Chromatin Starts to Come Clean

In an effort to identify a chromatin-associated pluripotent network, Rafiee et al., 2016Rafiee M. Girardot C. Sigismondo G. Krijgsveld J. Mol. Cell. 2016; 64 (this issue): 624-635Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar developed a powerful ChIP-MS technique and discovered a novel protein, TRIM24, enriched on OCT4-, SOX2-, and NANOG-associated chromatin, paving the way for future proteomic studies on chromatin. In an effort to identify a chromatin-associated pluripotent network, Rafiee et al., 2016Rafiee M. Girardot C. Sigismondo G. Krijgsveld J. Mol. Cell. 2016; 64 (this issue): 624-635Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar developed a powerful ChIP-MS technique and discovered a novel protein, TRIM24, enriched on OCT4-, SOX2-, and NANOG-associated chromatin, paving the way for future proteomic studies on chromatin. Protein interaction networks provide valuable insights into signaling and transcription regulation. Transcription occurs on chromatin, which encompasses DNA and histones, as well as other factors, and the dynamic features of chromatin reflect the transcription status. Traditional protein affinity purification cannot differentiate interactions either on or off chromatin. Recently, a variety of native or cross-linked chromatin immunoprecipitation mass spectrometry (ChIP-MS) methods has been developed to identify protein interactions on chromatin (Smolle et al., 2012Smolle M. Venkatesh S. Gogol M.M. Li H. Zhang Y. Florens L. Washburn M.P. Workman J.L. Nat. Struct. Mol. Biol. 2012; 19: 884-892Crossref PubMed Scopus (214) Google Scholar). These techniques apparently need further optimization, as most mass spectrometry-based affinity purification methods suffer from substantial contaminants, such that there is a designated web database, "CRAPome," to avoid false leads (Mellacheruvu et al., 2013Mellacheruvu D. Wright Z. Couzens A.L. Lambert J.P. St-Denis N.A. Li T. Miteva Y.V. Hauri S. Sardiu M.E. Low T.Y. et al.Nat. Methods. 2013; 10: 730-736Crossref PubMed Scopus (910) Google Scholar). Rafiee et al., 2016Rafiee M. Girardot C. Sigismondo G. Krijgsveld J. Mol. Cell. 2016; 64 (this issue): 624-635Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar addressed this issue head on. In this study, an interesting new method, ChIP-SICAP (selective isolation of chromatin-associated proteins), was developed. In addition to purifying chromatin-bound proteins exclusively, the main advantage of this new method is the minimalization of co-purified contaminants. This improvement entails an elegantly designed DNA affinity purification step. After formaldehyde cross-linked chromatin was immunoprecipitated (IP) with an antibody against the protein of interest (here: OCT4, SOX2, and NANOG [OSN]), the DNA was end-labeled with a biotin tag (Figure 1). A very stringent wash step (with 7.5% SDS) removed contaminants and the antibody from the chromatin. A subsequent IP with streptavidin further purified the biotin-labeled chromatin. As a result of this novel stringent method, contaminants, such as ribosomal proteins and ribonucleoproteins, were drastically reduced; yet, chromatin-associated proteins were enriched when compared to other methods. Utilizing quantitative stable isotope labeling, Rafiee et al., 2016Rafiee M. Girardot C. Sigismondo G. Krijgsveld J. Mol. Cell. 2016; 64 (this issue): 624-635Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar tested their new methodology on the naive and primed embryonic stem cell (ESC) pluripotency network (Ying et al., 2008Ying Q.L. Wray J. Nichols J. Batlle-Morera L. Doble B. Woodgett J. Cohen P. Smith A. Nature. 2008; 453: 519-523Crossref PubMed Scopus (2523) Google Scholar) with resounding results. Reinforcing the pluripotent circuitry, ChIP-SICAP against OSN identified 407 highly overlapping, common interactors, including the OSN triumvirate itself. In addition, under naive 2i conditions (two chemicals inhibiting mitogen-activated protein [MAP] kinase and GSK3 signaling), β-catenin, a transcription regulator that translocates into the nucleus after glycogen synthase kinase (GSK) inhibition, is enriched on chromatin. So too are many pluripotent factors, including PRDM14, ESRRB, TCF3, and TBX3. On the other hand, factors responsible for DNA methylation under serum conditions, i.e., DNMT3A, DNMT3L, and UHRF1, were depleted on chromatin under 2i conditions. This finding is in accordance with DNA methyltransferases being repressed under 2i conditions (Leitch et al., 2013Leitch H.G. McEwen K.R. Turp A. Encheva V. Carroll T. Grabole N. Mansfield W. Nashun B. Knezovich J.G. Smith A. et al.Nat. Struct. Mol. Biol. 2013; 20: 311-316Crossref PubMed Scopus (355) Google Scholar). In keeping with these observations, PRDM14, a pluripotent factor enriched in 2i chromatin, was previously reported to be upregulated and to directly repress DNMTs under 2i conditions (Grabole et al., 2013Grabole N. Tischler J. Hackett J.A. Kim S. Tang F. Leitch H.G. Magnúsdóttir E. Surani M.A. EMBO Rep. 2013; 14: 629-637Crossref PubMed Scopus (119) Google Scholar, Leitch et al., 2013Leitch H.G. McEwen K.R. Turp A. Encheva V. Carroll T. Grabole N. Mansfield W. Nashun B. Knezovich J.G. Smith A. et al.Nat. Struct. Mol. Biol. 2013; 20: 311-316Crossref PubMed Scopus (355) Google Scholar). Although the enrichment of PRDM14 and DNMTs on the respective chromatin could be reflective of their relative abundance under different culture conditions, Rafiee et al., 2016Rafiee M. Girardot C. Sigismondo G. Krijgsveld J. Mol. Cell. 2016; 64 (this issue): 624-635Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar found no major changes at the protein levels for the majority of the enriched factors, consistent with previous transcriptome data (Marks et al., 2012Marks H. Kalkan T. Menafra R. Denissov S. Jones K. Hofemeister H. Nichols J. Kranz A. Stewart A.F. Smith A. Stunnenberg H.G. Cell. 2012; 149: 590-604Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar). Rafiee et al., 2016Rafiee M. Girardot C. Sigismondo G. Krijgsveld J. Mol. Cell. 2016; 64 (this issue): 624-635Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar suggested that protein phosphorylation might contribute to such differences; however, alternative mechanisms are quite possible. The relative enrichment might reflect complex regulatory mechanisms on chromatin within the pluripotent network. In addition to known pluripotent factors that associate with OSN on chromatin, this promising new methodology also discovered novel proteins. From the 407 identified OSN chromatin-interacting proteins, two proteins stood out. One of them is TRIM24, a TRIM (tripartite motif) family protein involved in both oncogenesis and tumor suppression. To verify that TRIM24 is part of the OSN pluripotency network, Rafiee et al., 2016Rafiee M. Girardot C. Sigismondo G. Krijgsveld J. Mol. Cell. 2016; 64 (this issue): 624-635Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar examined its target sites genome wide and found a high degree of co-occupancy with OSN. The TRIM24 interaction appears functionally relevant as transcriptome analysis after shRNA-mediated TRIM24 knockdown points to its involvement in differentiation and cell proliferation. Lastly, as many pluripotent factors positively contribute to induced pluripotent stem (iPS) reprogramming, Rafiee et al., 2016Rafiee M. Girardot C. Sigismondo G. Krijgsveld J. Mol. Cell. 2016; 64 (this issue): 624-635Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar tested TRIM24 for its ability to promote this process, and indeed, TRIM24 addition led to a 12-fold enhancement in iPS cell reprogramming. Interestingly, TRIM24 is a chromatin reader that recognizes an unusual histone peptide H3K4me0-H3K23ac through its tandem PHD-Bromo domain (Tsai et al., 2010Tsai W.W. Wang Z. Yiu T.T. Akdemir K.C. Xia W. Winter S. Tsai C.Y. Shi X. Schwarzer D. Plunkett W. et al.Nature. 2010; 468: 927-932Crossref PubMed Scopus (321) Google Scholar). It is yet unknown whether this unique chromatin signature can foster transcription activation or repression. Further investigation of the status of other histone modifications at TRIM24 target loci will be informative. TRIM24 is not the only gem uncovered in this study. CBFA2T2, another novel protein in the OSN network was also identified. CBFA2T2 was recently discovered as being a PRDM14 interacting protein, involved in stabilizing OSN and PRDM14 on chromatin and contributing to pluripotency and germline development (Tu et al., 2016Tu S. Narendra V. Yamaji M. Vidal S.E. Rojas L.A. Wang X. Kim S.Y. Garcia B.A. Tuschl T. Stadtfeld M. Reinberg D. Nature. 2016; 534: 387-390Crossref PubMed Scopus (48) Google Scholar). This co-repressor does not bind to DNA directly, but its presence on chromatin is mediated through its interaction with PRDM14 and, most importantly, CBFA2T2 is necessary for the retention of associated pluripotent transcription factors, PRDM14 and OCT4, on chromatin. A knockin ESC line that harbors a CBFA2T2 mutant that lacks the capacity to form tetramers but retains PRDM14 binding activity is defective in chromatin binding and phenocopies the Cbfa2t2 knockout (KO) ESCs. This observation reinforces the role of this co-repressor in providing a scaffold for chromatin binding of such pluripotent factors and suggests an important function in regulating the off rate of OSN association with chromatin. We envision that ChIP-SICAP will be extensively used as a valuable first step toward identifying factors associated with chromatin. A streamlined scheme entailing ChIP-SICAP, followed by genomic and biological functional assays, will no doubt uncover more compelling factors, fleshing out the chromatin milieu. Another implication related to these studies, as implied above, is the issue of chromatin residence time. To bind chromatin is not enough; sufficient residence time might be needed for those factors to have a meaningful role in the transcription outcome. The recently designed single-cell, single-molecule, high-resolution imaging methodology will provide quantitative information, such as chromatin binding dynamics and residence time (Chen et al., 2014Chen J. Zhang Z. Li L. Chen B.C. Revyakin A. Hajj B. Legant W. Dahan M. Lionnet T. Betzig E. et al.Cell. 2014; 156: 1274-1285Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Finally, as ChIP-MS cannot dissect direct/indirect interactions, other biochemical and biophysical techniques are still needed to address this issue. Nonetheless, the field will no doubt benefit from this new technique that isolates chromatin for interrogation. We thank Dr. Lynne Vales for her invaluable comments. We regret not being able to cite all of the relevant references due to space limitations. Expanding the Circuitry of Pluripotency by Selective Isolation of Chromatin-Associated ProteinsRafiee et al.Molecular CellOctober 20, 2016In BriefRafiee et al. introduce ChIP-SICAP as a novel method to identify chromatin-bound proteins that colocalize with a bait protein on DNA. Applied to Oct4, Sox2, and Nanog in ESCs, they identify Trim24 as a novel protein that is functionally involved in the pluripotency network by promoting cellular reprogramming. Full-Text PDF Open Access