Small changes in bone structure of female a7 nicotinic acetylcholine receptor knockout mice

BACKGROUND: Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit a2 as positive regulator of bone mass accrual whereas of male mice deficient for a7-nAChR (a7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female a7KO compared to their corresponding wild-type mice (a7WT). METHODS: Vertebrae and long bones of female 16-week-old a7KO (n = 10) and a7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM). RESULTS: Bone of female a7KO revealed a significant increase in bending stiffness (p<0.05) and cortical thickness (p<0.05) compared to a7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N+ (p<0.05) and C4H8N+ (p<0.001) collagen fragments whereas a loss of osteoid was found by means of TEM. CONCLUSIONS: Our results on female a7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that a7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit a2 the a7-nAChR favours reduction of bone strength thereby showing similar effects as a7ß2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing a- and ß-subunits as well as the subunits a7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric a7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis

A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM

A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking.

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM

RNA interference (RNAi) identifies 78 factors inducing insulator reporter gene activity including NURF and dREAM components.

<p>(<b>A</b>) Workflow of the RNAi screen in 66×384-well plates from the DRSC. Knockdown of 13900 genes was done with <i>Drosophila</i> S2 cells with the integrated F8OF8L insulator reporter construct (F8, <i>Fab-8</i>; O, OpIE2 enhancer; L, <i>luciferase</i>). (<b>B</b>) Top GO-terms (determined via GeneCodis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765-CarmonaSaez1" target="_blank">[58]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765-TabasMadrid1" target="_blank">[60]</a>) for the 78 identified genes. (<b>C</b>) High-throughput data shown in a dotplot diagram. Z-scores are indicated for every well (well number). For many gene products several wells contain different dsRNA sequences targeting the same gene. Z-scores higher than two are highlighted in red. (<b>D</b>) Individual depletion of NURF and dREAM components and associated factors verify enhancer blocking function. S2 cell pools with the integrated F8OF8L insulator reporter (dark grey) or the control F8OL reporter construct (light grey) were incubated with dsRNA against factors of the NURF-complex (pink): ISWI, NURF-38, CAF1/p55, NURF301, Pzg, DREF or against the dREAM-complex (blue): CAF1/p55, Mip40, Mip130, E2F2. Reporter gene activity is expressed as fold change relative to control knockdown. Error bars indicate the standard deviation of three individual replicates. (p-values: *≤0.05, **≤0.01, ***≤0.001).</p

NURF and dREAM components co-localize with dCTCF/CP190.

<p>ChIP in S2 cells with antibodies against CTCF and CP190 and components of the NURF complex (ISWI, NURF301, Pzg and Chro) and dREAM complex (Mip40, Mip120, Mip130, E2F2) or CAF-1/p55. The genomic regions tested are indicated (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765.s013" target="_blank">Table S2</a>) and grouped into CTCF plus CP190, low CTCF plus CP190, low CTCF without CP190 and neither CTCF nor CP190. Error bars indicate the standard deviation of three independent experiments.</p

NURF and dREAM components co-localize with CP190 genome-wide.

<p>(<b>A</b>) Correlation analysis for genome-wide binding of CP190 with 215 profiles from S2 cells (modENCODE) and 5 profiles from Kc cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765-Georlette1" target="_blank">[33]</a>. Shown are the top 30 ranking factors. Components of the NURF complex are marked in pink and of the dREAM complex in blue. (<b>B</b>) Cluster heat map of 6,000 genomic regions with CP190 and/or CTCF sites compared with binding sites for components of NURF (NURF301, ISWI, Chro) and dREAM (E2F2, Lin-52, Mip120, Mip130, Myb) complexes. Each lane represents an 8 kb region. Scale represents binding (red) to no binding (blue).</p

Insulator specific effects of NURF and dREAM components.

<p>S2 cells pools with the integrated luciferase reporter constructs with different CTCF/CP190 binding sites located between the enhancer (O, OpIE2) and the promoter of the reporter gene (L, <i>luciferase</i>). (<b>A</b>) Luciferase activity after control knockdown of GFP shows the enhancer blocking activity of <i>Fab-8</i> (F8), bicoid (bcd), CG31472 and <i>Fab-6</i> (F6(2)), when compared to the CTCF binding site mutant (F8mut) (top). Error bars indicate the standard deviation of three biological replicates. The different insulator reporter constructs are depicted (bottom), the genomic fragments used are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765.s013" target="_blank">Table S2</a> and genome browser views are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107765#pone.0107765.s007" target="_blank">Figure S7</a>. (<b>B</b>) Knockdown experiments against CTCF, ISWI or NURF301 (top) and of CTCF, CAF1/p55 or triple-knockdown of Mip-factors (bottom). Fold change of luciferase activity is calculated relative to the control knockdown. Error bars indicate the standard error of three or more individual replicates (p-values: *≤0.05, **≤0.01, ***≤0.001; ND: not determined).</p

Purification of either CTCF or CP190 reveals NURF and dREAM binding to both insulator factors.

<p>(<b>A</b>) Interaction heatmap based on Mascot scores (dCTCF) or fold enrichment of normalized intensities (CP190), depicting associated factors identified by mass spectrometry after immunopurification of FLAG-dCTCF or FLAG-CP190 expressed in S2 cells. (<b>B</b>) Nuclear extracts from S2 cells (lanes 1–2, 7–10) and S2 cells stably expressing FLAG-CP190 (lanes 3–4) or FLAG-dCTCF (lanes 5–6) were precipitated with FLAG antibody (lanes 2, 4, 6), CP190 antibody (lane 9), dCTCF antibody (lane 10) or IgG (lane 8) as control. Antibodies used in Western blot are indicated on the right. Lanes 1, 3, 5 and 7: 1% Input.</p

Leukoencephalopathy with calcifications and cysts:Genetic and phenotypic spectrum

Biallelic mutations in SNORD118, encoding the small nucleolar RNA U8, cause leukoencephalopathy with calcifications and cysts (LCC). Given the difficulty in interpreting the functional consequences of variants in nonprotein encoding genes, and the high allelic polymorphism across SNORD118 in controls, we set out to provide a description of the molecular pathology and clinical spectrum observed in a cohort of patients with LCC. We identified 64 affected individuals from 56 families. Age at presentation varied from 3 weeks to 67 years, with disease onset after age 40 years in eight patients. Ten patients had died. We recorded 44 distinct, likely pathogenic, variants in SNORD118. Fifty two of 56 probands were compound heterozygotes, with parental consanguinity reported in only three families. Forty nine of 56 probands were either heterozygous (46) or homozygous (three) for a mutation involving one of seven nucleotides that facilitate a novel intramolecular interaction between the 5′ end and 3′ extension of precursor-U8. There was no obvious genotype–phenotype correlation to explain the marked variability in age at onset. Complementing recently published functional analyses in a zebrafish model, these data suggest that LCC most often occurs due to combinatorial severe and milder mutations, with the latter mostly affecting 3′ end processing of precursor-U8