1. Michiel Vermeulen  Is a corresponding author
  1. University Medical Center Utrecht, Netherlands

DNA methylation is a key mechanism used by higher eukaryotes to regulate gene expression. The addition of a methyl group to carbon atom number 5 within cytosine bases in DNA is known to repress the transcription of genes into messenger RNA molecules, thus reducing the production of the proteins coded by these genes. Most methylation occurs at CpG dinucleotides—cytosines that are paired with guanines—and these often cluster together to form CpG islands in the promoter regions of genes. In the late 1990s, it was discovered that transcription was repressed when methyl CpG binding proteins were recruited to methylated CpG islands (Hendrich and Bird, 1998).

Subsequent studies have confirmed that the binding of these proteins throughout the genome is proportional to the density of DNA methylation (Baubec et al., 2013), and have identified additional proteins with a high affinity for methylated CpG sites (reviewed in Defossez and Stancheva, 2011). Moreover, in recent years, other screening approaches based mainly on mass spectrometry have revealed that more proteins bind to methylated DNA than previously thought (Mittler et al., 2009; Bartke et al., 2010; Bartels et al., 2011; Spruijt et al., 2013). Now, in eLife, Heng Zhu and co-workers at the Johns Hopkins University School of Medicine—including Shaohui Hu as first author—use a high-throughput screening method to show that many human transcription factors also interact with genomic DNA sequences containing methylated CpG sites (Hu et al., 2013).

To this end, the Johns Hopkins researchers made use of a published protein microarray consisting of 1,321 transcription factors and 210 co-factors (Hu et al., 2009). Hu et al. incubated the array with 154 distinct human promoter sequences, each of which contained at least one methylated CpG dinucleotide.Their results revealed that 150 (97%) of the 154 methylated human promoter sequences showed specific binding to at least one protein on the microarray. Moreover, of the 1531 proteins, 47 (3%) showed binding to methylated cytosines within the promoters. Most of the proteins bound to methylated DNA in a sequence-dependent manner; however, a minority bound to many different methylated DNA probes, indicating that binding can sometimes occur independent of DNA sequence (Figure 1).

Some human transcription factors can bind to both methylated and non-methylated DNA sequences. Hu et al. examined the ability of 17 human transcription factors to bind to 150 different DNA motifs containing methylated or non-methylated CpG islands. Each row represents one transcription factor. For each motif, some transcription factors bound only to the methylated version (red), some to only the non-methylated version (blue); some to both methylated and non-methylated versions (green), and some to neither (grey). From Figure 2a in Hu et al., 2013.

A number of transcription factors, including KLF4—a recently identified methyl-CpG binding protein (Spruijt et al., 2013)—interacted with methylated sequences that did not resemble their known consensus DNA binding motifs. Using a technique based on electrophoresis, Hu et al. showed that KLF4 binds methylated and non-methylated DNA in a non-competitive manner: this suggests that different domains of the protein may be responsible for each type of binding, which they duly confirmed using molecular modeling and mutagenesis studies.

The Johns Hopkins researchers then mined published ChIP-sequencing data from stem cells to identify the target DNA sequences of KLF4, and compared these with data on genome-wide DNA methylation. Strikingly, KLF4 binding appears to be bimodal in nature throughout the genome, with 38% of KLF4 binding sites showing less than 20% methylation, and 48% showing methylation levels over 80%. Finally, Hu et al. used ChIP-bisulfite sequencing, which makes it possible to determine the methylation status of each cytosine within a target DNA sequence, to confirm that KLF4 also binds to both methylated and non-methylated DNA in vivo.

Hu et al. only profiled a small fraction of the complete human methylome for interactions with transcription factors; further proteins capable of binding genomic methyl CpG sequences surely await identification. The same holds true for interactions with methylated non-CpG sequences such as methyl-CpA (cytosine adjacent to adenine), which are fairly abundant in embryonic stem cells (Ramsahoye et al., 2000; Lister et al., 2009). To determine the physiological relevance of these interactions, it will be important to deduce the affinity with which proteins bind these sequences compared to their known targets; initial experiments along these lines are presented in the current eLife paper. Furthermore, recent evidence suggests that non-methylated CpG islands recruit activator proteins, many of which contain a CXXC motif (reviewed in Long et al., 2013). The transcription factor microarray approach used by the Johns Hopkins team, combined with quantitative mass spectrometry-based technology (Spruijt et al., 2013), could thus be used to identify the complete cellular complement of proteins that bind specifically to non-methylated CpG islands.

Finally, this study and other recently published papers force us to reconsider the mechanism(s) via which CpG methylation regulates transcription. Although DNA methylation is generally considered to be a repressive epigenetic modification, experiments presented by Hu et al. suggest that in some cases, methylation of a given promoter sequence can result in activation of transcription. Moreover, other work has revealed a temporal uncoupling of DNA methylation and transcriptional repression during Xenopus embryogenesis (Bogdanovic et al., 2011). Further experiments are therefore required to determine whether the functional readout of CpG methylation is affected by the repertoire and abundance of different DNA methylation ‘readers’ acting at any given time in a cell or a developing organism.

References

    1. Hendrich B
    2. Bird A
    (1998)
    Identification and characterization of a family of mammalian methyl-CpG binding proteins
    Mol Cell Biol 18:6538–6547.

Article and author information

Author details

  1. Michiel Vermeulen

    Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht, Netherlands
    For correspondence
    m.vermeulen-3@umcutrecht.nl
    Competing interests
    The author declares that no competing interests exist.

Publication history

  1. Version of Record published: September 3, 2013 (version 1)

Copyright

© 2013, Vermeulen

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 510
    views
  • 56
    downloads
  • 2
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Michiel Vermeulen
(2013)
Epigenetics: Making the most of methylation
eLife 2:e01387.
https://doi.org/10.7554/eLife.01387

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Christopher TA Lewis, Elise G Melhedegaard ... Julien Ochala
    Research Article

    Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77–107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Maximilian Nagel, Marco Niestroj ... Marc Spehr
    Research Article

    In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective ‘raw’ chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal’s sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary ‘secretome’, both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals.