Role of DNA methylation in WNT5A Promoter B expression in Osteosarcoma (SaOS-2) cells.

WNT5A, a member of the WNT family of secreted proteins, activates the non-canonical WNT pathway, regulates developmental events and is involved in tissue homeostasis. Misregulation of WNT5A has been associated with various types of cancer including pancreatic, colorectal, breast, lung and osteosarcoma. Recent studies have shown that WNT5A expression in cancer cells involves non-genetic (epigenetic) changes. The WNT5A gene has two similar transcription start sites (termed promoter A and promoter B in this study) and transcription from these sites give rise to two different messages that encode different protein isoforms. Epigenetic studies to date have focused on the WNT5A promoter A, however our preliminary analysis found that promoter B transcripts are nearly absent in osteosarcoma cells, but present in normal osteoblasts. The goal of this study was to determine if the decrease in promoter B transcripts in osteosarcoma was due to DNA methylation. We identified 6 CpG islands in the WNT5A intron 1 region which contains promoter B and exon 1B, and compared them with data available in NCBI epigenomics website. The NCBI database was also screened for data on the methylation status of these islands in various other cell lines and cancer types. The database analysis shows that there is little or no methylation of Regions 1 - 6 in cells, that normally express WNT5A (fibroblast, chondrocytes, and mesenchymal stem cells). However, in some colorectal tumor tissue there is extensive methylation within intron 1. In adjacent normal colon mucosa tissue, Regions 3 and 4 showed some degree of methylation. Methylation status of the CpG islands in normal osteoblasts and osteosarcoma (SaOS-2) was determined using Sodium bisulfite sequencing. The sequencing data showed that CpG Regions 1 and 2 are unmethylated. Regions 3, 4 and 5 are completely methylated and Region 6 is partially methylated. In normal osteoblasts all 6 regions were unmethylated. A demethylation experiment using 5-Aza-cytidine was performed to determine if decrease in methylation would increase promoter B transcripts. Treatment of SaOS-2 cells with 1µM 5-Aza-cytidine resulted in a 120 fold increase of promoter B transcripts. The methylation data of SaOS-2 cells treated with 1µM 5-Aza-cytidine shows that CpG Region 6 is more prone to demethylation when compared to CpG islands 3, 4, 5. Two clones of Region 6 showed that 4 out of 5 CpG's analyzed were demethylated. 5 clones of Region 3 and 4 showed that only 1-2 CpG's were demethylated out of 24 CpG's in Region 3 and 27 CpG's in Region 4. Overall these results suggest that promoter B transcription is down regulated by DNA methylation of CpG Region 6 and Region 6 is more prone to demethylation compared to Regions 3, 4 and 5

Rare variants in tenascin genes in a cohort of children with primary vesicoureteric reflux

Primary vesicoureteral reflux (PVUR) is the most common malformation of the kidney and urinary tract and reflux nephropathy is a major cause of chronic kidney disease in children. Recently, we reported mutations in tenascin XB (TNXB) as a cause of PVUR with joint hypermobility

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

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

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

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

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

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

<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

DNA methylation entropy as a measure of stem cell replication and aging

Abstract Background Epigenetic marks are encoded by DNA methylation and accumulate errors as organisms age. This drift correlates with lifespan, but the biology of how this occurs is still unexplained. We analyze DNA methylation with age in mouse intestinal stem cells and compare them to nonstem cells. Results Age-related changes in DNA methylation are identical in stem and nonstem cells, affect most prominently CpG islands and correlate weakly with gene expression. Age-related DNA methylation entropy, measured by the Jensen-Shannon Distribution, affects up to 25% of the detectable CpG sites and is a better measure of aging than individual CpG methylation. We analyze this entropy as a function of age in seven other tissues (heart, kidney, skeletal muscle, lung, liver, spleen, and blood) and it correlates strikingly with tissue-specific stem cell division rates. Thus, DNA methylation drift and increased entropy with age are primarily caused by and are sensors for, stem cell replication in adult tissues. Conclusions These data have implications for the mechanisms of tissue-specific functional declines with aging and for the development of DNA-methylation-based biological clocks