Quantitative Analysis of the DNA Methylation Sensitivity of Transcription Factor Complexes

Abstract

SUMMARY Although DNA modifications play an important role in gene regulation, the underlying mechanisms remain elusive. We developed EpiSELEX-seq to probe the sensitivity of transcription factor binding to DNA modification in vitro using massively parallel sequencing. Feature-based modeling quantifies the effect of cytosine methylation (5mC) on binding free energy in a position-specific manner. Application to the human bZIP proteins ATF4 and C/EBPβ, and three different Pbx-Hox complexes shows that 5mCpG can both increase and decrease affinity, depending on where the modification occurs within the protein-DNA interface. The TF paralogs tested vary in their methylation sensitivity, for which we provide a structural rationale. We show that 5mCpG can also enhance in vitro p53 binding, and provide evidence for increased in vivo p53 occupancy at methylated binding sites, correlating with primed-enhancer histone marks. Our results establish a powerful strategy for dissecting epigenomic modulation of protein-DNA interactions and their role in gene regulation.

Intrinsic DNA Shape Accounts for Affinity Differences between Hox-Cofactor Binding Sites

“…EMSAs and protein purification for EMSAs were performed as described previously (Kribelbauer et al, 2017; Slattery et al, 2011). The constructs used for EMSAs also correspond to those described previously, instead of the isolated homeodomain constructs that were used for crystallization.…”

Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells

“…Interestingly, the C/EBPβ–ATF4 heterodimer gained the ability to bind the non-canonical sequence 5′-CGATGCAA-3′ when the first cytosine was methylated. By contrast, ATF4 homodimers show decreased affinity for methylated DNA [82,84]. Furthermore, methyl-binding proteins may compete with the bZIP heterodimers for binding to methylated promoters [85].…”

Loss of Dnmt3a dependent methylation in inhibitory neurons impairs neural function through a mechanism that impacts Rett syndrome

“…These data highlight these proteins as primary candidates, though further studies are necessary to determine whether the other MBD-containing proteins read mCH in the brain, in vivo. However, there are other proteins involved in transcriptional regulation that do not possess a classic MBD and are sensitive to the methylation status of DNA, with some validated in vivo (57)(58)(59)(60)(61). For example, the DNA binding of many transcription factors is both positively and negatively affected by DNA methylation (58,59), a phenomena that is not exclusive to mammals (62).…”