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Characterization of Transcriptional and Post-transcriptional Regulation of lin-42/Period During Post-embryonic Development of C. elegans

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dc.contributor.authorJames, Tracyen
dc.contributor.committeechairBanerjee, Diyaen
dc.contributor.committeememberKuhn, Jeffrey R.en
dc.contributor.committeememberCimini, Danielaen
dc.contributor.committeememberSobrado, Pabloen
dc.contributor.departmentBiologyen
dc.date.accessioned2014-03-14T20:16:40Zen
dc.date.adate2012-10-23en
dc.date.available2014-03-14T20:16:40Zen
dc.date.issued2012-09-11en
dc.date.rdate2012-10-23en
dc.date.sdate2012-09-24en
dc.description.abstractPeriod, which is broadly conserved in metazoans, regulates circadian timing of neurophysiology as well as cell fate specification. Studies in mouse and humans indicate that period functions as a tumor suppressor and controls adult stem cell differentiation. However, regulation of period function in developmental pathways has not been characterized and appears to be different from its regulation and function in circadian pathways. lin-42 is the Caenorhabditis elegans ortholog of period and has both circadian and developmental timing functions. During post-embryonic larval development, cyclic expression and function of lin-42 controls stage-specific and reiterative cell fate choices of a subset of epidermal stem cells called seam cells. We are studying lin-42 regulation of seam cell fate during C. elegans larval development as a model for understanding the mechanisms of period regulation of adult stem cell fate in mammals. This dissertation describes the research undertaken to characterize the cis-regulatory elements and the trans-regulatory factors that control lin-42 expression. We used direct molecular interaction assays (Electrophoretic Mobility Shift Assay, EMSA) (Chapter 2) followed by an RNA interference (RNAi)-based genetic screen (Chapter 3) to identify lin-42 transcriptional regulators. Using the EMSA, we identified three 50 to 100 base pair regions (binding regions, BR1-3) in the lin-42 5รข noncoding sequences that were bound with specificity by C. elegans nuclear proteins. These binding regions represent putative cis-regulatory elements that may serve as transcription factor binding sites (TFBSs). We attempted to identify by mass spectrometry the proteins that bind to the BR sequences. We also used Phylogenetic Footprinting and bioinformatics screens to identify candidate C. elegans transcription factors (TFs) that may bind to putative TFBSs within the BR sequences. Using an RNAi-based screen, we tested the candidate TF genes for potential genetic interactions with lin-42. We identified ZTF-16, a member of the Hunchback/Ikaros zinc-finger transcription factor family, as a potential lin-42 activator and, using quantitative real-time PCR, confirmed that ztf-16 mutation results in down-regulation and loss of cycling expression of lin-42. We further determined that loss of ztf-16 results in seam cell development defects that phenocopy lin-42 loss-of-function, thus validating ZTF-16 as a transcriptional activator of lin-42.en
dc.description.degreePh. D.en
dc.identifier.otheretd-09242012-133234en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-09242012-133234/en
dc.identifier.urihttp://hdl.handle.net/10919/29083en
dc.publisherVirginia Techen
dc.relation.haspartJames_T_D_2012.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectRNAien
dc.subjectcircadian perioden
dc.subjectEMSAen
dc.subjectztf-16en
dc.subjectlin-42en
dc.subjectC. elegansen
dc.subjectpost-embryonicen
dc.subjecttranscriptionalen
dc.titleCharacterization of Transcriptional and Post-transcriptional Regulation of lin-42/Period During Post-embryonic Development of C. elegansen
dc.typeDissertationen
thesis.degree.disciplineBiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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