DNA Binding Site Selection Research Articles

Abstract 3582: PPAR gamma acetylation governs mammary adenocarcinoma tumor growth via acetylated residues that determine DNA sequence-specific binding

Abstract Background: HER2/neu overexpression in breast cancer confers a lipogenic phenotype. Although the introduction of anti-Her2 therapies has led to dramatic improvements in survival, nearly all patients with metastatic Her2-positive breast cancer will progress on treatment suggesting the importance of developing coextinction approaches targeting multiple pathways. Peroxisome proliferator-activated receptor γ (PPARγ), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. PPARγ binds additional DNA cis elements associated with other transcription factors, including C/EBPs, NFκB and AP-1 proteins, to promote non-canonical signaling. Defining the mechanisms governing the selection of canonical versus non-canonical PPARγ binding sequences may provide the opportunity to design regulators with distinct functions and side effects. PPARγ1 acetylation at K268/293 participates in the regulation of adipose tissue differentiation (1), and the conserved lysine residues (K154/155) govern lipogenesis in breast cancer cells (2). Although Cre based Pparγ1 gene deletion in mammary tumor oncomice showed Pparγ1 participates in the onset and progression of ErbB2-induced mammary tumorigenesis (3), the molecular mechanisms and the post-translational modifications of PPARγ governing Pparγ1 tumorigenic function remained to be determined. Methods: Herein, we defined the role of PPARγ1 acetylation in breast cancer growth in immune-deficient mice using distinct breast cancer cell lines (MCF10-Ras, MCF10A-NeuT). Furthermore, using ChIP and ChIP-Seq we show that the acetylated residues of Pparγ1 altered preference of cis-element binding in chromatin to augment Pparγ non-canonical binding. Results: Herein, the PPARγ1 acetylated residues K154/155 were shown to be essential for the induction of transcriptional modules governing growth factor signaling, cellular apoptosis, and autophagy in mice. The K154/155 residues determined the selection of genome-wide DNA binding sites, altering the selection from canonical to non-canonical (C/EBP) DNA sequence-specific binding. The gene signature reflecting the acetylation-dependent genomic occupancy provided predictive value in patient survival outcomes. Conclusions: Endogenous Pparγ1 promotes mammary tumor onset and progression. PPARγ1 breast tumor induction involves K154/155, which governs transcriptional programs (growth, apoptosis, and autophagy) via transcriptional complex binding to canonical vs. non-canonical regulatory elements. Pparγ1 acetylation participates in ErbB2-induced breast cancer tumor growth and inflammation and may represent a relevant target for therapeutic coextinction.

A Nitrate-Inducible GARP Family Gene Encodes an Auto-Repressible Transcriptional Repressor in Rice

Nitrogen is the most important macronutrient in plants and its supply induces responses in gene expression, metabolism and developmental processes. However, the molecular mechanisms underlying the nitrogen responses remain poorly understood. Here we show that the supply of nitrate but not ammonium immediately induces the expression of a transcriptional repressor gene in rice, designated NIGT1 (Nitrate-Inducible, GARP-type Transcriptional Repressor 1). The results of DNA-binding site selection experiments and electrophoretic mobility shift assays indicated that NIGT1 binds to DNA containing either of two consensus sequences, GAATC or GAATATTC. In transient reporter assays, NIGT1 was found to repress transcription from the promoters containing the identified NIGT1-binding sequences in vivo. Furthermore, NIGT1 repressed the activity of its own promoter, suggesting an autorepression mechanism. Consistently, nitrate-induced NIGT1 expression was found to be down-regulated after a transient peak during nitrate treatment, and the nitrate-induced expression of NIGT1 decreased in transgenic rice plants in which this gene was constitutively overexpressed. Furthermore, the chlorophyll content that could be a marker of nitrogen utilization was found to be decreased in NIGT1 overexpressors of rice grown with nitrate medium but not with ammonium medium. Thus, we propose NIGT1 as a nitrate-inducible and autorepressible transcriptional repressor that may play a role in the nitrogen response in rice. Taken together with the fact that the NIGT1-binding sites are conserved in promoter sequences of Arabidopsis NIGT1 homologs, our findings imply the presence of a time-dependent complex system for nitrate-responsive transcriptional regulation that is conserved in both monocots and dicots.

TaMYB13 is a transcriptional activator of fructosyltransferase genes involved in β‐2,6‐linked fructan synthesis in wheat

Fructans are soluble fructosyl-oligosaccharides deposited in many cool-season grass species as a carbon reserve; they are synthesised by fructosyltransferases. In wheat and barley fructans can accumulate in mature stems at a very high level and serve as an important carbon source for grain filling. Fructan synthesis in temperate cereals is regulated by sucrose level and developmental signals, and functions as a metabolic adjustment for carbon balance between carbon supply and sink demand. In this study the expression levels of a highly homologous group of Triticum aestivumMYB genes (TaMYB13-1, TaMYB13-2 and TaMYB13-3) were found to be positively correlated with the mRNA levels of sucrose:sucrose 1-fructosyltransferase (1-SST) and sucrose:fructan 6-fructosyltransferase (6-SFT) in wheat stems among recombinant inbred lines with a wide range of fructan concentrations through Affymetrix array expression analysis. This expression correction extended to expression profiles during stem development. TaMYB13 contains an R2R3-type MYB domain. Invitro random DNA-binding site selection followed by base substitution mutagenesis revealed that TaMYB13 bound to a (A/G/T)TT(A/T/C)GGT core sequence, which was present in the promoters of wheat Ta1-SST and Ta6-SFT genes as well as a barley Hv6-SFT gene. Transactivation analysis showed that TaMYB13 was a transcriptional activator and could markedly enhance the expression of 1-SST and 6-SFT promoter-driven reporter genes in wheat. Elimination of TaMYB13-binding sites in Ta6-SFT and Ta1-SST promoters markedly reduced TaMYB13-mediated reporter gene transactivation. These data suggest that TaMYB13 and its orthologues are positive regulators for controlling the expression of major fructosyltransferases involved in the fructan synthetic pathway in temperate cereals.

Whole-genome comparison of Leu3 binding in vitro and in vivo reveals the importance of nucleosome occupancy in target site selection

Sequence motifs that are potentially recognized by DNA-binding proteins occur far more often in genomic DNA than do observed in vivo protein-DNA interactions. To determine how chromatin influences the utilization of particular DNA-binding sites, we compared the in vivo genome-wide binding location of the yeast transcription factor Leu3 to the binding location observed on the same genomic DNA in the absence of any protein cofactors. We found that the DNA-sequence motif recognized by Leu3 in vitro and in vivo was functionally indistinguishable, but Leu3 bound different genomic locations under the two conditions. Accounting for nucleosome occupancy in addition to DNA-sequence motifs significantly improved the prediction of protein-DNA interactions in vivo, but not the prediction of sites bound by purified Leu3 in vitro. Use of histone modification data does not further improve binding predictions, presumably because their effect is already manifest in the global histone distribution. Measurements of nucleosome occupancy in strains that differ in Leu3 genotype show that low nucleosome occupancy at loci bound by Leu3 is not a consequence of Leu3 binding. These results permit quantitation of the epigenetic influence that chromatin exerts on DNA binding-site selection, and provide evidence for an instructive, functionally important role for nucleosome occupancy in determining patterns of regulatory factor targeting genome-wide.

UvrB Domain 4, an Autoinhibitory Gate for Regulation of DNA Binding and ATPase Activity

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.

In Vitro selection of DNA binding sites for transcription factor, PhaR, from Paracoccus denitrificans using genetic library on microbeads and flow cytometry

We attempted the selection of a site for DNA binding to a transcription factor, PhaR, from Paracoccus denitrificans expressed by cell-free protein synthesis, from a random oligonucleotide library on microbeads that was constructed by the emulsion PCR technique. PhaR is a repressor protein in P. denitrificans that binds to the phaP promoter region. We acquired three types of PhaR-binding DNA fragment using this system. The selected fragments contained a perfect PhaR binding consensus site (TGC I), a sequence similar to that of TGC I, and another PhaR binding site (TGC II).

DNA Binding Selectivity of MeCP2 Due to a Requirement for A/T Sequences Adjacent to Methyl-CpG

DNA methylation is interpreted by a family of methyl-CpG binding domain (MBD) proteins that repress transcription through recruitment of corepressors that modify chromatin. To compare in vivo binding of MeCP2 and MBD2, we analyzed immunoprecipitated chromatin from primary human cells. Genomic sites occupied by the two MBD proteins were mutually exclusive. As MeCP2 was unable to colonize sites vacated by depletion of MBD2, we tested the hypothesis that methyl-CpG alone is insufficient to direct MeCP2 binding. In vitro selection for MeCP2 bound DNA-enriched fragments containing A/T bases ([A/T] > or = 4) adjacent to methyl-CpG. [A/T] > or = 4 was found to be essential for high-affinity binding at selected sites and at known MeCP2 target regions in the Bdnf and Dlx6 genes. MBD2 binding, however, did not require an A/T run. The unexpected restriction of MeCP2 to a defined subset of methyl-CpG sites will facilitate identification of genomic targets that are relevant to Rett Syndrome.

In vitro selection of DNA binding sites for ABF1 protein from Saccharomyces cerevisiae

The autonomously replicating sequence-binding factor 1 (ABF1) from Sacchramoyces cerevisiae is known as a multifunctional DNA binding protein that is involved in transcriptional regulation, DNA-replication, and in restructuring of chromatin via nucleosome remodelling. ABF1 binds to DNA sequences found in ARS elements and in various transcriptional regulatory elements. This led to the early definition of the consensus motive 5′-CGTnnnnnnnGA(G/C)-3′. We have used a SELEX approach to expand and better characterize the DNA sequence requirements of ABF1. Starting from a pool of oligonucleotides randomized at a sequence of 30 nucleotides, we used EMSA to select for sequences with high affinity for ABF1. We obtained the sequences of 106 aptamers after the 15th SELEX round. A 16 nucleotide consensus was derived from this pool by analysis with the motif search programme MEME. Quantitative EMSA experiments verified our experimental approach since binding sequences which were bound with high affinity occurred more often in the pool and resembled the derived consensus to a higher degree. We found DNA sequences that are bound by ABF1 with nearly two-magnitude higher affinity as compared to the hitherto accepted ABF1 consensus sequence. This led us to postulate a strong recognition motive: 5′-TnnCGTnnnnnnTGAT-3′.

Characterization of Nkx3.2 DNA Binding Specificity and Its Requirement for Somitic Chondrogenesis

We have previously shown that Nkx3.2, a member of the NK class of homeoproteins, functions as a transcriptional repressor to promote somitic chondrogenesis. However, it has not been addressed whether Nkx3.2 can bind to DNA in a sequence-specific manner and whether DNA binding by Nkx3.2 is required for its biological activity. In this work, we employed a DNA binding site selection assay, which identified TAAGTG as a high affinity Nkx3.2 binding sequence. Sequence-specific binding of Nkx3.2 to the TAAGTG motif in vitro was confirmed by electrophoretic mobility shift assays, and mutagenesis of this sequence revealed that HRAGTG (where H represents A, C, or T, and R represents A or G) comprises the consensus DNA binding site for Nkx3.2. Consistent with these findings, the expression of a reporter gene containing reiterated Nkx3.2 binding sites was repressed in vivo by Nkx3.2 co-expression. In addition, we have generated a DNA nonbinding point mutant of Nkx3.2 (Nkx3.2-N200Q), which contains an asparagine to glutamine missense mutation in the homeodomain. Interestingly, despite being defective in DNA binding, Nkx3.2-N200Q still retains its intrinsic transcriptional repressor function. Finally, we demonstrate that unlike wild-type Nkx3.2, Nkx3.2-N200Q is unable to activate the chondrocyte differentiation program in somitic mesoderm, indicating that DNA binding by Nkx3.2 is critical for this factor to induce somitic chondrogenesis.

Kaposi's sarcoma-associated herpesvirus lytic origin (ori-Lyt)-dependent DNA replication: identification of the ori-Lyt and association of K8 bZip protein with the origin.

Herpesviruses utilize different origins of replication during lytic versus latent infection. Latent DNA replication depends on host cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin binding protein (OBP) that recruits the core replication machinery. In this report, we demonstrated that DNA sequences in two noncoding regions of the Kaposi's sarcoma-associated herpesvirus (KSHV) genome, between open reading frames (ORFs) K4.2 and K5 and between K12 and ORF71, are able to serve as origins for lytic cycle-specific DNA replication. The two ori-Lyt domains share an almost identical 1,153-bp sequence and a 600-bp downstream GC-rich repeat sequence, and the 1.7-kb DNA sequences are sufficient to act as a cis signal for replication. We also showed that an AT-palindromic sequence in the ori-Lyt domain is essential for the DNA replication. In addition, a virally encoded bZip protein, namely K8, was found to bind to a DNA sequence within the ori-Lyt by using a DNA binding site selection assay. The binding of K8 to this region was confirmed in cells by using a chromatin immunoprecipitation method. Further analysis revealed that K8 binds to an extended region, and the entire region is 100% conserved between two KSHV ori-Lyt's. K8 protein displays significant similarity to the Zta protein of Epstein-Barr virus (EBV), which is a known OBP of EBV. This notion, together with the ability of K8 to bind to the KSHV ori-Lyt, suggests that K8 may function as an OBP in KSHV.

Molecular structure of the GARP family of plant Myb-related DNA binding motifs of the Arabidopsis response regulators.

The B motif is a signature of type-B response regulators (ARRs) involved in His-to-Asp phosphorelay signal transduction systems in Arabidopsis. Homologous motifs occur widely in the GARP family of plant transcription factors. To gain general insight into the structure and function of B motifs (or GARP motifs), we characterized the B motif derived from a representative ARR, ARR10, which led to a number of intriguing findings. First, the B motif of ARR10 (named ARR10-B and extending from Thr-179 to Ser-242) possesses a nuclear localization signal, as indicated by the intracellular localization of a green fluorescent protein-ARR10-B fusion protein in onion epidermal cells. Second, the purified ARR10-B molecule binds specifically in vitro to DNA with the core sequence AGATT. This was demonstrated by several in vitro approaches, including PCR-assisted DNA binding site selection, gel retardation assays, and surface plasmon resonance analysis. Finally, the three-dimensional structure of ARR10-B in solution was determined by NMR spectroscopy, showing that it contains a helix-turn-helix structure. Furthermore, the mode of interaction between ARR10-B and the target DNA was assessed extensively by NMR spectroscopy. Together, these results lead us to propose that the mechanism of DNA recognition by ARR10-B is essentially the same as that of homeodomains. We conclude that the B motif is a multifunctional domain responsible for both nuclear localization and DNA binding and suggest that these insights could be applicable generally to the large GARP family of plant transcription factors.

Cyclin A-CDK phosphorylates Sp1 and enhances Sp1-mediated transcription.

Cyclin A-mediated activation of cyclin-dependent kinases (CDKs) is essential for cell cycle transversal. Cyclin A activity is regulated on several levels and cyclin A elevation in a number of cancers suggests a role in tumorigenesis. In the present study, we used a modified DNA binding site selection and PCR amplification procedure to identify DNA binding proteins that are potential substrates of cyclin A-CDK. One of the sequences identified is the Sp1 transcription factor binding site. Co-immunoprecipitation experiments show that cyclin A and Sp1 can interact physically. In vitro and in vivo phosphorylation studies indicate that cyclin A-CDK complexes can phosphorylate Sp1. The phosphorylation site is located in the N-terminal region of the protein. Cells overexpressing cyclin A have elevated levels of Sp1 DNA binding activity, suggesting that cyclin A-CDK-mediated phosphorylation augments Sp1 DNA binding properties. In co-transfection studies, cyclin A expression stimulated transcription from an Sp1-regulated promoter. Mutation of the phosphorylation site abrogated cyclin A-CDK-dependent phosphorylation, augmentation of Sp1 transactivation function and DNA binding activity.

Identification and characterization of members of the FKHR (FOX O) subclass of winged-helix transcription factors in the mouse.

Genetic alterations of FKHR (FOXO1), AF6q21 (FOXO2), and AFX (FOXO4), closely related members of the forkhead family of DNA binding proteins, in human cancers has suggested they play a role in the regulation of cellular differentiation or proliferation. In order to elucidate the function of this gene subfamily during mammalian development, we have identified and characterized three novel mouse genes; Fkhr1 (Foxo1), Fkhr2 (Foxo3), and Afxh (Foxo4), which are closely related to the human FKHR (Foxo1), AF6q21 (FOXO2), and AFX (FOXO4) genes, respectively. The genes are each expressed both during development and in the adult with distinct patterns ranging from ubiquitous [Fkhr2 (Foxo3)] to tissue-specific [Afxh (Foxo4)]. Selection of high-affinity DNA-binding sites from a pool of degenerate oligonucleotides demonstrated that the proteins encoded by these genes recognize a core sequence [(T/A) (A/T) A A C A] similar to that recognized by other forkhead domain-containing proteins. We have also identified additional FKHR-related genes expressed during development in both the chick and zebrafish. Further characterization will provide insight into the roles of members of the FKHR subfamily of forkhead-related genes during both normal and neoplastic development.

Selection and design of high affinity DNA ligands for mutant single-chain derivatives of the bacteriophage 434 repressor

Single-chain repressor RR(Tres) is a derivative of bacteriophage 434 repressor, which contains covalently dimerized DNA-binding domains (amino acids 1-69) of the phage 434 repressor. In this single-chain molecule, the wild type domain R is connected to the mutant domain R(TRES) by a recombinant linker in a head-to-tail arrangement. The DNA-contacting amino acids of R(TRES) at the -1, 1, 2, and 5 positions of the a3 helix are T, R, E, S respectively. By using a randomized DNA pool containing the central sequence -CATACAAGAAAGNNNNNNTTT-, a cyclic,in vitro DNA-binding site selection was performed. The selected population was cloned and the individual members were characterized by determining their binding affinities to RR(Tres) The results showed that the optimal operators contained the TTAC or TTCC sequences in the underlined positions as above, and that the Kd values were in the 1 x 10(-12) mol/L-1 x 10(11) mol/L concentration range. Since the affinity of the natural 434 repressor to its natural operator sites is in the 1 x 10(-9) mol/L range, the observed binding affinity increase is remarkable. It was also found that binding affinity was strongly affected by the flanking bases of the optimal tetramer binding sites, especially by the base at the 5' position. We constructed a new homodimeric single-chain repressor R(TRES)R(TRES) and its DNA-binding specificity was tested by using a series of new operators designed according to the recognition properties previously determined for the R(TREs) domain. These operators containing the consensus sequenceGTAAGAAARNTTACN orGGAAGAAARNTTCCN (R is A or G) were recognized by R(TRES)R(TRES) specifically, and with high binding affinity. Thus, by using a combination of random selection and rational design principles, we have discovered novel, high affinity protein-DNA interactions with new specificity. This method can potentially be used to obtain new binding specificity for other DNA-binding proteins.

Selective DNA binding and association with the CREB binding protein coactivator contribute to differential activation of alpha/beta interferon genes by interferon regulatory factors 3 and 7.

Recent studies implicate the interferon (IFN) regulatory factors (IRF) IRF-3 and IRF-7 as key activators of the alpha/beta IFN (IFN-alpha/beta) genes as well as the RANTES chemokine gene. Using coexpression analysis, the human IFNB, IFNA1, and RANTES promoters were stimulated by IRF-3 coexpression, whereas the IFNA4, IFNA7, and IFNA14 promoters were preferentially induced by IRF-7 only. Chimeric proteins containing combinations of different IRF-7 and IRF-3 domains were also tested, and the results provided evidence of distinct DNA binding properties of IRF-3 and IRF-7, as well as a preferential association of IRF-3 with the CREB binding protein (CBP) coactivator. Interestingly, some of these fusion proteins led to supraphysiological levels of IFN promoter activation. DNA binding site selection studies demonstrated that IRF-3 and IRF-7 bound to the 5'-GAAANNGAAANN-3' consensus motif found in many virus-inducible genes; however, a single nucleotide substitution in either of the GAAA half-site motifs eliminated IRF-3 binding and transactivation activity but did not affect IRF-7 interaction or transactivation activity. These studies demonstrate that IRF-3 possesses a restricted DNA binding site specificity and interacts with CBP, whereas IRF-7 has a broader DNA binding specificity that contributes to its capacity to stimulate delayed-type IFN gene expression. These results provide an explanation for the differential regulation of IFN-alpha/beta gene expression by IRF-3 and IRF-7 and suggest that these factors have complementary rather than redundant roles in the activation of the IFN-alpha/beta genes.

Sequence-specific High Mobility Group Box Factors Recognize 10–12-Base Pair Minor Groove Motifs

Sequence-specific High Mobility Group Box Factors Recognize 10–12-Base Pair Minor Groove Motifs

DNA binding site selection of dimeric and tetrameric Stat5 proteins reveals a large repertoire of divergent tetrameric Stat5a binding sites.

We have defined the optimal binding sites for Stat5a and Stat5b homodimers and found that they share similar core TTC(T/C)N(G/A)GAA interferon gamma-activated sequence (GAS) motifs. Stat5a tetramers can bind to tandemly linked GAS motifs, but the binding site selection revealed that tetrameric binding also can be seen with a wide range of nonconsensus motifs, which in many cases did not allow Stat5a binding as a dimer. This indicates a greater degree of flexibility in the DNA sequences that allow binding of Stat5a tetramers than dimers. Indeed, in an oligonucleotide that could bind both dimers and tetramers, it was possible to design mutants that affected dimer binding without affecting tetramer binding. A spacing of 6 bp between the GAS sites was most frequently selected, demonstrating that this distance is favorable for Stat5a tetramer binding. These data provide insights into tetramer formation by Stat5a and indicate that the repertoire of potential binding sites for this transcription factor is broader than expected.

DNA binding site selection by RNA polymerase II TAFs: a TAF(II)250-TAF(II)150 complex recognizes the initiator.

Basal transcription factor TFIID comprises the TATA-box-binding protein, TBP, and associated factors, the TAF(II)s. Previous studies have implicated TAF(II)250 and TAF(II)150 in core promoter selectivity of RNA polymerase II. Here, we have used a random DNA binding site selection procedure to identify target sequences for these TAFs. Individually, neither TAF(II)250 nor TAF(II)150 singles out a clearly constrained DNA sequence. However, a TAF(II)250-TAF(II)150 complex selects sequences that match the Initiator (Inr) consensus. When in a trimeric complex with TBP, these TAFs select Inr sequences at the appropriate distance from the TATA-box. Point mutations that inhibit binding of the TAF(II)250-TAF(II)150 complex also impair Inr function in reconstituted basal transcription reactions, underscoring the functional relevance of Inr recognition by TAFs. Surprisingly, the precise DNA sequence at the start site of transcription influences transcriptional regulation by the upstream activator Sp1. Finally, we found that TAF(II)150 specifically binds to four-way junction DNA, suggesting that promoter binding by TFIID may involve recognition of DNA structure as well as primary sequence. Taken together, our results establish that TAF(II)250 and TAF(II)150 bind the Inr directly and that Inr recognition can determine the responsiveness of a promoter to an activator.

Identification of a Novel DNA Binding Site for Nuclear Orphan Receptor OR1

The nuclear orphan receptor OR1 has been shown to bind as a heterodimer with retinoid X receptor (RXR) to direct repeat 4 (DR4) response elements. It remained unclear, however, whether this represents the only or the optimal binding site for this receptor. Therefore, we performed a DNA binding site selection assay that allows the identification of novel DNA binding sites for OR1 in an unbiased manner. While OR1 alone was not able to select a specific sequence from the pool of oligonucleotides, the OR1/RXR heterodimer selected a highly conserved DR1 element, termed DR1s, with two AGGTCA motifs spaced by one adenosine. The functional activity of the consensus binding site was verified in transient transfection assays and corroborated by in vitro studies. Based on the sequence of the consensus DR1s, we located putative natural binding sites in the 5'-promoter flanking regions of the rat S14 gene and the rat cholecystokinin type A receptor gene. Furthermore, we could show that although the OR1/RXR heterodimer has a distinct binding orientation on a DR4 element, it is able to bind in both orientations to the DR1s element. The OR1 paralog LXRalpha does not bind as a heterodimer with RXR to the DR1s element, indicating that these receptors, despite their homology, are involved in the regulation of different sets of genes.

Selection of DNA Binding Sites for Human Transcriptional Regulator GATA-6

The DNA-binding sites for transcriptional regulator GATA-6 were selected and amplified by means of a PCR-mediated random-site selection method involving filter binding and gel-mobility shift analysis using the zinc finger region of human GATA-6 fused with GST. Sequencing and comparison of the selected clones suggested that (A/T/C)GAT(A/T)(A) is the consensus binding sequence, which is similar to the GATA motif (A/T)GATA(A/G) and the gastric motif (G/C)PuPu(G/C)NGAT(A/T)PuPy. GATA-6 also binds to a similar sequence containing the GATC but not the GATG sequence. These results indicated that GATA-6 shows broader sequence specificity as to DNA binding. However, adenine is favored on both sides of the core for strong binding [AGAT(A/T)A], and the order of binding is GATA > GATT > GATC. Full-length human GATA-6 expressed in COS-1 cells showed essentially the same binding specificity, suggesting that the zinc finger region of GATA-6 mainly contributes to the selection of the binding sequence. Furthermore, the non-RI method presented in this paper is convenient for determination of the binding sites of other DNA-binding proteins.