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. 2013 Feb 28;3:35. doi: 10.3389/fonc.2013.00035

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Copyright © 2013 Yehezkel, Shaked, Sagie, Berkovitz, Shachar-Bener, Segev and Selig.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.

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Methylation of subtelomeric regions and other non-telomeric repetitive sequences, as determined by Southern analysis, following co-expression of human DNMT3B1 and DNMT3L in ICF fibroblasts. (A) RT-PCR analysis of DNMT3L mRNA expression in pCor + hTERT + DNMT3B1 fibroblasts introduced with either DNMT3L (DNMT3L) or with an empty plasmid (pBABE) (left panel). Size markers in kb appear to the left of the blot. β-actin was amplified as a control (right panel). (B) Western blot analysis of DNMT3L expression in pCor + hTERT + DNMT3B1 fibroblasts, which were transduced with either hDNMT3L or the empty plasmid (pBABE). α-Tubulin serves as a control for equal protein loading. (C) Methylation analysis of pCor + hTERT + DNMT3B1 + DNMT3L (DNMT3L) and pCor + hTERT + DNMT3B1 + pBABE (pBABE) fibroblasts with the subtelomeric NBL-1 probe following digestion of DNA samples with HpaII (H) and MspI (M). Arrows point to hybridization bands in samples that are hypomethylated and therefore appear in HpaII-digested DNA in addition to MspI-digested DNA. (D) Methylation analysis of pCor + hTERT + DNMT3B1 + DNMT3L (DNMT3L) and pCor + hTERT + DNMT3B1 + pBABE (pBABE) fibroblasts with the subtelomeric Hutel probe. DNA samples were digested with either Sau3AI (S) or MboI (Mb) and probed with the subtelomeric Hutel probe. Arrows point to hybridization bands in samples that are hypomethylated and therefore appear in Sau3AI-digested DNA in addition to MboI-digested DNA. (E) Methylation analysis of pCor + hTERT + DNMT3B1 + DNMT3L (DNMT3L) and pCor + hTERT + DNMT3B1 + pBABE (pBABE) fibroblasts with the p1A12 probe. DNA samples were digested initially with MseI, followed by redigestion with either MspI or HpaII and separated on a 2% agarose gel. Hypomethylation is visualized as an HpaII- hybridization pattern similar to that achieved with MspI- digestion. (F) Methylation analysis of pCor + hTERT + DNMT3B1 + DNMT3L (DNMT3L) and pCor + hTERT + DNMT3B1 + pBABE (pBABE) fibroblasts with the Satellite 2 repeat probe following digestion of DNA samples with BstBI. The hypomethylated samples are visualized by the appearance of bands at the lower molecular range.