Browsing by Subject "Expression"

The visual system in humans provides the ability to interpret our surroundings from many distances. This complex system serves as a powerful sense which can drastically impact the quality of life when threatened or eliminated. While the mechanisms involved in visual interpretation are largely understood, many of the mechanisms of ocular diseases remain elusive. The most common ocular disorders are refractive errors, where failure of normal growth processes results in eye components with shape and sizes that are not matched to provide uncorrected sharp visual acuity without correction. Myopia, or nearsightedness, is a refractive error with prevalence rates of epidemic proportions in some urban Asian settings, and rising in other developed countries. Pathological, or high myopia, has an increased risk for potentially blinding ocular morbidities which can be irreversible and further negatively impact quality of life. Myopia, like other common ocular disorders, results from a combination of environmental and genetic factors. Over 20 candidate genomic regions have been identified as involved in myopic development progression.

One such locus, MYP3, on chromosome 12q21-23 spans nearly 44 Mb with more than 200 protein-encoding genes mapped within. Sizable candidate disease genomic regions typically require refinement to identify genes or variants within them which may contribute to disease development. Without an understanding of the underlying mechanistic framework of a disease, as is the case with myopia, biological inferences are difficult to make in prioritizing candidates, which can make finding true disease causing variants seem like finding a needle in a haystack. A better understanding of human ocular growth, as it relates to refractive error, may lead to more knowledgeable approaches to identifying the cause(s) of myopic development and associated ocular diseases.

To identify genes involved in ocular growth and development, whole genome expression patterns were assessed in human ocular tissues of fetal versus adult eyes, and adult posterior versus peripheral tissues. No database exists of fetal ocular tissue gene expression. In addition to providing insights into expression patterns during ocular development, these tissues were also compared as a surrogate to study rapid eye growth states such as in myopia. Only ocular tissue types with clinical phenotypes associated with myopic development were considered. Human retina/retinal pigment epithelium (RPE), choroid, sclera, cornea* and optic nerve* tissues were isolated from fetal (N=9; *N= 6) and adult (N=6) normal donor eyes. The Illumina® whole genome expression microarray platform was used to assess differential expression of genes. Fetal tissues were compared to their adult counterparts while adult posterior tissues were compared to their peripheral counterparts, and the differences in each were assessed using Ingenuity Pathway Analysis (IPA) for enriched functional groups and canonical pathways. Statistical significance for all tissue comparisons was determined using the Benjamin and Hochberg False Discovery Rate (FDR, 5%). Differentially expressed genes were compared to previously identified candidates for myopic development.

Additionally, qualitative and quantitative association studies in a large family (N=82) based high myopia cohort by genotyping 768 single nucleotide polymorphisms (SNPs) in the peak linkage area was performed to fine map the MYP3 linkage peak. Qualitative testing for high myopia (≤ -5 diopter (D) affected, > -5 D unaffected) and quantitative testing on the average (avg) dioptric sphere (SPH) was performed. Five candidate SNPs were genotyped in a replicate high myopia cohort for independent validation. Additionally, the most significant SNPs were screened in a previously genotyped twin cohort as a second independent validation cohort.

Ocular growth expression data were used to help prioritize the resulting association candidates as supporting evidence and was not used on its own to identify or exclude candidates. Candidate genes (within 100 kilobases (kb) of highly associated SNPs) identified through either qualitative or quantitative association testing were screened in the most disease relevant tissues (retina/ retinal pigment epithelium (RPE), choroid and sclera) for differential expression during ocular growth and by physical regions of the tissues within the eye. Genes that were identified by microarray studies as being differentially expressed in one or more tissue were validated using quantitative real time PCR (RT-qPCR).

Significant gene expression changes with fold changes > 1.5 were found in adult versus fetal retina/RPE (N=1185), choroid (N=6446), sclera (N=1349), and cornea (N=3872), but not the optic nerve nor any of the central versus peripheral tissues. In all adult versus fetal tissues, differentially expressed genes belonging to cancer, development, and cell death/growth functional groups, as well as signaling canonical pathways were enriched. Seventeen genes previously associated with increased susceptibility for non-syndromic high myopia were in the most significant functional assignments for at least one adult versus fetal ocular tissue. In adult central versus peripheral tissues, there was considerably more variation by tissue in enriched functions and canonical pathways of differentially expressed genes. The only functional category shared by all three tissue types was development.

MYP3 association testing yielded several genetic markers as nominally significant in association with high myopia in qualitative testing including rs3803036 (p=9.1X10-4), a missense mutation in PTPRR; and rs4764971 (p = 6.1X10-4), an intronic SNP in UHRF1BP1L. After correction for multiple testing, quantitative tests found statistically significant SNPs rs4764971 (p = 3.1x10-6), also found by qualitative testing; rs7134216 (p = 5.4X10-7), in the 31 UTR of DEPDC4; and rs17306116 (p < 9X10-4), intronic within PPFIA2. The intronic SNP in UHRF1BP1L, rs4764971, was validated for association with the quantitative trait of sphere (SPH) using an independently collected non-syndromic, high myopia cohort. SNPs within PTPRR (for quantitative association) and PPFIA2 (for qualitative and quantitative association) both approached significance in the independent high myopia cohort.

As with screening genes previously implicated in myopic development, qualitative and quantitative association candidates were screened in the independent whole genome expression array analyses, comparing normal rapidly growing fetal to normal grown adult ocular tissues. PTPRR and PPFIA2, candidates from qualitative and quantitative association respectively, were both validated by RT-qPCR with differential expression in at least one disease relevant ocular tissue. PTPRR and PPFIA2 belong to the same gene family- that of protein tyrosine phosphatase (PTP) genes. This family of genes relays extracellular signals that regulate cell growth, division, maturation and function, and its differential expression is consistent with our myopia surrogate model.

Many genes implicated in either syndromic or non-syndromic myopia were present in the most significantly enriched adult versus fetal functional and/or canonical pathways together. The adult versus fetal choroid and cornea tissue types had the most overlap with known non-syndromic myopic-associated genes in the most significantly enriched functional groups. Further exploration of the connections amongst these known genes may elucidate possible mechanistic roles for disease progression and/or reveal related novel candidate genes. Differentially expressed genes in central versus peripheral tissues yielded minimal overlap with genes implicated in myopia; however, in addition to broadening our understanding of the spatial variances in these tissues they may contain clues to the development and/or progression of other ocular diseases such as retinopathy of prematurity development.

The overlap with previously identified myopia-associated genes supports the model of eye growth for studying myopic development in human tissues. This expression data can be used both in prioritizing candidate genes other proposed genomic myopia loci, and also in detailed pathway analyses to identify potential biological mechanisms for candidates within these loci. Our most strongly associated candidate gene both in the discovery and replicate cohort was UHRF1BP1L, which was not differentially expressed in our data; however, interacting genes regulate the expression of at least one differentially expressed gene, indicating a possibly pathway connection. It is possible that differential expression may have been missed by the microarray data, or it may not be differentially expressed and affects myopic development through alternative or indirect means. While the expression data is a useful tool in prioritizing and inferring mechanistic roles for candidates, it cannot be used to exclude candidates. Deeper study of the pathways of candidate genes for myopic development may reveal connections to genes involved in ocular growth. Despite these potential limitations, two of the three novel candidates, PTPRR and PPFIA2, were supported by genomic convergence with the expression data, in addition to our discovery genetic association data. The other novel candidate, UHRF1BP1L, was validated in an independent Caucasian high-grade myopia cohort. Further validation and refinement of these three novel MYP3 candidate genes is necessary to make further claims about their possible involvement in myopic progression.

Deciphering the molecular mechanisms driving the phenotypic differences between humans and primates remains a daunting challenge. Mutations found in protein coding DNA alone has not been able to explain these phenotypic differences. The hypothesis that mutations in non-coding regulatory DNA are responsible for altered gene expression leading to these phenotypic changes has now been widely supported by differential gene expression experiments. Yet, comprehensive identification of all regulatory DNA elements across different species has not been performed. To identify the genetic source of regulatory change, genome-wide DNaseI hypersensitivity assays, marking all types of active gene regulatory element sites, were performed in human, chimpanzee, macaque, orangutan, and mouse. Many DNaseI hypersensitive (DHS) sites were conserved among all 5 species, but we also identified hundreds of novel human- and chimpanzee-specific DHS gains and losses that showed signatures of positive selection. Species-specific DHS gains were enriched in distal non-coding regions, associated with active histone modifications, and positively correlated with increased expression - indicating that these are likely to be functioning as enhancers. Comparison to mouse DHS data indicate that human or chimpanzee DHS gains are likely to have been a result of single events that occurred primarily on the human- or chimpanzee-specific branch, respectively. In contrast, DHS losses are associated with events that occurred on multiple branches. At least one mechanism contributing to DHS gains and losses are species-specific variants that lead to sequence changes at transcription factor binding motifs, affecting the binding of TFs such as AP1. These variants were functionally verified by DNase footprinting and ChIP-qPCR analyses.

Animals must coordinate development with fluctuating nutrient availability. Nutrient availability governs post-embryonic development in Caenorhabditis elegans: larvae that hatch in the absence of food do not initiate post-embryonic development but enter "L1 arrest" (or "L1 diapause") and can survive starvation for weeks, while rapidly resume normal development once get fed. Insulin-like signaling (IIS) has been shown to be a key regulator of L1 arrest and recovery. However, the C. elegans genome encodes 40 insulin-like peptides (ILPs), and it is unknown which peptides participate in nutritional control of L1 arrest and recovery. Work in other contexts has identified putative receptor agonists and antagonists, but the extent of specificity versus redundancy is unclear beyond this distinction.

We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified 13 candidate agonists and 8 candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists was largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in systemic control of L1 development. Transcriptional regulation of candidate agonists was most significant in the intestine, as if nutrient uptake was a more important influence on transcription than sensory perception. Scanning in the 5' upstream promoter region of these 40 ILPs, We found that transcription factor PQM-1 and GATA putative binding sites are depleted in the promoter region of antagonists. A novel motif was also found to be over-represented in ILPs.

Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 recovery/developmental dynamics, though simultaneous disruption of ins-4 and daf-28 extended survival of L1 arrest without enhancing thermal tolerance, while overexpression of ins-4, ins-6 or daf-28 shortened L1 survival. Simultaneous disruption of several ILPs showed a temperature independent, transient dauer phenotype. These results revealed the relative redundancy and specificity among agonistic ILPs.

TGF- β and steroid hormone (SH) signaling have been reported to control the dauer formation along with IIS. Our preliminary results suggest they may also mediate the IIS control of L1 arrest and recovery, as the expression of several key components of TGF-β and SH signaling pathway genes are negatively regulated by DAF-16, and loss-of-function of these genes partially represses daf-16 null phenotype in L1 arrest, and causes a retardation in L1 development.

In summary, my dissertation study focused on the IIS, characterized the dynamics and sites of ILPs expression in response to nutrient availability, revealed the function of specific agonistic ILPs in L1 arrest, and suggested potential cross-regulation among IIS, TGF-β signaling and SH signaling in controlling L1 arrest and recovery. These findings provide insights into how post-embryonic development is governed by insulin-like signaling and nutrient availability.

Allelic imbalance, in which two alleles at a given locus exhibit differences in gene expression, chromatin composition and/or protein binding, is a widespread phenomenon in the human and other complex genomes. Most examples concern individual loci located more or less randomly around the genome and thus imply local and gene-specific mechanisms. However, genomic or chromosomal basis for allelic imbalance is supported by multi-locus examples such as those exemplified by domains of imprinted genes, spanning ~1-2 Mb, or by X chromosome inactivation, involving much of an entire chromosome. Recent studies have shown that genes on the two female X chromosomes exhibit a breadth of expression patterns ranging from complete silencing of one allele to fully balanced biallelic expression. Although evidence for heritability of allele-specific chromatin and expression patterns exists at individual loci, it is unknown whether heritability is also reflected in the chromosome-wide patterns of X inactivation.

The aim of this thesis is to elucidate the extent to which the widespread variable patterns of allelic imbalance on the human X chromosome in females are under genetic control and how access of the transcription machinery to the human inactive X chromosome in females is determined at a genomic level. For the set of variable genes examined in this study, the absence or presence of expression appears to be stochastic with respect to the population rather than abiding by strict genetic rules. Furthermore, variable gene expression that I have detected even among multiple clonal cell lines derived from a single individual suggests fluctuation in transcriptional machinery engagement. I find that, although expression at most genes on the human inactive X chromosome is repressed as a result of X inactivation, a number of loci are accessible to the transcriptional machinery. It appears that RNA Polymerase II is present at alleles on the inactive X even at the promoters of several silenced genes, indicating a potential for expression.

This thesis embodies a transition in the field of human X chromosome inactivation from gene by gene approaches used in the past to utilizing high-throughput technologies and applying follow-up analytic techniques to draw upon the vast data publicly available from large consortia projects.

Gene expression and regulation are major determinants of phenotypic traits displayed across species. Although the DNA sequence elements that control gene expression play a crucial role in determining species morphology, predicting cis-regulatory elements through sequence analysis alone remains a difficult task. A few regulatory elements, such as the TATA-box and Initiator sequence, have been known to exhibit overrepresentation at specific locations within the proximal promoter. However, the extent to which this occurs among cis-regulatory elements is not well understood. Here, we take a genome-wide approach towards detecting such functional sequence elements, using location-specific overrepresentation as a criterion for regulatory function. We provide evidence that a surprisingly large number of regulatory elements exhibit locational overrepresentation with respect to the transcription start site. We then utilize this characteristic to predict novel cis-regulatory elements overrepresented at particular locations within the proximal promoter.

Transcriptional regulation is most often controlled not by single protein factors acting in isolation, but instead multiple transcription factors acting together within multi-protein complexes. As protein-protein interactions are largely determined through protein structure, we would expect to see patterns of spatial preference between motif-pairs binding interacting factors. However, in the absence of methods to predict such spatial preferences between motifs, comprehensive assessments of such inter-relationships have not been previously conducted. As our model provides a general tool for detecting positional specificities of a motif relative to a given reference point, we expanded our model to measure distance preferences between pairs of motifs on a genome-wide scale. We show that there often exist patterns of spatial dependencies between pairs of sequence elements that bind interacting protein factors. We find that regulatory motifs binding interacting proteins often have multiple inter-motif distances at which they preferentially occur, and we show that the intervals between preferred distances are highly consistent across motif-pairs. This distance preference `phasing' was empirically found to occur at consistent intervals around ~8-10 bp, corresponding to approximately the number of nucleotides within a single turn of the DNA double-helix. This finding suggests a tendency for protein factor-pairs to interact in a specific orientation with respect to the turn of the DNA molecule, and offers a convenient method by which to determine motif-pairs binding interacting transcription factors de novo.

While little is known about the mechanisms by which individual cis-regulatory elements ultimately control gene expression, even less is known about how such elements evolve over time. A single transcription factor can potentially target hundreds of genes across the genome, and thus modifications in the binding affinities of such proteins must induce conversions at a multitude of functional sites in order to preserve the set of target genes that the trans-factor regulates. It is therefore commonly assumed that such changes occur rarely and at a slow rate over the course of evolution. Despite this widespread assumption, we find that a surprisingly large number of cis-regulatory elements have been subject to significant changes in consensus sequence in a lineage-specific manner. Here, we demonstrate that the genomic landscape is highly adaptable, rapidly adjusting to global changes in preferred regulatory consensus sequences. Focusing upon regulatory elements exhibiting location-specific overrepresentation, we find that a substantial fraction of regulatory elements have been subject to evolutionary modifications, even between closely related eutherians. These findings have broad implications regarding evolving phenotypes observed across species.