Eda-activated RelB recruits an SWI/SNF (BAF) chromatin-remodeling complex and initiates gene transcription in skin appendage formation Jian Simaa,1,2, Zhijiang Yana,1, Yaohui Chena, Elin Lehrmanna, Yongqing Zhanga, Ramaiah Nagarajaa, Weidong Wanga, Zhong Wangb, and David Schlessingera,2 aLaboratory of Genetics and Genomics, National Institute on Aging/NIH-Intramural Research Program, Baltimore, MD 21224; and bDepartment of Cardiac Surgery, Cardiovascular Research Center, University of Michigan, Ann Arbor, MI 48109 Edited by Elaine Fuchs, The Rockefeller University, New York, NY, and approved June 28, 2018 (received for review January 23, 2018) Ectodysplasin A (Eda) signaling activates NF-κB during skin ap- during organ development induce distinct BAF complexes to pendage formation, but how Eda controls specific gene transcrip- modulate gene expression. tion remains unclear. Here, we find that Eda triggers the formation Here, we report that skin-specific Eda signaling triggers the for- of an NF-κB–associated SWI/SNF (BAF) complex in which p50/RelB re- mation of a large BAF-containing complex that includes a BAF cruits a linker protein, Tfg, that interacts with BAF45d in the BAF com- complex, an NF-κB dimer of p50/RelB, and a specific linker pro- plex. We further reveal that Tfg is initially induced by Eda-mediated tein, Tfg (TRK-fusion gene). Thus, Eda/NF-κB signaling operates RelB activation and then bridges RelB and BAF for subsequent gene through a BAF complex to regulate specific gene expression in regulation. The BAF component BAF250a is particularly up-regulated in organ development, which may exemplify a more general paradigm skin appendages, and epidermal knockout of BAF250a impairs skin for gene-specific regulation in many other systems. appendage development, resulting in phenotypes similar to those of Eda-deficient mouse models. Transcription profiling identifies several Results target genes regulated by Eda, RelB, and BAF. Notably, RelB and the Eda Signaling Triggers Linkage of RelB to a BAF Complex. We hy- BAF complex are indispensable for transcription of Eda target genes, pothesized that tissue-specific Eda signaling might require NF- and both BAF complex and Eda signaling are required to open chro- κB activation to specify gene targets and a chromatin-remodeling matin of Eda targets. Our studies thus suggest that Eda initiates a complex to open chromatin at those sites. To test this hypothesis, signaling cascade and recruits a BAF complex to specific gene loci to we designed a workflow for the purification of BAF complexes facilitate transcription during organogenesis. from HaCaT keratinocytes before and after Eda stimulation (Fig. 1A), using a successful purification method described pre- ectodysplasin | RelB | SWI/SNF | BAF | chromatin remodeling viously (13, 19). We first generated a derivative of the HaCaT human keratinocyte stable cell line that expresses the Eda re- + ene regulation in development is orchestrated by signaling ceptor (Edar ). Immunoblotting demonstrated the expected ex- Gpathways, transcription factors (TFs), and epigenetic changes pression of Edar, and when conditioned medium (CM) containing in accessibility of genomic loci (1–3). In mammals, skin append- ages include hair, teeth, and several exocrine glands, and their Significance exquisite differentiation requires the ectodysplasin A (Eda) signaling pathway. Mutations in this pathway result in ectoder- mal dysplasia (EDA), characterized by defective formation of Specific gene regulation in organ development remains poorly understood. Here, we report that skin-specific ectodysplasin A hair follicles (HFs), sweat glands (SWs), teeth and Meibomian (Eda) signaling triggers the formation of a protein complex glands (MGs) (4). Ectodermal dysplasia is an X-linked heredi- that includes a BAF complex, an NF-kB dimer of p50/RelB, and tary genetic disorder. Notably, the X-linked skin phenotype was aspecific“linker” protein, Tfg. We further find that Eda- first described by Charles Darwin in 1875 (5). More than 100 y activated RelB recruits BAF complex to specific gene loci for later, people have identified three genes from patients with ec- local chromatin remodeling of target genes. These findings todermal dysplasia, Eda, Edar, and Edaradd, which constitute a may exemplify a more general model for specific gene regu- – – specific TNF ligand receptor adaptor family that is restricted to lation involving unique ligand–receptor complexes leading to skin appendages (6). Edar, like other TNF family receptors, selective activation of transcription factors, specific linkers, and κ activates NF- B (7). However, Eda activates a gene-expression tissue-specific chromatin-remodeling complex. profile that is distinct from that stimulated by classic TNF sig- naling in the immune system (8). How Eda activates a selective Author contributions: J.S., Z.Y., and D.S. designed research; J.S., Y.C., and E.L. performed cohort of NF-κB–mediated genes remains an open question. research; Z.Y., R.N., W.W., and Z.W. contributed new reagents/analytic tools; J.S., Y.Z., and One feature of selective activation of gene expression is genome D.S. analyzed data; and J.S. and D.S. wrote the paper. accessibility to TFs at specific sites in chromatin (9). Accessibility The authors declare no conflict of interest. of genomic DNA can be epigenetically modulated by several This article is a PNAS Direct Submission. chromatin-remodeling complexes, including the SWI/SNF (BAF) This open access article is distributed under Creative Commons Attribution-NonCommercial- complex (10). In mammals, BAF complexes contain 12–15 subunits NoDerivatives License 4.0 (CC BY-NC-ND). (11). The assembly of complexes containing mutually exclusive Data deposition: The data reported in this paper have been deposited in the Gene Ex- pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. subunits is highly divergent and may function in a cell-specific GSE97783). manner (12). Two distinct BAF complexes have been identified, 1J.S. and Z.Y. contributed equally to this work. BAF (13) and polybromo-associated BAF (PBAF) (14). In addi- 2To whom correspondence may be addressed. Email: [email protected] or tion, some tissue-specific BAFs have been reported, including em- [email protected]. bryonic stem cell BAF (esBAF) (15, 16), neuron-specific BAF This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. BIOLOGY (nBAF) (17), and l leukemic BAF (eukBAF) (18). However, it has 1073/pnas.1800930115/-/DCSupplemental. DEVELOPMENTAL not been determined whether tissue- or stage-specific cell signals Published online July 23, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1800930115 PNAS | August 7, 2018 | vol. 115 | no. 32 | 8173–8178 Downloaded by guest on September 25, 2021 Notably, an additional protein, Tfg, was coprecipitated, that had A B Superose 6 gel filtration (Edar-) cells (Edar+) cells been suggested as possibly involved in NF-κB action (21) but had 1719 21 2325 27 29 31 33 35 3739 4143 not been previously found in any BAF complex. It appeared in + or BAF250a both BAF170 and RelB immunoprecipitates from Edar cells (SI Eda-A1 CM BRG1 Appendix, Table S1), and the gel-filtration profile of Tfg was Nuclear protein extraction similar to that of RelB (Fig. 1B). Fully ECL-exposed immuno- BAF170 Edar (+) blots excluded the possibility that the absence of Tfg and RelB Superose 6 gel filtration RelB bands in the higher molecular weight position in the gel filtration − Eda-A1 (+) Tfg was due to their lower protein levels in Edar cells (SI Appendix, Affinity purification of Fig. S1B). BAF170 and RelB IP-Western blotting using total NE SWI/SNF fraction RelB further verified that the BAF complex, but not PBAF, had in- deed interacted with the NF-κB dimer RelB/p50 and did so only Mass spectral analysis of Edar (-) Tfg SWI/SNF components after activation of Eda signaling (Fig. 1D). In further urea de- Void 670 470 Kd naturation experiments, either p50/RelB or Tfg was still stable in its association with the BAF complex after treatment with more CDBAF170 IP RelB IP BAF170 IP )+( )+(radE than 2 M urea (SI Appendix, Fig. S1C). To rule out the possibility )-( )-( κ Kd t t that NF- B may indirectly associate with the BAF complex rad radE rad BAF250 u u + p p 250 GgI G through DNA, IP from NE of Edar cells was performed in the nI nI gI Eda-A1 (+) BRG1/Brm E E 150 BAF170 RelB presence of ethidium bromide (EtBr), a DNA-interacting drug BAF155 BAF170 that dissociates proteins from DNA. The amount of RelB in the 100 BRG1 BAF170 immunoprecipitate was not affected by the presence of 75 BAF60 BAF250a EtBr (SI Appendix, Fig. S1D), indicating that RelB complexes BAF57 with SWI/SNF through protein interaction. SNF5 BAF200 50 Actin BAF180 Direct protein–protein binding experiments in vitro further BAF45 p50 37 explored the possibility that Tfg might link the BAF complex to p52 NF-κB. We used a cell-free protein synthesis system to produce RelA individual proteins, including 10 components of the RelB- 25 Tfg associated BAF complex, five NF-kBs, and Tfg, detected by immunoblotting (SI Appendix, Fig. S1 E–G). Gel silver staining Fig. 1. SWI/SNF (BAF) complexes with NF-κB upon Eda signaling in kerati- confirmed the production and purity of key proteins (SI Ap- nocytes. (A) Schematic overview shows the workflow of BAF complex puri- pendix, Fig. S1H). Protein–protein binding assays showed that fication. (B) Immunoblotting shows gel-filtration profiles in the NE from the Tfg directly bound only RelB among the five NF-kBs and bound indicated cells and treatments. Black rectangles indicate fractions collected BAF45d but no other tested BAF (SI Appendix, Fig. S2 A and B). for IP-MS analysis. (C) A silver-stained gel shows the BAF complexes purified + by BAF170 IP from the NE of Edar cells. (D) BAF170 (Left) and RelB (Right)IP Reverse IP-Western blotting confirmed the direct binding of Tfg with total NE was followed by immunoblotting with the indicated anti- to both RelB and BAF45d (SI Appendix, Fig.