MAX is an epigenetic sensor of 5-carboxylcytosine and is altered in multiple myeloma

Abstract

The oncogenic transcription factor MYC and its binding partner MAX regulate gene expression by binding to DNA at enhancer-box (E-box) elements 5΄-CACGTG-3΄. In mammalian genomes, the central E-box CpG has the potential to be methylated at the 5-position of cytosine (5mC), or to undergo further oxidation to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), or 5-carboxyl (5caC) forms. We find that MAX exhibits the greatest affinity for a 5caC or unmodified C-containing E-box, and much reduced affinities for the corresponding 5mC, 5hmC or 5fC forms. Crystallization of MAX with a 5caC modified E-box oligonucleotide revealed that MAX Arg36 recognizes 5caC using a 5caC–Arg–Guanine triad, with the next nearest residue to the carboxylate group being Arg60. In an analysis of >800 primary multiple myelomas, MAX alterations occurred at a frequency of ∼3%, more than half of which were single nucleotide substitutions affecting a basic clamp-like interface important for DNA interaction. Among these, arginines 35, 36 and 60 were the most frequently altered. In vitro binding studies showed that whereas mutation of Arg36 (R36W) or Arg35 (R35H/L) completely abolished DNA binding, mutation of Arg60 (R60Q) significantly reduced DNA binding, but retained a preference for the 5caC modified E-box. Interestingly, MAX alterations define a subset of myeloma patients with lower MYC expression and a better overall prognosis. Together these data indicate that MAX can act as a direct epigenetic sensor of E-box cytosine modification states and that local CpG modification and MAX variants converge to modulate the MAX-MYC transcriptional network.

TET proteins in natural and induced differentiation

“…Pioneer transcription factors that are not sensitive to DNA modifications or nucleosome occupancy may be able to bind to these sites and recruit TET proteins to generate oxi-mC (Figure 1), with subsequent effects on other transcription factor binding such as basic helix-loop-helix (bHLH) family members binding E-box sites. Some E-box motifs contain a CpG and hemi-modified sequences containing C on one strand and 5caC on the other augmented the TF/DNA interaction [30]. At CpG containing sites, DNA containing symmetrical 5mC or 5hmC modifications impaired binding, while hemi-modified sequences were permitted [30,31].…”

“…Some E-box motifs contain a CpG and hemi-modified sequences containing C on one strand and 5caC on the other augmented the TF/DNA interaction [30]. At CpG containing sites, DNA containing symmetrical 5mC or 5hmC modifications impaired binding, while hemi-modified sequences were permitted [30,31]. In this scenario, a single base modification followed by DNA replication could allow binding at this site in both daughter cells.…”

Distinct pattern of epigenetic DNA modification in leukocytes from patients with colorectal carcinoma and individuals with precancerous conditions, benign adenoma and inflammatory bowel disease – a link to oxidative stress

“…It is also possible that different proteins/factors recognize the modifications and determine their fate [43]. Interestingly, some recent experiments demonstrated that transcription factors, Myc and Max, and perhaps also a number of other regulatory proteins, may specifically recognize 5-caCyt, but have lesser affinity to 5-fCyt, and show only a trace of affinity to 5-mCyt and 5-hmCyt [44]. Moreover, a recent study conducted by Xiong et al [45] showed that Sall4, an oncogenic protein being overexpressed in colon cancer [46], may further enhance TET2-catalyzed oxidation of 5-hmCyt.…”

Recognition of Oxidized 5‐Methylcytosine Derivatives in DNA by Natural and Engineered Protein Scaffolds

“…Finally, the MYC binding partner MAX (a basic helix‐loop‐helix protein) has been shown to bind its CpG‐containing enhancer target DNA sequence preferentially in the presence of a caC or C rather then mC, hmC or fC. A crystal structural analysis of MAX bound to its caC‐containing target DNA revealed a critical arginine involved in recognition of the caC‐paired G residue and a second arginine in ∼6 Å distance to the caC carboxyl group that may be involved in water‐mediated interactions with this group …”