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1.  Specific and Redundant Functions of Retinoid X Receptor/Retinoic Acid Receptor Heterodimers in Differentiation, Proliferation, and Apoptosis of F9 Embryonal Carcinoma Cells  
The Journal of Cell Biology  1997;139(3):735-747.
We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)α and retinoic acid receptor (RAR)α genes or the RXRα and RARγ genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRα−/−, RARα−/−, and RARγ−/− cells. RXRα−/−/ RARα−/− cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRα−/− cells. In contrast, RXRα−/−/RARγ−/− cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRα and RARγ represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype– and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.
PMCID: PMC2141719  PMID: 9348290
2.  Retinoid Regulated Association of Transcriptional Coregulators and the Polycomb Group Protein SUZ12 with the Retinoic Acid Response Elements of Hoxa1, RARβ2, and Cyp26A1 in F9 Embryonal Carcinoma Cells 
Journal of molecular biology  2007;372(2):298-316.
Hox gene expression is activated by all-trans retinoic acid (RA), through binding to Retinoic Acid Receptor-Retinoid X Receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) of target genes. The RARs and RXRs each have three isotypes (α, β, and γ), which are encoded by distinct genes. Hox genes are also repressed by polycomb group proteins (PcG), though how these proteins are targeted is unclear. We used chromatin immunoprecipitation assays to investigate the association of RXRα, RARγ, cofactors, and the PcG protein SUZ12 with the Hoxa1, RARβ2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells (teratocarcinoma stem cells) during RA treatment. We demonstrate that RARγ and RXRα are associated with RAREs prior to and during RA treatment. pCIP, p300, and RNA polymerase II levels increased at target RAREs upon exposure to RA. Conversely, SUZ12 was found associated with all RAREs studied and these associations were attenuated by treatment with RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2, and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalent domain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreased at target RAREs whereas H3ac and H3K4me2 levels remained constant. These studies provide insight into the dynamics of association of coregulators with RAREs and demonstrate a novel link between RA signaling and PcG repression.
doi:10.1016/j.jmb.2007.06.079
PMCID: PMC2972191  PMID: 17663992
SUZ12; polycomb; differentiation; retinoic acid receptors; epigenetic marks
3.  Gene Expression Profiling Elucidates a Specific Role for RARγ in the Retinoic Acid Induced Differentiation of F9 Teratocarcinoma Stem Cells 
Biochemical pharmacology  2007;75(5):1129-1160.
The biological effects of all-trans-retinoic acid (RA), a major active metabolite of retinol, are mainly mediated through its interactions with retinoic acid receptor (RARs α, β, γ) and retinoid X receptor (RXRs α, β, γ) heterodimers. RAR/RXR heterodimers activate transcription by binding to RA-response elements (RAREs or RXREs) in the promoters of primary target genes. Murine F9 teratocarcinoma stem cells have been widely used as a model for cellular differentiation and RA signaling during embryonic development. We identified and characterized genes that are differentially expressed in F9 wild type (Wt) and F9 RAR γ−/− cells, with and without RA treatment, through the use of oligonucleotide based microarrays. Our data indicate that RARγ, in the absence of exogenous RA, modulates gene expression. Genes such as Sfrp2, Tie1, Fbp2, Emp1, and Emp3 exhibited higher transcript levels in RA treated Wt, RARα−/− and RARβ2−/− lines than in RA-treated RARγ−/− cells, and represent specific RARγ targets. Other genes, such as Runx1, were expressed at lower levels in both F9 RARβ2−/− and RARγ−/− cell lines then in F9 Wt and RARα−/−. Genes specifically induced by RA at 6h with the protein synthesis inhibitor cycloheximide in F9 Wt, but not in RARγ−/− cells, included Hoxa3, Hoxa5, Gas1, Cyp26a1, Sfrp2, Fbp2, and Emp1. These genes represent specific primary RARγ targets in F9 cells. Several genes in the Wnt signaling pathway were regulated by RARγ. Delineation of the receptor specific actions of RA with respect to cell proliferation and differentiation should result in more effective therapies with this drug.
doi:10.1016/j.bcp.2007.11.006
PMCID: PMC2988767  PMID: 18164278
retinoic acid receptor; gene expression profiles; differentiation; retinol; sfrp5; Tie1
4.  Positive and Negative TAFII Functions That Suggest a Dynamic TFIID Structure and Elicit Synergy with TRAPs in Activator-Induced Transcription 
Molecular and Cellular Biology  2001;21(20):6882-6894.
Human transcription factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFIIs). To elucidate the structural organization and function of TFIID, we expressed and characterized the product of a cloned cDNA encoding human TAFII135 (hTAFII135). Comparative far Western blots have shown that hTAFII135 interacts strongly with hTAFII20, moderately with hTAFII150, and weakly with hTAFII43 and hTAFII250. Consistent with these observations and with sequence relationships of hTAFII20 and hTAFII135 to histones H2B and H2A, respectively, TFIID preparations that contain higher levels of hTAFII135 also contain higher levels of hTAFII20, and the interaction between hTAFII20 and hTAFII135 is critical for human TFIID assembly in vitro. From a functional standpoint, hTAFII135 has been found to interact strongly and directly with hTFIIA and (within a complex that also contains hTBP and hTAFII250) to specifically cooperate with TFIIA to relieve TAFII250-mediated repression of TBP binding and function on core promoters. Finally, we report a functional synergism between TAFIIs and the TRAP/Mediator complex in activated transcription, manifested as hTAFII-mediated inhibition of basal transcription and a consequent TRAP requirement for both a high absolute level of activated transcription and a high and more physiological activated/basal transcription ratio. These results suggest a dynamic TFIID structure in which the switch from a basal hTAFII-enhanced repression state to an activator-mediated activated state on a promoter may be mediated in part through activator or coactivator interactions with hTAFII135.
doi:10.1128/MCB.21.20.6882-6894.2001
PMCID: PMC99865  PMID: 11564872
5.  Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression. 
Molecular and Cellular Biology  1997;17(2):644-655.
Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.
PMCID: PMC231790  PMID: 9001218
6.  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.
doi:10.1128/MCB.22.9.3178-3193.2002
PMCID: PMC133751  PMID: 11940675
7.  Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping. 
Molecular and Cellular Biology  1994;14(1):287-298.
The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RAR alpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RAR alpha/RXR alpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RAR alpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RAR alpha conformation seems to allow RAR alpha/RXR alpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RAR alpha. Complexation of RAR beta, RAR gamma, and RXR alpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.
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PMCID: PMC358378  PMID: 8264595
8.  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.
doi:10.1128/MCB.21.15.5109-5121.2001
PMCID: PMC87236  PMID: 11438666
9.  RARγ is required for correct deposition and removal of Suz12 and H2A.Z in embryonic stem cells 
Journal of Cellular Physiology  2011;226(2):293-298.
Retinoic acid (RA) induces embryonic stem cell differentiation. The effects of RA are mediated by retinoic acid receptors (RARs) that promote epigenetic changes controlling gene transcription. We show here that RARγ, in the absence of the ligand RA, is required for deposition of the histone variant H2A.Z and the Polycomb protein Suz12 at RA target genes, and that both RARγ and Suz12 exist in a multi-protein complex in the absence of ligand. Addition of RA causes removal of H2A.Z and Suz12 from RARγ target genes when the genes are transcriptionally activated.
doi:10.1002/jcp.22420
PMCID: PMC3369573  PMID: 20857416
Polycomb group; epigenetic; gene expression; retinoic acid
10.  Adenovirus E1A functions as a cofactor for retinoic acid receptor beta (RAR beta) through direct interaction with RAR beta. 
Molecular and Cellular Biology  1995;15(11):5868-5878.
Transcription regulation by DNA-bound activators is thought to be mediated by a direct interaction between these proteins and TATA-binding protein (TBP), TFIIB, or TBP-associated factors, although occasionally cofactors or adapters are required. For ligand-induced activation by the retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimer, the RAR beta 2 promoter is dependent on the presence of E1A or E1A-like activity, since this promoter is activated by retinoic acid only in cells expressing such proteins. The mechanism underlying this E1A requirement is largely unknown. We now show that direct interaction between RAR and E1A is a requirement for retinoic acid-induced RAR beta 2 activation. The activity of the hormone-dependent activation function 2 (AF-2) of RAR beta is upregulated by E1A, and an interaction between this region and E1A was observed, but not with AF-1 or AF-2 of RXR alpha. This interaction is dependent on conserved region III (CRIII), the 13S mRNA-specific region of E1A. Deletion analysis within this region indicated that the complete CRIII is needed for activation. The putative zinc finger region is crucial, probably as a consequence of interaction with TBP. Furthermore, the region surrounding amino acid 178, partially overlapping with the TBP binding region, is involved in both binding to and activation by AF-2. We propose that E1A functions as a cofactor by interacting with both TBP and RAR, thereby stabilizing the preinitiation complex.
PMCID: PMC230838  PMID: 7565739
11.  Retinoic Acid Receptor-Mediated Induction of ABCA1 in Macrophages 
Molecular and Cellular Biology  2003;23(21):7756-7766.
ABCA1, the mutant molecule in Tangier Disease, mediates efflux of cellular cholesterol to apoA-I and is induced by liver X receptor (LXR)/retinoid X receptor (RXR) transcription factors. Retinoic acid receptor (RAR) activators (all-trans-retinoic acid [ATRA] and TTNPB) were found to increase ATP-binding cassette transporter 1 (ABCA1) mRNA and protein in macrophages. In cellular cotransfection assays, RARγ/RXR activated the human ABCA1 promoter, via the same direct repeat 4 (DR4) promoter element as LXR/RXR. Chromatin immunoprecipitation analysis in macrophages confirmed the binding of RARγ/RXR to the ABCA1 promoter DR4 element in the presence of ATRA, with weaker binding of RARα/RXR, and no binding of RARβ/RXR. However, in macrophages from RARγ−/− mice, TTNPB still induced ABCA1, in association with marked upregulation of RARα, suggesting that high levels of RARα can compensate for the absence of RARγ. Dose-response experiments with ATRA in mouse primary macrophages showed that other LXR target genes were weakly induced (ABCG1 and SREBP-1c) or not induced (apoE and LXRα). The more specific RAR activator TTNPB did not induce SREBP-1c in mouse primary macrophages or liver. These studies indicate a direct role of RARγ/RXR in induction of macrophage ABCA1.
doi:10.1128/MCB.23.21.7756-7766.2003
PMCID: PMC207565  PMID: 14560020
12.  Transcription factor TAFII250 promotes Mdm2-dependent turnover of p53 
Oncogene  2007;26(29):4234-4242.
The p53tumour suppressor is regulated mainly by Mdm2, an E3 ubiquitin ligase that promotes the ubiquitylation and proteasome-mediated degradation of p53. Many agents that induce p53 are inhibitors of transcription, suggesting that the p53 pathway can detect a signal(s) arising from transcriptional malfunction. Mdm2 associates with TAFII250, a component of the general transcription factor TFIID. Inactivation of TAFII250 in ts13 cells, which express a temperature-sensitive mutant of TAFII250, leads to the induction of p53 and cell cycle arrest. In the present study, we show that TAFII250 stimulates the ubiquitylation and degradation of p53 in a manner that is dependent upon Mdm2 and requires its acidic domain. Mechanistically, TAFII250 downregulates Mdm2 auto-ubiquitylation, leading to Mdm2 stabilization, and promotes p53-Mdm2 association through a recently defined second binding site in the acidic domain of Mdm2. These data provide a novel route through which TAFII250 can directly influence p53 levels and are consistent with the idea that the maintenance of p53 turnover is coupled to the integrity of RNA polymerase II transcription.
doi:10.1038/sj.onc.1210209
PMCID: PMC2695134  PMID: 17237821
p53; Mdm2; TAFII250; transcription factor; ubiquitylation
13.  Retinoic Acid Receptor γ1 (RARγ1) Levels Control RARβ2 Expression in SK-N-BE2(c) Neuroblastoma Cells and Regulate a Differentiation-Apoptosis Switch 
Molecular and Cellular Biology  1998;18(11):6482-6492.
Vitamin A and its derivatives (retinoids) have profound effects on the proliferation and differentiation of many cell types and are involved in a diverse array of developmental and physiological regulatory processes, including those responsible for the development of the mature nervous system. Retinoid signals are mediated by retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs), which show distinct spatio-temporal patterns of expression during development and in adult tissues. We have used SK-N-BE2(c) neuroblastoma cells to study the effects of reciprocal regulation of expression of various RARs. We show that in these cells RARγ1 acts as a repressor of RARβ2 transcription in the absence of an agonist. In the presence of RA, the expression of RARγ1 is reduced and that of RARβ2 is induced. Overexpression of RARγ1 neutralizes the effects of RA on RARβ induction. Expression of an RARγ1-specific antisense construct leads to the constitutive expression of RARβ2. Although both overexpression of RARγ1 and its reduction of expression can result in inhibition of cell proliferation, they induce different morphological changes. Reduction of RARγ1 (and induction of RARβ) leads to increased apoptosis, whereas RARγ1 overexpression leads to differentiation in the absence of apoptosis. Thus, RARγ1 appears to control a differentiation-apoptosis switch in SK-N-BE2(c) neuroblastoma cells.
PMCID: PMC109234  PMID: 9774664
14.  Prodos Is a Conserved Transcriptional Regulator That Interacts with dTAFII16 in Drosophila melanogaster 
Molecular and Cellular Biology  2001;21(2):614-623.
The transcription factor TFIID is a multiprotein complex that includes the TATA box binding protein (TBP) and a number of associated factors, TAFII. Prodos (PDS) is a conserved protein that exhibits a histone fold domain (HFD). In yeast two-hybrid tests using PDS as bait, we cloned the Drosophila TAFII, dTAFII16, as a specific PDS target. dTAFII16 is closely related to human TAFII30 and to another recently discovered Drosophila TAF, dTAFII24. PDS and dTAFII24 do not interact, however, thus establishing a functional difference between these dTAFs. The PDS-dTAFII16 interaction is mediated by the HFD motif in PDS and the N terminus in dTAFII16, as indicated by yeast two-hybrid assays with protein fragments. Luciferase-reported transcription tests in transfected cells show that PDS or an HFD-containing fragment activates transcription only with the help of dTAFII16 and TBP. Consistent with this, the eye phenotype of flies expressing a sev-Ras1 construct is modulated by PDS and dTAFII16 in a gene dosage-dependent manner. Finally, we show that PDS function is required for cell viability in somatic mosaics. These findings indicate that PDS is a novel transcriptional coactivator that associates with a member of the general transcription factor TFIID.
doi:10.1128/MCB.21.2.614-623.2001
PMCID: PMC86631  PMID: 11134347
15.  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
16.  Metabolism and growth inhibition of four retinoids in head and neck squamous normal and malignant cells 
British Journal of Cancer  2001;85(4):630-635.
Isotretinoin (13- cis -retinoic acid, 13cRA) has proven to be active in chemoprevention of head and neck squamous cell carcinoma (HNSCC). Moreover, both all-trans-retinoic acid (ATRA) and 13cRA induce objective responses in oral premalignant lesions. After binding of retinoids to retinoic acid receptors (RARs and RXRs) dimers are formed that are able to regulate the expression of genes involved in growth and differentiation. We compared the metabolism and level of growth inhibition of 13cRA with that of ATRA, 9cRA and retinol in four HNSCC cell lines and normal oral keratinocyte cultures (OKC). These retinoid compounds are known to bind with different affinities to the retinoic acid receptors. We observed that all retinoids were similar with respect to their capacity to induce growth inhibition. One HNSCC line could be ranked as sensitive, one as moderately sensitive and the remaining two were totally insensitive; OKC were moderately sensitive. The rate at which the cells were able to catabolize the retinoid was similar for all compounds. Retinoid metabolism in HNSCC cells resulted in a profile of metabolites that was unique for each retinoid. These metabolic profiles were different in OKC. Our findings indicate that differences in retinoid receptor selectivity of these retinoids do not influence the level of growth inhibition and rate of metabolism. © 2001 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2001.1952
PMCID: PMC2364086  PMID: 11506507
head and neck cancer; isotretinoin; keratinocytes; metabolism; retinoid; squamous
17.  Targeted disruption of retinoic acid receptor alpha (RAR alpha) and RAR gamma results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism. 
Molecular and Cellular Biology  1995;15(2):843-851.
F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions.
PMCID: PMC231962  PMID: 7823950
18.  Prevention of KLF4-mediated tumor initiation and malignant transformation by UAB30 rexinoid 
Cancer biology & therapy  2009;8(3):289-298.
The transcription factor KLF4 acts in post-mitotic epithelial cells to promote differentiation, and functions in a context-dependent fashion as an oncogene. In the skin KLF4 is co-expressed with the nuclear receptors RARγ and RXRα, and formation of the skin permeability barrier is a shared function of these three proteins. We utilized a KLF4-transgenic mouse model of skin cancer in combination with cultured epithelial cells to examine functional interactions between KLF4 and retinoic acid receptors. In cultured cells, activation of a conditional, KLF4-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in rapid upregulation of transcripts for nuclear receptors including RARγ and RXRα. We tested retinoids in epithelial cell transformation assays, including an RAR-selective agonist (all-trans RA), an RXR-selective agonist (9-cis UAB30, rexinoid), and a pan agonist (9-cis RA). Unlike for several other genes, transformation by KLF4 was inhibited by each retinoid, implicating distinct nuclear receptor heterodimers as modulators of KLF4 transforming activity. When RXRα expression was suppressed by RNAi in cultured cells, transformation was promoted and the inhibitory effect of 9-cis UAB30 was attenuated. Similarly as shown for other mouse models of skin cancer, rexinoid prevented skin tumor initiation resulting from induction of KLF4 in basal keratinocytes. Rexinoid permitted KLF4 expression and KLF4-induced cell cycling, but attenuated the KLF4-induced misexpression of cytokeratin 1 in basal cells. Neoplastic lesions including hyperplasia, dysplasia and squamous cell carcinoma-like lesions were prevented for up to 30 days. Taken together, the results identify retinoid receptors including RXRα as ligand-dependent inhibitors of KLF4-mediated transformation or tumorigenesis.
PMCID: PMC2776760  PMID: 19197145
KLF4; tumor initiation; squamous cell carcinoma; retinoid; rexinoid; nuclear receptors; chemoprevention
19.  Endogenous retinoid X receptors can function as hormone receptors in pituitary cells. 
Molecular and Cellular Biology  1994;14(11):7105-7110.
Retinoids regulate gene transcription by interacting with both retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs). Since unliganded RXRs can act as heterodimerization partners for RARs and other nuclear hormone receptors, it is unclear whether ligand binding by RXRs actually regulates the expression of naturally occurring genes. To address this issue, we synthesized the RXR-selective retinoid SR11237 and confirmed its specificity in transient transfection and proteolytic susceptibility assays before using it to assess the contribution of ligand-activated RXRs to retinoid action. Unlike RAR ligands, SR11237 did not increase endogenous RAR beta mRNA levels in F9 embryonal carcinoma cells, even though it activated transcription of an RXR-responsive reporter gene in these cells. Thus, it is likely that RARs mediate the induction of RAR beta gene expression by RA. In contrast, the RXR-specific ligand induced rat growth hormone mRNA in GH3 pituitary cells, indicating that the effects of RA on growth hormone gene expression at least in part involve ligand binding to endogenous RXRs in vivo. Our results indicate that in addition to serving as cofactors for other nuclear hormone receptors, endogenous RXRs can function as ligand-dependent regulators of gene expression, i.e., classical nuclear hormone receptors.
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PMCID: PMC359244  PMID: 7935425
20.  Redundant Role of Tissue-Selective TAFII105 in B Lymphocytes 
Molecular and Cellular Biology  2002;22(18):6564-6572.
Regulated gene expression is a complex process achieved through the function of multiple protein factors acting in concert at a given promoter. The transcription factor TFIID is a central component of the machinery regulating mRNA synthesis by RNA polymerase II. This large multiprotein complex is composed of the TATA box binding protein (TBP) and several TBP-associated factors (TAFIIs). The recent discovery of multiple TBP-related factors and tissue-specific TAFIIs suggests the existence of specialized TFIID complexes that likely play a critical role in regulating transcription in a gene- and tissue-specific manner. The tissue-selective factor TAFII105 was originally identified as a component of TFIID derived from a human B-cell line. In this report we demonstrate the specific induction of TAFII105 in cultured B cells in response to bacterial lipopolysaccharide (LPS). To examine the in vivo role of TAFII105, we have generated TAFII105-null mice by homologous recombination. Here we show that B-lymphocyte development is largely unaffected by the absence of TAFII105. TAFII105-null B cells can proliferate in response to LPS, produce relatively normal levels of resting antibodies, and can mount an immune response by producing antigen-specific antibodies in response to immunization. Taken together, we conclude that the function of TAFII105 in B cells is likely redundant with the function of other TAFII105-related cellular proteins.
doi:10.1128/MCB.22.18.6564-6572.2002
PMCID: PMC135626  PMID: 12192054
21.  Synergistic activation of retinoic acid (RA)-responsive genes and induction of embryonal carcinoma cell differentiation by an RA receptor alpha (RAR alpha)-, RAR beta-, or RAR gamma-selective ligand in combination with a retinoid X receptor-specific ligand. 
Molecular and Cellular Biology  1995;15(12):6481-6487.
Retinoic acid receptor (RAR)-retinoid X receptor (RXR) heterodimers bind to cognate response elements in vitro more efficiently than do RAR or RXR homodimers, and both RAR and RXR partners have been shown to activate various promoters in transiently transfected cells. We have now investigated whether ligand-dependent activation of both heterodimeric partners is involved in induced expression of endogenous RA-responsive genes and in P19 and F9 cell differentiation. On their own, low concentrations of retinoids selective for either RAR alpha, RAR beta, or RAR gamma did not induce or very inefficiently induced the expression of several RA target genes or triggered differentiation. An RXR-specific synthetic retinoid was similarly inefficient at any concentration. In contrast, at the same concentrations, various combinations of RAR (RAR alpha, RAR beta, or RAR gamma) and RXR selective retinoids resulted in synergistic induction of all retinoic acid (RA) target genes examined, as well as in cell differentiation. However, the magnitude of this synergistic activation varied depending on both the RAR-RXR combination and the promoter context of the responsive genes. Promiscuous activation of the three RARs, or concomitant activation of RAR alpha and RAR gamma, at selective retinoid concentrations also resulted in induction of gene expression and cell differentiation. Taken together, our results are consistent with the conclusion that the RAR and RXR partners of RAR-RXR heterodimers can synergistically activate transcription of RA-responsive genes and can induce differentiation of P19 and F9 cells. Our results also indicate that there is a significant degree of functional redundancy between the three RAR types which, however, varies with the nature of the RA target genes.
PMCID: PMC230900  PMID: 8524212
22.  Requirement for TAFII250 Acetyltransferase Activity in Cell Cycle Progression 
Molecular and Cellular Biology  2000;20(4):1134-1139.
The TATA-binding protein (TBP)-associated factor TAFII250 is the largest component of the basal transcription factor IID (TFIID). A missense mutation that maps to the acetyltransferase domain of TAFII250 induces the temperature-sensitive (ts) mutant hamster cell lines ts13 and tsBN462 to arrest in late G1. At the nonpermissive temperature (39.5°C), transcription from only a subset of protein encoding genes, including the G1 cyclins, is dramatically reduced in the mutant cells. Here we demonstrate that the ability of the ts13 allele of TAFII250 to acetylate histones in vitro is temperature sensitive suggesting that this enzymatic activity is compromised at 39.5°C in the mutant cells. Mutagenesis of a putative acetyl coenzyme A binding site produced a TAFII250 protein that displayed significantly reduced histone acetyltransferase activity but retained TBP and TAFII150 binding. Expression of this mutant in ts13 cells was unable to complement the cell cycle arrest or transcriptional defect observed at 39.5°C. These data suggest that TAFII250 acetyltransferase activity is required for cell cycle progression and regulates the expression of essential proliferative control genes.
PMCID: PMC85231  PMID: 10648598
23.  Reversal by RARα agonist Am580 of c-Myc-induced imbalance in RARα/RARγ expression during MMTV-Myc tumorigenesis 
Breast Cancer Research : BCR  2012;14(4):R121.
Introduction
Retinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently, the nuclear retinoic acid receptor (RAR) isotypes α, β and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance, RARγ appears to be involved in stem cell compartment expansion, while RARα and RARβ are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer, disrupts the balance between RARγ and RARα/β in favor of RARγ.
Methods
The effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium, mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (Am580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis.
Results
Modulation of the RARα/β to RARγ expression in mammary glands of normal mice, oncomice, and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation, survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist Am580 led to significant inhibition of mammary tumor growth (~90%, P<0.001), lung metastasis (P<0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice, RARα responsive genes such as Cyp26A1, E-cadherin, cellular retinol-binding protein 1 (CRBP1) and p27, were up-regulated. In contrast, the mammary gland tumors of mice that responded poorly to Am580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus, activation of the RARα pathway is linked to tumor growth inhibition, differentiation and cell death.
Conclusions
The functional consequence of the interplay between c-Myc oncogene expression and the RARγ to RARα/β balance suggests that prevalence of RARγ over-RARα/β expression levels in breast cancer accompanied by c-Myc amplification or over-expression in breast cancer should be predictive of response to treatment with RARα-isotype-specific agonists and warrant monitoring during clinical trials.
See related editorial by Garattini et al http://breast-cancer-research.com/content/14/5/111
doi:10.1186/bcr3247
PMCID: PMC3680916  PMID: 22920668
24.  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.
doi:10.1128/MCB.00756-09
PMCID: PMC2798310  PMID: 19884340
25.  Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis 
Genome Biology  2011;12(1):R2.
Background
Among its many roles in development, retinoic acid determines the anterior-posterior identity of differentiating motor neurons by activating retinoic acid receptor (RAR)-mediated transcription. RAR is thought to bind the genome constitutively, and only induce transcription in the presence of the retinoid ligand. However, little is known about where RAR binds to the genome or how it selects target sites.
Results
We tested the constitutive RAR binding model using the retinoic acid-driven differentiation of mouse embryonic stem cells into differentiated motor neurons. We find that retinoic acid treatment results in widespread changes in RAR genomic binding, including novel binding to genes directly responsible for anterior-posterior specification, as well as the subsequent recruitment of the basal polymerase machinery. Finally, we discovered that the binding of transcription factors at the embryonic stem cell stage can accurately predict where in the genome RAR binds after initial differentiation.
Conclusions
We have characterized a ligand-dependent shift in RAR genomic occupancy at the initiation of neurogenesis. Our data also suggest that enhancers active in pluripotent embryonic stem cells may be preselecting regions that will be activated by RAR during neuronal differentiation.
doi:10.1186/gb-2011-12-1-r2
PMCID: PMC3091300  PMID: 21232103

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