Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila

We have previously reported the identification of a nonhistone chromosomal protein (nhcp-19; now called HP1) preferentially associated with the heterochromatin of Drosophila melanogaster. A detailed study of the HP1 distribution pattern on polytene chromosomes by immunofluorescent staining, using monoclonal antibody C1A9, has been carried out. The results indicate that this protein is found within the centric beta-heterochromatin, in cytological regions 31, 41 and 80, and throughout polytene chromosome 4. Staining of telomeres is frequently observed, those of chromosome arms 2R and 3R and the X chromosome being the most conspicuous. Analysis of a fourth chromosome insertional translocation T(3;4)f/In(3L)P confirms an autonomous interaction with chromosome 4 material. Similarly, the beta-heterochromatin distal to light on chromosome arm 2L, moved to position 97D2 on chromosome arm 3R in the rearrangement ltx13, is prominently stained using the C1A9 antibody. Staining of intact salivary glands indicates that this rearranged segment of beta-heterochromatin is not associated with the polytene chromocenter, but provides an independent structural reference point. HP1 is not observed in the nuclei of the early syncytial embryo, but becomes concentrated in the nuclei at the syncytial blastoderm stage (ca. nuclear division cycle 10). This suggests that heterochromatin formation occurs at approximately the same stage at which nuclei first become transcriptionally competent. Thus, the C1A9 antibody may serve as a useful marker for both structural and functional studies of the Drosophila nucleus

Triangulating Abuse Liability Assessment for Flavoured Cigar Products Using Physiological, Behavioural Economic and Subjective Assessments: A Within-subjects Clinical Laboratory Protocol

Introduction In the USA, Food and Drug Administration regulations prohibit the sale of flavoured cigarettes, with menthol being the exception. However, the manufacture, advertisement and sale of flavoured cigar products are permitted. Such flavourings influence positive perceptions of tobacco products and are linked to increased use. Flavourings may mask the taste of tobacco and enhance smoke inhalation, influencing toxicant exposure and abuse liability among novice tobacco users. Using clinical laboratory methods, this study investigates how flavour availability affects measures of abuse liability in young adult cigarette smokers. The specific aims are to evaluate the effect of cigar flavours on nicotine exposure, and behavioural and subjective measures of abuse liability. Methods and analyses Participants (projected n=25) are healthy smokers of five or more cigarettes per day over the past 3 months, 18–25 years old, naive to cigar use (lifetime use of 50 or fewer cigar products and no more than 10 cigars smoked in the past 30 days) and without a desire to quit cigarette smoking in the next 30 days. Participants complete five laboratory sessions in a Latin square design with either their own brand cigarette or a session-specific Black & Mild cigar differing in flavour (apple, cream, original and wine). Participants are single-blinded to cigar flavours. Each session consists of two 10-puff smoking bouts (30 s interpuff interval) separated by 1 hour. Primary outcomes include saliva nicotine concentration, behavioural economic task performance and response to various questionnaire items assessing subjective effects predictive of abuse liability. Differences in outcomes across own brand cigarette and flavoured cigar conditions will be tested using linear mixed models

Molecular cloning of a human homologue of Drosophila heterochromatin protein HP1 using anti-centromere autoantibodies with anti-chromo specificity

We have identified a novel autoantibody specificity in scleroderma that we term anti-chromo. These antibodies recognize several chromosomal antigens with apparent molecular mass of between 23 and 25 kDa, as determined by immunoblots. Anti-chromo autoantibodies occur in 10-15% of sera from patients with anti-centromere antibodies (ACA). We used anti-chromo antibodies to screen a human expression library and obtained cDNA clones encoding a 25 kDa chromosomal autoantigen. DNA sequence analysis reveals this protein to be a human homologue of HP1, a heterochromatin protein of Drosophila melanogaster. We designate our cloned protein HP1Hs alpha. Epitope mapping experiments using both human and Drosophila HP1 reveal that anti-chromo antibodies target a region at the amino terminus of the protein. This region contains a conserved motif, the chromo domain (or HP1/Pc box), first recognized by comparison of Drosophila HP1 with the Polycomb gene product. Both proteins are thought to play a role in creating chromatin structures in which gene expression is suppressed. Anti-chromo thus defines a novel type of autoantibody that recognizes a conserved structural motif found on a number of chromosomal proteins

Electronic cigarettes: a survey of users

<p>Abstract</p> <p>Background</p> <p>Little is known about users of electronic cigarettes, or their opinions, satisfaction or how and why they use such products.</p> <p>Methods</p> <p>An internet survey of 81 ever-users of ecigarettes in 2009. Participants answered open-ended questions on use of, and opinions about, ecigarettes.</p> <p>Results</p> <p>Respondents (73 current and 8 former users) lived in France, Canada, Belgium or Switzerland. Most respondents (77%) were men; 63% were former smokers and 37% were current smokers. They had used e-cigarettes for 100 days (median) and drew 175 puffs per day (median). Participants used the ecigarette either to quit smoking (53 comments), to reduce their cigarette consumption (14 comments), in order not to disturb other people with smoke (20 comments), or in smoke-free places (21 comments). Positive effects reported with ecigarettes included their usefulness to quit smoking, and the benefits of abstinence from smoking (less coughing, improved breathing, better physical fitness). Respondents also enjoyed the flavour of ecigarettes and the sensation of inhalation. Side effects included dryness of the mouth and throat. Respondents complained about the frequent technical failures of ecigarettes and had some concerns about the possible toxicity of the devices and about their future legal status.</p> <p>Conclusions</p> <p>Ecigarettes were used mainly to quit smoking, and may be helpful for this purpose, but several respondents were concerned about potential toxicity. There are very few published studies on ecigarettes and research is urgently required, particularly on the efficacy and toxicity of these devices.</p

Heterochromatin: A Rapidly Evolving Species Barrier

Recent work has shown that changes in the sequence composition of heterochromatin, or in the factors that maintain that heterochromatin, may play an important role in speciation

The Chromatin Remodelling Factor dATRX Is Involved in Heterochromatin Formation

Despite extensive study of heterochromatin, relatively little is known about the mechanisms by which such a structure forms. We show that the Drosophila homologue of the human α-thalassemia and mental retardation X-linked protein (dATRX), is important in the formation or maintenance of heterochromatin through modification of position effect variegation. We further show that there are two isoforms of the dATRX protein, the longer of which interacts directly with heterochromatin protein 1 (dHP-1) through a CxVxL motif both in vitro and in vivo. These two proteins co-localise at heterochromatin in a manner dependent on this motif. Consistent with this observation, the long isoform of the dATRX protein localises primarily to the heterochromatin at the chromocentre on salivary gland polytene chromosomes, whereas the short isoform binds to many sites along the chromosome arms. We suggest that the establishment of a regular nucleosomal organisation may be common to heterochromatin and transcriptionally repressed chromatin in other locations, and may require the action of ATP dependent chromatin remodelling factors

Functional conservation of the Drosophila hybrid incompatibility gene Lhr

<p>Abstract</p> <p>Background</p> <p>Hybrid incompatibilities such as sterility and lethality are commonly modeled as being caused by interactions between two genes, each of which has diverged separately in one of the hybridizing lineages. The gene <it>Lethal hybrid rescue </it>(<it>Lhr</it>) encodes a rapidly evolving heterochromatin protein that causes lethality of hybrid males in crosses between <it>Drosophila melanogaster </it>females and <it>D. simulans </it>males. Previous genetic analyses showed that hybrid lethality is caused by <it>D. simulans Lhr </it>but not by <it>D. melanogaster Lhr</it>, confirming a critical prediction of asymmetry in the evolution of a hybrid incompatibility gene.</p> <p>Results</p> <p>Here we have examined the functional properties of <it>Lhr </it>orthologs from multiple Drosophila species, including interactions with other heterochromatin proteins, localization to heterochromatin, and ability to complement hybrid rescue in <it>D. melanogaster</it>/<it>D. simulans </it>hybrids. We find that these properties are conserved among most <it>Lhr </it>orthologs, including <it>Lhr </it>from <it>D. melanogaster</it>, <it>D. simulans </it>and the outgroup species <it>D. yakuba</it>.</p> <p>Conclusions</p> <p>We conclude that evolution of the hybrid lethality properties of <it>Lhr </it>between <it>D. melanogaster </it>and <it>D. simulans </it>did not involve extensive loss or gain of functions associated with protein interactions or localization to heterochromatin.</p

Structural Basis of the Chromodomain of Cbx3 Bound to Methylated Peptides from Histone H1 and G9a

HP1 proteins are highly conserved heterochromatin proteins, which have been identified to be structural adapters assembling a variety of macromolecular complexes involved in regulation of gene expression, chromatin remodeling and heterochromatin formation. Much evidence shows that HP1 proteins interact with numerous proteins including methylated histones, histone methyltransferases and so on. Cbx3 is one of the paralogues of HP1 proteins, which has been reported to specifically recognize trimethylated histone H3K9 mark, and a consensus binding motif has been defined for the Cbx3 chromodomain.Here, we found that the Cbx3 chromodomain can bind to H1K26me2 and G9aK185me3 with comparable binding affinities compared to H3K9me3. We also determined the crystal structures of the human Cbx3 chromodomain in complex with dimethylated histone H1K26 and trimethylated G9aK185 peptides, respectively. The complex structures unveil that the Cbx3 chromodomain specifically bind methylated histone H1K26 and G9aK185 through a conserved mechanism.The Cbx3 chromodomain binds with comparable affinities to all of the methylated H3K9, H1K26 and G9aK185 peptides. It is suggested that Cbx3 may regulate gene expression via recognizing both histones and non-histone proteins

Histone H3K9 Trimethylase Eggless Controls Germline Stem Cell Maintenance and Differentiation

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms