Abstract
Abstract 4796
Introduction:
Hβ-1 tubulin is essential for normal thrombopoiesis and constitutes the majority of β-tubulin within the platelet marginal band. Hβ-1 tubulin has the highest sequence diversity from other tubulin isotypes, and has expression restricted to hematologic tissues. The best-characterized expression occurs in megakaryocytes, where temporal expression is limited to specific stages of megakaryocyte maturation. While expression of Hβ-1 tubulin has been shown to be dependent on transcription factors such as NF-E2, these factors have a multitude of gene targets and do not provide a full explanation of mechanisms responsible for specific Hβ-1 tubulin expression. Epigenetic regulation of gene transcription is now widely accepted as a mechanism of tissue-specific expression of gene products; however, the role of epigenetic regulation in the expression of tubulin isotypes has not been explored. Given the highly regulated expression of Hβ-1 tubulin and the potential of epigenetic modifications to drive specific gene expression, we hypothesized that epigenetic modulation via DNA methylation may be a mechanism for regulation of Hβ-1 tubulin expression.
Methods:
We identified 3 CpG-dense areas upstream and within TUBB1 (which encodes Hβ-1 tubulin) based on Human hg19 genome assembly. These areas included the putative promoter region (-2000 to +1bp from the 5'UTR) upstream of the transcriptional start site; a 3kb region spanning most of intron 1; and a region within exon 4. We used Sequenom MassARRAY EpiTYPER on bisulphite-converted DNA to quantitatively determine percent methylation at each CpG within the three CpG-dense regions. Specifically, we extracted genomic DNA from three cell lines expressing Hβ-1 tubulin (K562, MEG-01, and HEL), and six non-expressing cell lines (two hematologic cell lines (REH, KCL-22) and four epithelial cell lines (H1299, PC3, LNCap2, MDA-MB-231)), and performed bisulphite conversion of DNA. A difference of >25% was considered significant when comparing individual CpGs; methylation differences between CpGs across cell lines are reported as mean ± SEM. For each region, median methylation for each CpG was calculated across cell lines within a group, and regional methylation differences compared using the Mann-Whitney test.
Results:
Between 50–75% of CpGs within the three CpG-dense regions were available for analysis. In exon 4, there was no difference in overall methylation or methylation at any individual CpG between the Hβ-1 tubulin-expressing and non-expressing cell lines. In contrast, within intron 1, Hβ-1 tubulin-expressing cell lines were significantly hypomethylated compared to non-expressing cell lines (p=0.002). This difference was localized to a 1.5kb region within intron 1; mean methylation difference at each CpG within this region was 59±6%. The upstream promoter region similarly showed significant hypomethylation in Hβ-1 tubulin-expressing cell lines (p=0.001); the differentially methylated CpGs were localized to a 350bp region just upstream of the transcription start site, and mean methylation difference was 60±16% at each CpG. Methylation patters were highly similar between CpGs within each group (i.e. Hβ-1 tubulin-expressing or non-expressing cell lines), with only 10% of individual CpGs showing >15% methylation difference between cell lines of the same group.
Conclusion:
We found significant extra- and intra-genic DNA methylation differences in TUBB1 between Hβ-1 tubulin-expressing and non-expressing cell lines. Methylation changes were localized to two CpG-dense regions, namely the upstream promoter region and intron 1 of TUBB1, while a third region in exon 4 showed no differences in methylation. The overall methylation differences within the regions were attributable to large methylation differences at individual CpGs localized to particular areas within those regions. Taken together, these results suggest that tissue-specific expression of Hβ-1 tubulin may be regulated in part by highly-specific changes in DNA methylation of TUBB1. To our knowledge, this is the first report of epigenetic modulation associated with tissue-specific tubulin isotype expression. Further work is underway to confirm these findings in normal primary hematopoietic tissues and to investigate their associations with temporal expression of Hβ-1 tubulin during megakaryocyte development.
Disclosures:
No relevant conflicts of interest to declare.
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