Ultraconserved elements in the human genome: association and transmission analyses of highly constrained single-nucleotide polymorphisms

Ultraconserved elements in the human genome likely harbor important biological functions as they are dosage sensitive and are able to direct tissue-specific expression. Because they are under purifying selection, variants in these elements may have a lower frequency in the population but a higher likelihood of association with complex traits. We tested a set of highly constrained SNPs (hcSNPs) distributed genome-wide among ultraconserved and nearly ultraconserved elements for association with seven traits related to reproductive (age at natural menopause, number of children, age at first child, and age at last child) and overall [longevity, body mass index (BMI), and height] fitness. Using up to 24,047 European-American samples from the National Heart, Lung, and Blood Institute Candidate Gene Association Resource (CARe), we observed an excess of associations with BMI and height. In an independent replication panel the most strongly associated SNPs showed an 8.4-fold enrichment of associations at the nominal level, including three variants in previously identified loci and one in a locus (DENND1A) previously shown to be associated with polycystic ovary syndrome. Finally, using 1430 family trios, we showed that the transmissions from heterozygous parents to offspring of the derived alleles of rare (frequency ≤0.5%) hcSNPs are not biased, particularly after adjusting for the rates of genotype missingness and error in the data. The lack of transmission bias ruled out an immediately and strongly deleterious effect due to the rare derived alleles, consistent with the observation that mice homozygous for the deletion of ultraconserved elements showed no overt phenotype. Our study also illustrated the importance of carefully modeling potential technical confounders when analyzing genotype data of rare variants.     

Acyl chains of phospholipase d transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry

Background

Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.

Methodology/Principal findings

Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.

Conclusions

MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.     

Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation

A multichaperone nucleosome-remodeling complex that contains the H1 linker histone chaperone nuclear autoantigenic sperm protein (NASP) has recently been described. Linker histones (H1) are required for the proper completion of normal development, and NASP transports H1 histones into nuclei and exchanges H1 histones with DNA. Consequently, we investigated whether NASP is required for normal cell cycle progression and development. We now report that without sufficient NASP, HeLa cells and U2OS cells are unable to replicate their DNA and progress through the cell cycle and that the NASP(-/-) null mutation causes embryonic lethality. Although the null mutation NASP(-/-) caused embryonic lethality, null embryos survive until the blastocyst stage, which may be explained by the presence of stored NASP protein in the cytoplasm of oocytes. We conclude from this study that NASP and therefore the linker histones are key players in the assembly of chromatin after DNA replication.     

Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.     

Disentangling environmental and host sources of fungal endophyte communities in an experimental beachgrass study

Disentangling the ecological factors that contribute to the assembly of the microbial symbiont communities within eukaryotic hosts is an ongoing challenge. Broadly speaking, symbiont propagules arrive either from external sources in the environment or from internal sources within the same host individual. To understand the relative importance of these propagule sources to symbiont community assembly, we characterized symbiotic fungal endophyte communities within the roots of three species of beachgrass in a field experiment. We manipulated two aspects of the external environment, successional habitat and physical disturbance. To determine the role of internal sources of propagules for endophyte community assembly, we used beachgrass individuals with different pre‐existing endophyte communities. Endophyte species richness and community composition were characterized using culture‐based and next‐generation sequencing approaches. Our results showed that external propagule sources associated with successional habitat, but not disturbance, were particularly important for colonization of most endophytic taxa. In contrast, internal propagule sources played a minor role for most endophytic taxa but were important for colonization by the dominant taxon Microdochium bolleyi. Our findings highlight the power of manipulative field experiments to link symbiont community assembly to its underlying ecological processes, and to ultimately improve predictions of symbiont community assembly across environments.