Expression of TAF4 splice variants and isoforms in human MSCs differentiated into adipocytes, osteoblasts or chondrocytes and treated with <i>TAF4</i> RNAi.

<p>(<b>A</b>) RT-PCR analysis of different individual hMSCs clones (hMSCs 1–5) using <i>TAF4_v1a</i> (full length) specific primers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074799#pone.0074799.s002" target="_blank">Table S2</a>). Expression of <i>GAPDH</i> mRNA is shown at the bottom. (<b>B</b>) Expression of <i>TAF4</i> ASVs encoding proteins with compromised hTAF4-TAFH domain is dominant in differentiated hMSCs. RT-PCR analysis using <i>TAF4</i> ASV-specific primers was performed 7 days after induction of differentiation of hMSCs towards adipo-, osteo- and chondrogenic lineages. <i>GAPDH</i> mRNA expression was used for the normalization. (<b>C</b>) Expression of <i>TAF4</i> ASVs following treatment of human MSCs with control and hTAF4-TAFH-domain targeting <i>TAF4</i> siRNAs. Cells were treated with <i>TAF4</i> or control siRNAs at the indicated time points and RT-PCR analysis performed using <i>TAF4</i> ASV-specific primers. Analysis of <i>GAPDH</i> mRNA expression was used for normalization. (<b>D</b>) siRNA-mediated silencing targeting <i>TAF4</i> exons V or VI induces changes in the expression of TAF4 protein isoforms as detected at 48 h post-treatment using Western blot analysis. The asterisk indicates the canonical form of TAF4 protein with the molecular weight of 135 kDa, two asterisks indicate TAF4_v2 isoform with a calculated molecular weight of about 73 kDa.</p

Alternative Splicing Targeting the hTAF4-TAFH Domain of TAF4 Represses Proliferation and Accelerates Chondrogenic Differentiation of Human Mesenchymal Stem Cells

<div><p>Transcription factor IID (TFIID) activity can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. Alternative splicing of TFIID subunits is often the result of external stimulation of upstream signaling pathways. We studied tissue distribution and cellular expression of different splice variants of TFIID subunit <i>TAF4</i> mRNA and biochemical properties of its isoforms in human mesenchymal stem cells (hMSCs) to reveal the role of different isoforms of TAF4 in the regulation of proliferation and differentiation. Expression of <i>TAF4</i> transcripts with exons VI or VII deleted, which results in a structurally modified hTAF4-TAFH domain, increases during early differentiation of hMSCs into osteoblasts, adipocytes and chondrocytes. Functional analysis data reveals that TAF4 isoforms with the deleted hTAF4-TAFH domain repress proliferation of hMSCs and preferentially promote chondrogenic differentiation at the expense of other developmental pathways. This study also provides initial data showing possible cross-talks between TAF4 and TP53 activity and switching between canonical and non-canonical WNT signaling in the processes of proliferation and differentiation of hMSCs. We propose that TAF4 isoforms generated by the alternative splicing participate in the conversion of the cellular transcriptional programs from the maintenance of stem cell state to differentiation, particularly differentiation along the chondrogenic pathway.</p></div