Identification and characterization of putative methylation targets in the MAOA locus using bioinformatic approaches

Abstract

Monoamine oxidase A (MAO A) is an enzyme that catalyzes the oxidation of neurotransmitter amines. A functional polymorphism in the human MAOA gene (high- and low-MAOA) has been associated with distinct behavioral phenotypes. To investigate directly the biological mechanism whereby this polymorphism influences brain function, we recently measured the activity of the MAO A enzyme in healthy volunteers. When found no relationship between the individual's brain MAO A level and the MAOA genotype, we postulated that there are additional regulatory mechanisms that control the MAOA expression. Given that DNA methylation is linked to the regulation of gene expression, we hypothesized that epigenetic mechanisms factor into the MAOA expression. Our underplaying assumption was that the differences in an individual's genotype play a key role in the epigenetic potential of the MAOA locus and, consequently, determine the individual's level of MAO A activity in the brain. As a first step towards experimental validation of the hypothesis, we performed a comprehensive bioinformatic analysis aiming to interrogate genomic features and attributes of the MAOA locus that might modulate its epigenetic sensitivity. Major findings of our analysis are the following: (1) the extended MAOA regulatory region contains two CpG islands (CGIs), one of which overlaps with the canonical MAOA promoter and the other is located further upstream; both CGIs exhibit sensitivity to differential methylation. (2) The uVNTR's effect on the MAOA's transcriptional activity might have epigenetic nature: this polymorphic region resides within the MAOA's CGI and itself contains CpGs, thus, the number of repeating increments effectively changes the number of methylatable cytosines in the MAOA promoter. An array of in silico analyses (the nucleosome positioning, the physical properties of the local DNA, the clustering of transcription-factor binding sites) together with experimental data on histone modifications and Pol 2 sites and data from the RefSeq mRNA library suggest that the MAOA gene might have an alternative promoter. Based on our findings, we propose a regulatory mechanism for the human MAOA according to which the MAOA expression in vivo is executed by the generation of tissue-specific transcripts initiated from the alternative promoters (both CGI-associated) where transcriptional activation of a particular promoter is under epigenetic control.

Figure 1. The human MAOA gene

1A –Panel A shows the human MAOA gene in a context of the chromosome X (the UCSC Genome Browser). 1B – This diagram shows a schematic presentation of the MAOA gene promoter region (from 2.5 kb upstream to 500 nucleotides downstream of the transcription start site). The green box represents the core promoter and the yellow color highlights translated sequence. The two VNTRs are boxed. The allelic variations of the uVNTR are shown as different length boxes below the sequence bar. The blue stripes indicate positions of the known SNPs in the region. 5′ UTRs of three representative human mRNAs are shown as bars the size of which reflects the length of transcript's 5′ UTR.

Figure 2. CpG islands mapping

2A –A screenshot of the UCSC Genome Browser represents two bona fide CGIs regions as blue bars. The hue of the bar corresponds to its epigenetic score. The Bona fide CGIs are in perfect positional overlap with CpG Islands predicted by Takai and Jones criteria (shown as green bars), but have extended boundaries. Note the position of the uVNTR polymorphism in the centre of the CGI_1059 (circled yellow). Further upstream of the CGI_1059 is another tandem-repeat region (circled red). 2B – Two sequences (outlined in blue) produced by the CpGcluster algorithm are mapped to the MAOA regulatory region. Both CpGcluster regions encompass tandem repeats. 2C - CG cluster annotation for MAOA locus shown as a magenta bar. The sequence of the CG cluster contains 2195 nucleotides and encompasses both CGIs and the novel tandem-repeated region. The table summarizes sequence features and characteristics of the two bona fide CGIs. 2D – Experimentally-derived data on the methylation status of the MAOA promoter region. Methylated CpG sites detected in normal colon cells (top) and in blood cells (bottom) as was established by the reduced representation bisulfite sequencing (RRBS) . The methylation status of the region is apparently different in two cell types where much less methylation was detected in blood cells (red circles indicate lack of methylation). The uVNTR is boxed to demonstrate that lack of methylation here is due to the experimental design of the study where repeating sequences were excluded from the analysis.

Figure 2. CpG islands mapping

2A –A screenshot of the UCSC Genome Browser represents two bona fide CGIs regions as blue bars. The hue of the bar corresponds to its epigenetic score. The Bona fide CGIs are in perfect positional overlap with CpG Islands predicted by Takai and Jones criteria (shown as green bars), but have extended boundaries. Note the position of the uVNTR polymorphism in the centre of the CGI_1059 (circled yellow). Further upstream of the CGI_1059 is another tandem-repeat region (circled red). 2B – Two sequences (outlined in blue) produced by the CpGcluster algorithm are mapped to the MAOA regulatory region. Both CpGcluster regions encompass tandem repeats. 2C - CG cluster annotation for MAOA locus shown as a magenta bar. The sequence of the CG cluster contains 2195 nucleotides and encompasses both CGIs and the novel tandem-repeated region. The table summarizes sequence features and characteristics of the two bona fide CGIs. 2D – Experimentally-derived data on the methylation status of the MAOA promoter region. Methylated CpG sites detected in normal colon cells (top) and in blood cells (bottom) as was established by the reduced representation bisulfite sequencing (RRBS) . The methylation status of the region is apparently different in two cell types where much less methylation was detected in blood cells (red circles indicate lack of methylation). The uVNTR is boxed to demonstrate that lack of methylation here is due to the experimental design of the study where repeating sequences were excluded from the analysis.

Figure 3. Empirical data on histone modifications in the MAOA promoter region

ENCODE Consortium data for two cell lines: GM 12878 (red) and K562 (blue) are shown within the context of the UCSC Browser. Panel A represents profiles of the H3K9ac and H3K27ac and panel B represents profiles of the H3K4me2, H3K4me3 and H3K27me3. Note the overlap between the “active” histone marks, Pol 2 sites, and the 5′ end of the distal CpG.

Figure 4. Regulatory sequences in the MAOA locus

3A - NXScore peaks around two alternative TSSs of the MAOA are denoted by dashed boxes; the peaks are localized within the bona fide CGIs (blue bars). While the heights of these peaks are very similar, profiles of the regions upstream of the peaks are distinct: an additional strong peak (red arrow) is mapped in 5′ to CGI_1059. The figure was prepared by uploading the NXScore results as a Custom Track on the UCSC Genome Browser and taking a snapshot with the Custom Track featuring the bona fide CGIs. 3B - CTCFBSDB contains information on validated insulator sites for the MAOA gene that are mapped in trans to the MAOA locus. Note the scale of the figure that represents about 30 kb in 5′ of the TSS. The cluster of the validated CTCF-binding sites (circled yellow) resides between the two domains positive for histone H3K27me3 methylation. The enlarged fragment shows the genomic region of the CTCF-binding sites, and the green arrow indicates the H3K27me3-negative sequence. The table summarizes the features of the two CTCF-binding sites that we predicted for the MAOA promoter region (cis-regulatory elements, custom sequence query, CTCFSDB).

Figure 4. Regulatory sequences in the MAOA locus

3A - NXScore peaks around two alternative TSSs of the MAOA are denoted by dashed boxes; the peaks are localized within the bona fide CGIs (blue bars). While the heights of these peaks are very similar, profiles of the regions upstream of the peaks are distinct: an additional strong peak (red arrow) is mapped in 5′ to CGI_1059. The figure was prepared by uploading the NXScore results as a Custom Track on the UCSC Genome Browser and taking a snapshot with the Custom Track featuring the bona fide CGIs. 3B - CTCFBSDB contains information on validated insulator sites for the MAOA gene that are mapped in trans to the MAOA locus. Note the scale of the figure that represents about 30 kb in 5′ of the TSS. The cluster of the validated CTCF-binding sites (circled yellow) resides between the two domains positive for histone H3K27me3 methylation. The enlarged fragment shows the genomic region of the CTCF-binding sites, and the green arrow indicates the H3K27me3-negative sequence. The table summarizes the features of the two CTCF-binding sites that we predicted for the MAOA promoter region (cis-regulatory elements, custom sequence query, CTCFSDB).

Figure 5. Conservation of the MAOA regulatory region

An output of the PAZAR database (ORCAtk) search for TFBSs in the conserved regions and annotations are shown as tracks in the UCSC Browser. 4A - Phylogenetic footprinting of the MAOA by multiple-species analysis identified regions with scores0000 above the cut off (default settings). Dark red bars indicate spanning of regulatory sequences, denoted here as pleiadesgenes_R. This figure combines a graphical display of the regulatory sequences (bars in the top) with the regions that have a high conservation score in multiple species analysis (red peaks). 4B - Regions with high conservation score in a pair-wise sequence comparison. The broadly spanned peaks reflect the evolutionally small distance between humans and primates. The regions of tandem repeats are not conserved: they are projected into the gaps between the peaks (green arrows).

Figure 6. Promoter prediction with CoreBoost_HM

6A - A region of multiple occurrences of promoter-associated motif M22 (TGCGCANK) was used for a BLAT search and visualized as “Your Sequence” in UCSC Browser and mapped to the 5′ flanking regions of the distal MAOA CGI (displayed in UCSC Browser). Note the good overlap of this region with regions detected by CoreBoost_HM on the figure's upper panel. 6B - Predicted CpG promoter (top) and non-CpG promoter (bottom) are shown as density plots where the heights of the peaks correspond to the calculated scores. Independently of the test set up, the highest peaks are consistently mapped to CGI_1059 region (green dashed box), whereas only modest ones are in the MAOA core promoter region (blue dashed box). Note precise positional overlap of the gap between the two peaks of the CGI_1060 and the motif M22 cluster (indicated by red arrow). 6C - This panel shows the MAOA transcripts (representing actual in vivo transcription) in respect to predicted MAOA promoters. Albeit most of the mRNA have relatively short 5′ UTR and are initiated from the canonical MAOA promoter (blue dashed box), the BC044797 mRNA is initiated from the predicted alternative promoter. Note that the BC044797 initiation site is projected to the gap between the peaks as it is demonstrated in validation tests of the CoreBoost_HM algorithm.