Update publication information. Abstract DNA modifications, represented by methylcytosine (5mC), and its oxidized derivatives, such as hydroxymethylcytosine (5hmC), formylcytosine (5fC), and carboxylcytosine (5caC), play important roles in epigenetic regulation of biological processes. The specific recognition of DNA modifications by the transcriptional protein machinery is thought to be a potential mechanism for epigenetic-driven gene regulation, and many modified DNA-specific binding proteins have been uncovered. However, the panoramic view of the roles of DNA modification readers at the proteome level remains largely unclear. Here, we employed a recently developed concatenated tandem array of consensus transcription factor (TF) response elements (catTFREs) approach to profile the binding activity of TFs at DNA modifications (5C, 5mC, 5hmC, and 5fC). Modified DNA-binding activity was quantified for 1,039 TFs, representing 70% of the TFs in the human genome. In addition, we monitored the modified DNA- binding activity of 600 TFs during the mouse brain development from the embryo to the adult stages. Readers of these DNA modifications were predicted, and the hierarchical networks between the transcriptional protein machinery and modified DNA were described. We further demonstrated that ZNF24 and ZSCAN21 are potential readers of 5fC-modified DNA. This study provides a landscape of TF–DNA modification interactions that can be used to elucidate the epigenetic-related transcriptional regulation mechanisms under physiological and pathological conditions.