The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

Uterine leiomyomas, or fibroids, are benign tumors that affect millions of women worldwide and that can cause considerable morbidity. To study the genetic basis of this tumor type, we examined 18 uterine leiomyomas derived from 17 different patients by exome sequencing and identified tumor-specific mutations in the mediator complex subunit 12 (MED12) gene in 10. Through analysis of 207 additional tumors, we determined that MED12 is altered in 70% (159 of 225) of tumors from a total of 80 patients. The Mediator complex is a 26-subunit transcriptional regulator that bridges DNA regulatory sequences to the RNA polymerase II initiation complex. All mutations resided in exon 2, suggesting that aberrant function of this region of MED12 contributes to tumorigenesis.

Cohesin is present in almost all active enhancer regions, where it is associated with transcription factors. Cohesin frequently colocalizes with CTCF (CCCTC-binding factor), affecting genomic stability, expression and epigenetic homeostasis. Cohesin subunits are mutated in cancer, but CTCF/cohesin-binding sites (CBSs) in DNA have not been examined for mutations. Here we report frequent mutations at CBSs in cancers displaying a mutational signature where mutations in A•T base pairs predominate. Integration of whole-genome sequencing data from 213 colorectal cancer (CRC) samples and chromatin immunoprecipitation sequencing (ChIP-exo) data identified frequent point mutations at CBSs. In contrast, CRCs showing an ultramutator phenotype caused by defects in the exonuclease domain of DNA polymerase ɛ (POLE) displayed significantly fewer mutations at and adjacent to CBSs. Analysis of public data showed that multiple cancer types accumulate CBS mutations. CBSs are a major mutational hotspot in the noncoding cancer genome.

Chromosome shattering and reassembly resembling chromothripsis (a single genomic event that results in focal losses and rearrangements in multiple genomic regions) is a major cause of chromosomal abnormalities in uterine leiomyomas; we propose that tumorigenesis occurs when tissue-specific tumor-promoting changes are formed through these events. Chromothripsis has previously been associated with aggressive cancer; its common occurrence in leiomyomas suggests that it also has a role in the genesis and progression of benign tumors. We observed that multiple separate tumors could be seeded from a single lineage of uterine leiomyoma cells. (Funded by the Academy of Finland Center of Excellence program and others.).

Nucleosomes cover most of the genome and are thought to be displaced by transcription factors (TFs) in regions that direct gene expression. However, the modes of interaction between TFs and nucleosomal DNA remain largely unknown. Here, we have systematically explored interactions between the nucleosome and 220 TFs representing diverse structural families. Consistently with earlier observations, we find that the majority of the studied TFs have less access to nucleosomal DNA than to free DNA. The motifs recovered from TFs bound to nucleosomal and free DNA are generally similar; however, steric hindrance and scaffolding by the nucleosome result in specific positioning and orientation of the motifs. Many TFs preferentially bind close to the end of nucleosomal DNA, or to periodic positions at its solvent-exposed side. TFs often also bind to nucleosomal DNA in a particular orientation. Some TFs specifically interact with DNA located at the dyad position where only one DNA gyre is wound, whereas other TFs prefer sites spanning two DNA gyres and bind specifically to each of them. Our work reveals striking differences in TF binding to free and nucleosomal DNA, and uncovers a rich interaction landscape between TFs and the nucleosome.