Aberrant DNA methylation and patterns of histone modifications at the WNT5A Promoter B in osteosarcoma cells

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Candie Rumph (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Karen Katula

Abstract: WNT5A is a secreted ligand involved in differentiation, proliferation, cell movement and apoptosis. WNT5A is often misregulated in cancer and is known to be involved in cancer metastasis. The focus of this study was on the possible epigenetic regulation of WNT5A expression in osteosarcoma cells. In this study, I analyzed WNT5A regulation from its two major transcription start sites, termed Promoter A and Promoter B. The levels of promoter B transcripts were reduced in the osteosarcoma cell line, U20S, and in primary osteosarcoma tissue of three individuals, compared to normal osteoblasts. To determine if this decrease in Promoter B activity in U20S is due to DNA methylation, I analyzed six CpG islands associated with Promoter B by bisulfite sequencing. Results show that Regions 1 and 2 are unmethylated, Regions 3, 4, 5 are methylated and Region 6, which includes the Promoter B start of transcription, is partially methylated. The DNA methylation pattern is similar to the pattern determined in another osteosarcoma cell line SaOS-2. However, Region 6 of U20S is only 25% methylated, whereas Region 6 of SaOS-2 is 62% methylated. The level of methylation is negatively correlated with Promoter B transcript levels, indicating that Region 6 methylation affects transcription. Histone modifications were examined for their involvement in Promoter A and Promoter B activity by chromatin immunoprecipitation (ChIP) assays. H3K4me3, an activating histone mark, showed high enrichment in Promoter A and reduced enrichment in Promoter B Region 6 of U20S cells, indicating that H3K4me3 has a role in the reduction of Promoter B activity. H3K27me3 and H3K9me3, repressive histone marks, were not differentially enriched on Promoters A and B. H3K27me3 was enriched on Regions 3 and 4 located upstream of the Promoter B start of transcription. Overall, these results suggest that WNT5A Promoter B activity is reduced in osteosarcoma by both histone modification and DNA methylation.

Additional Information

Language: English
Date: 2014
WNT5A, Osteosarcoma Cells, DNA methylation
Wnt proteins
DNA $x Methylation

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