Transcriptional regulation of the chicken MLC3f gene

The expression of the chicken fast skeletal myosin alkali light chain (MLC) 3f is subject to complex patterns of control by developmental and physiologic signals. Regulation over MLC3f gene expression is thought to be exerted primarily at the transcriptional level. The purpose of this dissertation was to identify cis-acting elements on the 5$\sp\prime$ flanking region of chicken MLC3f gene that are important for transcriptional regulation. The results show that the 5$\sp\prime$ flanking region of MLC3f gene contains multiple cis-acting elements. The nucleotide sequence of these elements demonstrates a high degree of conservation between different species and are also found in the 5$\sp\prime$ flanking regions of many muscle protein genes. The first regulatory region is located between $-$185 and $-$150 bp from the transcription start site and contains an AT-rich element. Linker scanner analyses have revealed that this element has a positive effect on transcription of the MLC3f promoter. Furthermore, when linked to a heterologous viral promoter, it can enhance reporter gene expression in a muscle-specific manner, independent of distance or orientation.^ The second regulatory region is located between $-$96 and $-$64 from the transcription start site. Sequences downstream of $-$96 have the capacity to drive muscle-specific reporter gene expression, although the region between $-$96 and $-$64 has no intrinsic enhancer-like activity. Linker scanner analyses have identified a GC-rich motif that required efficient transcription of the MLC3f promoter. Mutations to this region of DNA results in diminished capacity to drive reporter gene expression and is correlated with disruption of the ability to bind sequence-specific transcription factors. These sequence-specific DNA-binding proteins were detected in both muscle and non-muscle extracts. The results suggest that the mere presence or absence of transcription factors cannot be solely responsible for regulation of MLC3f expression and that tissue-specific expression may arise from complex interactions with muscle-specific, as well as more ubiquitous transcription factors with multiple regulatory elements on the gene. ^

ALLERGEN SENSITIZATION AND IMMUNOMODULATION BY SYNTHETIC LIGANDS, PUL-042, IN AN ALLERGEN-INDUCED ASTHMA MURINE MODEL

Allergen-induced asthma is the leading form of asthma and a chronic condition worldwide. Common allergens are known to contribute to the pathogenesis of this disease. Murine models of allergic asthma have mostly used an intraperitoneal route of sensitization (not airway) to study this disease. Allergic asthma pathophysiology involves the activation of TH2-specific cells, which triggers production of IgE antibodies, the up-regulation of TH2-specific cytokines (i.e. IL-4, IL-5, IL-9 and IL-13), increased airway eosinophilia, and mucin hypersecretion. Although there are several therapeutics currently treating asthmatic patients, some of these treatments can result in drug tolerance and may be linked to increased mortality. CpG oligodeoxynucleotides (ODNs) is a synthetic ligand that targets Toll-like Receptor (TLR) 9. It has been evaluated as a therapeutic agent for the treatment of cancer, infectious diseases, and for treating allergy and asthma. PUL-042 is also a synthetic TLR ligand and is composed of two agonists against TLR2/6 heterodimer and TLR9. Previous studies have evaluated PUL-042 for its ability to confer resistance against bacterial and viral lung infection. These findings, combined with studies performed using CpG ODNs, led to speculation that PUL-042 dampens the immune response in allergen-induced asthma. My thesis research investigated airway route sensitization and airway delivery of PUL-042 to evaluate its effects in reducing an allergen-induced asthma phenotype in a murine model. The results of this study contribute to the foundation for future investigations to evaluate the efficacy of PUL-042 as a novel therapy in allergic-asthma disease.

Characterization of the MLC box: A conserved {\it cis\/}-acting DNA element present in the chicken MLC1s/1c gene

The slow/cardiac alkali myosin light chain (MLC1s/1c) is a member of a multigene family whose protein products are essential for activation of the myosin ATPase. In the adult, the MLC1s/1c isoform is expressed in both cardiac and slow-twitch skeletal muscles, while it is expressed by all skeletal muscles during development.^ To elucidate the molecular mechanisms that underlie the transcriptional regulation of MLC1s/1c gene expression, the immediate 5$\sp\prime$ flanking region of the gene was isolated and shown to be capable of directing reporter gene expression. Analysis of this region revealed a 110 bp muscle-specific enhancer that includes a myocyte-specific enhancer-binding factor 2 (MEF-2) site, E-boxes, which are potential binding sites for the basic-helix-loop-helix proteins such as MyoD, and a MLC box. The focus of the thesis was to identify the role of the MLC box in expression of the MLC1s/1c gene.^ The MLC box is a member of the family of CArG box containing cis-acting DNA elements. Mutagenesis showed that the MLC box is necessary, but not sufficient, for the expression of a reporter gene linked to the 5$\sp\prime$ flanking region of the MLC1s/1c gene. Linker scanner and site-directed mutagenesis identified a number of potential sites within the 110 bp muscle-specific enhancer that may cooperate with the MLC box. These are the MEF-2 site, the E-box site, and a 10 bp element located upstream of the MEF-2 site that does not have sequence similarity with any known cis-acting element. The MLC box is capable of binding to factors present in muscle nuclear extracts, as well as to human recombinant serum response factor (SRF). Binding of SRF to the MLC box was correlated with the ability of the 5$\sp\prime$ flanking region of the MLC1s/1c gene to drive reporter gene expression. Results suggest a model in which binding of SRF to the MLC box activates expression of the MLC1s/1c gene while binding of the factors present in the nuclear extracts suppresses the expression of the gene. (Abstract shortened with permission of author.) ^