TINC — A Method to Dissect Regulatory Complexes at Single-Locus Resolution — Reveals an Extensive Protein Complex at the Nanog Promoter

Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general.Anja S. Knaupp, Monika Mohenska, Michael R. Larcombe, Ethan Ford, Sue Mei Lim, Kayla Wong, Joseph Chen, Jaber Firas, Cheng Huang, Xiaodong Liu, Trung Nguyen, Yu B.Y. Sun, Melissa L. Holmes, Pratibha Tripathi, Jahnvi Pflueger, Fernando J. Rossello, Jan Schro, der, Kathryn C. Davidson, Christian M. Nefzger, Partha P. Das, Jody J. Haigh, Ryan Lister, Ralf B. Schittenhelm, and Jose M. Pol

Transient and Permanent Reconfiguration of Chromatin and Transcription Factor Occupancy Drive Reprogramming

Somatic cell reprogramming into induced pluripotent stem cells (iPSCs) induces changes in genome architecture reflective of the embryonic stem cell (ESC) state. However, only a small minority of cells typically transition to pluripotency, which has limited our understanding of the process. Here, we characterize the DNA regulatory landscape during reprogramming by time-course profiling of isolated sub-populations of intermediates poised to become iPSCs. Widespread reconfiguration of chromatin states and transcription factor (TF) occupancy occurs early during reprogramming, and cells that fail to reprogram partially retain their original chromatin states. A second wave of reconfiguration occurs just prior to pluripotency acquisition, where a majority of early changes revert to the somatic cell state and many of the changes that define the pluripotent state become established. Our comprehensive characterization of reprogramming-associated molecular changes broadens our understanding of this process and sheds light on how TFs access and change the chromatin during cell-fate transitions