G-quadruplex DNA and their binding proteins

It has been speculated that G-rich DNA can form G-quadruplex structures in the promoter region of human genes. Recent results show that stabilization of G-quadruplex structures can silence DNA transcription in vitro, which strongly suggests that G-quadruplex structures and their binding proteins are directly involved in the regulation of gene expression. This dissertation focused on two G rich sequences: (1) the 2-repeat variant a sequence from the Insulin-Linked Polymorphic Region (ILPR) in the promoter region of the human insulin gene and close to the IGF-2 gene; and (2) the GGA-TCC repeat from the promoter region of the ERBB2 gene.;The ILPR is a regulatory sequence in the promoter region upstream of the human insulin gene and is widely recognized as a locus of type 1 diabetes susceptibility. Polymorphism of the ILPR sequence can affect expression of both insulin and the adjacent insulin-like growth factor 2 (IGF-2) genes. Several ILPR variants form G-quadruplex DNA structures in vitro that exhibit affinity binding to insulin and IGF-2. It has been suggested that the ILPR may form G-quadruplexes in vivo as well, raising the possibility that insulin and IGF-2 may bind to these structures in the ILPR in chromatin of live cells. The present work establishes the presence of insulin and IGF-2 in the nucleus of cells cultured from human fetal thymus and their association with the ILPR in the chromatin of these cells. Affinity MALDI-MS was used to test the hypothesis that G-quadruplex structures are involved in the binding interactions of the predominant ILPR sequence with IGF-2 and insulin. Very preliminary AFM images indicated possible G-quadruplex structures in chromatin fragments directly. These are the first studies of the binding of insulin and IGF-2 to the ILPR in vivo and they provide evidence in support of G-quadruplex formation in double-stranded DNA in vivo.;The 28 bp GGA-TCC repeat in the promoter region of ERBB2 has been related to down regulation of the protein erbB2, which is overexpressed in 30% of human breast cancers and is therefore treated as an important biomarker of breast cancer. The 28 bp GGA-TCC repeat is very similar to the GGA repeat of c-MYB, which is known to form a G-quadruplex structure in vitro. Based on the similarity of these two sequences, we hypothesized that the 28 bp GGA-TCC repeat in the ERBB2 promoter region could form a similar G-quadruplex structure and may bind with certain proteins to regulate erbB2 expression. Circular dichroism spectroscopy established that the Pu28-mer forms a (T:H:H:T) G-quadruplex structure that is similar to the structure reported in the literature for the GGA repeat of the c-MYB oncogene under the same experiment conditions. Further evidence is provided by in vitro capture and detection of recombinant MAZ (Zinc finger protein) by immobilized oligonucleotides containing the G-quadruplex forming ERBB2 GGA repeat sequence using an affinity MALDI-MS technique developed in our group. In addition, we identified several possible G-quadruplex binding proteins from nuclear extracts of MCF-7 cancer cells by mass spectrometric analysis. This is the first report of G-quadruplex formation in the GGA-TCC repeat of the ERBB2 gene. The results suggest that G-quadruplex formation in the G-rich region of the ERBB2 promoter plays a role in the regulation of expression of erbB2, the breast cancer biomarker.;Separate work was to increase in vitro and in vivo sample recovery of different cytokines by microdialysis sampling. Microdialysis sampling is a widely used method to sample from complex biological matrices. Cytokines are important signaling proteins that typically exhibit poor microdialysis sampling relative recovery. Heparin was included in the microdialysis perfusion fluid as an affinity agent to increase in vitro and in vivo recovery of different cytokines. The in vitro control and heparin-enhanced relative recoveries for five human cytokines using 0.1 muM heparin in the perfusion fluid flowing at 0.5 muL/min were (n=3): interleukin-4 (IL-4), 4.2+/-0.5% and 7.2+/-3.1%; interleukin-6 (IL-6), 1.4+/-0.8% and 3.6+/-1.3%; interleukin-7 (IL-7), 1.3+/-0.8% and 4.8+/-1.8%; monocyte chemoattractant protein-1 (MCP-1), 9.0+/-1.6% and 19.5+/-2.7%; tumor necrosis factor-alpha (TNF-alpha), 7.4+/-1.3% and 16.9+/-1.6%, respectively. In vivo experiments with control and heparin-perfused microdialysis probes implanted into the peritoneal cavity were performed using Sprague-Dawley rats that were administered lipopolysaccharide to elicit a cytokine response. Only IL-6 was obtained in measurable concentrations in all dialysates to allow relative recovery comparisons between the control vs. heparin-perfused probes. IL-6 in vivo recovery was increased by approximately two-fold with heparin-perfused probes. During in vivo collection detectable concentrations of MCP-1 were only recovered in heparin-perfused probes. Heparin successfully increased the microdialysis sampling relative recovery of human cytokines in vitro and rat cytokines in vivo.