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1.  Structural Basis of Natural Promoter Recognition by the Retinoid X Nuclear Receptor 
Scientific Reports  2015;5:8216.
Retinoid X receptors (RXRs) act as homodimers or heterodimerisation partners of class II nuclear receptors. RXR homo- and heterodimers bind direct repeats of the half-site (A/G)G(G/T)TCA separated by 1 nucleotide (DR1). We present a structural characterization of RXR-DNA binding domain (DBD) homodimers on several natural DR1s and an idealized symmetric DR1. Homodimers displayed asymmetric binding, with critical high-affinity interactions accounting for the 3′ positioning of RXR in heterodimers on DR1s. Differing half-site and spacer DNA sequence induce changes in RXR-DBD homodimer conformation notably in the dimerization interface such that natural DR1s are bound with higher affinity than an idealized symmetric DR1. Subtle changes in the consensus DR1 DNA sequence therefore specify binding affinity through altered RXR-DBD-DNA contacts and changes in DBD conformation suggesting a general model whereby preferential half-site recognition determines polarity of heterodimer binding to response elements.
PMCID: PMC4314640  PMID: 25645674
2.  Regulatory and Functional Connection of Microphthalmia-Associated Transcription Factor and Anti-Metastatic Pigment Epithelium Derived Factor in Melanoma1 
Neoplasia (New York, N.Y.)  2014;16(6):529-542.
Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor superfamily, has potent anti-metastatic effects in cutaneous melanoma through its direct actions on endothelial and melanoma cells. Here we show that PEDF expression positively correlates with microphthalmia-associated transcription factor (MITF) in melanoma cell lines and human samples. High PEDF and MITF expression is characteristic of low aggressive melanomas classified according to molecular and pathological criteria, whereas both factors are decreased in senescent melanocytes and naevi. Importantly, MITF silencing down-regulates PEDF expression in melanoma cell lines and primary melanocytes, suggesting that the correlation in the expression reflects a causal relationship. In agreement, analysis of Chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq) data sets revealed three MITF binding regions within the first intron of SERPINF1, and reporter assays demonstrated that the binding of MITF to these regions is sufficient to drive transcription. Finally, we demonstrate that exogenous PEDF expression efficiently halts in vitro migration and invasion, as well as in vivo dissemination of melanoma cells induced by MITF silencing. In summary, these results identify PEDF as a novel transcriptional target of MITF and support a relevant functional role for the MITF-PEDF axis in the biology of melanoma.
PMCID: PMC4198745  PMID: 25030625
Abbreviations; PEDF, pigment epithelium-derived factor; MITF, microphthalmia-associated transcription factor; RGP, radial growth phase of melanoma; VGP, vertical growth phase of melanoma; CM, cutaneous metastasis of melanoma; VM, visceral metastasis of melanoma; BRAF, v-raf murine sarcoma viral oncogene homolog B; NRAS, neuroblastoma RAS viral (v-ras) oncogene homolog; OIS, oncogene induced senescence; hnRNA, heterogeneous nuclear RNA
3.  SIRT1 promotes proliferation and inhibits the senescence-like phenotype in human melanoma cells 
Oncotarget  2014;5(8):2085-2095.
SIRT1 operates as both a tumor suppressor and oncogenic factor depending on the cell context. Whether SIRT1 plays a role in melanoma biology remained poorly elucidated. Here, we demonstrate that SIRT1 is a critical regulator of melanoma cell proliferation. SIRT1 suppression by genetic or pharmacological approaches induces cell cycle arrest and a senescence-like phenotype. Gain and loss of function experiments show that M-MITF regulates SIRT1 expression, thereby revealing a melanocyte-specific control of SIRT1. SIRT1 over-expression relieves the senescence-like phenotype and the proliferation arrest caused by MITF suppression, demonstrating that SIRT1 is an effector of MITF-induced proliferation in melanoma cells. Interestingly, SIRT1 level and activity are enhanced in the PLX4032-resistant BRAFV600E-mutated melanoma cells compared with their sensitive counterpart. SIRT1 inhibition decreases melanoma cell growth and rescues the sensibility to PLX4032 of PLX4032-resistant BRAFV600E-mutated melanoma cells. In conclusion, we provide the first evidence that inhibition of SIRT1 warrants consideration as an anti-melanoma therapeutic option.
PMCID: PMC4039147  PMID: 24742694
melanoma; MITF; SIRT1; PLX4032; treatment
4.  TAF4 Inactivation Reveals the 3 Dimensional Growth Promoting Activities of Collagen 6A3 
PLoS ONE  2014;9(2):e87365.
Collagen 6A3 (Col6a3), a component of extracellular matrix, is often up-regulated in tumours and is believed to play a pro-oncogenic role. However the mechanisms of its tumorigenic activity are poorly understood. We show here that Col6a3 is highly expressed in densely growing mouse embryonic fibroblasts (MEFs). In MEFs where the TAF4 subunit of general transcription factor IID (TFIID) has been inactivated, elevated Col6a3 expression prevents contact inhibition promoting their 3 dimensional growth as foci and fibrospheres. Analyses of gene expression in densely growing Taf4−/− MEFs revealed repression of the Hippo pathway and activation of Wnt signalling. The Hippo activator Kibra/Wwc1 is repressed under dense conditions in Taf4−/− MEFs, leading to nuclear accumulation of the proliferation factor YAP1 in the cells forming 3D foci. At the same time, Wnt9a is activated and the Sfrp2 antagonist of Wnt signalling is repressed. Surprisingly, treatment of Taf4−/− MEFs with all-trans retinoic acid (ATRA) restores contact inhibition suppressing 3D growth. ATRA represses Col6a3 expression independently of TAF4 expression and Col6a3 silencing is sufficient to restore contact inhibition in Taf4−/− MEFs and to suppress 3D growth by reactivating Kibra expression to induce Hippo signalling and by inducing Sfrp2 expression to antagonize Wnt signalling. All together, these results reveal a critical role for Col6a3 in regulating both Hippo and Wnt signalling to promote 3D growth, and show that the TFIID subunit TAF4 is essential to restrain the growth promoting properties of Col6a3. Our data provide new insight into the role of extra cellular matrix components in regulating cell growth.
PMCID: PMC3911972  PMID: 24498316
5.  Unique Aspects of Transcription Regulation in Male Germ Cells 
Spermatogenesis is a complex and ordered differentiation process in which the spermatogonial stem cell population gives rise to primary spermatocytes that undergo two successive meiotic divisions followed by a major biochemical and structural reorganization of the haploid cells to generate mature elongate spermatids. The transcriptional regulatory programs that orchestrate this process have been intensively studied in model organisms such as Drosophila melanogaster and mouse. Genetic and biochemical approaches have identified the factors involved and revealed mechanisms of action that are unique to male germ cells. In a well-studied example, cofactors and pathways distinct from those used in somatic tissues mediate the action of CREM in male germ cells. But perhaps the most striking feature concerns the paralogs of somatically expressed transcription factors and of components of the general transcription machinery that act in distinct regulatory mechanisms in both Drosophila and murine spermatogenesis.
Distinct regulatory mechanisms in Drosophila and murine spermatogenesis have been identified involving paralogs of somatically expressed transcription factors and components of the general transcription machinery (e.g., TAF proteins).
PMCID: PMC3119912  PMID: 21555408
6.  Phosphorylation of BRN2 Modulates Its Interaction with the Pax3 Promoter To Control Melanocyte Migration and Proliferation 
Molecular and Cellular Biology  2012;32(7):1237-1247.
MITF-M and PAX3 are proteins central to the establishment and transformation of the melanocyte lineage. They control various cellular mechanisms, including migration and proliferation. BRN2 is a POU domain transcription factor expressed in melanoma cell lines and is involved in proliferation and invasion, at least in part by regulating the expression of MITF-M and PAX3. The T361 and S362 residues of BRN2, both in the POU domain, are conserved throughout the POU protein family and are targets for phosphorylation, but their roles in vivo remain unknown. To examine the role of this phosphorylation, we generated mutant BRN2 in which these two residues were replaced with alanines (BRN2TS→BRN2AA). When expressed in melanocytes in vitro or in the melanocyte lineage in transgenic mice, BRN2TS induced proliferation and repressed migration, whereas BRN2AA repressed both proliferation and migration. BRN2TS and BRN2AA bound and repressed the MITF-M promoter, whereas PAX3 transcription was induced by BRN2TS but repressed by BRN2AA. Expression of the BRN2AA transgene in a Mitf heterozygous background and in a Pax3 mutant background enhanced the coat color phenotype. Our findings show that melanocyte migration and proliferation are controlled both through the regulation of PAX3 by nonphosphorylated BRN2 and through the regulation of MITF-M by the overall BRN2 level.
PMCID: PMC3302439  PMID: 22290434
7.  Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair 
Cell  2011;146(1):67-79.
DNA methylation is a major epigenetic mechanism for gene silencing. While methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here we show that either knockout or catalytic inactivation of the DNA repair enzyme Thymine DNA Glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific, developmentally- and hormonally-regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage-response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.
PMCID: PMC3230223  PMID: 21722948
embryonic lethality; base excision repair; CpG dinucleotides; DNA demethylation; promoter methylation profiles
8.  Interconversion between active and inactive TATA-binding protein transcription complexes in the mouse genome 
Nucleic Acids Research  2011;40(4):1446-1459.
The TATA binding protein (TBP) plays a pivotal role in RNA polymerase II (Pol II) transcription through incorporation into the TFIID and B-TFIID complexes. The role of mammalian B-TFIID composed of TBP and B-TAF1 is poorly understood. Using a complementation system in genetically modified mouse cells where endogenous TBP can be conditionally inactivated and replaced by exogenous mutant TBP coupled to tandem affinity purification and mass spectrometry, we identify two TBP mutations, R188E and K243E, that disrupt the TBP–BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not generally alter gene expression or genomic distribution of TBP, but positively or negatively affects TBP and/or Pol II recruitment to a subset of promoters. We identify promoters where wild-type TBP assembles a partial inactive preinitiation complex comprising B-TFIID, TFIIB and Mediator complex, but lacking TFIID, TFIIE and Pol II. Exchange of B-TFIID in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters completes preinitiation complex formation and recruits Pol II to activate their expression. We propose a novel regulatory mechanism involving formation of a partial preinitiation complex comprising B-TFIID that primes the promoter for productive preinitiation complex formation in mammalian cells.
PMCID: PMC3287176  PMID: 22013162
9.  The TIF1α-related TRIM cofactors couple chromatin modifications to transcriptional regulation, signaling and tumor suppression 
Transcription  2011;2(5):231-236.
TRIM24 (TIF1α), TRIM28 (TIF1β) and TRIM33 (TIF1γ) are related cofactors defining a subgroup of the tripartite motif (TRIM) superfamily comprising an N-terminal RING finger E3 ligase and a C-terminal PHD-Bromodomain chromatin interacting module. Increasing evidence highlights the important roles of these proteins as modulators of multiple signaling pathways during normal development and as tumor suppressors. The finding that they interact to form a multiprotein complex suggests new mechanisms to integrate multiple signaling pathways for tumor suppression.
PMCID: PMC3265781  PMID: 22231120
retinoic acid; transcriptional repression; hematopoeisis; TGFβ; SMAD4
10.  seqMINER: an integrated ChIP-seq data interpretation platform 
Nucleic Acids Research  2010;39(6):e35.
In a single experiment, chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq) provides genome-wide information about a given covalent histone modification or transcription factor occupancy. However, time efficient bioinformatics resources for extracting biological meaning out of these gigabyte-scale datasets are often a limiting factor for data interpretation by biologists. We created an integrated portable ChIP-seq data interpretation platform called seqMINER, with optimized performances for efficient handling of multiple genome-wide datasets. seqMINER allows comparison and integration of multiple ChIP-seq datasets and extraction of qualitative as well as quantitative information. seqMINER can handle the biological complexity of most experimental situations and proposes methods to the user for data classification according to the analysed features. In addition, through multiple graphical representations, seqMINER allows visualization and modelling of general as well as specific patterns in a given dataset. To demonstrate the efficiency of seqMINER, we have carried out a comprehensive analysis of genome-wide chromatin modification data in mouse embryonic stem cells to understand the global epigenetic landscape and its change through cellular differentiation.
PMCID: PMC3064796  PMID: 21177645
11.  Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells 
BMC Genomics  2010;11:530.
CREB and CREM are closely related factors that regulate transcription in response to various stress, metabolic and developmental signals. The CREMτ activator isoform is selectively expressed in haploid spermatids and plays an essential role in murine spermiogenesis.
We have used chromatin immunoprecipitation coupled to sequencing (ChIP-seq) to map CREM and CREB target loci in round spermatids from adult mouse testis and spermatogonia derived GC1-spg cells respectively. We identify more than 9000 genomic loci most of which are cell-specifically occupied. Despite the fact that round spermatids correspond to a highly specialised differentiated state, our results show that they have a remarkably accessible chromatin environment as CREM occupies more than 6700 target loci corresponding not only to the promoters of genes selectively expressed in spermiogenesis, but also of genes involved in functions specific to other cell types. The expression of only a small subset of these target genes are affected in the round spermatids of CREM knockout animals. We also identify a set of intergenic binding loci some of which are associated with H3K4 trimethylation and elongating RNA polymerase II suggesting the existence of novel CREB and CREM regulated transcripts.
We demonstrate that CREM and CREB occupy a large number of promoters in highly cell specific manner. This is the first study of CREM target promoters directly in a physiologically relevant tissue in vivo and represents the most comprehensive experimental analysis of CREB/CREM regulatory potential to date.
PMCID: PMC3091680  PMID: 20920259
12.  Cell-Specific Interaction of Retinoic Acid Receptors with Target Genes in Mouse Embryonic Fibroblasts and Embryonic Stem Cells▿ †  
Molecular and Cellular Biology  2009;30(1):231-244.
All-trans retinoic acid (RA) induces transforming growth factor beta (TGF-β)-dependent autocrine growth of mouse embryonic fibroblasts (MEFs). We have used chromatin immunoprecipitation to map 354 RA receptor (RAR) binding loci in MEFs, most of which were similarly occupied by the RARα and RARγ receptors. Only a subset of the genes associated with these loci are regulated by RA, among which are several critical components of the TGF-β pathway. We also show RAR binding to a novel series of target genes involved in cell cycle regulation, transformation, and metastasis, suggesting new pathways by which RA may regulate proliferation and cancer. Few of the RAR binding loci contained consensus direct-repeat (DR)-type elements. The majority comprised either degenerate DRs or no identifiable DRs but anomalously spaced half sites. Furthermore, we identify 462 RAR target loci in embryonic stem (ES) cells and show that their occupancy is cell type specific. Our results also show that differences in the chromatin landscape regulate the accessibility of a subset of more than 700 identified loci to RARs, thus modulating the repertoire of target genes that can be regulated and the biological effects of RA.
PMCID: PMC2798310  PMID: 19884340
13.  Abnormal Sperm in Mice Lacking the Taf7l Gene▿ †  
Molecular and Cellular Biology  2007;27(7):2582-2589.
TFIID is a general transcription factor required for transcription of most protein-coding genes by RNA polymerase II. TAF7L is an X-linked germ cell-specific paralogue of TAF7, which is a generally expressed component of TFIID. Here, we report the generation of Taf7l mutant mice by homologous recombination in embryonic stem cells by using the Cre-loxP strategy. While spermatogenesis was completed in Taf7l−/Y mice, the weight of Taf7l−/Y testis decreased and the amount of sperm in the epididymides was sharply reduced. Mutant epididymal sperm exhibited abnormal morphology, including folded tails. Sperm motility was significantly reduced, and Taf7l−/Y males were fertile with reduced litter size. Microarray profiling revealed that the abundance of six gene transcripts (including Fscn1) in Taf7l−/Y testes decreased more than twofold. In particular, FSCN1 is an F-action-bundling protein and thus may be critical for normal sperm morphology and sperm motility. Although deficiency of Taf7l may be compensated in part by Taf7, Taf7l has apparently evolved new specialized functions in the gene-selective transcription in male germ cell differentiation. Our mouse studies suggest that mutations in the human TAF7L gene might be implicated in X-linked oligozoospermia in men.
PMCID: PMC1899882  PMID: 17242199
14.  Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis 
The Journal of Cell Biology  2007;176(3):283-294.
During male germ cell postmeiotic maturation, dramatic chromatin reorganization occurs, which is driven by completely unknown mechanisms. For the first time, we describe a specific reprogramming of mouse pericentric heterochromatin. Initiated when histones undergo global acetylation in early elongating spermatids, this process leads to the establishment of new DNA packaging structures organizing the pericentric regions in condensing spermatids. Five new histone variants were discovered, which are expressed in late spermiogenic cells. Two of them, which we named H2AL1 and H2AL2, specifically mark the pericentric regions in condensing spermatids and participate in the formation of new nucleoprotein structures. Moreover, our investigations also suggest that TH2B, an already identified testis-specific H2B variant of unknown function, could provide a platform for the structural transitions accompanying the incorporation of these new histone variants.
PMCID: PMC2063955  PMID: 17261847
15.  Functional interaction between the homeoprotein CDX1 and the transcriptional machinery containing the TATA-binding protein 
Nucleic Acids Research  2006;35(1):175-185.
We have previously reported that the CDX1 homeoprotein interacts with the TATA-box binding protein (TBP) on the promoter of the glucose-6-phosphatase (G6Pase) gene. We show here that CDX1 interacts with TBP via the homeodomain and that the transcriptional activity additionally requires the N-terminal domain upstream of the homeodomain. CDX1 interacting with TBP is connected to members of the TFIID and Mediator complexes, two major elements of the general transcriptional machinery. Transcription luciferase assays performed using an altered-specificity mutant of TBP provide evidence for the functionality of the interaction between CDX1 and TBP. Unlike CDX1, CDX2 does not interact with TBP nor does it transactivate the G6Pase promoter. Swapping experiments between the domains of CDX1 and CDX2 indicate that, despite opposite functional effects of the homeoproteins on the G6Pase promoter, the N-terminal domains and homeodomains of both CDX1 and CDX2 have the intrinsic ability to activate transcription and to interact with TBP. However, the carboxy domains define the specificity of CDX1 and CDX2. Thus, intra-molecular interactions control the activity and partner recruitment of CDX1 and CDX2, leading to different molecular functions.
PMCID: PMC1802564  PMID: 17158164
16.  Cell-specific Nucleolar Localization of TBP-related Factor 2D⃞ 
Molecular Biology of the Cell  2004;15(10):4356-4368.
TATA-binding protein (TBP)-related factor 2 (TRF2) is one of four closely related RNA polymerase II transcription factors. We compared the intracellular localizations of TBP and TRF2 during the cell cycle and mitosis in HeLa cells. We show that during interphase, endogenous or exogenously expressed TRF2 is located almost exclusively in the nucleolus in HeLa or Cos cells. TRF2 localization is not affected by stress or mitotic stimuli, but TRF2 is rapidly released from the nucleolus upon inhibition of pol I transcription or treatment by RNase. These results suggest that localization of HeLa TRF2 requires a nucleolar-associated RNA species. In contrast, in 3T3 fibroblast cells, exogenously expressed TRF2 localizes to the nucleoplasm. Constitutive expression of ectopic TRF2 in 3T3 cells leads to a prolonged S phase of the cell cycle and reduced proliferation. Together with previous data, our results highlight the cell-specific localization and functions of TRF2. Furthermore, we show that during cell division, HeLa TRF2 and TBP are localized in the mitotic cytoplasm and TRF2 relocalizes into the nascent nucleoli immediately after mitosis, whereas TBP reassociates with the chromatin. Although partially contradictory results have been reported, our data are consistent with a model where only small proportion of the cellular TBP remains associated with specific promoter loci during mitosis.
PMCID: PMC519132  PMID: 15269281
17.  Distinct Mutations in Yeast TAFII25 Differentially Affect the Composition of TFIID and SAGA Complexes as Well as Global Gene Expression Patterns 
Molecular and Cellular Biology  2002;22(9):3178-3193.
The RNA polymerase II transcription factor TFIID, composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs), nucleates preinitiation complex formation at protein-coding gene promoters. SAGA, a second TAFII-containing multiprotein complex, is involved in transcription regulation in Saccharomyces cerevisiae. One of the essential protein components common to SAGA and TFIID is yTAFII25. We define a minimal evolutionarily conserved 91-amino-acid region of TAFII25 containing a histone fold domain that is necessary and sufficient for growth in vivo. Different temperature-sensitive mutations of yTAFII25 or chimeras with the human homologue TAFII30 arrested cell growth at either the G1 or G2/M cell cycle phase and displayed distinct phenotypic changes and gene expression patterns. Immunoprecipitation studies revealed that TAFII25 mutation-dependent gene expression and phenotypic changes correlated at least partially with the integrity of SAGA and TFIID. Genome-wide expression analysis revealed that the five TAFII25 temperature-sensitive mutant alleles individually affect the expression of between 18 and 33% of genes, whereas taken together they affect 64% of all class II genes. Thus, different yTAFII25 mutations induce distinct phenotypes and affect the regulation of different subsets of genes, demonstrating that no individual TAFII mutant allele reflects the full range of its normal functions.
PMCID: PMC133751  PMID: 11940675
18.  Chromatin-Dependent Cooperativity between Constitutive and Inducible Activation Domains in CREB 
Molecular and Cellular Biology  2001;21(23):7892-7900.
The cyclic AMP (cAMP)-responsive factor CREB induces target gene expression via constitutive (Q2) and inducible (KID, for kinase-inducible domain) activation domains that function synergistically in response to cellular signals. KID stimulates transcription via a phospho (Ser133)-dependent interaction with the coactivator paralogs CREB binding protein and p300, whereas Q2 recruits the TFIID complex via a direct association with hTAFII130. Here we investigate the mechanism underlying cooperativity between the Q2 domain and KID in CREB by in vitro transcription assay with naked DNA and chromatin templates containing the cAMP-responsive somatostatin promoter. The Q2 domain was highly active on a naked DNA template, and Ser133 phosphorylation had no additional effect on transcriptional initiation in crude extracts. Q2 activity was repressed on a chromatin template, however, and this repression was relieved by the phospho (Ser133) KID-dependent recruitment of p300 histone acetyltransferase activity to the promoter. In chromatin immunoprecipitation assays of NIH 3T3 cells, cAMP-dependent recruitment of p300 to the somatostatin promoter stimulated acetylation of histone H4. Correspondingly, overexpression of hTAFII130 potentiated CREB activity in cells exposed to cAMP, but had no effect on reporter gene expression in unstimulated cells. We propose that cooperativity between the KID and Q2 domains proceeds via a chromatin-dependent mechanism in which recruitment of p300 facilitates subsequent interaction of CREB with TFIID.
PMCID: PMC99956  PMID: 11689682
19.  The TFIID Components Human TAFII140 and Drosophila BIP2 (TAFII155) Are Novel Metazoan Homologues of Yeast TAFII47 Containing a Histone Fold and a PHD Finger 
Molecular and Cellular Biology  2001;21(15):5109-5121.
The RNA polymerase II transcription factor TFIID comprises the TATA binding protein (TBP) and a set of TBP-associated factors (TAFIIs). TFIID has been extensively characterized for yeast, Drosophila, and humans, demonstrating a high degree of conservation of both the amino acid sequences of the constituent TAFIIs and overall molecular organization. In recent years, it has been assumed that all the metazoan TAFIIs have been identified, yet no metazoan homologues of yeast TAFII47 (yTAFII47) and yTAFII65 are known. Both of these yTAFIIs contain a histone fold domain (HFD) which selectively heterodimerizes with that of yTAFII25. We have cloned a novel mouse protein, TAFII140, containing an HFD and a plant homeodomain (PHD) finger, which we demonstrated by immunoprecipitation to be a mammalian TFIID component. TAFII140 shows extensive sequence similarity to Drosophila BIP2 (dBIP2) (dTAFII155), which we also show to be a component of Drosophila TFIID. These proteins are metazoan homologues of yTAFII47 as their HFDs selectively heterodimerize with dTAFII24 and human TAFII30, metazoan homologues of yTAFII25. We further show that yTAFII65 shares two domains with the Drosophila Prodos protein, a recently described potential dTAFII. These conserved domains are critical for yTAFII65 function in vivo. Our results therefore identify metazoan homologues of yTAFII47 and yTAFII65.
PMCID: PMC87236  PMID: 11438666
20.  Coordinate regulation of RARgamma2, TBP, and TAFII135 by targeted proteolysis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells 
Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA) induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively) and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARγ2 receptor, and induction of target genes.
We show that the RNA polymerase II transcription factor TFIID subunits TBP and TAFII135 are selectively depleted in extracts from differentiated F9 cells. In contrast, TBP and TAFII135 are readily detected in extracts from differentiated F9 cells treated with proteasome inhibitors showing that their disappearance is due to targeted proteolysis. This regulatory pathway is not limited to F9 cells as it is also seen when C2C12 myoblasts differentiate into myotubes. Targeting of TBP and TAFII135 for proteolysis in F9 cells takes place coordinately with that previously reported for the RARγ2 receptor and is delayed or does not take place in RAR mutant F9 cells where differentiation is known to be impaired or abolished. Moreover, ectopic expression of TAFII135 delays proteolysis of the RARγ2 receptor and impairs primitive endoderm differentiation at an early stage as evidenced by cell morphology, induction of marker genes and apoptotic response. In addition, enhanced TAFII135 expression induces a novel differentiation pathway characterised by the appearance of cells with an atypical elongated morphology which are cAMP resistant.
These observations indicate that appropriately timed proteolysis of TBP and TAFII135 is required for normal F9 cell differentiation. Hence, in addition to transactivators, targeted proteolysis of basal transcription factors also plays an important role in gene regulation in response to physiological stimuli.
PMCID: PMC31370  PMID: 11285139
21.  Histone Folds Mediate Selective Heterodimerization of Yeast TAFII25 with TFIID Components yTAFII47 and yTAFII65 and with SAGA Component ySPT7 
Molecular and Cellular Biology  2001;21(5):1841-1853.
We show that the yeast TFIID (yTFIID) component yTAFII47 contains a histone fold domain (HFD) with homology to that previously described for hTAFII135. Complementation in vivo indicates that the yTAFII47 HFD is necessary and sufficient for vegetative growth. Mutation of highly conserved residues in the α1 helix of the yTAFII47 HFD results in a temperature-sensitive phenotype which can be suppressed by overexpression of yTAFII25, as well as by yTAFII40, yTAFII19, and yTAFII60. In yeast two-hybrid and bacterial coexpression assays, the yTAFII47 HFD selectively heterodimerizes with yTAFII25, which we show contains an HFD with homology to the hTAFII28 family We additionally demonstrate that yTAFII65 contains a functional HFD which also selectively heterodimerizes with yTAFII25. These results reveal the existence of two novel histone-like pairs in yTFIID. The physical and genetic interactions described here show that the histone-like yTAFIIs are organized in at least two substructures within TFIID rather than in a single octamer-like structure as previously suggested. Furthermore, our results indicate that ySPT7 has an HFD homologous to that of yTAFII47 which selectively heterodimerizes with yTAFII25, defining a novel histone-like pair in the SAGA complex.
PMCID: PMC86751  PMID: 11238921
22.  The Human TFIID Components TAFII135 and TAFII20 and the Yeast SAGA Components ADA1 and TAFII68 Heterodimerize to Form Histone-Like Pairs 
Molecular and Cellular Biology  2000;20(1):340-351.
It has been previously proposed that the transcription complexes TFIID and SAGA comprise a histone octamer-like substructure formed from a heterotetramer of H4-like human hTAFII80 (or its Drosophila melanogaster dTAFII60 and yeast [Saccharomyces cerevisiae] yTAFII60 homologues) and H3-like hTAFII31 (dTAFII40 and yTAFII17) along with two homodimers of H2B-like hTAFII20 (dTAFII30α and yTAFII61/68). However, it has not been formally shown that hTAFII20 heterodimerizes via its histone fold. By two-hybrid analysis with yeast and biochemical characterization of complexes formed by coexpression in Escherichia coli, we showed that hTAFII20 does not homodimerize but heterodimerizes with hTAFII135. Heterodimerization requires the α2 and α3 helices of the hTAFII20 histone fold and is abolished by mutations in the hydrophobic face of the hTAFII20 α2 helix. Interaction with hTAFII20 requires a domain of hTAFII135 which shows sequence homology to H2A. This domain also shows homology to the yeast SAGA component ADA1, and we show that yADA1 heterodimerizes with the histone fold region of yTAFII61/68, the yeast hTAFII20 homologue. These results are indicative of a histone fold type of interaction between hTAFII20-hTAFII135 and yTAFII68-yADA1, which therefore constitute novel histone-like pairs in the TFIID and SAGA complexes.
PMCID: PMC85089  PMID: 10594036
23.  Human TAFII55 Interacts with the Vitamin D3 and Thyroid Hormone Receptors and with Derivatives of the Retinoid X Receptor That Have Altered Transactivation Properties 
Molecular and Cellular Biology  1999;19(8):5486-5494.
We have identified novel interactions between the human (h)TATA-binding protein-associated factor TAFII55 and the ligand-binding domains (LBDs) of the nuclear receptors for vitamin D3 (VDR) and thyroid hormone (TRα). Following expression in Cos cells, hTAFII55 interacts with the VDR and TRα LBDs in a ligand-independent manner whereas no interactions with the retinoid X receptors (RXRs) or with other receptors were observed. Deletion mapping indicates that hTAFII55 interacts with a 40-amino-acid region spanning α-helices H3 to H5 of the VDR and TRα LBDs but not with the equivalent highly related region of RXRγ. TAFII55 also interacts with chimeric receptors in which the H3-to-H5 region of RXRγ has been replaced with that of the VDR or TRα. Furthermore, replacement of two single amino acids of the RXRγ LBD with their VDR counterparts allows the RXRγ LBD to interact with hTAFII55 while the corresponding double substitution allows a much stronger interaction. In transfection experiments, the single mutated RXRγ LBDs activate transcription to fivefold higher levels than wild-type RXRγ while the double mutation activates transcription to a level comparable to that observed with the VDR. There is therefore a correlation between the ability of the modified RXRs to interact with hTAFII55 and transactivation. These results strongly suggest that the TAFII55 interactions with the modified RXR LBDs modulate transcriptional activation.
PMCID: PMC84390  PMID: 10409738
24.  Synergistic Transcriptional Activation by TATA-Binding Protein and hTAFII28 Requires Specific Amino Acids of the hTAFII28 Histone Fold 
Molecular and Cellular Biology  1999;19(7):5050-5060.
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 α2-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 α-helix of hTAFII28 therefore mediates multiple interactions required for coactivator activity. We further show that mutation of specific residues in the H1′ α-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.
PMCID: PMC84343  PMID: 10373554

Results 1-24 (24)