The gene lin-42 is an ortholog of the mammalian period gene, a component of the circadian pathway that converts environmental stimuli into behavioral and physiological outputs over 24 hours. Mammalian period also regulates adult stem cell differentiation, although this function is poorly understood. The structure, function and expression of lin-42 are all similar to period. Therefore, we are studying lin-42 regulation and function during C. elegans larval development as a model for understanding period control of mammalian stem/progenitor cell development. Previous work has shown that ZTF-16 is a regulator of lin-42 transcription. The lin-42 locus encodes three isoforms, and we have characterized lin-42 isoform specific regulation by ZTF-16 through phenotypic assays and analysis of transcriptional reporter strains. Our data show that ZTF-16 regulates the cyclic expression of lin-42A and lin-42B during larval development. However, ztf-16 is not expressed during the adult stage and does not regulate lin-42C, which is expressed only in adults and may be responsible for the circadian functions of lin-42. We also show that ztf-16 reduction-of-function mutations phenocopy loss-of- function phenotypes of the lin-42A/B isoforms. Finally, we have found that deletion of a putative ZTF-16 transcription factor binding site within the lin-42BC promoter abolishes tissue-specific expression patterns. Together, these data indicate that ZTF-16 is required to regulate the expression of lin-42A/B during C. elegans development, and may do this by direct binding to the lin-42BC promoter. Our  findings pave the way for testing the possible regulation of period expression by HIL-family transcription factors in mammalian tissues. / Master of Science

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a growing problem worldwide due to the emergence of multi-drug resistant and extensively-drug resistant strains of the bacteria. A key to combatting the spread of these strains lies in the understanding of gene expression occurring in Mtb. This study focuses on the development and optimization of a luciferase-based bioluminescent transcriptional reporter that can be used to monitor gene expression in Mtb. The luminescent signal emitted from the reporter can be measured and correlated with the level of transcription of certain genes. This study focuses specifically on a gene called whiB7 which encodes a transcription factor known to contribute to the drug resistance of Mtb. The drug-inducible whiB7 promoter was cloned into various locations in the luciferase plasmid in order to determine the ideal configuration of the reporter for maximum luminescence. The optimized luciferase reporter was then compared with a fluorescent transcriptional reporter, mCherry, also under control of the whiB7 promoter. Fluorescent reporters present some disadvantages including delayed kinetics and inability to accurately reflect gene downregulation due to long half-life of reporter proteins. It was hypothesized that the luciferase reporter would solve these problems by offering a more sensitive and dynamic tool to monitor gene expression. Quantitative real-time PCR was used to measure whiB7 mRNA present in cultures containing either the luciferase or mCherry reporters. The luminescent and fluorescent signal given from these reporters was then compared to actual mRNA expression. It was observed that the signal from the luciferase reporter more closely matched mRNA expression at each timepoint, indicating that the luciferase reporter is a better gauge of actual gene expression levels than the mCherry reporter.

Interleukin-10 (IL-10) is an important regulator of immune responses secreted by different cell types. Previous results from our group suggested that the biological effects of this cytokine critically depend on its cellular source. Recent studies reported IL-10 dependent immunosuppressive functions of a specialized subset of regulatory B cells and mast cells. These results relied on adoptive cell transfers, a technique which can potentially introduce artifacts. Therefore, we aimed to readdress these questions in independent models using IL-10 transcriptional reporter mice and various conditional IL-10 mutant mice. Findings in IL-10 reporter system suggested prominent IL-10 transcription in regulatory B cells upon LPS administration. Exposure of mice to contact allergen revealed robust reporter expression in CD8 T cells, moderate to mild reporter expression in CD4 T cells and dendritic cells (DC) respectively, and lack of reporter expression in B cells, mast cells and NK cells in allergen challenged ears. We generated cell-type specific IL-10 mutants by Cre/LoxP-mediated conditional gene inactivation. Efficiency and specificity of Cre-mediated recombination was demonstrated by Southern blot and PCR methods. Various immunogenic challenges in conditional IL-10 mutants did not reveal a role for B cell-derived IL-10 in restraining innate TLR or T cell-dependent inflammatory responses. Likewise, mice with selective inactivation of the il10 gene in mast cells exhibited normal CHS responses and unaltered immune response to CpG oligodeoxynucleotides. On the other hand, DC-specific IL-10 mutants developed excessive inflammatory responses to contact allergens, while innate responses to TLR ligands were not altered. This indicates a non-redundant role for DC-derived IL-10 in contact allergy. Thus, the conditional IL-10 ‘‘knockout’’ mice combined with the novel transcriptional IL-10 reporter system can serve as ideal tools to understand the cell-type specific contributions to IL-10-mediated immune regulation.

Interleukin-10 (IL-10) is an important regulator of immune responses secreted by different cell types. Previous results from our group suggested that the biological effects of this cytokine critically depend on its cellular source. Recent studies reported IL-10 dependent immunosuppressive functions of a specialized subset of regulatory B cells and mast cells. These results relied on adoptive cell transfers, a technique which can potentially introduce artifacts. Therefore, we aimed to readdress these questions in independent models using IL-10 transcriptional reporter mice and various conditional IL-10 mutant mice. Findings in IL-10 reporter system suggested prominent IL-10 transcription in regulatory B cells upon LPS administration. Exposure of mice to contact allergen revealed robust reporter expression in CD8 T cells, moderate to mild reporter expression in CD4 T cells and dendritic cells (DC) respectively, and lack of reporter expression in B cells, mast cells and NK cells in allergen challenged ears. We generated cell-type specific IL-10 mutants by Cre/LoxP-mediated conditional gene inactivation. Efficiency and specificity of Cre-mediated recombination was demonstrated by Southern blot and PCR methods. Various immunogenic challenges in conditional IL-10 mutants did not reveal a role for B cell-derived IL-10 in restraining innate TLR or T cell-dependent inflammatory responses. Likewise, mice with selective inactivation of the il10 gene in mast cells exhibited normal CHS responses and unaltered immune response to CpG oligodeoxynucleotides. On the other hand, DC-specific IL-10 mutants developed excessive inflammatory responses to contact allergens, while innate responses to TLR ligands were not altered. This indicates a non-redundant role for DC-derived IL-10 in contact allergy. Thus, the conditional IL-10 ‘‘knockout’’ mice combined with the novel transcriptional IL-10 reporter system can serve as ideal tools to understand the cell-type specific contributions to IL-10-mediated immune regulation.