Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

Abstract

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the "open", transcriptionally active chromatin in developing and postmitotic mouse retina.

Epigenetic Landscape of HIV Infection in Primary Human Macrophage

“…6). A similar localization of 5hmc to the nuclear periphery was observed in developing mouse retinal photoreceptor cells (61). How the HIV-dependent degradation of TET2 is balanced with the enrichment and redistribution of 5hmc on the HIV genome remains to be elucidated.…”

Infrared nanospectroscopic mapping of a single metaphase chromosome

“…Upon the deposition on the substrate the chromosome arms may also bend and therefore the eu- and heterochromatin areas observed in the profiles could be shifted relative to one another. Moreover, fluorescence microscopy is limited because DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix (72). AFM-IR spectroscopy can detect the degree of methylation from the 3D volume of the sample under the AFM tip (73) as schematically presented in Figure 1.…”

Immunohistochemical detection of global epigenetic DNA modifications using antibodies to 5-methylcytosine and 5-hydroxymethylcytosine. Impact of antigen retrieval protocols.

“…The overall hyper-and hypomethylation status of an individual cell can be determined by using antibodies directed against 5-mC and 5-hmC in immunohisto(cyto)chemical procedures applying uorescence or bright eld microscopy (Singh et al 2018; Kato et al, 2020). Immunohistochemical studies targeting 5-mC and 5-hmC have been performed on different types of biological samples, including tumour tissues (Szulwach et al 2011;Singh et al 2018; Kato et al 2020). Discrepancies in staining patterns, the presence of positive nuclei adjacent to negative ones and the impact of immunocytochemical pretreatment protocols on the detection sensitivity of 5-mC and 5-hmC have been reported (Pendina et al 2017;Wang et al 2019; Estévez-Torres and Baigl 2011).…”

“…Reactivation of epigenetically silenced genes is initiated by the TET (ten-eleven translocation) enzyme family, which can convert 5-mC to 5-hydroxymethylcytosine (5-hmC) Detection methods for gene-speci c 5-mC and 5-hmC DNA modi cations are mostly based on methylation speci c polymerase chain reaction (PCR) assays or sequencing analyses of isolated DNA (Rein et al 1998;Storebjerg et al, 2018). The overall hyper-and hypomethylation status of an individual cell can be determined by using antibodies directed against 5-mC and 5-hmC in immunohisto(cyto)chemical procedures applying uorescence or bright eld microscopy (Singh et al 2018; Kato et al, 2020). Immunohistochemical studies targeting 5-mC and 5-hmC have been performed on different types of biological samples, including tumour tissues (Szulwach et al 2011;Singh et al 2018; Kato et al 2020).…”