A comprehensive in silico
analysis of epigenetic properties of the human MAOA
gene revealed new aspects of the epigenetic potential of this gene, previously unknown associations between different regulatory moieties and lead to some unexpected findings. This analysis was conceived to test our hypothesis suggesting that epigenetic mechanisms are involved in the control of the activity of the human MAOA
gene. Unarguably, the validation of the hypothesis necessitates experimental testing that affords direct measurement of the methylation state of this genomic region. There are numerous methods to detect and to quantify methylation; nevertheless all of the currently available assays have some limitations and drawbacks. For these reasons, we sought that the sequence-based analysis might add to comprehensive assessment of epigenetic potential of the region. In fact, the thorough bioinformatic analysis has its own merits; for example, a notable advantage of the in silico
analysis in that it is independent of the detection methods used 45
. Sequence-based analysis is neither contingent upon the nature of the biological sample used for the analysis, specifically, the cell type, tissue of origin, differentiation state etc., nor is influenced by transient nature of methylation state in vivo
We performed this analysis as a precede to the experimental testing of the MAOA methylation in primary genomic samples arguing that its results would help to properly design experiments, to minimize possible experimental bias, and thus, to improve validity of the experimental data.
Our findings support the original hypothesis of involvement of epigenetic mechanisms in regulation of the human MAOA gene. The principal findings of the analysis are the following: Firstly, the MAOA locus features multiple genomic targets exhibiting sensitivity to differential DNA methylation. Secondly, the uVNTR polymorphic region contributes to the epigenetic potential of the MAOA allele. Thirdly, the methylation status of the MAOA regulatory region in individual human genomes likely varies, whereby individual variations in the gene sequence might affect variations in methylation profile. Lastly, site-specific conditions, modulated by the epigenetic marks, can favor transcriptional initiation from one of multiple TSSs of the MAOA, so generating transcripts of different lengths, and thus adding a factor to the tissue-specific levels of the MAO A enzyme.
Proposing pivotal role of epigenetic factors in regulation of the MAOA
expression, we are constrain this paradigm to the human and the primate MAOA
gene. The comparative genomics approach is widely used to identify functional sequence elements and regulatory networks 96, 97
. Accordingly, in our study we employed both comparative and evolutionary analyses but their findings were unexpected. We found that the regulatory sequences of the MAOA
lack conservation (). Moreover, it turned out that the evolution of the regulatory MAOA
sequence was accompanied by gene conversion and results in considerable accumulation of G+C nucleotides (GC content in the mouse Maoa
promoter is 0.49 contrasting the 0.59 in human MAOA
). These findings require some explanation. Analysis of phylogenetic patterning suggested that variations in the CG content are associated with the emergence of the evolutionally novel GC-rich structural and functional transcription elements 98
. In this view, GC-bias conversion of the MAOA
promoter enables the action of additional regulatory mechanisms (epigenetic by nature) in human and primates. In addition, whilst the mouse Maoa
promoter is non-CGI, the chimp MAOA
promoter is CGI-overlapping which speaks to the possible involvement of epigenetic mechanisms in the MAOA
regulation in primates and human lineage.
We established that the regulatory region of the MAOA
gene spans over (-) 2 kb relative to the annotated TSS. This whole region was consistently marked as methylation-sensitive and contains CGIs. Interestingly, that the concept of “CpG Island shores” that was introduced in a recent paper 99
proposed the region about 2 kb in 5′ of TSS as functionally significant.
Established role of gene promoter methylation in expressional regulation 26
, prompted us explore properties of the MAOA
promoter in more details. Housekeeping genes 100
, including the MAOA
gene, commonly have a CGI-associated promoter and a ‘spike’ of GC content in the vicinity of the TSS. The characteristics of the CGI_1060 fulfil these criteria. Intrigued by detection of an additional CGI_1059 further upstream and by the fact that this CpG island encompasses uVNTR polymorphism with known effect on transcriptional rate 6
, we explored genomic features of this region and found that it might represent an additional promoter for the MAOA
. Implementation of several computational approaches including analysis of TF clusters, refinement of the boundaries of CisRed region (Figure S4
) and nucleosome positioning (), and analysis of physical properties of DNA (Figure S2
) – all these analyses consistently indicated the presence of an additional region with promoter characteristics upstream of the canonical MAOA promoter.
We performed several computational tests using promoter-searching programs that implement sequence-based predictions and predictions based on integration of different genomic annotations. Various experimental data, namely, ENCODE data on histone modifications and RNA polymerase II- binding sites collaborate our finding. The fact that the human MAOA
transcript BC044787 initiates about 1.5 kb upstream of the MAOA
TSS (initiation site is within the CGI_1059, Figure S6
, red arrow) demonstrates in vivo
utilization of the alternative MAOA
Most mammalian genes have multiple promoters containing multiple TSSs 101
, thus the possibility of alternative promoters for the MAOA
gene does not put this gene apart. An important aspect of our finding is that it offers new regulatory paradigm for the MAOA
gene since the putative promoters carry distinctly different information about gene expression, and therefore, may exhibit different sensitivity to the regulatory signals. Contrasting most human promoters where only one of alternative gene promoters is CGI-associated 102
, both putative alternative promoters of the MAOA
are CGI-promoters, suggesting ultimate role of methylation in regulation of the MAOA
. Intriguingly, the larger size of the distal CGI_1059 taken in the context of the observation that the CGIs overlapping with TSS are much longer comparing with these in other genic environments 103
, point to the dominance of this site as transcription initiation site.
The alternative MAOA
promoters have distinguishing properties, including occurrence and distribution of TFBSs, profiles of histone variants, and regional DNA folding, which can reflect their functionally distinct characteristics. For instance, we found dense nucleosome deposition directly upstream of the core TSS, while, in contrast, a large region upstream of the distal promoter is nucleosome-depleted (), pointing to the potential of these regions to initiate transcription under basic conditions. Also, Pol2 was detested only at the distal promoter region (, red arrows), suggesting that this promoter might maintain gene expression in a poised state 104
The concept of the alternative promoter for the MAOA
gene offers additional possible biological mechanisms to explain functional effect of the uVNTR polymorphism. The uVNTR region directly affects mRNA transcripts initiated from the alternative MAOA
determining their length as a function of increment. Thus, we suggest that compositional characteristics of the MAOA
promoter that encompasses uVNTR polymorphic region profoundly influence stability of the cognate mRNAs, for example due to differential stability (higher number of G4 structures was predicted for the longer transcripts). A similar regulatory mechanism has been demonstrated for the human insulin gene (ILPR). The minisatellite (INS
VNTR [MIM 125852]) upstream to the insulin gene promoter is related to gene's transcriptional activity and to the susceptibility for insulin-dependent diabetes mellitus (IDDM) (reviewed in 105
). Studies of the mechanisms which drive VNTR's effect on the gene expression had shown that the G-rich strand of the VNTR ILPR adopts an intra-molecularly folded hairpin G-quartet structure where the number of repeats defines the nature of DNA folding and regional stability 106
The concept of the alternative MAOA
promoter has several implications. For example, it is apparent that the transcription initiation from the distal MAOA
promoter would result in production of longer mRNAs that are thermodynamically more stable than shorter ones. Stability of the longer transcripts entails their effective translation into protein 107
. In addition, since most predicted localization motifs that enable intracellular trafficking of the mRNAs are mapped to 5′ UTR of the transcripts 108
, it is feasible that the longer transcripts would be more efficient in enabling targeting of the translational process to specific subcellular compartments, for instance, to the vicinity of neuronal terminals. In addition, well-established modulating effect of environment on the MAOA
expression might be exerted by the switch in transcription initiation, given that the alternative promoter is CGI-overlapping.
Conceptually, our hypothesis on the involvement of epigenetic mechanisms in the MAOA
regulation differs from the ones investigated in the other studies. Currently, most of the experimental and clinical studies investigate the role of DNA methylation in human diseases (see, for example a review 109
). As a result, a solid link between the aberrant epigenetic marks and the diseases has been established (reviewed in 110
). Less numerous research report an association of genomic polymorphisms and altered epigenetic status of a gene and only single ones present evidence supporting a role of DNA methylation in the regulation of normal gene expression 111, 108
. For example of the former is uncovered regulatory mechanism of the glucocorticoid receptor gene: Turner and others 112
showed that individualized epigenetic pattern of glucocorticoid receptor promoter which orchestrates the expression of this gene is influenced by the sequence variations. Akin, Candiloro and Dobrovic discovered that particular genotypes of the MGMT gene are associated with its methylation in healthy individuals 113
. We foresee that, in a similar way, genetic variations in the MAOA
promoter (including uVNTR) might be a contributing factor to the methylation state of the region. Considering these observations, we strongly suggest that the practical methylation analysis ultimately have to include sequencing of the primary genomic samples. Knowing the genetic variations in the samples will enable control for confounding effects due to heterogeneity of individual's genomes.
The emerging complex model of MAOA regulation offers a new perspective on the developmental dynamics and individual variations in the MAOA gene-expression, and ultimately, on regulation of the MAO A enzymatic activity in the human brain. We propose a novel regulatory paradigm for the MAOA gene in which its expression is governed in a highly coordinated fashion by the epigenetic mechanisms and the local transcriptional machinery, and is carried out by generation of tissue-specific alternative transcripts initiated from the alternative promoters. We will use the results of the analysis to guide our experimental investigation of the methylation status of the MAOA locus in individual genomes. Based on the findings, we will then explore an association between the MAOA methylation and the phenotype assessed by the level of the MAO A enzyme in the brain.