Search

Additional file 1. Supplementary Figures and Supplementary Tables.

Summary Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. DNA adenine methyltransferase identification (DamID) has emerged as a comprehensive method to map genome-wide occupancy of proteins of interest. A caveat of DamID is the specificity of Dam methyltransferase for GATC motifs that are not homogenously distributed in the genome. Here, we developed an optimized method named MadID, using proximity labeling of DNA by the methyltransferase M.EcoGII. M.EcoGII mediates N6-adenosine methylation in any DNA sequence context, resulting in deeper and unbiased coverage of the genome. We demonstrate, using m6A-specific immunoprecipitation and deep sequencing, that MadID is a robust method to identify protein-DNA interactions at the whole-genome level. Using MadID, we revealed contact sites between human telomeres, repetitive sequences devoid of GATC sites, and the nuclear envelope. Overall, MadID opens the way to identification of binding sites in genomic regions that were largely inaccessible., Graphical Abstract, Highlights • MadID: mapping of protein-DNA interactions in vivo using proximity labeling • Deeper and unbiased genome-wide coverage using M.EcoGII, a methyltransferase • Identification of binding sites in previously inaccessible regions of the genome • Identification of telomere-nuclear envelope contact sites, Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. Sobecki et al. developed an optimized method named MadID based on proximity labeling of DNA by the bacterial methyltransferase M.EcoGII. MadID results in deep and unbiased coverage for genome-wide mapping studies.

and Tables S1 and S2. (PDF 1589 kb)

Developmentally regulated genes are often controlled by distant enhancers, silencers and insulators, to implement their correct transcriptional programs. In recent years, the development of 3C and derived techniques (4C, 5C, HiC, ChIA- PET, etc.) has confirmed that chromatin looping is an important mechanism for the transfer of regulatory information in mammalian cells. At many developmentally regulated gene loci, transcriptional activation is indeed accompanied by the formation of chromatin loops between genes and distant enhancers. Similarly, dynamic looping between insulator elements and changes in local 3D organization may be observed upon variation in transcriptional activity. Chromatin looping also occurs at silent gene loci, where its function remains less understood. In lineage- committed cells, partial 3D configurations are detected at loci that are activated at later stages. However, these partial configurations usually lack promoter–enhancer loops that accompany transcriptional activation, suggesting they have structural functions. Definitive evidence for a repressive role of chromatin looping is still lacking. Chromatin loops have been reported at repressed loci but, alternatively, they may act as a distraction for active loops. Together, these mechanisms allow fine-tuning of regulatory programs, thus providing further diversity in the transcriptional control of developmentally regulated gene loci.

Genetic linkage analysis previously suggested that GKAP, a scaffold protein of the N-methyl-D-aspartate receptor (NMDAR), was a potential modifier of invasion in a mouse model of pancreatic neuroendocrine tumor (PanNET). Here, we establish that GKAP governs invasive growth and treatment response to NMDAR inhibitors of PanNET via its pivotal role in regulating NMDAR pathway activity. Combining genetic knockdown of GKAP and pharmacological inhibition of NMDAR, we implicate as downstream effectors FMRP and HSF1, which along with GKAP demonstrably support invasiveness of PanNET and pancreatic ductal adenocarcinoma cancer cells. Furthermore, we distilled genome-wide expression profiles orchestrated by the NMDAR-GKAP signaling axis, identifying transcriptome signatures in tumors with low/inhibited NMDAR activity that significantly associate with favorable patient prognosis in several cancer types.

Additional file 4. Review history.

Additional file 4. Review history.

SummaryTopologically Associating Domains (TADs) compartmentalize vertebrate genomes into sub-Megabase functional neighbourhoods for gene regulation, DNA replication, recombination and repair1-10. TADs are formed by Cohesin-mediated loop extrusion, which compacts the DNA within the domain, followed by blocking of loop extrusion by the CTCF insulator protein at their boundaries11-20. CTCF blocks loop extrusion in an orientation dependent manner, with both experimental and in-silico studies assuming that a single site of static CTCF binding is sufficient to create a stable TAD boundary21-24. Here, we report that most TAD boundaries in mouse cells are modular entities where CTCF binding clusters within extended genomic intervals. Optimized ChIP-seq analysis reveals that this clustering of CTCF binding does not only occur among peaks but also frequently within those peaks. Using a newly developed multi-contact Nano-C assay, we confirm that individual CTCF binding sites additively contribute to TAD separation. This clustering of CTCF binding may counter against the dynamic DNA-binding kinetics of CTCF25-27, which urges a re-evaluation of current models for the blocking of loop extrusion21-23. Our work thus reveals an unanticipatedly complex code of CTCF binding at TAD boundaries that expands the regulatory potential for TAD structure and function and can help to explain how distant non-coding structural variation influences gene regulation, DNA replication, recombination and repair5,28-34.