INTERROGATING THE REGULATION AND FUNCTION OF ZFP30 IN AIRWAY INFLAMMATION

Neutrophils play an integral role in the innate immune system, but robust recruitment of these cells can cause collateral host tissue damage. Neutrophils have been linked to the severity of a number of pulmonary diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, and acute respiratory distress syndrome. These diseases represent a significant economic and health burden, so increased understanding of neutrophil chemotaxis is of vital importance. In my dissertation, I have utilized molecular biology and mouse models to characterize Zfp30, a candidate regulator of the hallmark neutrophil chemokine CXCL1. I identified a specific SNP, rs51434084, that underlies a previously identified Zfp30 eQTL, and I demonstrated the mechanism through which this SNP impacts transcription: differential binding of the transcriptional repressor ZFP148. ZFP148 has a known regulatory role on the neutrophil chemokine CXCL5, and our results suggest an additional impact on CXCL1 via Zfp30. Additionally, I utilized an ex vivo model of airway epithelial cells generated from a Zfp30 knockout mouse strain on a C57BL/6J background to demonstrate that Zfp30 significantly modulates CXCL1 and IL-6 secretion in airway epithelial cells. In vivo models utilizing this knockout mouse strain revealed additional effects of Zfp30 knockout on IL-6 secretion in BALF, liver and spleen development, and blood glucose level. These studies explored and confirmed an impact of Zfp30 on the secretion of cytokines that modulate airway neutrophilia, and this body of work lays a foundation off of which future studies of Zfp30’s impact on development and diabetes may extend.Doctor of Philosoph

NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases

Differential Regulation of Zfp30 Expression in Murine Airway Epithelia Through Altered Binding of ZFP148 to rs51434084

Neutrophil chemotaxis to the airways is a key aspect of host response to microbes and a feature of multiple pulmonary diseases including asthma. Tight regulation of this recruitment is critical to prevent unwanted host tissue damage and inflammation. Using a mouse (Mus musculus) model of asthma applied to the Collaborative Cross population, we previously identified a lung gene expression quantitative trait locus (eQTL) for Zinc finger protein 30 (Zfp30) that was also a QTL for neutrophil recruitment and the hallmark neutrophil chemokine CXCL1. The Zfp30 eQTL is defined by three functionally distinct haplotypes. In this study, we searched for causal genetic variants that underlie the Zfp30 eQTL to gain a better understanding of this candidate repressor’s regulation. First, we identified a putative regulatory region spanning 500 bp upstream of Zfp30, which contains 10 SNPs that form five haplotypes. In reporter gene assays in vitro, these haplotypes recapitulated the three previously identified in vivo expression patterns. Second, using site-directed mutagenesis followed by reporter gene assays, we identified a single variant, rs51434084, which explained the majority of variation in expression between two out of three haplotype groups. Finally, using a combination of in silico predictions and electrophoretic mobility shift assays, we identified ZFP148 as a transcription factor that differentially binds to the Zfp30 promoter region harboring rs51434084. In conclusion, we provide evidence in support of rs51434084 being a causal variant for the Zfp30 eQTL, and have identified a mechanism by which this variant alters Zfp30 expression, namely differential binding of ZFP148

Structure–Activity Relationship Development Efforts towards Peripherally Selective Analogs of the Cannabinoid Receptor Partial Agonist BAY 59-3074

Selective modulation of peripheral cannabinoid receptors (CBRs) has potential therapeutic applications in medical conditions, including obesity, diabetes, liver diseases, GI disorders and pain. While there have been considerable efforts to produce selective antagonists or full agonists of CBRs, there has been limited reports on the development of partial agonists. Partial agonists targeting peripheral CBRs may have desirable pharmacological profiles while not producing centrally mediated dissociative effects. Bayer reported that BAY 59-3074 is a CNS penetrant partial agonist of both CB1 and CB2 receptors with efficacy in rat models of neuropathic and inflammatory pain. In this report, we demonstrate our efforts to synthesize analogs that would favor peripheral selectivity, while maintaining partial agonism of CB1. Our efforts led to the identification of a novel compound, which is a partial agonist of the human CB1 (hCB1) receptor with vastly diminished brain exposure compared to BAY 59-3074

Design, Synthesis, and Structure–Activity Relationship Studies of (4-Alkoxyphenyl)glycinamides and Bioisosteric 1,3,4-Oxadiazoles as GPR88 Agonists

International audienc

MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression

<div><p>MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10⁻¹⁶) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.</p></div

Higher expression of microRNAs forming triplex structures with duplex DNA is more frequently associated with increased gene expression.

<p>MicroRNA and mRNA expression were measured in leukemia cells (ALL) obtained at the time of diagnosis from two cohorts of patients (St. Jude Protocols Total 15 and Total 16). Genome-wide linear correlations between microRNA expression and mRNA expression calculated to form grade 1 triplex structures were assessed in each cohort separately and then a meta-analysis was performed. <b>(A)</b> The distributions of Spearman p-values for associations with positive or <b>(B)</b> negative correlations are shown. Over-representation of small p-values for positive associations was significantly enriched as compared to negative associations.</p

Detection of DNA-DNA and RNA-DNA triplexes by EMSA and NMR, and molecular modeling of miRNA-duplex DNA triplex.

<p><b>(A)</b> EMSA; 5’ ROX-labeled hairpin duplex DNA (0.1 μM) was incubated for 3-hrs at 22°C in the presence (lanes 2–11) or absence (lane 1) of 2.5 μM 483-opti DNA oligo, and increasing concentration (30, 60, 150 μM) of Hoogsteen bond-optimized hsa-miR-483-5p (483-opti, lanes 3–5), hsa-miR-483-5 (483, lanes 6–8), or a scrambled RNA oligo (Scramble, lanes 9–11). Duplexes and triplexes were resolved on a 20% non-denaturing acrylamide gel, and the ROX-signal visualized. Triplex of 483-opti DNA oligo and duplex DNA is readily detected (lane 2). The 483-opti RNA oligo competes with 483-opti DNA oligo for binding to duplex DNA which is evident by increased amounts of duplex DNA and decreased amounts of triplex (compare lanes 3–5 with lane 2). Hsa-miR-483-5p (483) and scrambled RNA, because of the fewer number of favorable Hoogsteen bonds, did not compete with the 483-opti DNA oligo for binding to duplex DNA (lanes 6–7 and 9–10, respectively). <b>(B-C)</b> NMR; Two-Dimensional (2D) [¹H, ¹H] TOCSY spectra of free single stranded hairpin duplex DNA (blue contours), hairpin duplex DNA combined with hsa-miR-483-5p RNA oligo (green contours; 1:1.5 ratio), and hairpin duplex DNA with single stranded DNA oligo with the same sequence as hsa-miR-483-5p (red contours; 1:1ratio). <b>(B)</b> Thymidine cross-peaks between H6 and H7 (methyl), and <b>(C)</b> cytosine cross-peaks between H5 and H6. Single stranded RNA (hsa-miR-483-5p) or single stranded DNA with hairpin duplex DNA show similar improvement in peak the intensities, and similar chemical shift perturbations/appearance of new peaks highlighted in blue boxes, suggesting that single stranded DNA and single stranded RNA of the same sequence bind to DNA duplex in a similar manner; the major differences (peaks in red boxes) are one peak among thymidine cross-peaks, showing an intermediate change (peak disappearing) with singe stranded RNA while saturated with hairpin duplex DNA, and two new peaks among cytosine cross-peaks showing much higher intensities with single stranded DNA, indicating that the latter DNA binds to duplex DNA duplex with higher binding affinity than RNA, consistent with the results obtained by EMSA. <b>(D)</b> Molecular model of hsa-miR-483-5p-DNA triplex. (I): the model of predicted miRNA and corresponding DNA duplex sequences (16 favorable Hoogsteen pairings). All predicted Hoogsteen base pairs are well maintained after removal of positional and distance restraints(II): negative control (antisense hsa-miR-483-5p) of model with 9 favorable Hoogsteen pairings. Both RNA and DNA duplex are largely twisted and nearly all predicted Hoogsteen pairings cannot be stably maintained. Residues in favor of Hoogsteen hydrogen bond formation are shown in red while the others are shown in blue.</p

MicroRNAs form triplex structures with DNA.

<p><b>(A)</b> Duplex DNA identified by genome-wide screens of binding sites was incubated in presence or absence of a synthesized hsa-miR-483-5p with a 3’ ROX label to perform a FRET assay to detect triplex formation (illustrated in <b>3B</b>). In the absence of ROX labeled hsa-miR-483-5p (<b>3A</b>, black line) a single emission peak at 520nm is observed which, with the addition of ROX labeled hsa-miR-483-5p (<b>3A</b>, red line), is diminished and a second FRET induced emission peak at 610nm is observed. <b>(C)</b> In a complementary surface plasmon resonance (SPR) based assay (illustrated in <b>3D</b>), a 3’ biotin labeled hsa-miR-483-5p was immobilized and duplex DNA was introduced in triplicate in a 2-fold dilution series starting at 20 nM.</p