PT  - JOURNAL ARTICLE
AU  - AS Knaupp
AU  - M Mohenska
AU  - MR Larcombe
AU  - E Ford
AU  - SM Lim
AU  - K Wong
AU  - J Chen
AU  - J Firas
AU  - C Huang
AU  - X Liu
AU  - T Nguyen
AU  - YBY Sun
AU  - ML Holmes
AU  - P Tripathi
AU  - FJ Rossello
AU  - J Schröder
AU  - CM Nefzger
AU  - PP Das
AU  - JJ Haigh
AU  - R Lister
AU  - RB Schittenhelm
AU  - JM Polo
TI  - TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novel <em>Nanog</em> regulators in mouse embryonic stem cells
AID  - 10.1101/2020.04.03.023200
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.04.03.023200
4099  - http://biorxiv.org/content/early/2020/04/04/2020.04.03.023200.short
4100  - http://biorxiv.org/content/early/2020/04/04/2020.04.03.023200.full
AB  - Cellular identity is ultimately controlled by transcription factors (TFs), which bind to specific regulatory elements (REs) within the genome to regulate gene expression and cell fate changes. While recent advances in genome-wide epigenetic profiling techniques have significantly increased our understanding of which REs are utilized in which cell type, it remains largely unknown which TFs and cofactors interact with these REs to modulate gene expression. A major hurdle in dissecting the whole composition of a multi-protein complex formed at a specific RE is the shortage of appropriate techniques. We have developed a novel method termed TALE-mediated Isolation of Nuclear Chromatin (TINC). TINC utilizes epitope-tagged TALEs to isolate a specific genomic region from the mammalian genome and includes a nuclei isolation and chromatin enrichment step for increased specificity. Upon cross-linking of the cells and isolation of the chromatin, the target region is purified based on affinity purification of the TALE and associated nucleic acid and protein molecules can be subjected to further analyses. A key TF in the pluripotency network and therefore in embryonic stem cells (ESCs) is NANOG. It is currently not fully understood how Nanog expression is regulated and consequently it remains unclear how the ESC state is maintained. Using TINC we dissected the protein complex formed at the Nanog promoter in mouse ESCs and identified many known and numerous novel factors.