Stem Cell Reports Article TINC— A Method to Dissect Regulatory Complexes at Single-Locus Resolution— Reveals an Extensive Protein Complex at the Nanog Promoter Anja S. Knaupp, 1,2,3,10 Monika Mohenska, 1,2,3,10 Michael R. Larcombe, 1,2,3,10 Ethan Ford, 4,5 Sue Mei Lim, 1,2,3 Kayla Wong, 1,2,3 Joseph Chen, 1,2,3 Jaber Firas, 1,2,3 Cheng Huang, 6 Xiaodong Liu, 1,2,3 Trung Nguyen, 4,5 Yu B.Y. Sun, 1,2,3 Melissa L. Holmes, 1,2,3 Pratibha Tripathi, 1,2 Jahnvi Pflueger, 4,5 Fernando J. Rossello, 1,2,3 Jan Schro ¨der, 1,2,3 Kathryn C. Davidson, 1,2,3 Christian M. Nefzger, 1,2,3 Partha P. Das, 1,2 Jody J. Haigh, 7,8,9 Ryan Lister, 4,5 Ralf B. Schittenhelm, 6, * and Jose M. Polo 1,2,3, * 1 Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia 2 Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC 3800, Australia 3 Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia 4 Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Perth, WA 6009, Australia 5 Harry Perkins Institute of Medical Research, Nedlands, WA 6009, Australia 6 Monash Proteomics and Metabolomics Facility, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia 7 Australian Centre for Blood Diseases, Monash University, Clayton, VIC 3004, Australia 8 Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, MB, Canada 9 Research Institute in Oncology and Hematology, CancerCare Manitoba, Winnipeg, MB, Canada 10 These authors contributed equally *Correspondence: ralf.schittenhelm@monash.edu (R.B.S.), jose.polo@monash.edu (J.M.P.) https://doi.org/10.1016/j.stemcr.2020.11.005 SUMMARY Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expres- sion. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. How- ever, 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-tun- ing 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. INTRODUCTION Pluripotent stem cells (PSCs) carry immense therapeutic po- tential as they can produce any cell type of the body and can self-renew indefinitely. The two most common in vitro PSCs are blastocyst-derived embryonic stem cells (ESCs) (Kauf- man et al., 1983; Martin 1981) and induced PSCs (iPSCs), which are obtained from somatic cells through expression of the transcription factors (TFs) OCT4, SOX2, KLF4, and C-MYC (Takahashi et al., 2007; Takahashi and Yamanaka 2006). Together with OCT4 and SOX2, NANOG forms the core transcriptional network in ESCs/iPSCs that mediates expression of self-renewal and pluripotency genes and repression of differentiation genes (Loh et al., 2006; Boyer et al., 2005; Chen et al., 2008; Marson et al., 2008; Kim et al., 2008). ESCs express Nanog heterogeneously and can be maintained upon Nanog deletion, although they are dif- ferentiation prone (Chambers et al., 2007). These NANOG fluctuations seem to play a role in lineage commitment with high levels impeding ESC differentiation (Abranches et al., 2014; Chambers et al., 2003; Kalmar et al., 2009). Like any gene, Nanog expression is regulated by TFs that interact with regulatory elements (REs) and other factors. At least two REs control Nanog in ESCs: the promoter and the 5 kb enhancer (Apostolou et al., 2013; Kagey et al., 2010). NANOG binds to both of these REs (Chen et al., 2008; Kim et al., 2008) and mediates positive (Wu et al., 2006) and negative (Fidalgo et al., 2012; Navarro et al., 2012) feedback loops. Positive transcriptional regula- tion has been associated with binding of OCT4 and SOX2 to the Nanog promoter (Rodda et al., 2005). Conversely, Nanog auto-repression has been linked to the interaction of NANOG with ZFP281 and the NuRD repressor complex (Fidalgo et al., 2012). Nevertheless, while some of the pro- teins that occupy these REs are known, the full complex composition remains largely elusive. This knowledge gap is partly due to the difficulty in dis- secting a specific regulatory complex. Chromatin immuno- precipitation (ChIP) has been invaluable to study in vivo DNA-protein interactions but only allows interrogation of one factor at a time, and requires a priori candidates and appropriate antibodies. Thus, being able to simultaneously analyze an entire protein complex is extremely advanta- geous but is challenging due to (1) a genomic region of low abundance (e.g., two copies per cell) has to be targeted specifically and (2) the interacting proteins have to be 1246 Stem Cell Reports j Vol. 15 j 1246–1259 j December 8, 2020 j ª 2020 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).