Published: August 16th, 2016
Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.
Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.
Methylation of cytosine bases in DNA (5mC) represents a major epigenetic mark found in vertebrates genomes associated with transcriptional silencing1. 5mC is being introduced and maintained by DNA methyltransferases2-5, and has been shown to play important roles in a number of biological processes including genomic imprinting, X-chromosome inactivation, cellular differentiation, and development3, 6. Consequently, the disruption of 5mC genomic patterns is associated with a number of diseases7, 8-11. Despite the progress in understanding 5mC role in development and disease, it is still remain largely unknown how this mark is being removed in developing and adult tissues. Several potential mechanisms of DNA demethylation have been recently proposed including active and passive demethylation mechanisms12, 13, 14, 15. Discovery of the products of 5mC sequential oxidation mediated by ten-eleven translocation enzymes (tet1/2/3) such as 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in eukaryotic DNA 16, 17, 18, 19 prompted speculations whether they may serve as intermediates of DNA demethylation processes or act as stable epigenetic marks in their own right13. While the demonstration that the component of base excision repair, thymine-DNA glycosylase (TDG) can bind and remove both 5fC and 5caC from DNA19, 20 suggests a role for the modified 5mC derivatives in an active DNA demethylation. Recent evidence showing that 5fC/5caC can modulate the rate of RNA II processivity points to potential involvement of these marks in transcriptional regulation29. Due to this potential biological importance of oxidized forms of 5mC, a range of biochemical and antibody based techniques have been employed for studying their genomic distribution and global content16, 19-24.
Given that most of the vertebrate organs consist of different cell types and that the distribution of modified cytosine bases is tissue and cell-type specific 16-18, 20, 23, 25-27, determining the spatial distribution of oxidized 5mC derivatives in different tissues becomes an important experimental task necessary for unveiling their biological functions. Most of the biochemical and antibody based approaches do not provide any spatial information regarding the distribution of the modified forms of 5mC in different tissue and cell-types. In contrast, immunochemistry based techniques can provide a rapid tool for assessing the spatial distribution and nuclear localization of 5mC28 and its oxidized derivatives20. That is said, the reported very low abundance of 5fC (20 in every 106 cytosine) and 5caC (3 in every 106 cytosine) in the mouse genome18 represents a significant challenge for standard immunochemistry.
Here, we describe a highly sensitive immunochemical method that provides robust and a rapid detection of oxidized form of cytosine in mammalian brain tissue. By incorporating peroxidase-conjugated secondary antibodies coupled with a signal amplification step, this method circumvents the challenges of detecting the very low amounts of 5fC and 5caC. In addition, this technique can be used to co-detect the modified forms of cytosine with lineage specific markers, effectively complementing other approaches in elucidating the biological functions of these epigenetic marks.
All the animal-involved procedures were performed in accordance with the University of Nottingham's ethical review board.
1. Selecting Suitable Tissue Preparation for Immunostaining
2. Dewaxing Paraffin Embedded Tissue Sections
3. Fixation and Permeabilization of Cryo- and Microtome Sections
4. Immunostaining for Oxi-5mC Derivatives
To determine the distribution of 5hmC in brain tissue sections, we performed co-detection of this epigenetic modification with a marker for post-mitotic neurons, NeuN, employing commercial anti-5hmC antibody that specifically interacts with this mark but not with other forms of modified cytosine20, 25. Immunohistochemical analysis of 5hmC and 5caC distributions in the adult brain revealed that whereas the prominent 5hmC staining co-localizes with NeuN positive cells, NeuN-negative glial cells possess lower levels of genomic 5hmC (Figure 1)20.
We recently showed that Tet-dependent 5mC oxidation is operative during lineage specification of neural stem cells (NSCs) 20. Despite being immunochemically undetectable in NSCs, 5fC and 5caC exhibit prominent immunostaining at early stages of NSCs differentiation towards neuronal and glial lineages. Both these marks transiently accumulate concurrently with appearance of the markers of early neuronal and glial differentiation20. To determine the distribution of 5caC in differentiating NSCs we performed co-staining of this mark with a glial marker GFAP on the fixed cultures of NSCs at 3 days of induction of glial differentiation. Unlike in NSCs or mature astrocytes (data not shown), we observed strong 5caC signal in relatively large proportion of the cells expressing GFAP in these cultures (Figure 2)20.
Figure 1. Co-immunostaining of 5hmC (green) with NeuN (red) in the Adult Brain Tissue Counterstained with DAPI (Blue). Individual channels and the merged view are shown. Scale bars are 25 µm. Please click here to view a larger version of this figure.
Figure 2. Co-immunostaining of 5caC with GFAP in the Culture of NSC at Day 3 of Glial Differentiation. Individual channels and the merged view are shown. Scale bars are 20 µm. Please click here to view a larger version of this figure.
Although the reported low abundance of 5mC oxidation derivatives, 5fC and 5caC in some tissues would present significant limitations for a standard immunochemistry protocol, the incorporation of peroxidase-conjugated secondary antibodies allowed the detection of these cytosine modifications in fixed tissues and cells (Figure 2). However, the optimal incubation time with the tyramide solution should be optimized experimentally for each individual batch of tyramide signal amplification kit where a linear relationship between signal intensity and duration of tyramide based signal amplification is observed, for details refer to Almeida et al. 201226. In addition, the signal/background ratio can be significantly enhanced by carrying the washes in a Coplin jar to allow efficient removal of excess antibodies. Following incubation with tyramide solution, it is important to immediately stop the reaction by washing in PBT solution to decrease background staining. It is critical not to allow the sections to dry at any point during the procedure.
The efficiency of DNA depurination can be improved by carrying out the depurination reaction at 37 °C. While using 4 N HCl instead of 2 N enhances the staining of the modified forms of 5mC using 4 N HCl for DNA depurination would not permit co-staining with DAPI, as it interacts exclusively with double stranded DNA.
Although this technique can provide robust semi-quantitative assessment of the modified forms of cytosine bases where detectable, it cannot be used for evaluating the absolute levels of 5mC or its oxidation derivatives. Therefore, we recommend the use of other complementary but quantitative approaches16, 19 -24. Since the discovery of 5mC oxidation derivatives in mammalian genomes, several approaches have been developed to study their biological roles16, 19-24. Although these approaches can be valuable in determining the absolute levels of 5mC oxidation derivatives, they do not provide information concerning their spatial distribution20, 26. We have successfully used the method described here to map the spatial distribution and localization of 5mC oxidation derivatives in different cell types of the developing and adult brain20
By revealing their spatial distribution20, this technique can be crucial to understanding the biological implications and the fate of oxidized derivatives of 5mC in various biological contexts where these modified derivatives of 5mC can be detected including cellular differentiation, development and disease. In addition, the method we describe here can give semi-quantitative assessments of the oxidized derivatives of 5mC in different tissues by assessing the kinetics of peroxidase reaction (which is proportional to the staining intensity) at different incubation times with tyramide26.
The authors declare no conflict of interest.
We thank the Advanced Microscopy Unit (School of Life Sciences, University of Nottingham) and the Histology team of MRC Human Reproductive Sciences Unit (Edinburgh), the Institute of Neuroscience at the ULB and the FNRS for help and support. This work was supported by MRC (MR/L001047/1 to A.D.J.).
|Use only in Class II safety cabinet
|Phosphate buffer saline (PBS)
|pPH 7.5, filtered before use
|4% paraformaldehyde (PFA)
|Once made can be stored at -20°C in aliquotes for several months
|4% formaldehyde (FA)
|Once made can be stored at -20°C in aliquotes for several months
|Ethanol, anhydrous denatured, histological grade
|95, 75, 50 % in PBS
|0.01 % Tween 20 in PBS
|0.5% Triton X-100 in PBS
|2 N HCl
|caution extremely toxic
|100 mM Tris-HCl
|PH adjusted to 8.5
|hydrophobic barrier pen
|Slide moisture chamber
|Scientific Device Laboratory
|anti-5mC mouse antibody
|monoclonal primary antibody
|rabbit polyclonal primary antibody
|rabbit polyclonal primary antibody
|Peroxidase-conjugated anti-rabbit seconday antibody
|555-congjuated goat anti-mouse antibody
|22x32mm Glass cover slips
|Tyramide Signal Amplification System
|Other fluorochome conjugated seconday antibodies can be used for co-detection of oxi-5mC
|Mounting Medium with DAPI
|Colourless nail polish
|chicken polyclonal anti-GFAP polyclonal antibody
|anti-NeuN mouse monoclonal antibody
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