Mutations In DNA-binding Domain Research Articles

Abstract P2-07-04: HER2 overexpressing breast cancers are more frequent in carriers of TP53 DNA-binding domain mutations than in carriers of oligomerization-site mutations

Abstract Germline TP53 mutations are associated with Li-Fraumeni Syndrome (LFS), an autosomal dominant disorder characterized by predisposition to multiple early-onset cancers including breast cancer (BC), the most prevalent tumor among women. The majority of germline TP53 mutations are missense substitutions clustered within the DNA-binding domain of the gene that disrupt the structure and function of the protein. Recently, the germline mutation p.R337H (c.1010G>A), lying in the oligomerization domain of p53, was reported at a high frequency in Southern and Southeastern Brazil. This mutation appears to result in a more subtle defect in the protein, which becomes functionally deficient only under particular conditions and specific tissues. Recent reports suggest that BC are HER2-postive in a high proportion (63-83%) of TP53 mutation carriers. Histopathologic features of breast cancers among carriers of oligomerization domain mutations have not been reported. Our objective was to analize HER2 status in breast cancers from carriers of germline TP53 mutations, both in the DNA-binding and oligomerization domains. We retrospectively reviewed the immunohistochemistry data of breast cancers from 64 carriers of TP53 p.R337H and 6 carriers of nonfunctional germline TP53 mutations (p.G245S, p.R273H, p.G244D or c.672+1G>T). All patients were recruited from the cancer genetics clinic at Hospital de Clínicas de Porto Alegre, Brazil. Germline TP53 mutations were identified by Sanger sequencing of the entire coding sequence and flanking intronic regions. Immunohistochemistry (IHC) was performed using standard procedures. HER2 expression was assessed using a 0 to 3+ scale according the ASCO-CAP Guidelines. All of the six patients with nonfunctional TP53 mutations showed strong (3+) HER2 staining by IHC in their breast tumors. In the group of p.R337H carriers, 23.4% showed significant HER2 overexpression (3+), 21.9% presented ambiguous HER2 results (2+), and 54.7% showed weak or no HER2 expression (1+ or 0). Thus, HER2 overexpressing breast cancers were more frequent in carriers of TP53 nonfunctional (DNA binding domain) mutations (p≤0.001) than in those carrying the oligomerization domain mutation. Preliminary results from this study suggest that germline mutations in different domains of the TP53 gene may predispose to breast cancer through different mechanisms. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-04.

GFI1B Mutation Causes a Platelet Function Defect With Reduced Alpha-Granule Content and Abnormal Aggregation Response

GFI1B is a transcription factor important for erythropoiesis and megakaryocyte development, but previously unknown to be associated with human disease. We have identified a family with an autosomal dominant bleeding disorder associated with thrombocytopenia, red cell anisopoikilocytosis and platelet dysfunction. The severity of bleeding is variable with some affected individuals experiencing spontaneous bleeding while other family members only exhibit abnormal bleeding with surgery. All affected individuals had a moderate thrombocytopenia ranging from 72-149 x109/L with an elevated mean platelet volume. Platelet function was perturbed with prolonged collagen/epinephrine closure times on the PFA-100 (294 vs 125 sec, P <0.001). Platelet aggregation studies were abnormal, with an absent collagen response and primary aggregation only with ADP, arachidonic acid and adrenaline. Genetic linkage analysis and massively parallel sequencing were performed on 16 family members to localize the mutation causing the disease phenotype to chromosome 9. A single nucleotide insertion was identified in GFI1B that predicts a frameshift mutation in the fifth zinc finger DNA-binding domain. This mutation alters the transcriptional activity of the protein as determined by luciferase production from the TGFBR3 and GFI1B promoters. Aberrant transcriptional activity was associated with a marked reduction in platelet alpha-granule content as measured by electron microscopy (1.3 vs 3.1 alpha-granules per 1µm2, P<0.001) with similar changes in platelet protein expression. Proteomic analysis of platelet lysates demonstrated altered levels of cytoskeletal proteins, including talin, vinculin and myosin light chain 6. GFI1B mutation represents a novel human bleeding disorder and the described phenotype identifies GFI1B as a critical regulator of platelet number and function. Disclosures:No relevant conflicts of interest to declare.

Mutations In The Zinc Finger Domain Of Human and Mouse KLF1 Cause Congenital Dyserythropoietic Anemia (CDA) Via Promiscuous DNA Binding and Ectopic Target Gene Expression

Krûppel-like factor-1 (KLF1) is an essential erythroid-specific transcription factor1, 2. A number of studies have shown up to ∼700 genes are poorly expressed when KLF1 is absent3-6. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice, and partly responsible for congenital dyserythropoietic anemia type IV (CDA-IV) observed in humans with dominant mutations in the DNA-binding domain of KLF17. Recently an ENU-generated mouse model of neonatal anemia, ‘nan', was also reported to harbour a mutation in the second zinc-finger of KLF18. Remarkably, the ‘nan' mutation (E339D) resides at exactly the same amino acid which results in human CDA IV (i.e. E325 in humans). Unlike loss of function point mutations in KLF1, this mutation leads to a more severe phenotype than the KLF1 null allele, suggesting it is an unusual dominant mutation9.To investigate how this mutation might cause disease, we introduced tamoxifen-inducible versions of KLF1 and KLF1nan into an erythroid cell line derived from Klf1-/- fetal liver cells10. We performed ChIP-seq to determine genome occupancy site preferences for KLF1 and KLF1nan. We identified about 4-fold the number of binding sites within the genome for KLF1nan versus KLF1; many of these are ectopic or promiscuous. Using de novo motif discovery11, we find KLF1nan binds a slightly degenerate CACC box element (CCMNGCCC) in comparison with wild type KLF1 (CCMCRCCC). This specificity is novel with respect to known TFs, so we think it represents specificity not normally present in mammals. The degenerate motif is consistent with models of how the second zinc finger of KLF1 specifically interacts with the 9bp consensus binding site12,13. We also isolated nascent RNA from wild type and mutant cells, to identify primary transcriptional targets of KLF1 and aberrant targets of the KLF1nanmutation. We performed primary transcript RNA-seq and validation using RT-PCR of pre-processed nuclear transcripts. Together the RNA-seq and ChIP-seq studies have provided a novel explanation for how mutations in KLF1 result in dominant anemia in mice and man. This mechanism, whereby a transcription factor DNA-binding domain mutation leads to promiscuous binding, activation of an aberrant transcriptional program and subsequent derailing of co-ordinated differentiation, is novel.

GFI1B mutation causes a bleeding disorder with abnormal platelet function

GFI1B is a transcription factor important for erythropoiesis and megakaryocyte development but previously unknown to be associated with human disease. A family with a novel bleeding disorder was identified and characterized. Genetic linkage analysis and massively parallel sequencing were used to localize the mutation causing the disease phenotype on chromosome 9. Functional studies were then performed in megakaryocytic cell lines to determine the biological effects of the mutant transcript. We have identified a family with an autosomal dominant bleeding disorder associated with macrothrombocytopenia, red cell anisopoikilocytosis, and platelet dysfunction. The severity of bleeding is variable with some affected individuals experiencing spontaneous bleeding while other family members exhibit only abnormal bleeding with surgery. A single nucleotide insertion was identified in GFI1B that predicts a frameshift mutation in the fifth zinc finger DNA-binding domain. This mutation alters the transcriptional activity of the protein, resulting in a reduction in platelet α-granule content and aberrant expression of key platelet proteins. GFI1B mutation represents a novel human bleeding disorder, and the described phenotype identifies GFI1B as a critical regulator of platelet shape, number, and function.

Requirement of estrogen receptor alpha DNA-binding domain for HPV oncogene-induced cervical carcinogenesis in mice

Cervical cancer is caused by human papillomavirus (HPV) in collaboration with other non-viral factors. The uterine cervix is hormone responsive and female hormones have been implicated in the pathogenesis of the disease. HPV transgenic mice expressing HPV16 oncogenes E6 (K14E6) and/or E7 (K14E7) have been employed to study a mechanism of estrogen and estrogen receptor α (ERα) in cervical carcinogenesis. A chronic exposure to physiological levels of exogenous estrogen leads to cervical cancer in the HPV transgenic mice, which depends on ERα. The receptor is composed of multiple functional domains including a DNA-binding domain (DBD), which mediates its binding to estrogen-responsive elements (EREs) on target genes. A transcriptional control of genes by ERα is mediated by either DBD-dependent (classical) or DBD-independent (non-classical) pathway. Although molecular mechanisms of ERα in cancer have been characterized extensively, studies investigating importance of each pathway for carcinogenesis are scarce. In this study, we employ knock-in mice expressing an ERα DBD mutant (E207A/G208A) that is defective specifically for ERE binding. We demonstrate that the ERα DBD mutant fails to support estrogen-induced epithelial cell proliferation and carcinogenesis in the cervix of K14E7 transgenic mice. We also demonstrate that cervical diseases are absent in K14E7 mice when one ERα DBD mutant allele and one wild-type allele are present. We conclude that the ERα classical pathway is required for cervical carcinogenesis in a mouse model.

Association of TP53 Mutational Status and Gender with Survival after Adjuvant Treatment for Stage III Colon Cancer: Results of CALGB 89803

The TP53 tumor suppressor is frequently mutated in colon cancer, but the influence of such mutations on survival remains controversial. We investigated whether mutations in the DNA-binding domain of TP53 are associated with survival in stage III colon cancer. The impact of TP53 genotype was prospectively evaluated in Cancer and Leukemia Group B 89803, a trial that randomized stage III colon cancer patients to receive adjuvant 5-fluorouracil/leucovorin (5FU/LV) or 5FU/LV with irinotecan (IFL). TP53 mutations were identified in 274 of 607 cases. The presence of any TP53 mutation did not predict disease-free survival (DFS) or overall survival with either adjuvant regimen when men and women were considered together or as separate groups. However, outcome differences among women became apparent when tumor TP53 genotype was stratified as wild-type versus zinc- or non-zinc-binding mutations in the TP53 DNA-binding domain. DFS at 5 years was 0.59, 0.52, and 0.78 for women with TP53 wild-type tumors, and tumors with zinc- or non-zinc-binding mutations, respectively. Survival at 5 years for these same women was 0.72, 0.59, and 0.90, respectively. No differences in survival by TP53 genotype were observed in men. The presence of any TP53 mutation within the DNA-binding domain did not predict survival in stage III colon cancer. However, TP53 genotype was predictive of survival in women following adjuvant therapy. Future colon cancer therapeutic trials, with inclusion of correlative molecular markers, should be designed to permit evaluation of survival and/or response to treatment in women separately from men.

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved β-loop of the helicase domain.

OP095: Novel TP53 coding region mutation in the patients of oral squamous cell carcinoma (OSCC) of Pakistan

Purpose The major risk factor for developing OSCC in countries like Pakistan is the excessive chewing habit of paan (betel quid), chhaliya (betel nut), tobacco, niswar (type of dipping tobacco, made from fresh tobacco leaves, calcium oxide, and wood ash), gutka (the preparation of crushed betel nut, tobacco, catechu, lime, saffron, spices, menthol, and sweet or savory flavors), and manpuri (the powder of betel nut, tobacco and slaked lime). It has been reported that exon 4–9 were the hot spots of the mutation in TP53. This study aims to find out the loss of TP53 functions due to mutation/polymorphism caused by genomic alteration and interaction with tobacco and its related ingredients in Pakistan. Materials and methods A total of 260 OSCC patient’s tissue and blood specimens were collected with informed consent from local hospitals of Karachi and compared with blood samples of 260 age and sex matched controls. Mutations in exons 2–11 of TP53 were examined by polymerase chain reaction and single stranded conformational polymorphisms (PCR-SSCP). The samples showing mobility shift were purified and directly sequenced. Results The novel “AGT” to “ACT” missence mutation was identified at position 719 in exon 7 ofTP53. This change substitutes the amino acid serine with threonine at position 240 of p53 protein suggesting that the threonine allele of p53 gene is associated with mutation in conserved region. This mutation was observed in the tumors of the OSCC patients. The blood samples of the patients and the controls have not shown the nucleotide change in this region. Conclusions This novel missence mutations in DNA binding domain indicated that the DNA structure may be damaged by the use of exogeneous DNA-damaging agents including tobacco related carcinogens present in gutka, niswar and manpuri, which may result in the loss of cellular processes and p53 protein function.

Abstract 880: Analysis of NF-κB as a prediction marker of 5-FU based adjuvant chemotherapy for adenocarcinoma of the stomach.

Abstract Introduction: The relapse rate of gastrointestinal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy is approximately 40%. Therefore, it is important to identify prediction markers of the chemotherapeutic efficacy. Recently, we identified NF-κB as a candidate relapse prediction biomarker from a cell-based screening followed by an immunohistochemical retrospective study (Ishida, et al PLoSONE, 2012). To evaluate the biological significance of NF-κB in the context of 5-FU-based chemotherapy, we analyzed the biological response of NF-κB stimulated by 5-FU. Materials and Methods: Human gastric cancer cell lines (MKN45, MKN74, KE39, GSS, KatoIII) were used. Induction and localization of NF-κB in response to 5-FU by western blot and immunohistochemistry were observed. Transcriptional products induced by 5-FU were investigated using a DNA microarray. Knockdown of p65 and p53 genes was performed to examine their transcriptional machinery. Both DNA binding domain mutation and codon72 polymorphism in p53 gene were also investigated. Pro/Pro variant from codon72 affects the binding efficacy for NF-κB subunit p65. Results: By applying 5-FU, both total p65 and phosphorylated-p65 levels were increased in the nucleus. By DNA microarray analysis, 10 transcript products induced by 5-FU were identified. Interestingly, 5 of 10 transcripts have been known as that of p53 downstream. Theoretical prediction of promoter binding sites of p53 and NF-κB revealed that p53 binding sequences were located closer to the transcription start site than those of NF-κB. However, the density of the consensus sequences of NF-κB was higher than that of p53. Interestingly, there were many binding sequences of NF-κB in TP53 promoter, but only a few of those of p53 in p65 promoter. Knockdown of p65 gene also decreased p53 protein as well as its downstream gene expression, such as p21. In contrast, NF-κB was not affected by TP53 knockdown. MKN45 and GSS, whose codon72 variant is Pro/Pro are relatively resistant to 5-FU, but Arg/Arg variant of KE39 and MKN74 is &amp;gt;10times sensitive to 5-FU. KatoIII with a large deletion of TP53 is resistant to 5-FU. These facts may suggest that NF-κB and p53 binding is relevant to 5-FU sensitivity. Conclusion: Present results suggest that: (i) NF-κB plays a more important role than p53 in response to 5-FU; and (ii) NF-κB may play a compensatory function in p53 mutant cells. From a viewpoint of predicting 5-FU based chemotherapeutic efficacy, NF-κB is considered to be a 5-FU-chemosensitivity prediction marker. Citation Format: Fumitaka Endo, Satoshi Nishizuka, Kazushige Ishida, Kohei Kume, Taishi Ide, Hirokatsu Katagiri, Kaoru Ishida, Takeshi Iwaya, Hisataka Fujiwara, Keisuke Koeda, Go Wakabayashi. Analysis of NF-κB as a prediction marker of 5-FU based adjuvant chemotherapy for adenocarcinoma of the stomach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 880. doi:10.1158/1538-7445.AM2013-880

Number of rare germline CNVs and TP53 mutation types

BackgroundThe Li-Fraumeni syndrome (LFS), an inherited rare cancer predisposition syndrome characterized by a variety of early-onset tumors, is caused by different highly penetrant germline mutations in the TP53 gene; each separate mutation has dissimilar functional and phenotypic effects, which partially clarifies the reported heterogeneity between LFS families. Increases in copy number variation (CNV) have been reported in TP53 mutated individuals, and are also postulated to contribute to LFS phenotypic variability. The Brazilian p.R337H TP53 mutation has particular functional and regulatory properties that differ from most other common LFS TP53 mutations, by conferring a strikingly milder phenotype.MethodsWe compared the CNV profiles of controls, and LFS individuals carrying either p.R337H or DNA binding domain (DBD) TP53 mutations by high resolution array-CGH.ResultsAlthough we did not find any significant difference in the frequency of CNVs between LFS patients and controls, our data indicated an increased proportion of rare CNVs per genome in patients carrying DBD mutations compared to both controls (p=0.0002***) and p.R337H (0.0156*) mutants.ConclusionsThe larger accumulation of rare CNVs in DBD mutants may contribute to the reported anticipation and severity of the syndrome; likewise the fact that p.R337H individuals do not present the same magnitude of rare CNV accumulation may also explain the maintenance of this mutation at relatively high frequency in some populations.

Gain-of-Phosphorylation Mutations in Coiled-Coil and DNA-Binding Domain of STAT1 Identified in Japanese Patients with Chronic Mucocutaneous Candidiasis

Abstract 4831Chronic mucocutaneous candidiasis (CMCD) is a rare congenital disorder characterized by persistent or recurrent skin, nails and mucosal membranes infections caused by Candida albicans. Several studies suggest that impairment of development in Th17 lineage and/or IL-17 signaling could be responsible for development of CMCD and seven responsible genes, CARD9, STAT3, IL12B, IL12RB1, IL17RA, IL17F, and AIRE have been identified. Recently, heterozygous mutations in coiled-coil domain (CCD) and DNA-binding domain (DBD) of STAT1 are identified in approximately 40% of patients with CMCD. Signal transducer and activation of transcription 1 (STAT1) is a DNA-binding factor which regulates specific gene transcription. IFN-γ stimulation results in phosphorylation of STAT1 at Tyr701 (pSTAT1) to induce the homodimerization, a gamma-activated factor (GAF), through the conformational change and the nuclear import. The GAF binds to the gamma-activated sequence (GAS) to induce the transcriptional activities. STAT1 mutations identified in patients with CMCD are gain-of-phosphorylation, gain-of-GAF DNA binding and gain-of-GAS transcription activity in response to IFN-γ, IFN-α and IL-27. Based on the results of transient gene experiments, impairment in dephosphorylation of STAT1 has been considered to be a molecular pathogenesis underlying the increased pSTAT1.Here we report six heterozygous missense mutations in CCD and DBD of STAT1, including two novel heterozygous STAT1 mutation, 1060C>A (L354M) and 986C>T (P329L), in two sporadic and four multiplex cases with CMCD in Japan. We investigated functional significance of these mutations by transient gene expression experiments using U3C STAT1 null fibrosarcoma cells. Similar to the previous studies, all mutant proteins showed increased pSTAT1 in response to IFN-α and IFN-γ. Increased GAF-DNA binding and GAS transcription activity were observed in mutant expressed cells. Thus, these mutations are gain-of-function mutations against GAF mediated transcription activity.We then studied dephosphorylation of STAT1 using peripheral blood mononuclear cells (PBMCs) from the patients. The PBMCs were incubated with Staurosporine, tyrosine kinase inhibitor, followed by IFN-γ stimulation and analyzed by flowcytometry and immunoblot. In both experiments, PBMCs from the patients showed increased pSTAT1 after IFN-γ stimulation. Furthermore, we observed persistent pSTAT1 after Staurosporine treatment. These findings suggest that excess pSTAT1 is caused by an impairment of dephosphorylation. The flowcytometry analysis showed no overlap in mean flow intensity of pSTAT1 between 6 patients and 11 healthy controls after Staurosporine treatment. Therefore, this method can be useful for rapid test of STAT1 function in patients with CMCD. Disclosures:No relevant conflicts of interest to declare.

Chronic Mucocutaneous Candidiasis Caused by a Gain-of-Function Mutation in the STAT1 DNA-Binding Domain

Chronic mucocutaneous candidiasis (CMC) is a heterogeneous group of primary immunodeficiency diseases characterized by chronic and recurrent Candida infections of the skin, nails, and oropharynx. Gain-of-function mutations in STAT1 were very recently shown to be responsible for autosomal-dominant or sporadic cases of CMC. The reported mutations have been exclusively localized in the coiled-coil domain, resulting in impaired dephosphorylation of STAT1. However, recent crystallographic analysis and direct mutagenesis experiments indicate that mutations affecting the DNA-binding domain of STAT1 could also lead to persistent phosphorylation of STAT1. To our knowledge, this study shows for the first time that a DNA-binding domain mutation of c.1153C>T in exon 14 (p.T385M) is the genetic cause of sporadic CMC in two unrelated Japanese patients. The underlying mechanisms involve a gain of STAT1 function due to impaired dephosphorylation as observed in the coiled-coil domain mutations.

HPV E6 and E7 Mediated Regulation of HPV(–) HNSCC Cell Lines

Objective: 1) Determine the capacity of HPV16 E6 and E7 viral oncoproteins to regulate cellular levels of p53 and Rb in the absence of the complete viral genome. 2) Understand the interaction between viral E6 and endogenous p53 by observing the effect of E6 on mutant p53. Method: Two HPV negative HNSCC cell lines with DNA binding domain mutations of p53 were infected with lentiviral vectors containing either HPV16 E6, E7, and luciferase or luciferase alone. Naturally infected HPV16 and18 positive cell lines were evaluated as controls. Expression of p53 and Rb was evaluated with PCR and immunohistochemistry. Cell proliferation was monitored by time to confluence. Results: PCR analysis of cDNA confirmed expression of E6 and E7 in the transduced and naturally infected cell populations and the absence of E6 and E7 expression in the HPV negative cell lines. E6 and E7 transduced cell lines demonstrated reduced staining intensity for p53 and Rb compared to the HPV negative parental cells. Naturally infected HPV positive cell line controls stained weakly for Rb and p53 as expected. Cell proliferation was modestly decreased in E6 and E7 transduced cell lines compared to the parental controls. Conclusion: E6 and E7 viral oncoproteins affect cellular levels of p53 and Rb in the absence of a complete HPV genome. Viral E6 maintains the capacity to target p53 for degradation in HPV negative HNSCC cell lines with mutant p53. Individual oncoproteins can modify the behavior of HPV negative cell lines.

Multiple DNA Binding Domains Mediate the Function of the ERCC1-XPF Protein in Nucleotide Excision Repair

ERCC1-XPF is a heterodimeric, structure-specific endonuclease that cleaves single-stranded/double-stranded DNA junctions and has roles in nucleotide excision repair (NER), interstrand crosslink (ICL) repair, homologous recombination, and possibly other pathways. In NER, ERCC1-XPF is recruited to DNA lesions by interaction with XPA and incises the DNA 5' to the lesion. We studied the role of the four C-terminal DNA binding domains in mediating NER activity and cleavage of model substrates. We found that mutations in the helix-hairpin-helix domain of ERCC1 and the nuclease domain of XPF abolished cleavage activity on model substrates. Interestingly, mutations in multiple DNA binding domains were needed to significantly diminish NER activity in vitro and in vivo, suggesting that interactions with proteins in the NER incision complex can compensate for some defects in DNA binding. Mutations in DNA binding domains of ERCC1-XPF render cells more sensitive to the crosslinking agent mitomycin C than to ultraviolet radiation, suggesting that the ICL repair function of ERCC1-XPF requires tighter substrate binding than NER. Our studies show that multiple domains of ERCC1-XPF contribute to substrate binding, and are consistent with models of NER suggesting that multiple weak protein-DNA and protein-protein interactions drive progression through the pathway. Our findings are discussed in the context of structural studies of individual domains of ERCC1-XPF and of its role in multiple DNA repair pathways.

Ensemble-Based Computational Approach Discriminates Functional Activity of p53 Cancer and Rescue Mutants

The tumor suppressor protein p53 can lose its function upon single-point missense mutations in the core DNA-binding domain (“cancer mutants”). Activity can be restored by second-site suppressor mutations (“rescue mutants”). This paper relates the functional activity of p53 cancer and rescue mutants to their overall molecular dynamics (MD), without focusing on local structural details. A novel global measure of protein flexibility for the p53 core DNA-binding domain, the number of clusters at a certain RMSD cutoff, was computed by clustering over 0.7 µs of explicitly solvated all-atom MD simulations. For wild-type p53 and a sample of p53 cancer or rescue mutants, the number of clusters was a good predictor of in vivo p53 functional activity in cell-based assays. This number-of-clusters (NOC) metric was strongly correlated (r2 = 0.77) with reported values of experimentally measured ΔΔG protein thermodynamic stability. Interpreting the number of clusters as a measure of protein flexibility: (i) p53 cancer mutants were more flexible than wild-type protein, (ii) second-site rescue mutations decreased the flexibility of cancer mutants, and (iii) negative controls of non-rescue second-site mutants did not. This new method reflects the overall stability of the p53 core domain and can discriminate which second-site mutations restore activity to p53 cancer mutants.

Differential and Interactive Effects of Ligand-Bound Progesterone Receptor A and B Isoforms on Tyrosine Hydroxylase Promoter Activity

The classical progesterone receptors (PRs) are expressed in some hypothalamic dopaminergic and brainstem noradrenergic neurones. Progesterone influences prolactin and luteinising hormone release from the anterior pituitary gland, in part by regulating the activity of these catecholaminergic neurones. The present study aimed to determine the effects of PRs on tyrosine hydroxylase (TH) promoter activity. When CAD, SK-N-SH and CV-1 cells were transfected with TH promoter constructs and PR-A or PR-B expression vectors, progesterone treatment caused three- to six-fold increases in TH-9.0 kb promoter activity in PR-B expressing cells, although only a modest increase or no change in PR-A expressing cells. Using CAD cells, deletional analysis mapped the site of PR action to the -1403 to -1304 bp region of the TH promoter. Mutational analysis of putative regulatory sequences in this region indicated that multiple DNA elements are required for complete PR-B transactivation. Electrophoretic mobility shift assays were unable to demonstrate direct PR-B binding to TH promoter DNA sequences. However, chromatin immunoprecipitation analysis indicated PR-B was recruited to the TH promoter. Two different PR-B DNA binding domain mutants had opposing effects on PR-B-mediated TH promoter activation. A GS to AA mutation located in the p-box of the first zinc finger of PR-B inhibited progesterone transactivation of the TH promoter, whereas a C to A mutation in the zinc finger increased transactivation. PR-A was able to inhibit PR-B transactivation in a dose-dependent manner, although the degree of PR-A inhibition was dependent on the TH promoter deletion construct. These data indicate that ligand-bound PR-B is recruited to DNA elements in the TH promoter and acts as a transcriptional activator of the TH gene, and also that changes in the ratio of PR-A to PR-B may affect the ability of progesterone to increase TH expression.

Intramolecular signal transmission in a tetrameric repressor of the IclR family

Members of the IclR family control bacterial genes involved in a number of physiological processes. The IclR-family member TtgV crystallizes as a tetramer, with each TtgV monomer consisting of two domains--a DNA binding domain and an effector recognition domain, which are interconnected by an extended α-helix. When bound to DNA, a kink is introduced so that the extended helix is split in two α-helices (helix-4 and -5). Differential scanning calorimetry studies revealed that TtgV unfolds in a single event, suggesting that the two domains unfold cooperatively. When mutations are introduced in helix-5 that disrupt interactions between Arg98 and Glu102, the thermal unfolding of the TtgV domains becomes uncoupled without compromising effector binding. Two of these mutants (TtgVE102R and TtgVE102A) showed impaired release from target DNA, suggesting that these mutations alter signal transmission. By combining various mutants, we found that the mutations in the connecting α-helix exhibited a dominant effect over mutations in DNA binding and effector binding domains. We propose a model in which the loss of cooperativity of unfolding of TtgV reflects perturbed interdomain communication, and that the transition from the continuous to discontinuous helix may mediate interdomain communication necessary for the proper functioning of TtgV.

Balancing cell numbers during organogenesis: Six1a differentially affects neurons and sensory hair cells in the inner ear

While genes involved in the differentiation of the mechanosensory hair cells and the neurons innervating them have been identified, genes involved in balancing their relative numbers remain unknown. Six1a plays a dual role by promoting hair cell fate while inhibiting neuronal fate in these two lineages. Genes homologous to six1a act as either transcriptional activators or repressors, depending on the partners with which they interact. By assaying the in vivo and in vitro effects of mutations in presumptive protein-protein interacting and DNA-binding domains of Six1a, we show that, in the developing zebrafish inner ear, Six1a promotes hair cell fate by acting as a transcriptional activator and inhibits neuronal fate by acting as a transcriptional repressor. We also identify several potential partners for Six1a that differ between these two lineages. The dual role of Six1a in the developing otocyst provides a mechanism for balancing the relative number of hair cells and neurons during organogenesis of the inner ear.

P1-S1.40 Emerging molecular mutations of reduced susceptibility to third-generation Cephalosporins in Neisseria gonorrhoeae isolates from Saskatchewan, Canada

BackgroundThird-generation cephalosporins (eg, ceftriaxone, Cro) are the antimicrobials of choice for the treatment of gonorrhoea. The molecular mechanisms causing reduced susceptibility to these antibiotics in Neisseria gonorrhoeae (Ng) isolates differ...

Differential altered stability and transcriptional activity of ΔNp63 mutants in distinct ectodermal dysplasias

Heterozygous mutations of p63, a key transcription factor in epithelial development, are causative in a variety of human ectodermal dysplasia disorders. Although the mutation spectrum of these disorders displays a striking genotype-phenotype association, the molecular basis for this association is only superficially known. Here, we characterize the transcriptional activity and protein stability of ΔNp63 mutants (that is, mutants of a p63 isoform that lacks the N-terminal transactivation domain) that are found in ectrodactyly-ectodermal dysplasia-cleft syndrome (EEC), ankyloblepharon-ectodermal dysplasia-clefting syndrome (AEC) and nonsyndromic split-hand/split-foot malformation (SHFM). DNA-binding and sterile alpha motif (SAM) domain mutants accumulate in the skin of EEC and AEC syndrome patients, respectively, and show extended half lives in vitro. By contrast, C-terminal mutations found in SHFM patients have half-lives similar to that of the wild-type protein. The increased half-life of EEC and AEC mutant proteins was reverted by overexpression of wild-type ΔNp63. Interestingly, the mutant proteins exhibit normal binding to and degradation by the E3 ubiquitin ligase Itch. Finally, EEC and AEC mutant proteins have reduced transcriptional activity on several skin-specific gene promoters, whereas SHFM mutant proteins are transcriptionally active. Our results, therefore, provide evidence for a regulatory feedback mechanism for p63 that links transcriptional activity to regulation of protein homeostasis by an unknown mechanism. Disruption of this regulatory mechanism might contribute to the pathology of p63-related developmental disorders.