Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs

The 5' terminus of the CyI actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2.5 kb of 5' flanking sequence, the transcribed leader sequence, and the first few codons of the CyI gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was micro-injected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous CyI gene first appear. The exogenous CyI.CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient CyI.CAT genes. The amounts of CAT enzyme produced by embryos bearing CyI.CAT deletions that lack various regions of the CyI sequence were measured. As little as 254 nucleotides of upstream CyI sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining less than 850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete CyI.CAT fusion construct

Association of Accelerometry-Measured Physical Activity and Cardiovascular Events in Mobility-Limited Older Adults: The LIFE (Lifestyle Interventions and Independence for Elders) Study.

BACKGROUND:Data are sparse regarding the value of physical activity (PA) surveillance among older adults-particularly among those with mobility limitations. The objective of this study was to examine longitudinal associations between objectively measured daily PA and the incidence of cardiovascular events among older adults in the LIFE (Lifestyle Interventions and Independence for Elders) study. METHODS AND RESULTS:Cardiovascular events were adjudicated based on medical records review, and cardiovascular risk factors were controlled for in the analysis. Home-based activity data were collected by hip-worn accelerometers at baseline and at 6, 12, and 24 months postrandomization to either a physical activity or health education intervention. LIFE study participants (n=1590; age 78.9±5.2 [SD] years; 67.2% women) at baseline had an 11% lower incidence of experiencing a subsequent cardiovascular event per 500 steps taken per day based on activity data (hazard ratio, 0.89; 95% confidence interval, 0.84-0.96; P=0.001). At baseline, every 30 minutes spent performing activities ≥500 counts per minute (hazard ratio, 0.75; confidence interval, 0.65-0.89 [P=0.001]) were also associated with a lower incidence of cardiovascular events. Throughout follow-up (6, 12, and 24 months), both the number of steps per day (per 500 steps; hazard ratio, 0.90, confidence interval, 0.85-0.96 [P=0.001]) and duration of activity ≥500 counts per minute (per 30 minutes; hazard ratio, 0.76; confidence interval, 0.63-0.90 [P=0.002]) were significantly associated with lower cardiovascular event rates. CONCLUSIONS:Objective measurements of physical activity via accelerometry were associated with cardiovascular events among older adults with limited mobility (summary score >10 on the Short Physical Performance Battery) both using baseline and longitudinal data. CLINICAL TRIAL REGISTRATION:URL: http://www.clinicaltrials.gov. Unique identifier: NCT01072500

Inactivation of the <i>WNT5A</i> Alternative Promoter B Is Associated with DNA Methylation and Histone Modification in Osteosarcoma Cell Lines U2OS and SaOS-2

<div><p><i>WNT5A</i> is a secreted ligand involved in Wnt pathway signaling and has a role in cell movement and differentiation. Altered <i>WNT5A</i> expression is associated with various cancers, although in most studies the focus has been on only one of the known <i>WNT5A</i> isoforms. In this study, we analyzed expression from two of the major <i>WNT5A</i> promoters, termed promoter A and promoter B, in normal human osteoblasts, SaOS-2 and U2OS osteosarcoma cell lines, and osteosarcoma tumor tissue. We found that both promoters A and B are active in normal osteoblasts with nearly 11-fold more promoter B than A transcripts. Promoter B but not promoter A transcripts are decreased or nearly undetectable in the SaOS-2 and U2OS cell lines and osteosarcoma tumor tissues. Transient transfection of promoter A and promoter B reporter constructs confirmed that SaOS-2 cells have the necessary factors to transcribe both promoters. Bisulfite sequencing analysis revealed that three CpG enriched regions upstream of the promoter B exon 1βare highly methylated in both SaOS-2 and U2OS cells. The CpG island sub-region R6 located in promoter B exon 1β was approximately 51% methylated in SaOS-2 and 25% methylated in U2OS. Region 3 was approximately 28% methylated in normal osteoblasts, whereas the others were unmethylated. Promoter B was re-activated by treatment of SaOS-2 cells with 1 μM 5-azacytidine, which was associated with only a small insignificant change in methylation of sub-region R6. ChIP analysis of U2OS and SaOS-2 cells indicated that the promoter B region is less enriched in the active histone mark H3K4me3, in comparison to promoter A and that there is increased enrichment of the repressive mark H3K27me3 in association with the promoter B genomic region in the cell line SaOS-2. These findings show that epigenetic inactivation of the <i>WNT5A</i> promoter B involves both DNA methylation and histone modifications and suggest that differential expression of the <i>WNT5A</i> alternative promoters A and B is a characteristic of osteosarcomas.</p></div

Similar pattern of CpG island methylation in the <i>WNT5A</i> intron 1 of SaOS-2 and U2OS osteosarcoma cell lines.

<p>Shown at the top are the CpG enriched region (R1–R6) locations in <i>WNT5A</i> intron 1. The numbers indicate the relative base pair location within the intron sequence with the first base pair of intron 1 designated as number 1. The total numbers of CpG’s per region are given in parenthesis. Bisulfite sequencing analysis for CpG region R1–R6 for A) normal osteoblasts, B) U2OS and C) SaOS-2 osteosarcoma cell lines. Closed circle = methylated CpG; open circle = unmethylated CpG. Each row is an individual sequenced clone. Only partial clones could be obtained for some regions. The percent methylated for region R6 CpG’s 23–26 and region R5 CpG’s 21–25 based on the clones shown for U2OS and SaOS-2 are given in parenthesis. The percent methtylated for the entire region R4 is shown for both U2OS and SaOS-2 and region R3 for osteoblast.</p

Promoter A and promoter B luciferase reporter constructs are expressed in SaOS-2 cells.

<p>A) Genomic region used for the construction of the luciferase reporter vectors. The numbered boxes are exon sequences. Black boxes are coding regions; gray boxes are noncoding regions. Lines between boxes are intron sequences. Cloned sequences of promoter A are upstream of exon 1. Promoter B cloned sequences include sequences downstream and upstream of exon 1β, located within the promoter A 6061 bp intron 1. B) Promoter A and C) promoter B reporter constructs used for transient transfection assays into SaOS-2 cells. The plasmid numbers are base pair upstream from the beginning of exon 1 (promoter A) as defined by the cDNA RefSeq NM_003392 or from exon 1β (promoter B) as defined by cDNA sequence AK290869. Below each set of constructs are the results of the transfection assays, expressed as firefly luciferase per <i>Renilla</i> control luciferase. Bars are plus/minus standard error of the mean, n = 4. Pairwise comparisons were made between all constructs (ANOVA). Only comparisons between a given construct and those longer than itself, showing a significant differences (p<0.05), are indicated (numbers above the bars). Constructs are numbered to the left, below the x-axis, along with their names.</p

Promoter B is reactivated in SaOS-2 cells by treatment with 5-azacytidine (5-aza).

<p>A) SaOS-2 cells were treated with 0 and 1 μM 5-aza for 4 days. Promoter A and promoter B specific transcripts were quantified by qPCR using standard curves. Bars are plus/minus standard error of the mean, n = 4, **p<0.001. B) Bisulfite sequencing was completed for CpG regions R3 to R6 using DNA isolated from SaOS-2 cells treated with 1 μM 5-aza for 4 days. The percent methylated for region R6 comparing 5-aza treated to untreated (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151392#pone.0151392.g004" target="_blank">Fig 4</a>) is shown in parenthesis.</p

Histone modifications in <i>WNT5A</i> promoter A, promoter B, and intron 1 of U2OS and SaOS-2 cells.

<p>A) Map of the <i>WNT5A</i> intron 1 genomic region showing the locations of the promoter A and promoter B ChIP primers (dark bars above line except for B1, which is below the line) relative to the CpG regions (R1–R6) and Exon 1 (promoter A) and Exon 1β (promoter B), shown as red boxes downstream of the arrows. B) Chromatin prepared from U2OS and SaOS-2 cells was analyzed by chromatin immunoprecipitation (ChIP) using antibodies against H3K27me3, H3K4me3, and H3K9me3 and IgG as a control. The % INPUT was determined by qPCR as described in the Materials and Methods. Black columns represent the averages of 6 to 12 values for SaOS-2 and 3 to 6 values for U2OS. Gray bars are the averages for the IgG control values. Bars are standard error of the mean. * indicates comparisons of the anti-histone antibody to IgG control values that are significantly different (p<0.05).</p

<i>WNT5A</i> promoter A and promoter B transcript levels in RNA isolated from A) normal human osteoblasts, B) U2OS and C) SaOS-2 human osteosarcoma cells lines, and D) osteosarcoma patient tumor tissue.

<p>Transcript copy numbers were quantified by qPCR using standard curves generated from purified promoter A and promoter B specific PCR products and expressed per μg RNA. Bars are plus/minus standard error of the mean (n = 3). Asterisk indicates promoter A and promoter B levels significantly different from one another at p<0.05 (unpaired Student’s t-test).</p

Human <i>WNT5A</i> genomic and primary transcripts.

<p>Shown is the genomic structure and two of the coding transcripts generated from the <i>WNT5A</i> gene region on chr3. The top diagram shows the relative locations of the <i>WNT5A</i> promoter A and promoter B starts of transcription (arrows). The black boxes are exon sequences. Shown below this are the derived promoter A and promoter B primary transcripts. Promoter A has a unique exon 1. Promoter B generates a primary transcript with a unique exon 1β and intron 1 (B). Exons 2, 3, 4, and 5 are common to both transcripts. Black boxes are coding exon sequences; open boxes are non coding exon sequences. The numbers in parentheses are sizes in base pair. The diagram was modified from Ensembl <i>WNT5A</i> ENSG00000114251 and Bauer et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151392#pone.0151392.ref031" target="_blank">31</a>].</p