I'm designing BSP primers for DNA methylation for rat BDKRB1 gene. Since gene methylation in promoter region has a regulatory effect on gene transcription, I looked up promoter regions for the gene and I did CpG island prediction to find islands. However, no CpG islands were found in the promoter region. Moreover, BSP primer design criteria entail that the primer pair must span a gene target with maximal number of islands without containing a CpG island in the primer sequence itself.
So, my question is:
Is it useful to look at methylation status for gene expression inside the transcribed region? does DNA methylation outside of the promoter region affect gene transcription/expression?
Methylation on distal regulatory elements (enhancers/silencers) affect gene expression. here is an exemple:
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Down-regulation of PU.1 by methylation of distal regulatory elements and the
promoter is required for myeloma cell growth.
Tatetsu H, Ueno S, Hata H, Yamada Y, Takeya M, Mitsuya H, Tenen DG, Okuno Y.
Cancer Res. 2007 Jun 1;67(11):5328-36.
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Several groups focus there research on methylation on enhancer (google: DNA methylation enhancer)
yes, there are several examples of gene expression affected by DNa methylation in coding regions.
this is classic example
Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8334-9.
Methylation of coding region alone inhibits gene expression in plant protoplasts.
Hohn T, Corsten S, Rieke S, Müller M, Rothnie H.
Jordi and Touati gave you a correct answer.
I would just add that this type of regulation is common for imprinted genes in mammal (e.g. of IGF2 and H19 regulated by a common Differentially Methylated Region).
Also in the cancer field, methylation found in the vicinity of CpG island but not within has been associated with change in expression. For more details you can refer to the work on CpG Island Shores performed by A. Feinberg's team.
It is actually for this reason that most of the recent genome wide methods for methylation tend to incorporate CpG dinuleotides outside CpG Island.
Also for your gene of interest you might want to treat cells with demethylating agent to see if your gene is overexpressed after treatment. Methylation might not play a role in the transcription regulation of this gene.
Dear Dr Ludovic Barault which demethylating agent do you recommend?
Thank you so much for your help.
Well I, myself never used them, but in mammal people usually work with Azacitidine or Decitabine.
Dear Mohamad
I work at NXT-Dx, a company specialized in epigenetic and epigenomics testing services. We have run a whole range of epigenomic/epigenetic experiments on a wide variety of species and are in most cases heavily involved in the bio-informatics and data-analysis of such projects. This means we have a lot of experience in this and therefore know first hand that DNA methylation isn't only affecting expression at promotor regions.
In several of our experiments we have seen that DNA methylation outside the promotor has an effect on expression by causing alternative splicing events. Obviously this is only revealed by looking at both expression (RNA-seq) and DNA methylation.
If you need any help in your project (running the experiment, analyzing the data, etc), please let me know. Also if you want some more information please let me know.
Maarten Braspenning
NXT-Dx
Also, these guys (see the reference below) showed that
" The majority of tissue-specific differentially methylated regions (tDMRs), in both relative and absolute terms, occurred in CpG-poor regions. Further analysis revealed that these regions were associated with alternative transcription events (alternative first exons, mutually exclusive exons and cassette exons). Only a minority of tDMRs mapped to gene-body CpG islands (13%) or CpG islands shores (25%) suggesting a less prominent role for these regions than indicated previously. Implementation of ENCODE annotations showed enrichment of tDMRs in DNase hypersensitive sites and transcription factor binding sites."
Slieker RC, Bos SD, Goeman JJ, Bovee JV, Talens RP, van der Breggen R, Suchiman HE, Lameijer EW, Putter H, van den Akker EB, Zhang Y, Jukema JW, Slagboom PE, Meulenbelt I & Heijmans BT (2013) Identification and systematic annotation of tissue-specific differentially methylated regions using the Illumina 450k array. Epigenetics & chromatin 6(1):26.
Doi: 10.1186/1756-8935-6-26
We published a review on that: Article Whole genome DNA methylation: Beyond genes silencing
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