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Modeling methyl-sensitive transcription factor motifs with an expanded epigenetic alphabet

By Coby Viner, Charles A. Ishak, James Johnson, Nicolas J. Walker, Hui Shi, Marcela K. Sjöberg-Herrera, Shu Yi Shen, Santana M. Lardo, David J. Adams, Anne C. Ferguson-Smith, Daniel D. De Carvalho, Sarah J Hainer, Timothy L. Bailey, Michael M. Hoffman

Posted 15 Mar 2016
bioRxiv DOI: 10.1101/043794

Introduction. Many transcription factors initiate transcription only in specific sequence contexts, providing the means for sequence specificity of transcriptional control. A four-letter DNA alphabet only partially describes the possible diversity of nucleobases a transcription factor might encounter. For instance, cytosine is often present in a covalently modified form: 5-methylcytosine (5mC). 5mC can successively oxidize to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Just as transcription factors distinguish one unmodified nucleobase from another, some distinguish between unmodified and covalently modified bases. Modification-sensitive transcription factors provide a mechanism for widespread changes in DNA methylation and hydroxymethylation to shift active gene expression programs. Methods. To understand the effect of modified nucleobases on gene regulation, we developed methods to discover motifs and identify transcription factor binding sites in DNA with covalent modifications. Our models expand the standard A/C/G/T DNA alphabet, adding m (5mC) h (5hmC), f (5fC), and c (5caC). We additionally add symbols to encode guanine complementary to these modified cytosine nucleobases, as well as symbols to represent states of ambiguous modification. We adapted the well-established position weight matrix (PWM) model of transcription factor binding affinity to an expanded DNA alphabet. Further, we developed a program, Cytomod, to create a modified sequence. We also enhanced the Multiple EM for Motif Elicitation (MEME) Suite, adding the capacity to handle custom alphabets. These versions permit users to specify new alphabets, anticipating future alphabet expansions. Results. We created expanded-alphabet sequences, using whole-genome maps of 5mC and 5hmC in mouse T cells and 5mC in human K562 cells. Using this sequence and chromatin immunoprecipitation-sequencing (ChIP-seq) data from the Encyclopedia of DNA Elements (ENCODE) Project and others, we identified modification-sensitive cis-regulatory modules. We elucidated various known methylation binding preferences, including the preference of ZFP57 and C/EBP\textbeta for methylated motifs and the preference of c-Myc for unmethylated E-box motifs. We determined cutoffs for modified base calling using hypothesis testing across different thresholds. Using these known binding preferences to tune model parameters, we discovered novel modified motifs, across a wide array of transcription factors. We clustered modified-unmodified hypothesis pairs, revealing a complex interplay of modification preferences. After predicting methylated and hydroxymethylated binding preferences of OCT4, we validated them with the first conjoint cleavage under targets and release using nuclease (CUT&RUN) experiments across conventional, methylation-, and hydroxymethylation-enriched sequences. Discussion. Hypothesis testing of motif central enrichment provides a natural means of differentially assessing modified versus unmodified binding affinity, without most of the limitations of a de novo analysis. Our approach readily extends to other DNA modifications, when provided with genome-wide single-base resolution data. As more high-resolution epigenomic data becomes available, we expect this method to continue to yield insights into altered transcription factor binding affinities across a variety of modifications.

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