Decreased Rate of β-Globin_L1 Allele Transcription Due to Intronic LINE-1 Insertion In the β-Globin gene Is Associated with β-globin_L1 Promoter and Enhancer Hypermethylation Which Is Not Reverted by Decitabine

Abstract 2073

We have previously described a unique etiology of β-thalassemia due retrotransposon LINE-1 (L1) insertion into the intron-2 of the β-globin gene in a mother and daughter of Ukrainian descent who exhibited typical laboratory features of β-thalassemia trait (1, 2). The affected β-globin_L1 gene generated roughly 10–15% of the total β−globin message (1). In order to unravel how the intronic insertion of transposable element attenuates the β-globin expression, interspecific hybrids of the propositus′ lymphocytes and mouse erythroleukemia (MEL) cells were generated (laboratory of Dr. T. Papayannopoulou, Seattle, WA) and used for expression studies. The total RNA from differently treated (emetine, 5-aza-2′-deoxycytidine (Sigma)) in vitro cultured cell hybrids were isolated and β-globin transcripts were analyzed by Real-Time qPCR with gene-specific primers. Nuclei from hybrid MEL cells were isolated to performed nuclear run-on assay. Bisulfite modification was done on genomic DNA from MEL hybrids and the promoter and enhancer regions of β-globin gene were amplified, PCR products were subcloned and sequenced. We demonstrated that the observed reduction in steady-state level of β-globin mRNA is partially caused by aberrant splicing followed by activation of nonsense-mediated decay (NMD) pathway, leading to increased degradation of aberrant β-globin mRNA variants. Reduction in expression of β-globin mRNA from β-globin_L1 allele comes also from altered rate of transcription. We performed PCR-based nuclear run-on assay and forty minutes of in vitro transcription revealed 30% decrease in β-globin_L1 allele transcription rate compared to wild-type β-globin allele. It is known that L1 regulatory regions are highly methylated (3) but there is no evidence so far demonstrating retrotransposon-mediated epigenetic control of neighboring genes. Therefore we determined the methylation status of 6 CpGs between −415 bp and +110 bp (bases upstream and downstream of transcription start) on the promoter region and 3 CpGs between +442 bp to +592 bp on the enhancer region (bases downstream of the poly(A) signal) of the mutated and normal β−globin alleles. Enhancer region of the mutated allele was 100% methylated (100% Me-CpG versus 9% Me-CpG in control), and promoter region was partially methylated (≈ 65% Me−CpG versus 35% Me-CpG in control). Surprisingly, treatment of induced hybrid MEL cells with demethylation agent (5-aza-2′-deoxycytidine, decitabine) did not change the methylation profile and consequently did not increase the expression of β-globin_L1 mRNA whereas the expression of γ-globin gene, which was used as an internal control, was increased. We therefore hypothesize that decreased rate of transcription from β-globin_L1 allele is associated with altered DNA topology caused by the L1 insertion and β-globin_L1 promoter-enhancer displacement. Resulting chromatin modifications lead to permanent β-globin_L1 gene silencing and hypermethylation of regulatory sequence refractory to demethylating agent.

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