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 TAF4 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 TAF4 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.

BackgroundNeuropathological findings support an autoimmune etiology as an underlying factor for loss of orexin-producing neurons in spontaneous narcolepsy type 1 (narcolepsy with cataplexy; sNT1) as well as in Pandemrix influenza vaccine-induced narcolepsy type 1 (Pdmx-NT1). The precise molecular target or antigens for the immune response have, however, remained elusive.MethodsHere we have performed a comprehensive antigenic repertoire analysis of sera using the next-generation phage display method - mimotope variation analysis (MVA). Samples from 64 children and adolescents were analyzed: 10 with Pdmx-NT1, 6 with sNT1, 16 Pandemrix-vaccinated, 16 H1N1 infected, and 16 unvaccinated healthy individuals. The diagnosis of NT1 was defined by the American Academy of Sleep Medicine international criteria of sleep disorders v3.FindingsOur data showed that although the immunoprofiles toward vaccination were generally similar in study groups, there were also striking differences in immunoprofiles between sNT1 and Pdmx-NT1 groups as compared with controls. Prominent immune response was observed to a peptide epitope derived from prostaglandin D2 receptor (DP1), as well as peptides homologous to B cell lymphoma 6 protein. Further validation confirmed that these can act as true antigenic targets in discriminating NT1 diseased along with a novel epitope of hemagglutinin of H1N1 to delineate exposure to H1N1.InterpretationWe propose that DP1 is a novel molecular target of autoimmune response and presents a potential diagnostic biomarker for NT1. DP1 is involved in the regulation of non-rapid eye movement (NREM) sleep and thus alterations in its functions could contribute to the disturbed sleep regulation in NT1 that warrants further studies. Together our results also show that MVA is a helpful method for finding novel peptide antigens to classify human autoimmune diseases, possibly facilitating the design of better therapies.

Changes in alternative splicing have been linked to cancer development. We hypothesized that changes occurring in tumor tissue can also be detected in the peripheral blood of cancer patients leading to discovery of blood biomarkers of breast cancer. Alternative splicing profiles of 94 genes were examined in cancerous breast tissue. Discriminating splice variants were analyzed in the peripheral blood of early stage (BCI/II) (stage I-II; n = 26), neoadjuvant receiving locally advanced breast cancer patients (LABC) (stage IIb-IIIa, b; n = 10) and healthy volunteers (n = 26) using qRT-PCR analysis. Changes in marker expression during neoadjuvant therapy were analyzed at 15 timepoints. High expression of REST-N50, the alternatively spliced variant of REST, was detected in the blood of LABC patients but not in BCI/II and healthy controls (p = 0.0032 and p = 0.0029, respectively). Expression levels of DOPEY1v2, the alternative splice variant of DOPEY1, in the blood could differentiate cancer from healthy controls (p = 0.024) and discriminate between patient groups (BCI/II vs LABC, p = 0.002). Positive response to neoadjuvant therapy of REST-N50-positive LABC patients correlated with a decrease in REST-N50 levels (p < 0.0001). Assessment of REST-N50 and DOPEY1v2 may prove useful in diagnostic blood tests of breast cancer. REST-N50 shows a high potential as a blood biomarker for evaluating the effectiveness of therapy in the neoadjuvant setting.