High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation

Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. We investigate this relationship for the well-studied Twist factor in Drosophila melanogaster embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identified previously studied Twist-dependent CRMs and robustly predicted new CRM activity in transgenesis, with newly identified Twist-occupied regions supporting diverse spatiotemporal patterns (>74% positive, n = 31). Some, but not all, candidate CRMs require Twist for proper expression in the embryo. The Twist motifs most favored in genome ChIP data (in vivo) differed from those most favored by Systematic Evolution of Ligands by EXponential enrichment (SELEX) (in vitro). Furthermore, the majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif located within 50 bp of the ChIP summit and, of these, CACATG was most prevalent. Mutagenesis experiments demonstrated that different Twist E-box motif types are not fully interchangeable, suggesting that the ChIP-derived consensus (CABVTG) includes sites having distinct regulatory outputs. Further analysis of position, frequency of occurrence, and sequence conservation revealed significant enrichment and conservation of CABVTG E-box motifs near Twist ChIP-seq signal summits, preferential conservation of ±150 bp surrounding Twist occupied summits, and enrichment of GA- and CA-repeat sequences near Twist occupied summits. Our results show that high resolution in vivo occupancy data can be used to drive efficient discovery and dissection of global and local cis-regulatory logic

Rapid Evolution of cis-Regulatory Architecture and Activity in the Drosophila Yellow Gene.

In the last 10 years, an increasing number of case studies showed that changes in cis-regulatory elements, mainly enhancers, are one of the main causes of altered phenotypes, but the mechanisms underlying enhancer evolution remain to be elucidated. More specifically, what is the relationship between changes in enhancer sequence, transcription factor binding and activity? In this thesis, I used evolution of yellow enhancers among Drosophila species as a model to shed light onto how cis-regulatory architecture and activity change over time. I first identified the enhancer activities lying in the 5’ intergenic and intronic regions of yellow from six Drosophila species spanning the Drosophila evolutionary history, using a reporter gene assay. I found that yellow epidermal-cell and wing-vein, but not bristle enhancers, have different positions, with respect to the coding sequence, in different Drosophila species. This was the first systematic demonstration of altered enhancer position between species and suggested that enhancer position can be labile. Sequence comparisons failed to show any indication of translocation or duplication suggesting gradual compensatory changes in the transcription factor binding profiles of yellow enhancers is the likely mechanism underlying altered enhancer position. Subsequent subdivision of yellow 5’ intergenic and intronic regions showed a complex distribution of enhancer activities among sub-elements, where some drove expression in patterns that were not part of the expression pattern driven by the full region. Existence of such “cryptic” epidermal-cell enhancer activities suggests that yellow cis-regulatory regions were primed for facilitating the rapid evolutionary changes in the position and activities of this enhancer. Lastly, for the first time, I identified a large set of candidate transcription factors binding to yellow enhancers. This thesis shows that position and activity of yellow enhancers diverged rapidly among species, perhaps by taking advantage of the cryptic activities lying in the yellow cis-regulatory sequences. Further in vitro and in vivo tests validating the direct binding of the identified transcription factors on yellow enhancers and characterizing their functional effects on yellow expression among species can elucidate the evolutionary changes underlying altered position and activity of the particular yellow enhancers.Ph.D.Molecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91435/1/gizemkly_1.pd

Design flexibility in cis-regulatory control of gene expression: synthetic and comparative evidence

In early Drosophila embryos, the transcription factor Dorsal regulates patterns of gene expression and cell fate specification along the dorsal–ventral axis. How gene expression is produced within the broad lateral domain of the presumptive neurogenic ectoderm is not understood. To investigate transcriptional control during neurogenic ectoderm specification, we examined divergence and function of an embryonic cis-regulatory element controlling the gene short gastrulation (sog). While transcription factor binding sites are not completely conserved, we demonstrate that these sequences are bona fide regulatory elements, despite variable regulatory architecture. Mutation of conserved sequences revealed that putative transcription factor binding sites for Dorsal and Zelda, a ubiquitous maternal transcription factor, are required for proper sog expression. When Zelda and Dorsal sites are paired in a synthetic regulatory element, broad lateral expression results. However, synthetic regulatory elements that contain Dorsal and an additional activator also drive expression throughout the neurogenic ectoderm. Our results suggest that interaction between Dorsal and Zelda drives expression within the presumptive neurogenic ectoderm, but they also demonstrate that regulatory architecture directing expression in this domain is flexible. We propose a model for neurogenic ectoderm specification in which gene regulation occurs at the intersection of temporal and spatial transcription factor inputs

Natural sequence variation as a tool to dissect gene expression regulation in Drosophila melanogaster

Genetic variation is a major cause of differences between individuals and it represents a powerful tool to study gene regulation. By interfering with cis- Regulatory Modules (CRMs), variants can unravel CRM function. On the other hand, predicting the effect of variants on phenotype by the DNA sequence has proven to be challenging. In this thesis, I use Drosophila embryonic development as a model system to study diversity in gene regulation at the transcriptional level. CRMs can be characterized using multiple genome-wide techniques such as DNase hypersensitivity. However, despite having comprehensive CRM maps, it is still difficult to predict what are the genes regulated by each CRM. Functional methods, such as mutagenesis, are effective but poorly scalable. To address this issue, I developed an eQTL method (called DHS-eQTL) that makes use of naturally occurring genetic variation, to associate CRMs with the genes they regulate. The results reveal 2,967 DHS-eQTLs and indicate a high extent of CRM sharing between genes. We validated the results with in silico and in vitro approaches and I discuss upcoming in vivo experiments. We observed long-range enhancer regulation suggesting that commonly used methods to associate genes and enhancers underestimate their distance. Also, the DHS-eQTLs show that promoter-proximal CRMs have widespread distal activity. The separation between populations causes an increase in genetic differences by drift and adaptation to different environments. We investigated gene expression differences between Drosophila populations from five continents by performing RNA-Seq on 80 inbred fly lines. We performed multiple quality-control tests to ensure that the gene expression dataset is of high quality. Gene expression profiles show detectable diversity among the fly lines from different continents and confirm what has been observed at the genetic level. In particular, the African population is the most separated, while the American, European and Australian ones show less diversity. In addition, we identified 903 gene and 2,021 exon eQTLs. Genetic variants can interfere with Transcription Factor Binding Sites (TFBS) and this might, in turn, lead to changes in chromatin accessibility. We applied LS-GKM (an SVM method that uses gapped k-mers) to learn sequence features of tissue-specific accessible chromatin and predict the impact of natural sequence variation on accessibility. We train LS-GKM on six tissue-specific training sets: neuroectodermal, mesodermal and double negative CRMs divided in promoter-proximal and promoter-distal. The method unbiasedly recovers tissue-specific TFBS and shows good performance despite the small training sets. Finally, we score variants from groups of inbred Drosophila lines. Interestingly, rare variants have a higher impact on accessibility

Metabolic and Chaperone Gene Loss Marks the Origin of Animals: Evidence for Hsp104 and Hsp78 Sharing Mitochondrial Clients

The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i) sixteen distinct biosynthetic functions; (ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii) six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones.Comment: This is a reformatted version from the recent official publication in PLoS ONE (2015). This version differs substantially from first three arXiV versions. This version uses a fixed-width font for DNA sequences as was done in the earlier arXiv versions but which is missing in the official PLoS ONE publication. The title has also been shortened slightly from the official publicatio

chroGPS, a global chromatin positioning system for the functional analysis and visualization of the epigenome

Development of tools to jointly visualize the genome and the epigenome remains a challenge. chroGPS is a computational approach that addresses this question. chroGPS uses multidimensional scaling techniques to represent similarity between epigenetic factors, or between genetic elements on the basis of their epigenetic state, in 2D/3D reference maps. We emphasize biological interpretability, statistical robustness, integration of genetic and epigenetic data from heterogeneous sources, and computational feasibility. Although chroGPS is a general methodology to create reference maps and study the epigenetic state of any class of genetic element or genomic region, we focus on two specific kinds of maps: chroGPSfactors, which visualizes functional similarities between epigenetic factors, and chroGPSgenes, which describes the epigenetic state of genes and integrates gene expression and other functional data. We use data from the modENCODE project on the genomic distribution of a large collection of epigenetic factors in Drosophila, a model system extensively used to study genome organization and function. Our results show that the maps allow straightforward visualization of relationships between factors and elements, capturing relevant information about their functional properties that helps to interpret epigenetic information in a functional context and derive testable hypotheses

Enhancer sharing promotes neighborhoods of transcriptional regulation across eukaryotes

Enhancers physically interact with transcriptional promoters, looping over distances that can span multiple regulatory elements. Given that enhancer–promoter (EP) interactions generally occur via common protein complexes, it is unclear whether EP pairing is predominantly deterministic or proximity guided. Here, we present cross-organismic evidence suggesting that most EP pairs are compatible, largely determined by physical proximity rather than specific interactions. By reanalyzing transcriptome datasets, we find that the transcription of gene neighbors is correlated over distances that scale with genome size. We experimentally show that nonspecific EP interactions can explain such correlation, and that EP distance acts as a scaling factor for the transcriptional influence of an enhancer. We propose that enhancer sharing is commonplace among eukaryotes, and that EP distance is an important layer of information in gene regulation

The Genetic Mechanisms Underlying the Concerted Expression of the yellow and tan Genes in Complex Patterns on the Abdomen and Wings of Drosophila guttifera

How complex morphological patterns form is an intriguing question in developmental biology. However, the mechanisms that generate complex patterns remain largely unknown. Here, we sought to identify the genetic mechanisms that regulate the tan (t) gene in a multi-spotted pigmentation pattern on the abdomen and wings of Drosophila guttifera. Previously, we showed that yellow (y) gene expression completely prefigures the abdominal and wing pigment patterns of this species. In the current study, we demonstrate that the t gene is co-expressed with the y gene in nearly identical patterns, both transcripts foreshadowing the adult abdominal and wing melanin spot patterns. We identified cis-regulatory modules (CRMs) of t, one of which drives reporter expression in six longitudinal rows of spots on the developing pupal abdomen, while the second CRM activates the reporter gene in a spotted wing pattern. Comparing the abdominal spot CRMs of y and t, we found a similar composition of putative transcription factor binding sites that are thought to regulate the complex expression patterns of both terminal pigmentation genes y and t. In contrast, the y and t wing spots appear to be regulated by distinct upstream factors. Our results suggest that the D. guttifera abdominal and wing melanin spot patterns have been established through the co-regulation of y and t, shedding light on how complex morphological traits may be regulated through the parallel coordination of downstream target genes

Reverse transcriptional profiling: non-correspondence of transcript level variation and proximal promoter polymorphism

BACKGROUND: Variation in gene expression between two Drosophila melanogaster strains, as revealed by transcriptional profiling, seldom corresponded to variation in proximal promoter sequence for 34 genes analyzed. Two sets of protein-coding genes were selected from pre-existing microarray data: (1) those whose expression varied significantly and reproducibly between strains, and (2) those whose transcript levels did not vary. Only genes whose regulation of expression was uncharacterized were chosen. At least one kB of the proximal promoters of 15–19 genes in each set was sequenced and compared between strains (Oregon R and Russian 2b). RESULTS: Of the many promoter polymorphisms, 89.6% were SNPs and 10.4% were indels, including homopolymer tracts, microsatellite repeats, and putative transposable element footprints. More than half of the SNPs were changes within a nucleotide class. Hypothetically, genes differing in expression between the two strains should have more proximal promoter polymorphisms than those whose expression is similar. The number, frequency, and type of polymorphism, however, were the same in both sets of genes. In fact, the promoters of six genes with significantly different mRNA expression were identical in sequence. CONCLUSION: For these genes, sequences external to the proximal promoter, such as enhancers or in trans, must play a greater role than the proximal promoter in transcriptomic variation between D. melanogaster strains