Abstract
Reprogramming of somatic cells has become a versatile tool for biomedical research and for regenerative medicine. In the current study, we show that manipulating alternative splicing (AS) is a highly potent strategy to produce cells for therapeutic applications. We demonstrate that silencing of hTAF4-TAFH activity of TAF4 converts human facial dermal fibroblasts to melanocyte-like (iMel) cells. iMel cells produce melanin and express microphthalmia-associated transcription factor (MITF) and its target genes at levels comparable to normal melanocytes. Reprogramming of melanoma cells by manipulation with hTAF4-TAFH activity upon TAFH RNAi enforces cell differentiation towards chondrogenic pathway, whereas ectoptic expression of TAF4 results in enhanced multipotency and neural crest-like features in melanoma cells. In both cell states, iMels and cancer cells, hTAF4-TAFH activity controls migration by supporting E- to N-cadherin switches. From our data, we conclude that targeted splicing of hTAF4-TAFH coordinates AS of other TFIID subunits, underscoring the role of TAF4 in synchronised changes of Pol II complex composition essential for efficient cellular reprogramming. Taken together, targeted AS of TAF4 provides a unique strategy for generation of iMels and recapitulating stages of melanoma progression.
Highlights
It is commonly known that mechanisms of cellular reprogramming share similar features with cancer initiation[18]
To assess whether alternative splicing (AS) of TAF4 exons V, VI and VII encoding the hTAF4-TAFH (Fig. 1a) is prevailing in cells of neural crest (NC) origin, we examined the expression of TAF4 ASVs in facial dermal fibroblasts, melanocytes and melanomas
Using transcript-specific RT-PCR approach, we exclusively examined the splicing of TAF4 exons encoding hTAF4-TAFH domain
Summary
It is commonly known that mechanisms of cellular reprogramming share similar features with cancer initiation[18]. Pluripotency transcription factors c-MYC and KLF4 are commonly known as proto-oncogenes[19]; similar signalling pathways are active in cancer development and upon generation of iPSCs18,20. We provide a new concept for cell reprogramming, where instead of changing the transcription regulatory networks by forced expression of lineage-specific transcription factors or use of different miRNAs, we advocate for targeted AS of the core components of RNA Pol II transcription machinery. We targeted the activity of TAF4 by TAFH-specific RNAi to examine the potential of this approach for reprogramming of human dermal fibroblasts. Data presented here allow us to conclude that targeting AS of TAF4 affects the entire TFIID complex, providing a unique model system to induce iMels and reprogram tumour cells to less or more aggressive cancer phenotypes
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