THE COMPLEX EVOLUTIONARY HISTORY OF SEEING RED: MOLECULAR PHYLOGENY AND THE EVOLUTION OF AN ADAPTIVE VISUAL SYSTEM IN DEEP‐SEA DRAGONFISHES (STOMIIFORMES: STOMIIDAE)

The vast majority of deep‐sea fishes have retinas composed of only rod cells sensitive to only shortwave blue light, approximately 480–490 nm. A group of deep‐sea dragonfishes, the loosejaws (family Stomiidae), possesses far‐red emitting photophores and rhodopsins sensitive to long‐wave emissions greater than 650 nm. In this study, the rhodopsin diversity within the Stomiidae is surveyed based on an analysis of rod opsin‐coding sequences from representatives of 23 of the 28 genera. Using phylogenetic inference, fossil‐calibrated estimates of divergence times, and a comparative approach scanning the stomiid phylogeny for shared genotypes and substitution histories, we explore the evolution and timing of spectral tuning in the family. Our results challenge both the monophyly of the family Stomiidae and the loosejaws. Despite paraphyly of the loosejaws, we infer for the first time that far‐red visual systems have a single evolutionary origin within the family and that this shift in phenotype occurred at approximately 15.4 Ma. In addition, we found strong evidence that at approximately 11.2 Ma the most recent common ancestor of two dragonfish genera reverted to a primitive shortwave visual system during its evolution from a far‐red sensitive dragonfish. According to branch‐site tests for adaptive evolution, we hypothesize that positive selection may be driving spectral tuning in the Stomiidae. These results indicate that the evolutionary history of visual systems in deep‐sea species is complex and a more thorough understanding of this system requires an integrative comparative approach.     

INFERRING THE PAST AND PRESENT CONNECTIVITY ACROSS THE RANGE OF A NORTH AMERICAN LEAF BEETLE: COMBINING ECOLOGICAL NICHE MODELING AND A GEOGRAPHICALLY EXPLICIT MODEL OF COALESCENCE

The leaf beetle Chrysomela aeneicollis occurs across Western North America, either at high elevation or in small, isolated populations along the coast, and thus has a highly fragmented distribution. DNA sequence data (three loci) were collected from five regions across the species range. Population connectivity was examined using traditional ecological niche modeling, which suggested that gene flow could occur among regions now and in the past. We developed geographically explicit coalescence models of sequence evolution that incorporated a two‐dimensional representation of the hypothesized ranges suggested by the niche‐modeling estimates. We simulated sequence data according to these models and compared them to observed sequences to identify most probable scenarios regarding the migration history of C. aeneicollis. Our results disagreed with initial niche‐modeling estimates by clearly rejecting recent connectivity among regions, and were instead most consistent with a long period of range fragmentation, extending well beyond the last glacial maximum. This application of geographically explicit models of coalescence has highlighted some limitations of the use of climatic variables for predicting the present and past range of a species and has explained aspects of the Pleistocene evolutionary history of a cold‐adapted organism in Western North America.     

Side-Chain to Main-Chain Hydrogen Bonding Controls the Intrinsic Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix

Many transmembrane helices contain serine and/or threonine residues whose side chains form intrahelical H-bonds with upstream carbonyl oxygens. Here, we investigated the impact of threonine side-chain/main-chain backbonding on the backbone dynamics of the amyloid precursor protein transmembrane helix. This helix consists of a N-terminal dimerization region and a C-terminal cleavage region, which is processed by γ-secretase to a series of products. Threonine mutations within this transmembrane helix are known to alter the cleavage pattern, which can lead to early-onset Alzheimer’s disease. Circular dichroism spectroscopy and amide exchange experiments of synthetic transmembrane domain peptides reveal that mutating threonine enhances the flexibility of this helix. Molecular dynamics simulations show that the mutations reduce intrahelical amide H-bonding and H-bond lifetimes. In addition, the removal of side-chain/main-chain backbonding distorts the helix, which alters bending and rotation at a diglycine hinge connecting the dimerization and cleavage regions. We propose that the backbone dynamics of the substrate profoundly affects the way by which the substrate is presented to the catalytic site within the enzyme. Changing this conformational flexibility may thus change the pattern of proteolytic processing.     

Opposing Roles of Dnmt1 in Early- and Late-Stage Murine Prostate Cancer

Previous studies have shown that tumor progression in the transgenic adenocarcinoma of mouse prostate (TRAMP) model is characterized by global DNA hypomethylation initiated during early-stage disease and locus-specific DNA hypermethylation occurring predominantly in late-stage disease. Here, we utilized Dnmt1 hypomorphic alleles to examine the role of Dnmt1 in normal prostate development and in prostate cancer in TRAMP. Prostate tissue morphology and differentiation status was normal in Dnmt1 hypomorphic mice, despite global DNA hypomethylation. TRAMP; Dnmt1 hypomorphic mice also displayed global DNA hypomethylation, but were characterized by altered tumor phenotype. Specifically, TRAMP; Dnmt1 hypomorphic mice exhibited slightly increased tumor incidence and significantly increased pathological progression at early ages and, conversely, displayed slightly decreased tumor incidence and significantly decreased pathological progression at advanced ages. Remarkably, hypomorphic Dnmt1 expression abrogated local and distant site macrometastases. Thus, Dnmt1 has tumor suppressor activity in early-stage prostate cancer, and oncogenic activity in late stage prostate cancer and metastasis. Consistent with the biological phenotype, epigenomic studies revealed that TRAMP; Dnmt1 hypomorphic mice show dramatically reduced CpG island and promoter DNA hypermethylation in late-stage primary tumors compared to control mice. Taken together, the data reveal a crucial role for Dnmt1 in prostate cancer and suggest that Dnmt1-targeted interventions may have utility specifically for advanced and/or metastatic prostate cancer.Changes in DNA methyltransferase (Dnmt) expression and DNA methylation are observed in human prostate cancer (3, 38, 41). Of particular interest, genes with tumor suppressive function become hypermethylated and silenced, which correlates with the development of specific disease phenotypes (2, 3, 38). Although an association between prostate cancer and alterations in DNA methylation has been established, in vivo models are required to determine whether these changes functionally contribute to the disease. In this context, studies in which pharmacological inhibitors of Dnmts were shown to inhibit prostate cancer in murine models have proven informative (34, 56). However, it remains unknown whether genetic disruption of epigenetic components, such as Dnmts, also impacts prostate cancer development. This is a critical question since the pharmacological inhibitors of Dnmts have pleiotropic effects, including those unrelated to activation of methylation-silenced genes (21, 23, 31). Moreover, no studies to date have examined whether Dnmts or DNA methylation play roles in normal prostate development; this information is vital to fully understanding the effects that inhibiting DNA methylation may have on prostate cancer.Dnmt1 is a maintenance DNA methyltransferase that propagates preexisting DNA methylation patterns in genomic DNA (44). Dnmt1 also is involved in de novo DNA methylation in cancer cells and interacts with other key epigenetic control molecules, including histone-modifying enzymes (11, 19). Murine models have been used to investigate the in vivo functions of Dnmt1. Complete genetic knockout of Dnmt1 is embryonic lethal in mice (29). However, hypomorphic expression of Dnmt1 allows murine development to proceed but causes global DNA hypomethylation and impacts cancer development and progression (7, 14, 28). Specifically, hypomorphic expression of Dnmt1 can lead to the development of lymphoma (14). Furthermore, crossing Dnmt1 hypomorphic mice with murine tumor models alters tumor progression, resulting in either increased or decreased tumor development, depending on the disease stage and tissue site (1, 7, 53). For example, reduced expression of Dnmt1 dramatically decreases intestinal polyp formation in Apc^Min/+ mice, either alone or in combination with 5-aza-2′-deoxycytidine treatment (7, 27). However, it was later noted that reduced expression of Dnmt1 has a dual effect on intestinal cancer in Apc^Min/+ mice, in which the development of early stage intestinal microadenomas is accelerated, whereas the formation of adenomatous polyps is significantly reduced (53). In addition, Apc^Min/+ Dnmt1 hypomorphic mice develop liver cancer associated with the loss of heterozygosity of Apc (53). Similarly, in Dnmt1 hypomorphic mice crossed to Mlh1^−/− mice, a dual effect was noted wherein mice developed fewer intestinal cancers but displayed increased T- and B-cell lymphomas (52). In addition, a recent study demonstrated that hypomorphic Dnmt1 expression is associated with reduced squamous cell carcinoma of the tongue and esophagus, resulting in decreased invasive cancer (1). Taken together, the data suggest that Dnmt1 has diverse effects on cancer development, which are dependent on tissue context and tumor stage.TRAMP is a well-established transgenic prostate cancer model driven by prostate-specific expression of the simian virus 40 (SV40) T/t oncogenes (16). TRAMP mice are characterized by Dnmt mRNA and protein overexpression, altered DNA methylation, and altered gene expression during prostate cancer development (2, 33, 35, 37). Of the three enzymatically active Dnmts, Dnmt1 shows the greatest level of overexpression in TRAMP, and this correlates with Rb inactivation, a key genetic event driving prostate cancer in the model (37). Most critically, global DNA hypomethylation occurs during early and late disease stages, while DNA hypermethylation occurs primarily at late disease stages in TRAMP (35).Here, we utilized Dnmt1 hypomorphic mice and the TRAMP model to assess the role of DNA methylation in both normal prostatic development and prostate cancer. The Dnmt1 hypomorphic mouse model used involves two different hypomorphic alleles (N and R), resulting in four genotypes with progressively reduced DNA methylation (Dnmt1^+/+, Dnmt1^R/+, Dnmt1^N/+, and Dnmt1^N/R) (7, 52). The N allele consists of a PGK-Neo insertion that deletes a portion of exon 4 of Dnmt1, resulting in severely reduced Dnmt1 expression, while the R allele involves a lacO insertion into intron 3 of Dnmt1, which partially reduces Dnmt1 expression (7, 52). Based on our previous work establishing the timing of DNA hypomethylation and DNA hypermethylation in TRAMP, we hypothesized that hypomorphic Dnmt1 expression in TRAMP may have tumor-promoting effects at early disease stages and tumor-inhibitory effects at later stages of prostate cancer progression. Our data are consistent with this hypothesis and, more importantly, reveal a critical and unanticipated role for Dnmt1 in prostate cancer metastasis.     

Relaxin-3 and receptors in the human and rhesus brain and reproductive tissues

Evidence suggests that relaxin-3 may have biological functions in the reproductive and central nervous systems. To date, however, relaxin-3 biodistribution has only been investigated in the mouse, rat, pig and teleost fish. Characterizing relaxin-3 gene structure, expression patterns, and function in non-human primates and humans is critical to delineating its biological significance. Experiments were performed to clone the rhesus macaque orthologues of the relaxin-3 peptide hormone and its cognitive receptors (RXFP1 and RXFP4). An investigation of rhesus relaxin-3 bioactivity and RXFP1 binding properties was also performed. Next we sought to investigate relaxin-3 immunoreactivity in human and rhesus macaque tissues. Immunohistofluorescence staining for relaxin-3 in the brain, testis, and prostate indicated predominant immunostaining in the ventral and dorsal tegmental nuclei, interstitial space surrounding the seminiferous tubules, and prostatic stromal cells, respectively. Further, in studies designed towards exploring biological functions, we observed neuroprotective actions of rhesus relaxin-3 on human neuronal cell cultures. Taken together, this study broadens the significance of relaxin-3 as a peptide involved in both neuronal cell function and reproductive tissues in primates.