs accompanied with generally repressed gene expression. Removal of another RNA splicing factor Zrsr2 promoted the acquisition of CM molecular features in CFs and mouse embryonic fibroblasts (MEFs) at both protein and mRNA levels. Moreover, a consistent increase of reprogramming efficiency was observed in CFs and MEFs treated with shRNAs targeting Bcor (component of BCOR complex superfamily) or Stag2 (component of cohesin complex). Our work thus reveals several additional epigenetic and splicing factors that are either inhibitory to or required for iCM reprogramming and highlights the importance of epigenetic regulation and RNA splicing process during cell fate conversion.

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