We have inactivated transcription factor TFIID subunit TBP-associated factor 4 (TAF4) in mouse embryonic fibroblasts. Mutant taf4 À/À cells are viable and contain intact TFIID comprising the related TAF4b showing that TAF4 is not an essential protein. TAF4 inactivation deregulates more than 1000 genes indicating that TFIID complexes containing TAF4 and TAF4b have distinct target gene specificities. However, taf4 À/À cell lines have altered morphology and exhibit serum-independent autocrine growth correlated with the induced expression of several secreted mitotic factors and activators of the transforming growth factor b signalling pathway. In addition to TAF4 inactivation, many of these genes can also be induced by overexpression of TAF4b. A competitive equilibrium between TAF4 and TAF4b therefore regulates expression of genes controlling cell proliferation. We have further identified a set of genes that are regulated both by TAF4 and upon adaptation to serum starvation and which may be important downstream mediators of serum-independent growth. Our study also shows that TAF4 is an essential cofactor for activation by the retinoic acid receptor and CREB, but not for Sp1 and the vitamin D3 receptor.

The TEAD (1-4) transcription factors comprise the conserved TEA/ATTS DNA-binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy among the family members. Expression of the TEA/ATTS DNA-binding domain that acts as a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, whereas selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation. RNA-seq identifies a set of genes whose expression is strongly reduced upon TEAD4 knockdown among which are structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF (connective tissue growth factor) to promote differentiation. Together these results show that TEAD factor activity is essential for normal C2C12 cell differentiation and suggest a role for TEAD4 in regulating expression of the unfolded protein response genes. The TEAD transcription factors make a highly conserved family of 4 DNA-binding proteins 1,2 containing the TEA (Yeast (TEC-1), Aspergillus nidulans (AbaA) and Drosophilla (scalloped))/ATTS (Aspergillus nidulans (AbaA), Yeast (TEC-1), human TEF1, and Drosophilla (scalloped)) DNA-binding domain (DBD). 3,4 The TEA domain comprises a three-helix bundle with a homeodomain fold and binds a consensus MCAT (5 0 -CATTCCA/ T-3 0 ) element originally defined as the GT-II motif of the simian virus 40 (SV40) enhancer. 5 Mammalian TEADs are widely expressed with prominent expression in the nervous system and muscle. In-vitro, cell-based, knockout and transgenic studies have addressed the role of TEAD factors in regulation of muscle-expressed genes. 6-8 Cardiac troponin T, myosin, heavy polypeptide 7, cardiac muscle, beta (b-MHC) and Myocardin, have functional MCAT motifs in their regulatory regions. 2 Stimulation of a1-adrenergic signalling has been shown to induce cardiac hypertrophy and activate transcription of the b-MHC and skeletal a-actin genes in an MCAT-and TEAD-dependent manner in cultured neonatal rat cardiomyocytes. 9 Cardiac muscle-specific overexpression of TEAD4 in transgenic mice has been shown to induce arhythmias in vivo. 8 TEAD4 is specifically expressed in developing skeletal muscle in mouse embryos. 1 Chromatin immunoprecipitation-array hybridization (ChIP-chip) showed that TEAD4 is a direct target of the MYOD1 and MYOG transcription factors in C2C12 cells. 10 Although TEAD4 upregulation by MYOD1 and MYOG during differentiation is thought to activate transcription of muscle structural genes, mouse knockouts do not show any evident role for TEAD4 i...

Somatic oncogenic mutation of BRAF coupled with inactivation of PTEN constitute a frequent combination of genomic alterations driving the development of human melanoma. Mice genetically engineered to conditionally express oncogenic Braf V600E and inactivate Pten in melanocytes following tamoxifen treatment rapidly develop melanoma. While early-stage melanomas comprised melanin-pigmented Mitf and Dct-expressing cells, expression of these and other melanocyte identity genes was lost in later stage tumours that showed histological and molecular characteristics of de-differentiated neural crest type cells. Melanocyte identity genes displayed loss of active chromatin marks and RNA polymerase II and gain of heterochromatin marks, indicating epigenetic reprogramming during tumour progression. Nevertheless, late-stage tumour cells grown in culture re-expressed Mitf, and melanocyte markers and Mitf together with Sox10 coregulated a large number of genes essential for their growth. In this melanoma model, somatic inactivation that the catalytic Brg1 (Smarca4) subunit of the SWI/SNF complex and the scaffolding Bptf subunit of the NuRF complex delayed tumour formation and deregulated large and overlapping gene expression programs essential for normal tumour cell growth. Moreover, we show that Brg1 and Bptf coregulated many genes together with Mitf and Sox10. Together these transcription factors and chromatin remodelling complexes orchestrate essential gene expression programs in mouse melanoma cells.

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IntroductionIn recent work we could show that sensitized human T lymphocytes reveal altered STAT6/STAT1 in terms of DNA binding balance compared to normal controls [1,2]. As STAT3 is specific for Th17 signal transduction, and this cell type can play a dominant role in B cell regulation and induction of allergic, inflammatory and autoimmune disorders [3, for review 4], the aim of this paper was to investigate STAT3 activation in the down stream regulation of circulating human lymphocytes ex vivo. Material and methodsHuman PBMC from 5 atopic/non-atopic volunteers (IgE > 500 IU/ml, normal age matched controls: < 50 IU/ml) were stimulated by PHA and anti-CD3/IL-2 (1 ng/ml) in 3-day cultures. Allergic patients were suffering from seasonal hay fever and chronic atopic eczema. IL-23 (10 ng/ml) and histamine (2.5 mM) were added 4 hours post-plated. Cytokines were measured by enzyme linked immune sorbent assay (ELISA) and STAT-DNA interaction by electrophoretic mobility shift assay (EMSA), respectively. For EMSA, the infrared dye labelled double stranded DNA oligonucleotide STAT3 5'GAT CCT TCT GGG AAT TCC TAG ATC 3' (LI-COR Nebraska USA) was applied. PBMC differentiation was identified using cytoflow/FACS analyses. EMSA results were processed using the Gelscan V 5.1 software: P < 0.05 was considered statistically significant. Results and discussionIt could be shown that IL-17E as well as IL-4 was constitutively increased in the atopic samples and that anti-CD3 was a more effective stimulator than PHA compared to the non-atopic matched controls (Tab. 1). For IL-4, PHA was more effective than both anti-CD3 and IFN-g in the non-atopic group (p < 0.05, n = 5, Student t). The most augmented IL-17E-responses could be demonstrated in non-atopics using IL-23 as a stimulator (Tab. 1). The contrary effect was observed in the atopic group, where the initially high levels of IL-17E were down-regulated corresponding to the increase of Th1 (IFN-g) polarization. This is in a good accordance to recent work, where the IL-23/Th17-axis was shown to be the predominant regulator of inflammatory and allergic disorders [5]. In terms of a potential negative feedback of histamine it could be demonstrated that the lymphocyte proliferation and Th1 activity (IFN-g) was increased, thereby inducing down-regulation of the Th2/Th17-IL-17E activity (data not shown). This is in a good agreement to the recent observation indicating that histamine feedback responses were strongly concentration-dependent in human lymphocytes ex vivo [6]. Surprisingly, anti-CD3-stimulated DNA binding of STAT3 was suppressed after Th17-stimulatory IL-23 activation in the presence of IL-1ß with an inverse effect in the atopic group (Fig. 1). After addition of histamine (2.5 mM) the responses turned into the contrary. There was an increase in the atopic group but inhibition in the non-atopic group as it was demonstrated also for STAT1 in recent work [6].From these results it could be concluded that Th17 showed downstream regulatory potency with specific differences in atopic pat...