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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. [ABSTRACT FROM AUTHOR]

In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is specifically localized on the mate X chromosome to increase its expression ∼2-fold. We recently proposed a model for the targeted assembly of the MSL complex, in which initial binding occurs at ∼35 dispersed chromatin entry sites, followed by spreading in cis into flanking regions. Here, we analyze one of the chromatin entry sites, the roX1 gene, to determine which sequences are sufficient to recruit the MSL complex. We found association and spreading of the MSL complex from roX1 transgenes in the absence of detectable roX1 RNA synthesis from the transgene. We mapped the recruitment activity to a 217 bp roX1 fragment that shows mate-specific DNase hypersensitivity and can be preferentially cross-linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is sufficient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kitobases into neighboring regions. [ABSTRACT FROM AUTHOR]

Super-enhancers (SEs) are stretches of enhancers ensuring a high level of expression of key genes associated with cell function. The identification of cancer-specific SE-driven genes is a powerful means for the development of innovative therapeutic strategies. Here, we identify a MITF/SOX10/TFIIH-dependent SE promoting the expression of BAHCC1 in a broad panel of melanoma cells. BAHCC1 is highly expressed in metastatic melanoma and is required for tumor engraftment, growth, and dissemination. Integrative genomics analyses reveal that BAHCC1 is a transcriptional regulator controlling expression of E2F/KLF-dependent cell-cycle and DNA-repair genes. BAHCC1 associates with BRG1-containing remodeling complexes at the promoters of these genes. BAHCC1 silencing leads to decreased cell proliferation and delayed DNA repair. Consequently, BAHCC1 deficiency cooperates with PARP inhibition to induce melanoma cell death. Our study identifies BAHCC1 as an SE-driven gene expressed in melanoma and demonstrates how its inhibition can be exploited as a therapeutic target., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. Tissue-specific inactivation of Taf4 in murine intestinal epithelium during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells. In adults, Taf4 loss impacted the stem cell compartment and associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge. Taf4 inactivation ex vivo in enteroids prevented budding formation and maintenance and caused broad chromatin remodeling and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). Treatment of Taf4-mutant enteroids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Taf4 loss also led to increased PRC2 activity in cells of adult crypts associated with modification of the immune/inflammatory microenvironment that potentiated Apc-driven tumorigenesis. Our results reveal a novel function of Taf4 in antagonizing PRC2-mediated repression of the stem cell gene expression program to assure normal development, homeostasis, and immune-microenvironment of the intestinal epithelium., (© 2023. The Author(s).)

Tumor heterogeneity is a key feature of melanomas that hinders development of effective treatments. Aiming to overcome this, we identified LINC00518 (LENOX; lincRNA-enhancer of oxidative phosphorylation) as a melanoma-specific lncRNA expressed in all known melanoma cell states and essential for melanoma survival in vitro and in vivo. Mechanistically, LENOX promoted association of the RAP2C GTPase with mitochondrial fission regulator DRP1, increasing DRP1 S637 phosphorylation, mitochondrial fusion, and oxidative phosphorylation. LENOX expression was upregulated following treatment with MAPK inhibitors, facilitating a metabolic switch from glycolysis to oxidative phosphorylation and conferring resistance to MAPK inhibition. Consequently, combined silencing of LENOX and RAP2C synergized with MAPK inhibitors to eradicate melanoma cells. Melanomas are thus addicted to the lncRNA LENOX, which acts to optimize mitochondrial function during melanoma development and progression., Significance: The lncRNA LENOX is a novel regulator of melanoma metabolism, which can be targeted in conjunction with MAPK inhibitors to eradicate melanoma cells., (©2022 The Authors; Published by the American Association for Cancer Research.)

Malignant melanoma is a major public health issue displaying frequent resistance to targeted therapy and immunotherapy. A major challenge lies in better understanding how melanoma cells evade immune elimination and how tumor growth and metastasis is facilitated by the tumor microenvironment. Here, we show that expression of the cytokine thymic stromal lymphopoietin (TSLP) by epidermal keratinocytes is induced by cutaneous melanoma in both mice and humans. Using genetically engineered models of melanoma and tumor cell grafting combined with TSLP-KO or overexpression, we defined a crosstalk between melanoma cells, keratinocytes, and immune cells in establishing a tumor-promoting microenvironment. Keratinocyte-derived TSLP is induced by signals derived from melanoma cells and subsequently acts via immune cells to promote melanoma progression and metastasis. Furthermore, we show that TSLP signals through TSLP receptor-expressing (TSLPR-expressing) DCs to play an unrecognized role in promoting GATA3+ Tregs expressing a gene signature including ST2, CCR8, ICOS, PD-1, CTLA-4, and OX40 and exhibiting a potent suppressive activity on CD8+ T cell proliferation and IFN-γ production. An analogous population of GATA3-expressing Tregs was also identified in human melanoma tumors. Our study provides insights into the role of TSLP in programming a protumoral immune microenvironment in cutaneous melanoma.

Regulation of gene expression involves a complex and dynamic dialogue between transcription factors, chromatin remodelling and modification complexes and the basal transcription machinery. To address the function of the Taf4 subunit of general transcription factor TFIID in the regulation of insulin signalling, it was inactivated in adult murine pancreatic beta cells. Taf4 inactivation impacted the expression of critical genes involved in beta-cell function leading to increased glycaemia, lowered plasma insulin levels and defective glucose-stimulated insulin secretion. One week after Taf4-loss, single-cell RNA-seq revealed cells with mixed beta cell, alpha and/or delta cell identities as well as a beta cell population trans-differentiating into alpha-like cells. Computational analysis of single-cell RNA-seq defines how known critical beta cell and alpha cell determinants may act in combination with additional transcription factors and the NuRF chromatin remodelling complex to promote beta cell trans-differentiation., (© 2021. The Author(s).)

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.

In two independent consanguineous families each with two children affected by mild intellectual disability and microcephaly, we identified two homozygous missense variants (c.119T>A [p.Met40Lys] and c.92T>A [p.Leu31His]) in TATA-box-binding-protein-associated factor 13 (TAF13). Molecular modeling suggested a pathogenic effect of both variants through disruption of the interaction between TAF13 and TAF11. These two proteins form a histone-like heterodimer that is essential for their recruitment into the general RNA polymerase II transcription factor IID (TFIID) complex. Co-immunoprecipitation in HeLa cells transfected with plasmids encoding TAF11 and TAF13 revealed that both variants indeed impaired formation of the TAF13-TAF11 heterodimer, thus confirming the protein modeling analysis. To further understand the functional role of TAF13, we performed RNA sequencing of neuroblastoma cell lines upon TAF13 knockdown. The transcriptional profile showed significant deregulation of gene expression patterns with an emphasis on genes related to neuronal and skeletal functions and those containing E-box motives in their promoters. Here, we expand the spectrum of TAF-associated phenotypes and highlight the importance of TAF13 in neuronal functions., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation.

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a(-/-) embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a(-/-) embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a(-/-) ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis.

There is a paucity of new therapeutic targets to control allergic reactions and forestall the rising trend of allergic diseases. Although a variety of immune cells contribute to allergy, cytokine-secreting αβ(+)CD4(+) T-helper 2 (TH2) cells orchestrate the type-2-driven immune response in a large proportion of atopic asthmatics. To identify previously unidentified putative targets in pathogenic TH2 cells, we performed in silico analyses of recently published transcriptional data from a wide variety of pathogenic TH cells [Okoye IS, et al. (2014) Proc Natl Acad Sci USA 111(30):E3081-E3090] and identified that transcription intermediary factor 1 regulator-alpha (Tif1α)/tripartite motif-containing 24 (Trim24) was predicted to be active in house dust mite (HDM)- and helminth-elicited Il4(gfp+)αβ(+)CD4(+) TH2 cells but not in TH1, TH17, or Treg cells. Testing this prediction, we restricted Trim24 deficiency to T cells by using a mixed bone marrow chimera system and found that T-cell-intrinsic Trim24 is essential for HDM-mediated airway allergy and antihelminth immunity. Mechanistically, HDM-elicited Trim24(-/-) T cells have reduced expression of many TH2 cytokines and chemokines and were predicted to have compromised IL-1-regulated signaling. Following this prediction, we found that Trim24(-/-) T cells have reduced IL-1 receptor (IL-1R) expression, are refractory to IL-1β-mediated activation in vitro and in vivo, and fail to respond to IL-1β-exacerbated airway allergy. Collectively, these data identify a previously unappreciated Trim24-dependent requirement for IL-1R expression on TH2 cells and an important nonredundant role for T-cell-intrinsic Trim24 in TH2-mediated allergy and antihelminth immunity.

MIcrophthalmia-associated Transcription Factor (MITF) regulates melanocyte and melanoma physiology. We show that MITF associates the NURF chromatin-remodelling factor in melanoma cells. ShRNA-mediated silencing of the NURF subunit BPTF revealed its essential role in several melanoma cell lines and in untransformed melanocytes in vitro. Comparative RNA-seq shows that MITF and BPTF co-regulate overlapping gene expression programs in cell lines in vitro. Somatic and specific inactivation of Bptf in developing murine melanoblasts in vivo shows that Bptf regulates their proliferation, migration and morphology. Once born, Bptf-mutant mice display premature greying where the second post-natal coat is white. This second coat is normally pigmented by differentiated melanocytes derived from the adult melanocyte stem cell (MSC) population that is stimulated to proliferate and differentiate at anagen. An MSC population is established and maintained throughout the life of the Bptf-mutant mice, but these MSCs are abnormal and at anagen, give rise to reduced numbers of transient amplifying cells (TACs) that do not express melanocyte markers and fail to differentiate into mature melanin producing melanocytes. MSCs display a transcriptionally repressed chromatin state and Bptf is essential for reactivation of the melanocyte gene expression program at anagen, the subsequent normal proliferation of TACs and their differentiation into mature melanocytes.

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

The functions of the TAF subunits of mammalian TFIID in physiological processes remain poorly characterised. In this study, we describe a novel function of TAFs in directing genomic occupancy of a transcriptional activator. Using liver-specific inactivation in mice, we show that the TAF4 subunit of TFIID is required for post-natal hepatocyte maturation. TAF4 promotes pre-initiation complex (PIC) formation at post-natal expressed liver function genes and down-regulates a subset of embryonic expressed genes by increased RNA polymerase II pausing. The TAF4-TAF12 heterodimer interacts directly with HNF4A and in vivo TAF4 is necessary to maintain HNF4A-directed embryonic gene expression at post-natal stages and promotes HNF4A occupancy of functional cis-regulatory elements adjacent to the transcription start sites of post-natal expressed genes. Stable HNF4A occupancy of these regulatory elements requires TAF4-dependent PIC formation highlighting that these are mutually dependent events. Local promoter-proximal HNF4A-TFIID interactions therefore act as instructive signals for post-natal hepatocyte differentiation.

The major core promoter-binding factor in polymerase II transcription machinery is TFIID, a complex consisting of TBP, the TATA box-binding protein, and 13 to 14 TBP-associated factors (TAFs). Previously we found that the histone H2A-like TAF paralogs TAF4 and TAF4b possess DNA-binding activity. Whether TAF4/TAF4b DNA binding directs TFIID to a specific core promoter element or facilitates TFIID binding to established core promoter elements is not known. Here we analyzed the mode of TAF4b.TAF12 DNA binding and show that this complex binds DNA with high affinity. The DNA length required for optimal binding is approximately 70 bp. Although the complex displays a weak sequence preference, the nucleotide composition is less important than the length of the DNA for high affinity binding. Comparative expression profiling of wild-type and a DNA-binding mutant of TAF4 revealed common core promoter features in the down-regulated genes that include a TATA-box and an Initiator. Further examination of the PEL98 gene from this group showed diminished Initiator activity and TFIID occupancy in TAF4 DNA-binding mutant cells. These findings suggest that DNA binding by TAF4/4b-TAF12 facilitates the association of TFIID with the core promoter of a subset of genes.

The TAF4 subunit of transcription factor TFIID was inactivated in the basal keratinocytes of foetal and adult mouse epidermis. Loss of TAF4 in the foetal epidermis results in reduced expression of the genes required for skin barrier function, leading to early neonatal death. By contrast, TAF4 inactivation in adult epidermis leads to extensive fur loss and an aberrant hair cycle characterised by a defective anagen phase. Although the mutant epidermis contains few normal anagen-phase hair follicles, many genes expressed at this stage are strongly upregulated indicating desynchronized and inappropriate gene expression. The TAF4 mutant adult epidermis also displays interfollicular hyperplasia associated with a potent upregulation of several members of the EGF family of mitogens. Moreover, loss of TAF4 leads to malignant transformation of chemically induced papillomas and the appearance of invasive melanocytic tumours. Together, our results show that TAF4 is an important regulator of keratinocyte proliferation and has cell-autonomous and non-cell-autonomous tumour suppressor activity.

Monozygotic twin brothers with a subtelomeric 6q deletion presented with mental retardation, microcephaly, seizures, an enlarged cisterna magna, dimpling at elbows, a high arched palate and a thin upper lip. The same subtelomeric deletion was detected in the mother of the patients, presenting with a milder phenotype. We narrowed down the breakpoint to a region of approximately 100 kb and estimated the size of the terminal deletion to be 1.2 Mb. This region contains four known and seven putative genes. Comparison of the deletion with other reported patients showed TBP was the most plausible candidate gene for the mental retardation in this syndrome. We verified that the TBP gene expression was halved in our patients using real-time PCR. Cognitive and behavioural tests performed on previously described heterozygous tbp mice suggested that TBP is potentially involved in cognitive development.

RNA polymerase II general transcription factor TFIID is a macromolecular complex comprising the TATA-binding protein, TBP and 13-14 evolutionary conserved TBP-associated factors, TAFs. Although genetic experiments have shown that TAFs are essential for cell cycle progression in yeast and in rapidly proliferating vertebrate cells in vitro, new experiments indicate they may be dispensible in specific developmental and physiological processes. Moreover, the TAF4 subunit of TFIID negatively regulates proliferation by inhibiting activation of the TGFbeta signalling pathway by its paralogue TAF4b. TAF4 is however essential in the retinoic acid and cAMP signalling pathways acting as a cofactor for CREB and the retinoic acid receptor, but is a negative regulator of the ATF7 transcription factor.

We have inactivated transcription factor TFIID subunit TBP-associated factor 4 (TAF4) in mouse embryonic fibroblasts. Mutant taf4(-/-) 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(-/-) 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 beta 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.

Dosage compensation in flies involves doubling the transcription of genes on the single male X chromosome to match the combined expression level of the two female X chromosomes. Crucial for this activation is the acetylation of histone H4 by the histone acetyltransferase (HAT) MOF. In male cells, MOF resides in a complex (dosage compensation complex, DCC) with MSL proteins and noncoding roX RNA. Previous studies suggested that MOF's localization to the X chromosome was largely RNA-mediated. We now found that contact of the MOF chromo-related domain with roX RNA plays only a minor role in correct targeting to the X chromosome in vivo. Instead, a strong, direct interaction between a conserved MSL1 domain and a zinc finger within MOF's HAT domain is crucial. The functional consequences of this interaction were studied in vitro. Simultaneous contact of MOF with MSL1 and MSL3 led to its recruitment to chromatin, a dramatic stimulation of HAT activity and to improved substrate specificity. Activation of MOF's HAT activity upon integration into the DCC may serve to restrict the critical histone modification to the male X chromosome.

Transcription regulation in male germ cells can involve specialised mechanisms and testis-specific paralogues of the general transcription machinery. Here we describe TAF7L, a germ-cell-specific paralogue of the TFIID subunit TAF7. TAF7L is expressed through most of the male germ-cell differentiation programme, but its intracellular localisation is dynamically regulated from cytoplasmic in spermatogonia and early spermatocytes to nuclear in late pachytene spermatocytes and haploid round spermatids. Import of TAF7L into the nucleus coincides with decreased TAF7 expression and a strong increase in nuclear TBP expression, which suggests that TAF7L replaces TAF7 as a TFIID subunit in late pachytene spermatocytes and in haploid cells. In agreement with this, biochemical experiments indicate that a subpopulation of TAF7L is tightly associated with TBP in both pachytene and haploid cells and TAF7L interacts with the TFIID subunit TAF1. We further show that TAF3, TAF4 and TAF10 are all strongly expressed in early spermatocytes, but that in contrast to TBP and TAF7L, they are downregulated in haploid cells. Hence, different subunits of the TFIID complex are regulated in distinct ways during male germ-cell differentiation. These results show for the first time how the composition of a general transcription factor such as TFIID and other TAF-containing complexes are modulated during a differentiation programme highlighting the unique nature of the transcription regulatory machinery in spermatogenesis.

MSL complexes bind the single male X chromosome in Drosophila to increase transcription approximately 2-fold. Complexes contain at least five proteins and two noncoding RNAs, roX1 and roX2. The mechanism of X chromosome binding is not known. Here, we identify a 110 bp sequence in roX2 characterized by high-affinity MSL binding, male-specific DNase I hypersensitivity, a shared consensus with the otherwise dissimilar roX1 gene, and conservation across species. Mutagenesis of evolutionarily conserved sequences diminishes MSL binding in vivo. MSL binding to these sites is roX RNA dependent, suggesting that complexes become competent for binding only after incorporation of roX RNAs. However, the roX RNA segments homologous to the DNA binding sites are not required, ruling out simple RNA-DNA complementarity as the primary targeting mechanism. Our results are consistent with a model in which nascent roX RNA assembly with MSL proteins is an early step in the initiation of dosage compensation.

Coexpression of the human TATA-binding protein (TBP)-associated factor 28 (hTAFII28) with the altered-specificity mutant TBP spm3 synergistically enhances transcriptional activation by the activation function 2 of the nuclear receptors (NRs) for estrogen and vitamin D3 from a reporter plasmid containing a TGTA element in mammalian cells. This synergy is abolished by mutation of specific amino acids in the alpha2-helix of the histone fold in the conserved C-terminal region of hTAFII28. Critical amino acids are found on both the exposed hydrophilic face of this helix and the hydrophobic interface with TAFII18. This alpha-helix of hTAFII28 therefore mediates multiple interactions required for coactivator activity. We further show that mutation of specific residues in the H1' alpha-helix of TBP either reduces or increases interactions with hTAFII28. The mutations which reduce interactions with hTAFII28 do not affect functional synergy, whereas the TBP mutation which increases interaction with hTAFII28 is defective in its ability to synergistically enhance activation by NRs. However, this TBP mutant supports activation by other activators and is thus specifically defective for its ability to synergize with hTAFII28.

Determination of the crystal structure of the human TBP-associated factor (hTAF(II))28/hTAF(II)18 heterodimer shows that these TAF(II)s form a novel histone-like pair in the TFIID complex. The histone folds in hTAF(II)28 and hTAF(II)18 were not predicted from their primary sequence, indicating that these TAF(II)s define a novel family of atypical histone fold sequences. The TAF(II)18 and TAF(II)28 histone fold motifs are also present in the N- and C-terminal regions of the SPT3 proteins, suggesting that the histone fold in SPT3 may be reconstituted by intramolecular rather than classical intermolecular interactions. The existence of additional histone-like pairs in both the TFIID and SAGA complexes shows that the histone fold is a more commonly used motif for mediating TAF-TAF interactions than previously believed.

Human papillomavirus 8 (HPV8) is one of the oncogenic HPV types specifically associated with skin cancers of epidermodysplasia verruciformis patients. The early gene products of this virus exert functions in transformation (E2, E6, E7), replication (E1, E2) and in the control of viral transcription (E2, E7). Many viral and cellular transactivators of transcription have been shown to interact selectively and directly with a number of TATA-box-binding protein (TBP)-associated factors (TAFIIs), which then play a role as coactivators. Using glutathione-S-transferase (GST) pull-down experiments, we tested in vitro interactions between GST-HPV8-E1, -E2, -E6 and -E7 and 7 in-vitro-translated TAFIIs in the human (h) system (hTAFII18, hTAFII20, hTAFII28, hTAFII30, hTAFII55, hTAFII100, hTAFIIDeltaN135) or TBP. We could show that GST-HPV8-E2 interacts directly at least with hTAFII55 and TBP. Deletion analysis indicated that a domain overlapping with the C-terminal moiety of HPV8-E2 is required for binding to TBP, whereas determinants for interactions with hTAFII55 are in the central and C-terminal part of the E2 protein. In similar binding studies, GST-HPV8-E6 interacted with hTAFII28, hTAFIIDeltaN135 and TBP, and more weakly with hTAFII20, whereas GST- HPV8-E7 bound to hTAFII20, hTAFII28, hTAFII55, hTAFIIDeltaN135 and TBP. Deletion analysis revealed that the C-terminal part of HPV8-E7 is required for the interaction with these hTAFIIs. In contrast, no interactions were observed between GST-HPV8-E1 and in-vitro-translated hTAFIIs.

The Tax protein encoded by human T cell leukemia virus type I transactivates the viral promoter by forming a complex with several cellular factors bound to three repeats of a specific upstream regulatory sequence. We have shown that transactivation by Tax was correlated with its ability to interact with the C-terminal moiety of the TATA box-binding protein (TBP). In the present study, the ability of Tax to interact with several human TBP-associated factors (TAF(II)s) was analyzed. We show that Tax interacts selectively with hTAF(II)28 in transfected HeLa cells. A direct interaction between Tax and hTAF(II)28 was also observed in vitro with purified proteins. In transient expression studies we show that overexpression of hTAF(II)28 significantly increased transactivation by Tax, both in the absence and in the presence of overexpressed TBP. The ability of hTAF(II)28 to potentiate transactivation correlated with the ability of Tax to interact with hTAF(II)28 and also with the ability of hTAF(II)28 to interact with TBP. Coexpression of TBP and hTAF(II)28 resulted in an additive increase in transactivation by Tax. From these observations we propose that transcriptional activation by Tax involves multiple interactions with TFIID via its TBP and hTAF(II)28 subunits.