How epigenetic changes in DNA can be good or bad
Swedish scientists can now explain how some 'master' proteins activate regions of our genes which are normally not active, all as a result of epigenetic changes. This information gives us a better idea of what regulates genes in embryo development and even diseases such as cancer.
A DNA molecule carries information in a sequence of four bases, adenine (A), cytosine (C), guanine (G) and thymine (T), letters forming the language of genes. Short sequences of these letters form 'DNA words' that say when and where proteins are made in our bodies. Most of our cells contain these letters in precisely the same order. But as needed, specific letter patterns are regulated to become active (expressed) allowing cells to make proteins which change cells into brain, nerves, eye, muscle and so forth.
Key to the regulation of genes are specialised proteins known as transcription factors. They bind to letter sequences to either activate or repress their expression and are known to be DNA-binding.
In one example, the DNA letter C exists in two forms, cytosine and methylcytosine. It can be thought of as the same letter with and without an accent (C versus Ç). The methy form of this DNA base was made by an epigenetic modification outside the gene which created a biochemical change - not altering the original DNA sequence - but producing a different outcome. This change to a base is known as methylation.
The two variations of C have no effect on the kind of proteins made, but when and where the proteins are made. Previous research has shown gene regions where C is methylated (Ç) are usually inactive. Many transcription factors cannot bind to sequences with a methylated Ç.
The work appears in Science, May 5, 2017.
By analysing hundreds of different human transcription factors, scientists at Karolinska Institute, Sweden, found some transcription factors actually prefer to bind to methylated Ç.
These transcription factors are not only important in embryo development, but to development of prostate and colorectal cancers as well.
"This study identifies how the modification of DNA structure affects binding by transcription factors, and this increases our understanding of how genes are regulated in cells and further aids us in deciphering the grammar of DNA", says Professor Jussi Taipale, Karolinska Institute.
These results may crack the genetic code which controls the expression of our genes, broadly affecting our understanding of human development and disease.
"These results suggest that such 'master' regulatory factors could activate regions of the genome that are normally inactive, leading to the formation of organs during development, OR the initiation of pathological changes in cells that lead to diseases such as cancer."
Jussi Taipale PhD, Division of Functional Genomics and Systems Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; Genome-Scale Biology Program, University of Helsinki, Helsinki, Finland, and leader of the research
Nearly all cells in the human body share the same primary genome sequence consisting of four nucleotide bases. One of the bases, cytosine, is commonly modified by methylation of its 5 position in CpG dinucleotides (mCpG). Most CpG dinucleotides in the human genome are methylated, but the level of CpG methylation varies with genetic location (promoter versus gene body), whether genes are active versus silenced, and cell type. Research has shown that the maintenance of a particular cellular state after cell division is dependent on faithful transmission of methylated CpGs, as well as inheritance of the mother cellsí repertoire of transcription factors by the daughter cells. These two mechanisms of epigenetic inheritance are linked to each other; the binding of transcription factors can be affected by cytosine methylation, and cytosine methylation can, in turn, be added or removed by proteins that associate with transcription factors.
The genetic and epigenetic language, which imparts when and where genes are expressed, is understood at a conceptual level. However, a more detailed understanding is needed of the genomic regulatory mechanism by which methylated cytosines affect transcription factor binding. Because cytosine methylation changes DNA structure, it has the potential to affect binding of all transcription factors. However, a systematic analysis of binding of a large collection of transcription factors to all possible DNA sequences has not previously been conducted.
Globally characterize the effect of cytosine methylation on transcription factor binding, we systematically analyzed binding specificities of full-length transcription factors and extended DNA binding domains to unmethylated and CpG-methylated DNA by using methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment). We evaluated binding of 542 transcription factors and identified a large number of previously uncharacterized transcription factor recognition motifs. Binding of most major classes of transcription factors, including bHLH, bZIP, and ETS, was inhibited by mCpG. In contrast, transcription factors such as homeodomain, POU, and NFAT proteins preferred to bind methylated DNA. This class of binding was enriched in factors with central roles in embryonic and organismal development.
The observed binding preferences were validated using several orthogonal methods, including bisulfite-SELEX and protein-binding microarrays. In addition, the preference of the pluripotency factor OCT4 to bind to a mCpG-containing motif was confirmed by chromatin immunoprecipitation analysis in mouse embryonic stem cells with low or high levels of CpG methylation (due to deficiency in all enzymes that methylate cytosines or contribute to their removal, respectively). Crystal structure analysis of the homeodomain proteins HOXB13, CDX1, CDX2, and LHX4 revealed three key residues that contribute to the preference of this developmentally important family of transcription factors for mCpG. The preference for binding to mCpG was due to direct hydrophobic interactions with the 5-methyl group of methylcytosine. In contrast, inhibition of binding of other transcription factors to methylated sequences was found to be caused by steric hindrance.
All authors: Yimeng Yin, Ekaterina Morgunova, Arttu Jolma, Eevi Kaasinen, Biswajyoti Sahu, Syed Khund-Sayeed, Pratyush K. Das, Teemu Kivioja, Kashyap Dave, Fan Zhong, Kazuhiro R. Nitta, Minna Taipale, Alexander Popov, Paul A. Ginno, Silvia Domcke, Jian Yan, Dirk Schübeler, Charles Vinson, and Jussi Taipale. 'Impact of cytosine methylation on DNA binding specificities of human transcription factors'. Science, 5 May 2017.
The study was supported by the Academy of Finland Center of Excellence in Cancer Genetics and the ERA SynBio project MirrorBio, Karolinska Institutet's Center for Innovative Medicine, Knut and Alice Wallenberg Foundation, Göran Gustafsson Foundation, and the Swedish Research Council.
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System diagram analysing the impact of CpG methylation on transcription factor binding.
Bottom left panel shows (ORANGE) fraction of transcription factors that prefer methylated or those
(TEAL) unmethylated CpG sites. AFFECTED in multiple ways (YELLOW), NOT AFFECTED (GREEN),
or (GREY) do not have a CpG in their pattern, as determined by methylation-sensitive SELEX (top left).
The structure and logos on the right highlight how HOXB13 recognizes mCpG
(BLUE shading indicates a CpG affected by methylation).
Image Credit: Karolinska Institutet, Stockholm, Sweden