Miniaturized HOXB13 Mimetics are Sequence-Specific, Methyl-Sensitive DNA Binders

Homeobox protein Hox-B13 (HOXB13) is an oncogenic transcription factor associated with prostate cancer risk. Unlike most transcription factors, HOXB13 is methyl-sensitive and has been shown to be preferentially recruited to DNA prostate cancer risk loci. Here we miniaturized HOXB13 to create metal-stapled mimetics that can target its cognate primary DNA binding site in a sequence-specific, methyl-sensitive manner. Targeted profiling of 5-methylcytosine (5mC) recognition using V269X mimetic mutants revealed that Leucine can further enhance both methyl-sensitivity and methylspecificity through methyl-methyl contact with 5mC and decreased methyl-pi interactions with cytosine. Collectively, our study revealed novel insights into the molecular interactions of HOXB13 with methylated DNA, and new tools that will provide a structural basis for future development of a new class of sequence-specific methylated DNA binders.