Evolution of Genes Involved in Mammalian Reproduction and Sex Determination.

Traits that influence reproductive success and reproductive isolation in plants and animals have been the central focus of evolutionary biology since Darwin. Many genes were recently identified as important players during various phases of fertilization and sex determination. Although our understanding of the mechanism underlying the evolution of these genes is not complete, the emerging trend indicates that genes involved in reproduction are often rapidly evolving. Elucidating the mechanisms and factors that influence this fast pace of evolution has broad implications to human health and fertility, as well as the process of speciation, which is of fundamental relevance to the theory of evolution. Herein, I present case studies on three genes CATSPER1, SED1 and Sry, which are essential in sperm motility, sperm adhesion, and sex determination, respectively, and propose mechanisms by which natural selection has shaped their evolution. CATSPER1 is a calcium voltage-gated ion channel expressed on the plasma membrane of the principal tailpiece of spermatozoa and is essential for sperm motility. My findings document the first known case of positive Darwinian selection acting on the length of a protein (Podlaha and Zhang 2003). Specifically, natural selection promotes fixation of insertions and deletions in the CATSPER1 N-terminus, both in primates and rodents (Podlaha et al. 2005), affecting the ion channel’s rate of inactivation. Through the length variation of the N-terminus, channel’s rate of inactivation may have direct impact on sperm motility, which is an important determinant of sperm competition. SED1 is an important sperm-egg binding protein. My study provides evidence for a functional shift in SED1 due to a lineage specific loss of a protein-protein binding domain (Podlaha, Webb, and Zhang 2006). This domain loss was accompanied by positive selection in other parts of the protein in the ancestor of New World and Old World monkeys. The multifunctional nature of SED1 protein, however, obscures identification of the selective agent underlying this functional shift. Lastly, my analysis of the sex determination gene Sry addresses questions about the molecular mechanism that gives rise to fertile sex reversed females in akodont rodents.Ph.D.Ecology and Evolutionary BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/57630/2/opodlaha_1.pd

Histone Modifications Are Associated with Transcript Isoform Diversity in Normal and Cancer Cells

Mechanisms that generate transcript diversity are of fundamental importance in eukaryotes. Although a large fraction of human protein-coding genes and lincRNAs produce more than one mRNA isoform each, the regulation of this phenomenon is still incompletely understood. Much progress has been made in deciphering the role of sequence-specific features as well as DNA-and RNA-binding proteins in alternative splicing. Recently, however, several experimental studies of individual genes have revealed a direct involvement of epigenetic factors in alternative splicing and transcription initiation. While histone modifications are generally correlated with overall gene expression levels, it remains unclear how histone modification enrichment affects relative isoform abundance. Therefore, we sought to investigate the associations between histone modifications and transcript diversity levels measured by the rates of transcription start-site switching and alternative splicing on a genome-wide scale across protein-coding genes and lincRNAs. We found that the relationship between enrichment levels of epigenetic marks and transcription start-site switching is similar for protein-coding genes and lincRNAs. Furthermore, we found associations between splicing rates and enrichment levels of H2az, H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K9me3, H3K27ac, H3K27me3, H3K36me3, H3K79me2, and H4K20me, marks traditionally associated with enhancers, transcription initiation, transcriptional repression, and others. These patterns were observed in both normal and cancer cell lines. Additionally, we developed a novel computational method that identified 840 epigenetically regulated candidate genes and predicted transcription start-site switching and alternative exon splicing with up to 92% accuracy based on epigenetic patterning alone. Our results suggest that the epigenetic regulation of transcript isoform diversity may be a relatively common genome-wide phenomenon representing an avenue of deregulation in tumor development

Large-scale viral genome analysis identifies novel clinical associations between hepatitis B virus and chronically infected patients

Chronic hepatitis B; HBeAg status; Viral genome variationHepatitis B crónica; Estado de HBeAg; Variación del genoma viralHepatitis B crònica; Estat de HBeAg; Variació del genoma viralDespite the high global prevalence of chronic hepatitis B (CHB) infection, datasets covering the whole hepatitis B viral genome from large patient cohorts are lacking, greatly limiting our understanding of the viral genetic factors involved in this deadly disease. We performed deep sequencing of viral samples from patients chronically infected with HBV to investigate the association between viral genome variation and patients’ clinical characteristics. We discovered novel viral variants strongly associated with viral load and HBeAg status. Patients with viral variants C1817T and A1838G had viral loads nearly three orders of magnitude lower than patients without those variants. These patients consequently experienced earlier viral suppression while on treatment. Furthermore, we identified novel variants that either independently or in combination with precore mutation G1896A were associated with the transition from HBeAg positive to the negative phase of infection. These observations are consistent with the hypothesis that mutation of the HBeAg open reading frame is an important factor driving CHB patient’s HBeAg status. This analysis provides a detailed picture of HBV genetic variation in the largest patient cohort to date and highlights the diversity of plausible molecular mechanisms through which viral variation affects clinical phenotype

Wings, Horns, and Butterfly Eyespots: How Do Complex Traits Evolve?

Do novel complex traits evolve when pre-existent complex developmental networks are recruited into new places in the body? Here we look closely at the genomic fingerprints that are produced as a result of gene network co-option

The Microevolution of V1r Vomeronasal Receptor Genes in Mice

Vomeronasal sensitivity is important for detecting intraspecific pheromonal cues as well as environmental odorants and is involved in mating, social interaction, and other daily activities of many vertebrates. Two large families of seven-transmembrane G-protein–coupled receptors, V1rs and V2rs, bind to various ligands to initiate vomeronasal signal transduction. Although the macroevolution of V1r and V2r genes has been well characterized throughout vertebrates, especially mammals, little is known about their microevolutionary patterns, which hampers a clear understanding of the evolutionary forces behind the rapid evolutionary turnover of V1r and V2r genes and the great diversity in receptor repertoire across species. Furthermore, the role of divergent vomeronasal perception in enhancing premating isolation and maintaining species identity has not been evaluated. Here we sequenced 44 V1r genes and 25 presumably neutral noncoding regions in 14 wild-caught mice belonging to Mus musculus and M. domesticus, two closely related species with strong yet incomplete reproductive isolation. We found that nucleotide changes in V1rs are generally under weak purifying selection and that only ∼5% of V1rs may have been subject to positive selection that promotes nonsynonymous substitutions. Consistent with the low functional constraints on V1rs, 18 of the 44 V1rs have null alleles segregating in one or both species. Together, our results demonstrate that, despite occasional actions of positive selection, the evolution of V1rs is in a large part shaped by purifying selection and random drift. These findings have broad implications for understanding the driving forces of rapid gene turnovers that are often observed in the evolution of large gene families

Pogostick: A New Versatile piggyBac Vector for Inducible Gene Over-Expression and Down-Regulation in Emerging Model Systems

Non-traditional model systems need new tools that will enable them to enter the field of functional genetics. These tools should enable the exploration of gene function, via knock-downs of endogenous genes, as well as over-expression and ectopic expression of transgenes.We constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ line transformation by the piggyBac transposable element. Pogostick can be found at www.addgene.org, a non-profit plasmid repository. The vector currently uses the heat-shock promoter Hsp70 from Drosophila to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. We detail how to clone candidate genes into this vector and test its functionality in Drosophila by targeting a gene coding for the fluorescent protein DsRed. By cloning a single DsRed copy into the vector, and generating transgenic lines, we show that DsRed mRNA and protein levels are elevated following heat-shock. When cloning a second copy of DsRed in reverse orientation into a flanking site, and transforming flies constitutively expressing DsRed in the eyes, we show that endogenous mRNA and protein levels drop following heat-shock. We then test the over-expression vector, containing the complete cDNA of Ultrabithorax (Ubx) gene, in an emerging model system, Bicyclus anynana. We produce a transgenic line and show that levels of Ubx mRNA expression rise significantly following a heat-shock. Finally, we show how to obtain genomic sequence adjacent to the Pogostick insertion site and to estimate transgene copy number in genomes of transformed individuals.This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles

Testing the Chromosomal Speciation Hypothesis for Humans and Chimpanzees

Fixed differences of chromosomal rearrangements between isolated populations may promote speciation by preventing between-population gene flow upon secondary contact, either because hybrids suffer from lowered fitness or, more likely, because recombination is reduced in rearranged chromosomal regions. This chromosomal speciation hypothesis thus predicts more rapid genetic divergence on rearranged than on colinear chromosomes because the former are less porous to gene flow. A number of studies of fungi, plants, and animals, including limited genetic data of humans and chimpanzees, support the hypothesis. Here we reexamine the hypothesis for humans and chimpanzees with substantially more genomic data than were used previously. No difference is observed between rearranged and colinear chromosomes in the level of genomic DNA sequence divergence between species. The same is also true for protein sequences. When the gorilla is used as an outgroup, no acceleration in protein sequence evolution associated with chromosomal rearrangements is found. Furthermore, divergence in expression pattern between orthologous genes is not significantly different for rearranged and colinear chromosomes. These results, showing that chromosomal rearrangements did not affect the rate of genetic divergence between humans and chimpanzees, are expected if incipient species on the evolutionary lineages separating humans and chimpanzees did not hybridize

Hypothetical Illustration of Gene Network Co-Option and De Novo Network Evolution

<p>(A) Modular gene network where gene <i>X</i>, at the top of a regulatory network, directs expression of gene <i>Y</i>, which in turn directs expression of gene <i>Z</i>. All these genes are expressed in the tip of an appendage (e.g., leg) depicted on the right. (B) The modular gene network is co-opted into a new tissue by the evolution of a novel CRE in gene <i>X</i>. The <i>Y</i> and <i>Z</i> genes, which only receive inputs from <i>X</i> and <i>Y</i> genes, respectively, are also turned on in the new tissue (e.g., eyespot centers in a butterfly larval wing). The CREs of the <i>Y</i> and <i>Z</i> genes now have a dual function in directing gene expression in two separate developmental contexts, e.g., they are pleiotropic. (C) De novo network evolution where elements of the same non-modular gene network, <i>X</i>, <i>Y</i>, and <i>Z</i>, each evolve a separate CRE that drives gene expression in the novel developmental context.</p

Examples of Pleiotropic CREs

<div><p>A schematic representation of putatively pleiotropic CREs is shown for: (A) The <i>spalt</i> (<i>sal</i> and <i>salr</i>) gene complex; (B) <i>spineless</i> (<i>ss</i>); (C) <i>yellow</i> (<i>y</i>); (D) <i>odd-skipped</i> (<i>odd</i>). Gene orientation is marked by arrows. Ovals show approximate position of CREs surrounding the protein-coding genes. Checkmarks of tissue/organs above CREs represent the multiple domains of gene expression driven by the same CRE. Modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b024" target="_blank">24</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b026" target="_blank">26–28</a>]. The multiple CREs that drive gene expression in the same tissue or organ mostly drive gene expression in distinct cell populations.</p> <p>Abbreviations: CNS, central nervous system; PNS, peripheral nervous system.</p></div

Similar Gene Expression in Various Non-Homologous Structures

<p>The transcription factor Distal-less (Dll) is expressed (A) in the horn primordium of the African dung beetle, Onthophagus nigriventris(modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b009" target="_blank">9</a>]); (B) in the eyespot focus of the squinting bush brown butterfly, Bicyclus anynana (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b040" target="_blank">40</a>]); and (C) in the leg imaginal disc of the fruit fly, D. melanogaster (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b041" target="_blank">41</a>]). The transcription factor Spalt (Sal) is expressed (D) in the antenna imaginal disc of D. melanogaster (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b042" target="_blank">42</a>]); and (E) in the eyespot field of B. anynana pupal wings (modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1000037#pbio-1000037-b012" target="_blank">12</a>]).</p