DNA Methylation Readers and Cancer: Mechanistic and Therapeutic Applications

Abstract

DNA methylation is a major epigenetic process that regulates chromatin structure which causes transcriptional activation or repression of genes in a context-dependent manner. In general, DNA methylation takes place when methyl groups are added to the appropriate bases on the genome by the action of "writer" molecules known as DNA methyltransferases. How these methylation marks are read and interpreted into different functionalities represents one of the main mechanisms through which the genes are switched "ON" or "OFF" and typically involves different types of "reader" proteins that can recognize and bind to the methylated regions. A tightly balanced regulation exists between the "writers" and "readers" in order to mediate normal cellular functions. However, alterations in normal methylation pattern is a typical hallmark of cancer which alters the way methylation marks are written, read and interpreted in different disease states. This unique characteristic of DNA methylation "readers" has identified them as attractive therapeutic targets. In this review, we describe the current state of knowledge on the different classes of DNA methylation "readers" identified thus far along with their normal biological functions, describe how they are dysregulated in cancer, and discuss the various anti-cancer therapies that are currently being developed and evaluated for targeting these proteins.

Keywords: DNA methylation; MBD; cancer; epigenetics; methyl-binding proteins.

Figure 1

Classification of methyl-binding proteins (MBPs). The proteins with methyl-CpG binding abilities are broadly classified into three families based on the functional domains used for binding to methylated DNA. The “MBD-containing proteins” were the first group of MBPs to be identified and are further classified into three subfamilies (MeCP2-MBD, HMT-MBD, and HAT-MBD) based on the presence of functional domains other than MBD. The members of the HMT-MBD and HAT-MBD subfamilies have protein methyltransferase and acetylase activities respectively. The “Methyl-CpG binding Zinc finger proteins” have at least 8 members (Kaiso, ZBTB4, ZBTB38, ZFP57, KLF4, EGR1, WT1, CTCF) that can bind to methylated region using the Zinc finger motifs while the third family of MBPs consisting of UHRF1 and UHRF2 proteins uses their Set and RING-associated (SRA) domain to bind methylated DNA.

Figure 2

Schematic representation of the major domains present in different MBD-containing proteins. All members of this family of proteins have the MBD domain. However, not all 11 members under this family can bind methylated DNA but were classically grouped under the same family because of having the MBD domain. Each member of this family has domains other than MBD that gives them unique characteristics to carry out different cellular functionalities. The MeCP2, MBD1, and MBD2 contain a TRD domain that helps them to recruit chromatin remodeling corepressors and thereby cause transcriptional silencing. In addition, the MBD1 protein may have two or three CXXC-domains due to splicing of its gene. The first two CXXC domains of MBD1 bind to methylated DNA while the third CXXC domain binds to unmethylated DNA. MBD2 has unique G/R rich domain that allows protein-protein interaction as well as post-translational modification. Due to a mutation in the region of the gene encoding the MBD domain of MBD3, it cannot bind to methylated DNA. However, MBD3 can interact with other chromatin remodeling complex and play a role in the regulation of gene expression. The glycosylase domain at the C-terminal end of MBD4 provides it with the unique function of having DNA glycosylase activity while the N-terminal MBD helps it to bind methylated DNA. MBD5 and MBD6 cannot bind to methylated DNA but can interact with the mammalian polycomb deubiquitinase complex PR-DUB. In addition, the PWWP (Pro-Try-Try-Pro) motif of MBD5 helps it bind to the methylated histones. The SET domain provides SETDB1 and SETDB2 the ability to act as protein methyltransferase and the Tudor domains help SETDB1 to bind to methylated histones. In addition, these proteins also have PreSET domain located N-terminus to the SET domain that functions in stabilizing the SET domain. Both BAZ2A and BAZ2B contains a PHD domain to bind unmodified histone while bromodomain and DDT domains allow them to recognize the acetylated histones and DNA binding abilities respectively. (Figure not drawn to the exact scale of the proteins).

Figure 3

Characteristic domain architecture and function of different MBD2 isoforms. MBD2a is the canonical isoform containing four domains while MBD2b lacks the N-terminal G/R-repeat due to the use of alternative start site during translation. On the other hand, the MBD2c isoform is formed due to the inclusion of an alternative third exon which produces a premature stop codon, and as a result, the MBD2c lacks the TRD and CC domains (Figure not drawn to the exact scale of the proteins).

Figure 4

Schematic representation of the domains present in different Methyl-CpG Binding Zinc finger proteins. All members of this family contain C-terminal Zinc finger motifs that allow them to bind to both methylated and unmethylated DNA. In addition, Kaiso, ZBTB4, and ZBTB38 contain the BTB/POZ domain while ZFP57, WT1, EGR1 contain the Krüppel associated box (KRAB), Proline-Glutamine rich (Pro-Glu-rich), and repressor domains (RD) respectively. On the other hand, the KLF4 protein contains an activation domain (AD), repressor domain (RD), and an NLS (nuclear localization signal/sequence) apart from the Zinc fingers (Figure not drawn to the exact scale of the proteins).

Figure 5

Schematic representation of the domains present in different SRA-domain containing proteins. The SRA domain allows them to bind to hemi-methylated DNA, the Ubl and RING domains are involved in ubiquitination, and the TTD and PHD domains allow to interact with the histones (Figure not drawn to the exact scale of the proteins).

Figure 6

Schematic representation of the currently described anti-cancer strategies against different MBPs. Polyphenols obtained from natural compounds can downregulate the aberrantly expressed MeCP2, UHRF1 in cancer cells via differential regulation of cancer-related signaling pathways. The naturally occurring physiologic compound S-adenosylmethionine as well as anti-sense oligonucleotides can downregulate the elevated expression of MBD2 gene and cause inhibition of tumor growth, invasion, and metastasis. Immunotherapy against WT1 antigen has shown promising effects in clinical trials for several malignancies.