Colon cancer-associated DNA polymerase β variant induces genomic instability and cellular transformation

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer.     

Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes

In Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila.     

The fanconi anemia pathway limits human papillomavirus replication

High-risk human papillomaviruses (HPVs) deregulate epidermal differentiation and cause anogenital and head and neck squamous cell carcinomas (SCCs). The E7 gene is considered the predominant viral oncogene and drives proliferation and genome instability. While the implementation of routine screens has greatly reduced the incidence of cervical cancers which are almost exclusively HPV positive, the proportion of HPV-positive head and neck SCCs is on the rise. High levels of HPV oncogene expression and genome load are linked to disease progression, but genetic risk factors that regulate oncogene abundance and/or genome amplification remain poorly understood. Fanconi anemia (FA) is a genome instability syndrome characterized at least in part by extreme susceptibility to SCCs. FA results from mutations in one of 15 genes in the FA pathway, whose protein products assemble in the nucleus and play important roles in DNA damage repair. We report here that loss of FA pathway components FANCA and FANCD2 stimulates E7 protein accumulation in human keratinocytes and causes increased epithelial proliferation and basal cell layer expansion in the HPV-positive epidermis. Additionally, FANCD2 loss stimulates HPV genome amplification in differentiating cells, demonstrating that the intact FA pathway functions to restrict the HPV life cycle. These findings raise the possibility that FA genes suppress HPV infection and disease and suggest possible mechanism(s) for reported associations of HPV with an FA cohort in Brazil and for allelic variation of FA genes with HPV persistence in the general population.     

The structure and functioning of the couplon in the mammalian cardiomyocyte

The couplons of the cardiomyocyte form nanospaces within the cell that place the L-type calcium channel (Ca_v1.2), situated on the plasmalemma, in opposition to the type 2 ryanodine receptor (RyR2), situated on the sarcoplasmic reticulum. These two molecules, which form the basis of excitation–contraction coupling, are separated by a very limited space, which allows a few Ca²⁺ ions passing through Ca_v1.2 to activate the RyR2 at concentration levels that would be deleterious to the whole cell. The limited space also allows Ca²⁺ inactivation of Ca_v1.2. We have found that not all couplons are the same and that their properties are likely determined by their molecular partners which, in turn, determine their excitability. In particular, there are a class of couplons that lie outside the RyR2-Ca_v1.2 dyad; in this case, the RyR2 is close to caveolin-3 rather than Ca_v1.2. These extra-dyadic couplons are probably controlled by the multitude of molecules associated with caveolin-3 and may modulate contractile force under situations such as stress. It has long been assumed that like the skeletal muscle, the RyR2 in the couplon are arranged in a structured array with the RyR2 interacting with each other via domain 6 of the RyR2 molecule. This arrangement was thought to provide local control of RyR2 excitability. Using 3D electron tomography of the couplon, we show that the RyR2 in the couplon do not form an ordered pattern, but are scattered throughout it. Relatively few are in a checkerboard pattern—many RyR2 sit edge-to-edge, a configuration which might preclude their controlling each other's excitability. The discovery of this structure makes many models of cardiac couplon function moot and is a current avenue of further research     

Genome-wide association genetics of an adaptive trait in lodgepole pine

Pine cones that remain closed and retain seeds until fire causes the cones to open (cone serotiny) represent a key adaptive trait in a variety of pine species. In lodgepole pine, there is substantial geographical variation in serotiny across the Rocky Mountain region. This variation in serotiny has evolved as a result of geographically divergent selection, with consequences that extend to forest communities and ecosystems. An understanding of the genetic architecture of this trait is of interest owing to the wide-reaching ecological consequences of serotiny and also because of the repeated evolution of the trait across the genus. Here, we present and utilize an inexpensive and time-effective method for generating population genomic data. The method uses restriction enzymes and PCR amplification to generate a library of fragments that can be sequenced with a high level of multiplexing. We obtained data for more than 95,000 single nucleotide polymorphisms across 98 serotinous and nonserotinous lodgepole pines from three populations. We used a Bayesian generalized linear model (GLM) to test for an association between genotypic variation at these loci and serotiny. The probability of serotiny varied by genotype at 11 loci, and the association between genotype and serotiny at these loci was consistent in each of the three populations of pines. Genetic variation across these 11 loci explained 50% of the phenotypic variation in serotiny. Our results provide a first genome-wide association map of serotiny in pines and demonstrate an inexpensive and efficient method for generating population genomic data.     

Associations between retinol-binding protein 4 and cardiometabolic risk factors and subclinical atherosclerosis in recently postmenopausal women: Cross-sectional analyses from the KEEPS Study

ABSTRACT: BACKGROUND: The published literature regarding the relationships between retinol-binding protein 4 (RBP4) and cardiometabolic risk factors and subclinical atherosclerosis is conflicting, likely due, in part, to limitations of frequently used RBP4 assays. Prior large studies have not utilized the gold-standard Western Blot analysis of RBP4 levels. METHODS: Full-length serum RBP4 levels were measured by Western Blot in 709 postmenopausal women screened for the Kronos Early Estrogen Prevention Study. Cross-sectional analyses related RBP4 levels to cardiometabolic risk factors, carotid artery intima-media thickness (CIMT), and coronary artery calcification (CAC). RESULTS: The mean age of women was 52.9 (+/- 2.6) years, and the median RBP4 level was 49.0 (interquartile range 36.9-61.5) ug/mL. Higher RBP4 levels were weakly associated with higher triglycerides (age, race, and smoking-adjusted partial Spearman correlation coefficient= 0.10; P=0.01), but were unrelated to blood pressure, cholesterol, C-reactive protein, glucose, insulin, and CIMT levels (all partial Spearman correlation coefficients [less than or equal to]0.06, P>0.05). Results suggested a curvilinear association between RBP4 levels and CAC, with women in the bottom and upper quartiles of RBP4 having higher odds of CAC (odds ratio [95% confidence interval] 2.10 [1.07-4.09], 2.00 [1.02-3.92], 1.64 [0.82-3.27] for the 1st, 3rd, and 4th RBP4 quartiles vs. the 2nd quartile). However, a squared RBP4 term in regression modeling was non-significant (P=0.10). CONCLUSIONS: In these healthy, recently postmenopausal women, higher RBP4 levels were weakly associated with elevations in triglycerides and with CAC, but not with other risk factors or CIMT. These data using the gold standard of RBP4 methodology only weakly support the possibility that perturbations in RBP4 homeostasis may be an additional risk factor for subclinical coronary atherosclerosis. Trial Registration: ClinicalTrials.gov number NCT00154180.     

The Aromatase Gene (CYP19A1) Variants and Circulating Hepatocyte Growth Factor in Postmenopausal Women

Background

Estrogen and androgen have been linked to the regulation of circulating hepatocyte growth factor (HGF), an adipose tissue-derived cytokine. It is possible that the CYP19A1 gene which alters sex hormones production may influence HGF levels. We examined the association between the CYP19A1 gene variants and plasma HGF concentrations.

Design

We evaluated 45 common and putative functional variants of CYP19A1 and circulating levels of HGF among 260 postmenopausal women who later developed colorectal cancer from the Women''s Health Initiative Observational Cohort. As the distribution of HGF levels was highly skewed, we transformed HGF concentrations for all women into a log-, ranked-, or normal score-scale value. Multiple linear regression with adjustment for age was used to evaluate the associations.

Results

We observed an association between the rs7172156, rs1008805, rs6493494, rs749292, and rs11636639 variants and HGF levels in ranked and normal score scales (corrected p values ≤0.02), although the association of these 5 SNPs with log-scale HGF was not significant (corrected p values ≥0.16). The associations remained unchanged after additional adjustment for hormone therapy use and estradiol levels. These 5 SNPs, which were in linkage disequilibrium (pairwise D′≥97%, r²≥56%), constituted a block with 2 common haplotypes accounting for 82% frequency. The most common haplotype, TCCCA, was associated with lower ranked- or normal score-transformed HGF levels (corrected p values ≤0.001), whereas the second most common haplotype, CTTCA, was associated with higher ranked- or normal score-transformed HGF levels (corrected p values ≤0.02).

Conclusion

Our findings of a potential association between the CYP19A1 variants and circulating HGF levels warrant confirmation in studies with larger sample size.     

The human gastric cancer-associated DNA polymerase β variant D160N is a mutator that induces cellular transformation

Approximately 30% of human tumors sequenced to date harbor mutations in the POLB gene that are not present in matched normal tissue. Many mutations give rise to enzymes that contain non-synonymous single amino acid substitutions, several of which have been found to have aberrant activity or fidelity and transform cells when expressed. The DNA Polymerase β (Pol β) variant Asp160Asn (D160N) was first identified in a gastric tumor. Expression of D160N in cells induces cellular transformation as measured by hyperproliferation, focus formation, anchorage-independent growth and invasion. Here, we show that D160N is an active mutator polymerase that induces complex mutations. Our data support the interpretation that complex mutagenesis is the underlying mechanism of the observed cellular phenotypes, all of which are linked to tumorigenesis or tumor progression.