An insight into the various regulatory mechanisms modulating human DNA methyltransferase 1 stability and function

Abstract

DNA methylation is one of the principal epigenetic signals that participate in cell specific gene expression in vertebrates. DNA methylation plays a quintessential role in the control of gene expression, cellular differentiation and development. It also plays a central role in the preservation of chromatin structure and chromosomal integrity, parental imprinting, X-chromosome inactivation, aging and carcinogenesis. The foremost contributor in the mammalian methylation scheme is DNMT1, a maintenance methyltransferase that faithfully copies the pre-existing methyl marks onto hemimethylated daughter strands during DNA replication to maintain the established methylation patterns across successive cell divisions. The ever-changing cellular physiology and the significant part that DNA methylation plays in genome regulation necessitate rigid management of this enzyme. In mammalian cells, a host of intrinsic and extrinsic mechanisms regulate the expression, activity and stability of DNMT1. Transcriptional regulation, post-transcriptional auto-inhibitory controls and post-translational modifications of the enzyme are responsible for the efficient inheritance of DNA methylation patterns. Also, a large number of intra- and intercellular signaling cascades and numerous interactions with other modulator molecules that affect the catalytic activity of the enzyme at multiple levels function as major checkpoints of the DNMT1 control system. An in-depth understanding of the DNMT1 enzyme, its targeting and function is crucial for comprehending how DNA methylation is coordinated with other critical developmental and physiological processes. This review aims to provide a comprehensive account of the various regulatory mechanisms and interactions of DNMT1 so as to elucidate its function at the molecular level and understand the dynamics of DNA methylation at the cellular level.

Figure 1. The schematic diagram of the different structural domains of DNMT1 and their functions. The N-terminal domain contains (1) a charge-rich DMAP domain which helps in interaction with the transcriptional repressor DMAP1 as well as controls the stability and binding of DNMT1 to DNA at CpG sites, (2) a PBD domain (Proliferating Cell Nuclear Antigen (PCNA) Binding Domain), which mediates the interaction of DNMT1 with PCNA and targets DNMT1 to the replication foci, (3) at least three independent functional NLS (Nuclear Localization Signal) sequences, (4) a RFTS [Replication Foci Targeting Sequence,also called TS (Targeting Sequence)] domain that targets the DNMT1 to replication foci and also mediates dimerization of DNMT1, (5) a zinc domain, also called as CXXC domain which is necessary for the catalytic activity of the enzyme and (6) a PBHD domain (Polybromo Homology Domain) containing two adjacent BAH sequence [BAH1 and BAH2 (Bromo-adjacent homology 1 and 2)]. The PBHD domain has been proposed to act as a protein–protein interaction module specialized in gene silencing. The C-terminal catalytic domain of DNMT1 contains ten characteristic sequence motifs (i.e., conserved motifs I–X), and the spacing sequences between VIII and IX motif is referred to as TRD. The coordination between N-terminal and C-terminal domains is essential for efficient catalytic activity as well as for its interactions with other protein regulators.

Figure 2. The figure depicts transcriptional regulation of DNMT1 expression by different cellular signaling pathways. The expression and activity of DNMT1 is synchronized by a number of signal transduction pathways that include cancer prone cellular signaling pathways such as RAS and PI3K/PKB as well as developmentally inclined networks such as HH and TGFβ-SMAD pathways. These signaling pathways control the expression of DNMT1 and, consequently, the functioning of the DNA methylation machinery to maintain a homeostatic pattern of gene expression in accordance to the cellular needs. P1, P2, P3 and P4 represents the transcription initiation sites and AP1, AP2, AP3 represents the three AP (activator protein) sites. Blue dots downstream of P1 site represent the three c-Jun-dependent enhancers.

Figure 3. The figure represents the various auto-inhibitory mechanisms that prevent any unforeseen and un-programmed de novo methylation by DNMT1.This arrangement acts as a failsafe against uncontrolled methylation by DNMT1 to protect the genome from being transcriptionally silenced at several gene loci that are crucial for growth and differentiation. (A) When DNMT1 bind with fully methylated DNA, the RFTS domain in the N-terminal region is inserted deeply into the active site in the catalytic region; thus, inhibiting the enzyme from needlessly methylating the genome de novo. (B) When DNMT1 binds to an unmethylated site, the CXXC domain acts as a sensor for the methylation status of the substrate excludes the substrate from the catalytic center, thus avoiding any erroneous methylation in previously unmethylated regions.

Figure 4. The figure shows the various interactions of DNMT1. DNMT1 participates in a number of collaborations with a host of regulatory proteins and nuclear re-programmers that simultaneously affect DNMT1 activity as well as its functional exchange. DNMT1 works in concert with proteins found at DNA replication forks—PCNA, proteins participating in chromatin re-organization—DNA methyltransferase DNMT3A and DNMT3B, HDAC1, HDAC2, DNA binding proteins—MeCp2, MBD2, MBD3 and other chromatin binding proteins—UHRF1 and polycomb proteins, proteins associated with cell cycle regulation or response to DNA damage and tumor suppressors—p21 (WAF), Rb protein, p53 protein, PARP1—and, finally, a number of transcription factors and regulators involved in DNA methylation inheritance.

Figure 5. The figure depicts the various post-translational modifications that affect the stability and activity of DNMT1. The major PTMs include methylation, acetylation, ubiquitination, sumoylation and phosphorylation. All of these post-translational regulatory operations are interlinked to maintain a rigid control over the stability, abundance and activity of DNMT1 in a cell-cycle dependent manner.