Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

DNA methylation, a well-documented mechanism associated with transcriptional regulation entails modification of cytosine residues in a 5'-cytosine-phosphate-guanine-3' (CpG) dinucleotide context via the addition of a methyl group (-CH₃) to the 5- carbon atom of the cytosine pyrimidine ring to form 5-methylcytosine (5mC)¹. In mammals, approximately 70% of CpGs are methylated which constitutes only 1% of their genomes as they are depleted of this palindromic sequence owing to 5mC mutagenic propensity to spontaneously deaminate to thymine². Presence of methyl groups on gene promoter sequences show strong correlation with transcriptional repression in vertebrates^3,4,5. Addition of these methyl groups is catalyzed by highly conserved DNA methyltransferase (DNMT) enzymes DNMT3a, 3b and 3L, and DNMT1 which modify CpG cytosines in de novo and maintenance methylation contexts respectively⁶. DNMT3A/B expression is elevated during development in embryonic stem cells and epiblast; however its diminished expression is observed following pluripotent cell lineage commitment to somatic fates during differentiation⁸. Whilst sharing functional redundancy, DNMT3a and 3b display tissue-specific expression patterns, with 3a detected uniformly in mouse embryos but 3b predominantly localized to neuroectoderm and chorionic ectoderm tissues⁸.

Methylation signatures can be inherited during mitosis and meiosis¹⁰. Maintenance methylation involves DNMT1 facilitated modification of CpG cytosine residues existing in hemi-methylated palindromes on double-stranded DNA¹¹. DNMT1 binds DNA at replication forks¹¹ and consequently, genomic methylation levels peak during S phase of the cell cycle¹². DNMT1 methylates unmodified cytosines thereby distinguishing newly synthesized DNA strands, promoting X-chromosome inactivation and maintaining transcriptional repression profiles¹³. Through recognition of hemi-methylated DNA CpG sequences, DNMT1 maintains established patterns of methylation demarcated by de novo methylation e.g. repression of Long Interspersed Nuclear Element 1 (LINE1) retrotransposon promoters to inhibit its potentially carcinogenic propagation¹⁴. Although possessing hemi-methylated DNA preferential binding affinity, DNMT1 can methylate unmethylated CpG island (CGI) sequences in DNMT3a/b ^-/- mutant cells, fulfilling an emergency de novo methylation role¹⁵. Thus owing to the semi-conservative nature of DNA replication¹⁶, maintenance methylation can faithfully recapitulate methylation signatures from parent to daughter cell¹⁷.

However, for gene expression to be dynamically modulated, repressive methylation modification must be erased, which can be achieved by passive and active demethylation mechanisms¹⁸. The Ten-Eleven Translocase (TET) proteins belonging to a conserved family of dioxygenase proteins are capable of iterative oxidation of methyl groups on CpG residues¹⁹. These Tet proteins, homologous to J-Base binding proteins (JBP) discovered in Trypanosome bruceii, recognise and bind modified DNA bases e.g. 5mC and oxygenate these residues to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)²⁰. Tet protein facilitated oxidation of 5mC results in stepwise conformation change from methyl to hydroxyl, carbonyl and carboxylate configurations, however 5fC and 5caC modifications can be synthesized directly from 5mC oxidation^21,22,23,24.

An informative indication of TET protein activity in understanding their regulation is studying the distribution and abundance of oxi-methyl-cytosine marks. Significant 5mC presence at CpG poor promoters is detectable in contrast to unmethylated CpG rich regions, the latter being characteristic of CpG islands²⁵. Across tissues, highest tissue specific methylation is observed in brain, testis and blood whilst oral mucosa exhibit greatest hypomethylation, indicating a differential methylation pattern occurring at tissue specific promoters²⁶.

Through utilizing sensitive anti-5mC and anti-5hmC antibodies in selective methyl/hydroxymethyl DNA immunoprecipitation (meDIP/hmeDIP), and subsequent high throughput sequencing, Ficz et al. demonstrated high 5hmC occupancy at promoters, exons and LINE-1 retrotransposon sequences which correlated with reduced 5mC levels at these locations in mouse embryonic stem cells²⁷. Inversely, greatest 5mC enrichment was observed at repetitive satellite sequences where 5hmC presence was limited²⁸. Studies performed on human frontal lobe brain tissue reveal highly significant 5hmC enrichment, four fold higher than in mouse embryonic stem cells²⁸. In concordance with previous observations, high throughput sequencing of frontal lobe tissue illustrated majority 55-59% of 5hmC signal localized at low density CpG promoter regions, 35-38% within gene bodies and approximately 6% occupancy at intergenic regions. In contrast, 5mC was enriched at intergenic regions (25-26%) and higher within gene bodies (52-55%) but reduced (22-24%) at promoter sequences²⁹. These studies indicate abundance of 5hmC in embryonic stem cells and somatic tissue, particularly the brain, however investigations on 5fC and 5caC distributions are limited.

Interestingly, recently discovered in eukaryotes, methylation of adenine residues at the position 6 nitrogen (N⁶) (6mA) display a genomic abundance profile inverse to that of 5mC³⁰. Observations from liquid chromatography coupled tandem mass spectrometry reveal 6mA absolute levels to exist in excess in zebra fish and porcine early embryos compared to sperm with its levels (0.003% of genomic adenines) increasing steadily upon fertilization, peaking at the morula developmental stage (33 fold higher than sperm) and reaching steady state somatic levels of 0.004% of genomic adenines³¹. Immunoprecipitation of 6mA enriched DNA sequences has demonstrated predominant occupancy (approximately 80% enrichment) of this mark at repetitive element regions and transcriptional start sites³². These observations contextualize and validate the discovery of 6mA demethylase-null embryonic stem cells exhibiting accumulated 6mA mediated LINE-1 retrotransposon silencing compared to transcriptionally active elements in wildtype cells. These data suggest a transcriptional regulatory function for 6mA³³.

Whilst conjugated biochemical tags coupled to subsequent DIP assays indicate presence or absence of oxidized methylcytosine derivatives (oxi-mCs), they cannot impart spatial distribution or quantifiable information of these marks^34,35. A protocol for sensitive immunochemical detection of 5hmC and 5caC was recently developed³⁶. This fluorophore conjugated secondary antibody-based immunostaining method coupled with utilizing scanning laser confocal microscopy possesses the unique advantage of providing visual localization of these DNA modifications within the cells, thus, emphasizing individual positively or negatively stained cells corresponding with the heterogeneous presence of these marks. The 5hmC and 5caC absolute signal intensities as amplified by the conjugated antibodies enable semi-quantitative interpretations to be proposed about the magnitude and positions of these marks within the nucleus e.g. heterochromatic and euchromatic regions^37,38. Here a technique for the computational analysis of confocal microscopy images is described. The generation of 2.5D spatial distribution plots for displaying distinct 5hmC and 5caC signal peaks per pixel and their locations within nuclei is demonstrated. Histogram plots of 5hmC and 5caC signal intensity profiles can illustrate trends in abundance of these marks as the peaks and troughs are plotted as separate non-overlapping channels. Finally, by implementing the colocalization function, the degree of proximity of one signal to another can be determined and as a result of this, their respective genomic coordinates can be identified.