A reexamination of spreading of position-effect variegation in the white-roughest region of Drosophila melanogaster

In Drosophila, heterochromatin causes mosaic silencing of euchromatic genes brought next to it by chromosomal rearrangements. Silencing has been observed to "spread": genes closer to the heterochromatic rearrangement breakpoint are silenced more frequently than genes farther away. We have examined silencing of the white and roughest genes in the variegating rearrangements In(1)w(m4), In(1)w(mMc), and In(1)w(m51b). Eleven stocks bearing these chromosomes differ widely in the strength of silencing of white and roughest. Stock-specific differences in the relative frequencies of inactivation of white and roughest were found that map to the white-roughest region or the adjacent heterochromatin. Most stock-specific differences did not correlate with gross differences in the heterochromatic content of the rearranged chromosomes; however, two stocks, In(1)w(m51b) and In(1)w(mMc), were found to have anomalous additional heterochromatin that may act in trans to suppress variegating alleles. In comparing different stocks, the frequency of silencing of the roughest gene, which is more distant from heterochromatin, does not correlate with the frequency of silencing of the more proximal white gene on the same chromosome, in contradiction to the expectation of models of continuous linear propagation of silencing. We frequently observed rough eye tissue that is pigmented, as though an active white gene is skipped.     

The HP1 chromo shadow domain binds a consensus peptide pentamer

Heterochromatin-associated protein 1 (HP1) is thought to affect chromatin structure through interactions with other proteins in heterochromatin. Chromo domains located near the amino (amino chromo) and carboxy (chromo shadow) termini of HP1 may mediate such interactions, as suggested by domain swapping, in vitro binding and 3D structural studies . Several HP1-associated proteins have been reported, providing candidates that might specifically complex with the chromo domains of HP1. However, such association studies provide little mechanistic insight and explore only a limited set of potential interactions in a largely non-competitive setting. To determine how chromo domains can selectively interact with other proteins, we probed random peptide phage display libraries using chromo domains from HP1. Our results demonstrate that a consensus pentapeptide is suffident for specific interaction with the HP1 chromo shadow domain. The pentapeptide is found in the amino acid sequence of reported HP1-associated proteins, including the shadow domain itself. Peptides that bind the shadow domain also disrupt shadow domain dimers. Our results suggest that HP1 dimerization, which is thought to mediate heterochromatin compaction and cohesion, occurs via pentapeptide binding. In general, chromo domains may function by avidly binding short peptides at the surface of chromatin-associated proteins.     

Repetitive arrays containing a housekeeping gene have altered polytene chromosome morphology in Drosophila

Much of our understanding of gene and chromatin organization has been developed from observation of polytene chromosomes. We describe an experimental approach using transgenes that has allowed us to observe local changes in polytene morphology. A composite P transposon that contains a fusion between the regulatory region of Prat, a purine synthesis gene, and brown (bw), an eye pigment reporter, was transformed into the 65A10 polytene band and subjected to P-transposase mutagenesis. Arrays of up to 320 kb at 65A10 were recovered by selection for increased pigment, and pigment levels were found to be proportional to numbers of copies. In polytene chromosomes, the original transformant was found to split 65A10 into two thin bands separated by an interband. With increases in copy number, the interband became progressively denser, eventually forming a dark, amorphous, deformable structure unlike any previously reported. The persistence of Prat expression in development, together with the cytological appearance of these large arrays, suggest that the state of the Prat promoter is affecting polytene structure. Because this unique structure is distinct from bands, interbands, puffs, and the chromocenter, which comprise polytene chromosomes, we suggest that it is composed of an altered form of chromatin.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

The structure, catalytic mechanism and regulation of adenylyl cyclase

The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity.     

Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat

Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.     

Association of 25-Hydroxyvitamin D status and genetic variation in the vitamin D metabolic pathway with FEV1 in the Framingham Heart Study

Background

Vitamin D is associated with lung function in cross-sectional studies, and vitamin D inadequacy is hypothesized to play a role in the pathogenesis of chronic obstructive pulmonary disease. Further data are needed to clarify the relation between vitamin D status, genetic variation in vitamin D metabolic genes, and cross-sectional and longitudinal changes in lung function in healthy adults.

Methods

We estimated the association between serum 25-hydroxyvitamin D [25(OH)D] and cross-sectional forced expiratory volume in the first second (FEV₁) in Framingham Heart Study (FHS) Offspring and Third Generation participants and the association between serum 25(OH)D and longitudinal change in FEV₁ in Third Generation participants using linear mixed-effects models. Using a gene-based approach, we investigated the association between 241 SNPs in 6 select vitamin D metabolic genes in relation to longitudinal change in FEV₁ in Offspring participants and pursued replication of these findings in a meta-analyzed set of 4 independent cohorts.

Results

We found a positive cross-sectional association between 25(OH)D and FEV₁ in FHS Offspring and Third Generation participants (P = 0.004). There was little or no association between 25(OH)D and longitudinal change in FEV₁ in Third Generation participants (P = 0.97). In Offspring participants, the CYP2R1 gene, hypothesized to influence usual serum 25(OH)D status, was associated with longitudinal change in FEV₁ (gene-based P < 0.05). The most significantly associated SNP from CYP2R1 had a consistent direction of association with FEV₁ in the meta-analyzed set of replication cohorts, but the association did not reach statistical significance thresholds (P = 0.09).

Conclusions

Serum 25(OH)D status was associated with cross-sectional FEV₁, but not longitudinal change in FEV₁. The inconsistent associations may be driven by differences in the groups studied. CYP2R1 demonstrated a gene-based association with longitudinal change in FEV₁ and is a promising candidate gene for further studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0238-y) contains supplementary material, which is available to authorized users.