Modulate DNA Binding Research Articles

DNA binding activity of Helicobacter pylori DnaB helicase: the role of the N-terminal domain in modulating DNA binding activities

Replicative helicases are major motor proteins essential for chromosomal DNA replication in prokaryotes. Usually hexameric in solution, their DNA binding property must have different roles at stages ranging from the loading onto a branched structure at initiation from the origin to the highly processive translocation during elongation. Here, we have analysed the DNA binding activity of Helicobacter pylori (Hp) replicative helicase, DnaB. The results indicate that while the C-terminal region is important for its DNA binding activity, the N-terminus appears to dampen the protein's affinity for DNA. The masking activity of the N-terminus does not require ATP or hexamerization of HpDnaB and can be overcome by deleting the N-terminus. It can also be neutralized by engaging the N-terminus in an interaction with a partner like the C-terminus of DnaG primase. The inhibitory effect of the N-terminus on DNA binding activity is consistent with the 3D homology model of HpDnaB. Electron microscopy of the HpDnaB-ssDNA complex showed that HpDnaB preferentially bound at the ends of linear ssDNA and translocated along the DNA in the presence of ATP. These results provide an insight into the stimulatory and inhibitory effects of different regions of HpDnaB on DNA binding activities that may be central to the loading and translocation functions of DnaB helicases.

Mutations in CDON, Encoding a Hedgehog Receptor, Result in Holoprosencephaly and Defective Interactions with Other Hedgehog Receptors

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDON's ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.

ERK/p90RSK/14-3-3 signalling has an impact on expression of PEA3 Ets transcription factors via the transcriptional repressor capicúa

Compounds that inhibit signalling upstream of ERK (extracellular-signal-regulated kinase) are promising anticancer therapies, motivating research to define how this pathway promotes cancers. In the present study, we show that human capicúa represses mRNA expression for PEA3 (polyoma enhancer activator 3) Ets transcription factors ETV1, ETV4 and ETV5 (ETV is Ets translocation variant), and this repression is relieved by multisite controls of capicúa by ERK, p90RSK (p90 ribosomal S6 kinase) and 14-3-3 proteins. Specifically, 14-3-3 binds to p90RSK-phosphorylated Ser173 of capicúa thereby modulating DNA binding to its HMG (high-mobility group) box, whereas ERK phosphorylations prevent binding of a C-terminal NLS (nuclear localization sequence) to importin α4 (KPNA3). ETV1, ETV4 and ETV5 mRNA levels in melanoma cells are elevated by siRNA (small interfering RNA) knockdown of capicúa, and decreased by inhibiting ERK and/or expressing a form of capicúa that cannot bind to 14-3-3 proteins. Capicúa knockdown also enhances cell migration. The findings of the present study give further mechanistic insights into why ETV1 is highly expressed in certain cancers, indicate that loss of capicúa can desensitize cells to the effects of ERK pathway inhibitors, and highlight interconnections among growth factor signalling, spinocerebellar ataxias and cancers.

Structural basis for the transcriptional regulation of membrane lipid homeostasis

DesT is a transcriptional repressor that regulates the genes which control the unsaturated:saturated fatty acid ratio available for membrane lipid synthesis. DesT bound to unsaturated acyl-CoA has a high affinity for its cognate palindromic DNA-binding site, whereas DesT bound to saturated acyl-CoA does not bind to this site. Structural analyses of the DesT–oleoyl-CoA–DNA and DesT–palmitoyl-CoA complexes reveal that acyl chain shape directly influences the packing of hydrophobic core residues within the DesT ligand-binding domain. These changes are propagated to the paired DNA-binding domains via conformational changes to modulate DNA binding. These structural interpretations are supported by the in vitro and in vivo characterization of site-directed mutants. The regulation of DesT by the unsaturated:saturated ratio of acyl chains rather than the concentration of a single ligand is a paradigm for understanding transcriptional regulation of membrane lipid homeostasis.

Insight into the induction mechanism of the GntR/HutC bacterial transcription regulator YvoA

YvoA is a GntR/HutC transcription regulator from Bacillus subtilis implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its 2.4-Å crystal structure reveals a homodimeric assembly with each monomer displaying a two-domain fold. The C-terminal domain, which binds the effector N-acetylglucosamine-6-phosphate, adopts a chorismate lyase fold, whereas the N-terminal domain contains a winged helix–turn–helix DNA-binding domain. Isothermal titration calorimetry and site-directed mutagenesis revealed that the effector-binding site in YvoA coincides with the active site of related chorismate lyase from Escherichia coli. The characterization of the DNA- and effector-binding properties of two disulfide-bridged mutants that lock YvoA in two distinct conformational states provides for the first time detailed insight into the allosteric mechanism through which effector binding modulates DNA binding and, thereby regulates transcription in a representative GntR/HutC family member. Central to this allosteric coupling mechanism is a loop-to-helix transition with the dipole of the newly formed helix pointing toward the phosphate of the effector. This transition goes in hand with the emergence of internal symmetry in the effector-binding domain and, in addition, leads to a 122° rotation of the DNA-binding domains that is best described as a jumping-jack-like motion.

Redox-Dependent Translocation of p53 to Mitochondria or Nucleus in Human Melanocytes after UVA- and UVB-Induced Apoptosis

The p53 protein is an important transcription factor and tumor suppressor that is induced in response to many forms of cellular stress. UVA irradiation of human melanocytes caused generation of reactive oxygen species, which altered the intracellular redox balance and was accompanied by translocation of p53 to mitochondria. In contrast, UVB did not affect the redox status and p53 was translocated to the nucleus. Although different intracellular location of p53, UVA/B induced apoptosis through the intrinsic pathway detected as translocation of Bax to mitochondria, release of cytochrome c, and activation of caspases. These events were all prevented by inhibition of p53 with pifithrin-alpha. Furthermore, inhibition of p53 prevented lysosomal membrane permeabilization, detected as translocation of cathepsins to the cytosol, after UVB exposure, whereas UVA-induced lysosomal release was unaffected by inhibition of p53. In control cells, p53 coimmunoprecipitated with the antiapoptotic proteins Bcl-2 and Bcl-x(L) and upon UVA exposure the interaction was replaced by binding to the proapoptotic proteins Bax, Noxa, and Puma. Our findings suggest that UVA-induced apoptosis is caused by extensive oxidative damage leading to p53-regulated mitochondrial release, whereas UVB induces DNA damage and apoptosis signaling upstream of lysosomal membrane permeabilization.

ATP Binding and Hydrolysis by Mcm2 Regulate DNA Binding by Mcm Complexes

The essential minichromosome maintenance (Mcm) proteins Mcm2 through Mcm7 likely comprise the replicative helicase in eukaryotes. In addition to Mcm2–7, other subcomplexes, including one comprising Mcm4, Mcm6, and Mcm7, unwind DNA. Using Mcm4/6/7 as a tool, we reveal a role for nucleotide binding by Saccharomyces cerevisiae Mcm2 in modulating DNA binding by Mcm complexes. Previous studies have shown that Mcm2 inhibits DNA unwinding by Mcm4/6/7. Here, we show that interaction of Mcm2 and Mcm4/6/7 is not sufficient for inhibition; rather, Mcm2 requires nucleotides for its regulatory role. An Mcm2 mutant that is defective for ATP hydrolysis (K549A), as well as ATP analogues, was used to show that ADP binding by Mcm2 is required to inhibit DNA binding and unwinding by Mcm4/6/7. This Mcm2-mediated regulation of Mcm4/6/7 is independent of Mcm3/5. Furthermore, the importance of ATP hydrolysis by Mcm2 to the regulation of the native complex was apparent from the altered DNA binding properties of Mcm2 KA–7. Moreover, together with the finding that Mcm2 K549A does not support yeast viability, these results indicate that the nucleotide-bound state of Mcm2 is critical in regulating the activities of Mcm4/6/7 and Mcm2–7 complexes.

Synthesis, Cytotoxicity, and DNA Interactions of New Cisplatin Analogues Containing Substituted Benzimidazole Ligands

Six new platinum(II) complexes with 1-H or methyl-2-chloromethyl or acetoxymethyl or 2'-hydroxyethylbenzimidazole carrier ligands were synthesized and evaluated for their reactivity against model nucleophile I(-), cellular uptake, and in vitro antiproliferative activities against the human MCF-7 breast and HeLa cervix cancer cell lines. The effect of the compounds on pBR322 plasmid DNA was studied by gel electrophoretic mobility measurements. Flow cytometric analysis was also carried out to study the effect of representative compounds 1 and 2, bearing 2-chloromethyl or -acetoxymethylbenzimidazole carrier ligands, on the cell cycle distribution of MCF-7 and HeLa cells, respectively. In general, it was found that Pt(II) complexes were less cytotoxic than cisplatin and were comparable to carboplatin. The results of the plasmid DNA interaction and the restriction studies suggest that changing the chemical structure of the benzimidazole ligands may modulate DNA binding mode and the sequence selectivity. Compounds 1 and 2 had no significant effect on the cell cycle profile of the cells used. However, compound 2 induced a significant increase in the SubG1 cell population at a concentration of 20 microM.

Multiple Intrinsically Disordered Sequences Alter DNA Binding by the Homeodomain of the Drosophila Hox Protein Ultrabithorax

During animal development, distinct tissues, organs, and appendages are specified through differential gene transcription by Hox transcription factors. However, the conserved Hox homeodomains bind DNA with high affinity yet low specificity. We have therefore explored the structure of the Drosophila melanogaster Hox protein Ultrabithorax and the impact of its nonhomeodomain regions on DNA binding properties. Computational and experimental approaches identified several conserved, intrinsically disordered regions outside the homeodomain of Ultrabithorax that impact DNA binding by the homeodomain. Full-length Ultrabithorax bound to target DNA 2.5-fold weaker than its isolated homeodomain. Using N-terminal and C-terminal deletion mutants, we demonstrate that the YPWM region and the disordered microexons (termed the I1 region) inhibit DNA binding approximately 2-fold, whereas the disordered I2 region inhibits homeodomain-DNA interaction a further approximately 40-fold. Binding is restored almost to homeodomain affinity by the mostly disordered N-terminal 174 amino acids (R region) in a length-dependent manner. Both the I2 and R regions contain portions of the activation domain, functionally linking DNA binding and transcription regulation. Given that (i) the I1 region and a portion of the R region alter homeodomain-DNA binding as a function of pH and (ii) an internal deletion within I1 increases Ultrabithorax-DNA affinity, I1 must directly impact homeodomain-DNA interaction energetics. However, I2 appears to indirectly affect DNA binding in a manner countered by the N terminus. The amino acid sequences of I2 and much of the I1 and R regions vary significantly among Ultrabithorax orthologues, potentially diversifying Hox-DNA interactions.

Cardioplegia and Diazoxide Modulate STAT3 Activation and DNA Binding

Previously, we have shown that magnesium supplemented potassium (DSA) cardioplegia and DSA containing diazoxide (DSA+DZX) significantly decrease apoptosis after ischemia. The mechanism for this enhanced cardioprotection was unknown, but we believed that alterations in signal transducers and activators of transcription (STATs) may play a role. To investigate this hypothesis, we examined the effects of DSA and DSA+DZX cardioplegia on STAT1/3 phosphorylation and DNA binding in the in situ blood perfused pig heart model. Pigs (32 to 42 kg) undergoing total cardiopulmonary bypass underwent left anterior descending coronary artery occlusion for 30 minutes. The aorta was crossclamped and DSA (n = 6) or DSA+DZX (n = 6) cardioplegia was administered, followed by 30 minutes of global ischemia and 120 minutes of reperfusion. Control hearts (n = 3) received cardiopulmonary bypass and sham reperfusion only. Tissue samples from regional and global ischemia zones were harvested and used for Western blot and electrophoretic mobility shift assay. Regional and global ischemia significantly increase proapoptotic STAT1 tyrosine phosphorylation. This increase is significantly greater in the regional as compared with the global ischemia zone. Tyrosine phosphorylation of antiapoptotic STAT3 is increased in the global ischemic zone but is significantly decreased in the regional ischemic zone and is associated with increased apoptosis. The DSA+DZX significantly increases tyrosine phosphorylation of antiapoptotic STAT3 and DNA binding in the regional ischemia zone and significantly decreases apoptosis. The addition of diazoxide to DSA cardioplegia significantly decreases apoptosis by significantly increasing tyrosine phosphorylation of STAT3 and its DNA binding and represents an additional modality for enhancing myocardial protection.

DNA and Protein Footprinting Analysis of the Modulation of DNA Binding by the N-Terminal Domain of the Saccharomyces cerevisiae TATA Binding Protein

Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by "protein footprinting" with hydroxyl radical (*OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.

Transcriptional Repression by the T-box Proteins Tbx18 and Tbx15 Depends on Groucho Corepressors

Tbox18 (Tbx18) and Tbox15 (Tbx15) encode a closely related pair of vertebrate-specific T-box (Tbx) transcription factors. Functional analyses in the mouse have proven the requirement of Tbx15 in skin and skeletal development and of Tbx18 in the formation of the vertebral column, the ureter, and the posterior pole of the heart. Despite the accumulation of genetic data concerning the embryological roles of these genes, it is currently unclear how Tbx18 and Tbx15 exert their function on the molecular level. Here, we have initiated a molecular analysis of Tbx18 and Tbx15 proteins and have characterized functional domains for nuclear localization, DNA binding, and transcriptional modulation. We show that both proteins homo- and heterodimerize, bind to various combinations of T half-sites, and repress transcription in a Groucho-dependent manner. Competition with activating T-box proteins may constitute one mode of action as we show that Tbx18 interacts with Gata4 and Nkx2-5 and competes Tbx5-mediated activation of the cardiac Natriuretic peptide precursor type a-promoter and that ectopic expression of Tbx18 down-regulates Tbx6-activated Delta-like 1 expression in the somitic mesoderm in vivo.

Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova

In Sterkiella nova, alpha and beta telomere proteins bind cooperatively with single-stranded DNA to form a ternary alpha.beta.DNA complex. Association of telomere protein subunits is DNA-dependent, and alpha-beta association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, alpha and DNA first form a stable alpha.DNA complex followed by the addition of beta in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different, with DeltaCp = -305 +/- 3 cal mol(-1) K(-1) for alpha binding with DNA and DeltaCp = -2010 +/- 20 cal mol(-1) K(-1) for the addition of beta to complete the alpha.beta.DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed alpha.beta complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of beta. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length beta or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of beta.

β-(1→3)-D-glucan modulates DNA binding of nuclear factors κB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1β/IL-1 receptor antagonist ratio

Backgroundβ-1→3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses. Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal β-1→3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1).ResultsDespite an activation of nuclear factor (NF)κB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1β, IL-6, tumor necrosis factor α or interferon γ induced by glucan phosphate. Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h: 5000 pg/ml), likely due to binding of NFκB to a consensus site in the IL-8 promoter. An increase in IL-1receptor antagonist(RA) production (peak at 24 h: 12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels. Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments. When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFκB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA. Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1β and IL-6. As a consequence, glucan phosphate shifted the TSST-1-induced IL-1β/IL-1RA ratio towards an anti-inflammatory phenotype. Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2.ConclusionThus, β-1→3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction. Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a β-1→3-D-glucan.

Close‐up of a replisome

The assembly of proteins, the replisome, that replicates DNA is comprised of molecular motors and switches that coordinate reactions through multiple interactions. Although differing in complexity, the basic machinery of all replication systems is remarkably conserved throughout evolution. The economy of proteins found in the replication system of bacteriophage T7 has made possible the analysis of a replisome at the molecular level. Four proteins of known crystal structure are sufficient for the basis reactions: T7 DNA polymerase and its processivity factor (E. coli thioredoxin), T7 helicase-primase, and T7 ssDNA binding protein. A reconstituted replisome effects DNA synthesis in which leading and lagging strand synthesis are coordinated and processive. Nascent Okazaki fragments are found within a loop of lagging strand DNA. Occupancy of the nucleotide binding sites of the hexameric helicase, located between adjacent subunits, modulate DNA binding via conformational changes. The transfer of ssDNA from subunit to subunit is coupled to the sequential hydrolysis of dTTP. The recycling of the lagging strand DNA polymerase requires multiple steps and interactions. Interactions between the helicase and primase domains result in the pausing of leading strand synthesis observed during primer synthesis in single molecule experiments. Thioredoxin binds to a unique 76 amino acid fragment located in the thumb sub-domain of T7 DNA polymerase initiating conformational changes that lead to interactions with the primer-template, the helicase-primase, and the ssDNA binding protein. The acidic C-termini of the latter two proteins are essential for these interactions. Thus a three-way switch mediates the interactions of the multiple components during DNA synthesis. Supported by USPHS Grant GM 54397.

Determining the mechanism of allosteric regulation of NikR binding to DNA activated by Ni 2+

E. coli NikR is a nickel and DNA binding transcriptional repressor protein that responds to elevated levels of intracellular nickel by binding to the nikABCDE operator and repressing nickel permease production. NikR is a homotetramer composed of two distinct domains. The two N-terminal ribbon-helix-helix (RHH) domains are made up of intertwined dimers and bind to DNA while the C-terminal regulatory domain (an ACT type fold) is responsible for tetramerization and contains one high affinity nickel binding site per chain (1 Ni2+/monomer, Kd ~ 1 pM). Metal binding controls the DNA-binding capability of NikR. One Ni2+ per monomer activates NikR tetramer binding to nikABCDE operator DNA with ~5nM affinity. The mechanism by which nickel modulates DNA binding affinity is not known. Specific DNA binding also causes a change in high-affinity Ni2+coordination geometry (4- to 6- coordinate), suggesting a conformational connection between nickel and DNA binding sites. We have utilized equilibrium molecular dynamics (MD) analyses and conformational clustering strategies to explore the structural and energetic connections between NikR domains. In addition we have used coarse-grained modeling techniques to elucidate large-scale NikR structural transitions. We have also applied hydrogen/deuterium (H/D) exchange measured by mass spectrometry to experimentally measure conformational changes caused by metal and DNA binding. These three strategies are combined to suggest residues that are important for transducing the Ni2+ binding signal into activation of the DNA binding competent state of NikR. Site-directed mutagenesis of E. coli NikR and subsequent biophysical characterization will reveal the role of these residues in allosteric activation of this regulatory protein.

The Deinococcus radiodurans-Encoded HU Protein Has Two DNA-Binding Domains

Deinococcus radiodurans can reconstitute its genome from double-strand breaks, most likely due to unusually efficient DNA repair and recombination. Factors that may contribute to such processes include the histone-like protein HU. The D. radiodurans-encoded HU (DrHU), which binds preferentially to DNA recombination intermediates, contains a 47-amino acid extension preceding the fold characteristic of HU proteins. Here we use electrophoretic mobility shift assays and DNA footprinting to show that the DrHU N-terminal domain significantly modulates DNA binding. The truncated DrHU (deltaDrHU), comprising only the conserved DNA-binding fold, has a site size of approximately 11 bp in contrast to full-length DrHU which does not stably engage DNA shorter than approximately 50 bp. Unlike wild-type DrHU, deltaDrHU distinguishes between linear DNA and DNA with nicks or gaps. DeltaDrHU also binds preferentially to four-way junction (4WJ) DNA, with half-maximal saturation of 1.4 +/- 0.4 nM compared to 20 +/- 2 nM for 37 bp duplex DNA. However, in contrast to full-length protein which binds the junction arms, deltaDrHU primarily protects the junction crossover. Evidently, the DrHU N-terminus changes the mode of binding to both 4WJ DNA, duplex DNA, and DNA with nicks or gaps, thereby resulting in DrHU binding preferentially only to 4WJ structures. Combined with Western blots that confirm the presence of the extended form of DrHU in vivo, our data provide mechanistic insight into discrimination between 4WJ DNA and other distorted DNA constructs and suggest that an in vivo role of DrHU may be to stabilize DNA junctions.

This Month in Gastroenterology

This Month in Gastroenterology

Intra-domain communication between the N-terminal and DNA-binding domains of the androgen receptor: modulation of androgen response element DNA binding

The androgen receptor (AR) is a ligand-activated transcription factor that recognises and binds to specific DNA response elements upon activation by the steroids testosterone or dihydrotestosterone. In vitro, two types of response element have been characterised - non-selective elements that bind the androgen, glucocorticoid and progesterone receptors, and androgen receptor-selective sequences. In the present study, the allosteric effects of DNA binding on the receptor amino-terminal domain (NTD) were studied. Binding to both types of DNA response element resulted in changes in the intrinsic fluorescence emission spectrum for four tryptophan residues within the AR-NTD and resulted in a more protease-resistant conformation. In binding experiments, it was observed that the presence of the AR-NTD reduced the affinity of receptor polypeptides for binding to both selective and non-selective DNA elements derived from the probasin, PEM and prostatin C3 genes respectively, without significantly altering the protein-base pair contacts. Taken together, these results highlight the role of intra-domain communications between the AR-NTD and the DNA binding domain in receptor structure and function.

In vitro analysis of partial loss-of-function ZIC2 mutations in holoprosencephaly: alanine tract expansion modulates DNA binding and transactivation

Heterozygous loss-of-function mutations in ZIC2 result in the severe brain malformation known as holoprosencephaly (HPE), indicating that forebrain development is exquisitely sensitive to the activity of this poorly understood transcription factor. To identify the regions of ZIC2 that are essential for activity, we have assessed the ability of a variety of ZIC2 mutant proteins to function in in vitro assays. Two sources of information were used to design relevant mutations. First, phenotype producing mutations in human and in mouse ZIC2 were mimicked and secondly, a comparative sequence analysis of the C-terminal was carried out. Analysis of these mutations suggests that either a decrease or an increase in ZIC2 mediated transcriptional activity can produce a forebrain phenotype. In addition, the analysis reveals that the C-terminal of ZIC2 contains both activation and repression domains. This region of ZIC2 contains an alanine-tract, and expansion of this domain is associated with HPE. In vitro analysis of proteins with alterations in alanine-tract length illustrates that the C-terminal alanine-tract of ZIC2 influences the strength of DNA binding and alters transcriptional activity in a promoter-specific manner. This finding provides a possible mechanism by which alanine-tract expansion mutations could alter the function of other transcription factors.