[Epigenetics 2:4, 227-236; October/November/December 2007]; ©2007 Landes Bioscience
Research Paper
Correlation of DNA Methylation with Histone Modifications Across the HNRPA2B1-CBX3 Ubiquitously-Acting Chromatin Open Element (UCOE) Marianne Lindahl Allen Michael Antoniou*
Abstract
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*Correspondence to: Michael Antoniou; Nuclear Biology Group; Department of Medical and Molecular Genetics; King’s College London School of Medicine; King’s College London-Guy’s Campus; 8th Floor Guy’s Tower; Guy’s Hospital; London, SE1 9RT UK; Tel: +44.207.188.3708; Fax: +44.207.188.2585; Email:
[email protected]
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Nuclear Biology Group; Department of Medical and Molecular Genetics; King’s College London School of Medicine; Guy’s Hospital; London, UK
The methylation‑free CpG island encompassing the divergently transcribed promoters of the HNRPA2B1 and CBX3 housekeeping genes possesses a dominant ubiquitously‑ acting chromatin opening element (UCOE) capability. This element allows reproducible and stable transgene expression including from within centromeric heterochromatin. We present an investigation of DNA methylation and histone modification marks across the HNRPA2B1‑CBX3 locus in primary peripheral blood mononuclear cells (PBMCs) to characterise the chromatin structure that underlies UCOE activity. The CpG methylation‑ free region associated with the UCOE extends into the central areas of HNRPA2B1 and CBX3, with a total length of ~5 kb. However, the DNA in the 3' half of both genes is methylated. Histone H4 lysine (K) acetylation shows a broad distribution across both genes, whilst histone H3 lysine acetylation peaks around the transcriptional start sites and drops to background levels at the 3' ends. Higher levels of H3K4 di‑methylation are present at the 3' end of the genes in contrast to H3K4 tri‑methylation which peaks around the transcriptional start sites. Therefore, methylated DNA in transcribed regions of these genes has been shown here to co-exist with active histone modification marks, indicating that these functionally opposing epigenetic signatures can overlap. This suggests that an extended large region of unmethylated DNA in combination with distinct histone modification patterns are at the basis of UCOE function.
Original manuscript submitted: 06/29/07 Manuscript accepted: 10/31/07
Acknowledgements
Introduction
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We would to thank E. Splinter, D. Cousins and D. Chow for help and advice on various methods used in this paper. M.L.A was supported by a Medical Research Council (UK) PhD studentship (award number G78/7909).
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DNA methylation, chromatin, UCOE, gene regulation, CpG island, micrococcal nuclease
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Key words
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Previously published as an Epigenetics E-publication: http://www.landesbioscience.com/journals/epigenetics/article/5231
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CpG dinucleotides are vastly underrepresented within the genome with exceptions being short stretches of GC‑rich DNA, known as CpG islands.1 The ����������������������� generally accepted definition of what constitutes a CpG island was proposed by Gardiner‑Garden and Frommer2 as being a 200 bp stretch of DNA with a C + G content of 50% and an observed CpG/expected CpG ratio in excess of 0.6. The ��������������������������������� promoters of all known housekeeping and approximately 40% of tissue specific genes are associated with CpG islands, which are in general between 1–2 kb in length and extend into the first intron but not usually beyond this point.3 As a result, CpG islands have been used to predict the number of genes within the genome.4 Approximately 70% of the CpG sites within the mammalian genome are methylated. CpG islands are an exception to this rule and when associated with actively transcribed gene promoters they are invariantly unmethylated, contain nucleosomes with hyperacetylated histones H3 and H4 and lack histone H1.5 At present there is only a modest knowledge about the true relationship between active histone post‑translational modification marks and underlying DNA methylation status.6 Methylated DNA is commonly associated with a lack of histone acetylation and a closed chromatin config-uration.7 In contrast, an open transcriptionally competent or active chromatin structure is associated with hyperacetylation of histones8 and generally lacks CpG methylation. We have previously shown that the methylation‑free CpG islands that span the closely spaced, dual divergently transcribed promoters of the human TBP‑PSMB1 and HNRPA2B1‑CBX3 housekeeping genes are able to confer reproducible, stable expression upon transgenes, even when integrated into centromeric heterochromatin.9‑11 Furthermore, the CBX3‑HNRPA2B1 dual promoter CpG island region is able to not only enhance expression but also more importantly completely negate DNA‑methylation mediated transcriptional silencing of human cytomegalovirus (hCMV) promoter/ enhancer driven transgenes in stable transfected tissue culture cells.11 Furthermore,
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Supplemental information can be found at: www.landesbioscience.com/supplement/ AllenEPI2-4-sup.pdf
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Epigenetics
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DNA Methylation and Histone Marks Across the UCOE
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by digestion with RNase‑Free DNase (Qiagen, Crawley, UK). Oligo‑primed cDNA was generated by using the Superscript II RNase H Reverse Transcriptase kit (Invitrogen). Real‑time PCR were performed and analyzed on a LightCyclerTM 1.2 (Roche Applied Science, Lewes, UK) using the QuantiTect SYBR Green PCR Master Mix system (Qiagen).14 Cycling conditions were as follows: 15 minutes 95°C followed by 40 cycles of 95°C 15 seconds, 59°C 20 seconds and 72°C 15 seconds. Primers used for PCR amplification are shown in Supplementary Table ST1. CT values for no reverse transcriptase controls at site CBX3 were at least ten‑fold higher than experimental samples, corresponding to
