Submitted to the faculty of Indiana University in partial fulfillment of the requirements for the degree Master of Science in the School of Informatics, December 2004 / The process of generating RNA message from a gene is termed "transcription," and is performed by aptly named "transcription factors." These transcription factors can function singly or in conjunction with other factors to regulate gene expression, and thereby control, at one level, the amount of protein product which a gene can produce. This transcriptional regulation can have profound biological effects. Identification of the DNA sequences bound by transcription factors (regulatory elements) is a challenge as these sequences can be very short and may be located large distances from the target gene. Also, many current approaches for discovering regulatory elements in genomic DNA are limited to those sequences previously identified as consensus sequences for transcription factors. We have developed a novel tool, ReguLocator, for the identification of putative regulatory element. It uses the Smith-Waterman algorithm to identify short, conserved DNA sequences (ranked by p-value) derived from 2 large DNA sequences of interest. No weighting is applied for spatial alignment. Although speed must be improved to make use of ReguLocator practical, initial comparisons to similar tools provide promising results, and may open the door to better regulatory element identification.

When the human genome project was completed, it revealed a surprising result. 98% of the genome did not code for protein of which more than 50% are repeats— later known as ”Junk DNA”. However, comparative genomics unveiled that many noncoding elements are evolutionarily constrained; thus luckily to have a role in genome stability and regulation. Though, their exact functions remained largely unknown. Several large international consortia such as the Functional Annotation of Mammalian Genomes (FANTOM) and the Encyclopedia of DNA Elements (ENCODE) were set to understand the structure and the regulation of the genome. Specifically, these endeavors aim to measure and reveal the transcribed components and functional elements of the genome. One of the most the striking findings of these efforts is that most of the genome is transcribed, including non-conserved noncoding elements and repeat elements. Specifically, we investigated the evolution and epigenetic properties of noncoding elements. 1. We compared genomes of evolutionarily distant species and showed the ubiquity of constrained noncoding elements in metazoa. 2. By integrating multi-omic data (such as transcriptome, nucleosome profiling, histone modifications), I conducted a comprehensive analysis of epigenetic properties (chromatin states) of conserved noncoding elements in insects. We showed that those elements have distinct and protective sequence features, undergo dynamic epigenetic regulation, and appear to be associated with the structural components of the chromatin, replication origins, and nuclear matrix. 3. I focused on the relationship between enhancers and repetitive elements. Using Cap Analysis of Gene Expression (CAGE) and RNASeq, I compiled a full catalog of active enhancers (a class of noncoding elements) during myogenesis of human primary cells of healthy donors and donors affected by Duchenne muscular dystrophy (DMD). Comparing the two time-courses, a significant change in the epigenetic landscape in DMD was observed that lead to global dysregulation of enhancers and associated repetitive elements.

In my dissertation I investigate two genera of geckos (Cyrtodactylus and Cnemaspis) that are distributed across Southeast Asia with an emphasis on Cnemaspis. In Chapter 1 I use a multilocus dataset, ancestral area analyses, and molecular clock dating to generate a species level time calibrated phylogeny to test the monophyly of Cyrtodactylus and to identify major biogeographical patterns. I identified that Cyrtodactylus is monophyletic only if the the Sri Lankan genus often recognized as Geckoella is included. The results of the Biogeographical analyses reveal a west to east pattern of diversification. Chapter 2 I use a traditional morphological dataset to describe a new species of Cnemaspis from Peninsular Malaysia. In Chapter 3 my colleagues and I use a multilocus dataset and morphological characters to revise the taxonomy of all Cnemaspis species and put this in a phylogenetic context. This resulted in the description of eight new species and allowed us to generate hypotheses relating to parallel evolution, diversity, and biogeography. In Chapter 4 I use additional taxon sampling of Cnemaspis from Thailand to generate a more complete phylogeny and use an integrative taxonomic approach to describe three new species. In Chapter 5 I used high throughput sequencing of sequence capture and Ultra Conserved Elements to test for a rapid radiation in Cnemaspis and to investigate biogeographic hypotheses relating to the diversification and evolution of Cnemaspis. I determined that there was a temporal rapid radiation of Cnemaspis that coincides with the temporal diversification of other terrestrial vertebrates across Sundaland. The results of these studies indicate that the species diversity of Cnemaspis is underestimated and that both genera have experienced similar temporal and spatial diversification coinciding with major vicariant events during the Eocene and the Oligocene-Miocene transition.

In my dissertation I investigate two genera of geckos (Cyrtodactylus and Cnemaspis) that are distributed across Southeast Asia with an emphasis on Cnemaspis. In Chapter 1 I use a multilocus dataset, ancestral area analyses, and molecular clock dating to generate a species level time calibrated phylogeny to test the monophyly of Cyrtodactylus and to identify major biogeographical patterns. I identified that Cyrtodactylus is monophyletic only if the the Sri Lankan genus often recognized as Geckoella is included. The results of the Biogeographical analyses reveal a west to east pattern of diversification. Chapter 2 I use a traditional morphological dataset to describe a new species of Cnemaspis from Peninsular Malaysia. In Chapter 3 my colleagues and I use a multilocus dataset and morphological characters to revise the taxonomy of all Cnemaspis species and put this in a phylogenetic context. This resulted in the description of eight new species and allowed us to generate hypotheses relating to parallel evolution, diversity, and biogeography. In Chapter 4 I use additional taxon sampling of Cnemaspis from Thailand to generate a more complete phylogeny and use an integrative taxonomic approach to describe three new species. In Chapter 5 I used high throughput sequencing of sequence capture and Ultra Conserved Elements to test for a rapid radiation in Cnemaspis and to investigate biogeographic hypotheses relating to the diversification and evolution of Cnemaspis. I determined that there was a temporal rapid radiation of Cnemaspis that coincides with the temporal diversification of other terrestrial vertebrates across Sundaland. The results of these studies indicate that the species diversity of Cnemaspis is underestimated and that both genera have experienced similar temporal and spatial diversification coinciding with major vicariant events during the Eocene and the Oligocene-Miocene transition.