CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair

ARTICLE https://doi.org/10.1038/s41467-020-17955-2 OPEN CTCF-mediated chromatin looping in EGR2 regulation and SUZ12 recruitment critical for peripheral myelination and repair 1234567890():,; Jincheng Wang 1,4, Jiajia Wang2,4, Lijun Yang2,4, Chuntao Zhao2, Laiman Natalie Wu2, Lingli Xu2, Feng Zhang2, Qinjie Weng 1 ✉, Michael Wegner 3 & Q. Richard Lu 2 ✉ Chromatin organization is critical for cell growth, differentiation, and disease development, however, its functions in peripheral myelination and myelin repair remain elusive. In this report, we demonstrate that the CCCTC-binding factor (CTCF), a crucial chromatin organizer, is essential for Schwann cell myelination and myelin regeneration after nerve injury. Inhibition of CTCF or its deletion blocks Schwann cell differentiation at the pro-myelinating stage, whereas overexpression of CTCF promotes the myelination program. We find that CTCF establishes chromatin interaction loops between enhancer and promoter regulatory elements and promotes expression of a key pro-myelinogenic factor EGR2. In addition, CTCF interacts with SUZ12, a component of polycomb-repressive-complex 2 (PRC2), to repress the transcriptional program associated with negative regulation of Schwann cell maturation. Together, our findings reveal a dual role of CTCF-dependent chromatin organization in promoting myelinogenic programs and recruiting chromatin-repressive complexes to block Schwann cell differentiation inhibitors to control peripheral myelination and repair. 1 Center for Drug Safety Evaluation and Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China. 2 Department of Pediatrics, Brain Tumor Center, Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA. 3 Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. 4These authors contributed equally: Jincheng Wang, Jiajia Wang, Lijun Yang. ✉email: ; NATURE COMMUNICATIONS | (2020)11:4133 | https://doi.org/10.1038/s41467-020-17955-2 | www.nature.com/naturecommunications 1 ARTICLE H NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17955-2 igh-order chromatin organization and remodeling are critical for fundamental biological processes1–3. Local chromatin environments that modulate recruitment of transcriptional complexes to regulatory elements are highly dynamic and depend on stage- or cell-type-specific nucleosome positions or chromatin looping1–3. The chromatin reorganization process enables long-range interactions such as those between promoters and enhancers that activate gene transcription. In addition, insulator-mediated contacts can organize the genome into functionally distinct domains1–3. Defects in chromatin structural organization or looping can cause aberrant transcriptional regulation, leading to various diseases including intellectual disabilities, cancer, and aging4–6. Schwann cells (SCs) are myelinating glia in the peripheral nervous system (PNS) that form myelin sheaths around axons to optimize the saltatory nerve conduction. Defects in SCs lead to various peripheral neuropathies including motor and sensory disabilities7,8. SC-lineage development includes the specification of neural crest cells to SC precursors that give rise to immature SCs, which further differentiate into mature myelinating SCs9. The process of SC development is regulated by various intrinsic and extrinsic cues. Among intrinsic factors, transcriptional regulators such as SOX10, OCT6 (a.k.a. POU3F1), and EGR2 (a.k.a. KROX20) are required for sequential progression from immature to promyelinating SCs, and eventually into myelinating SCs10–12. SC development is coordinated by a hierarchy of transcriptional regulators with a main axis that SOX10 activates OCT6, and then cooperates with OCT6 to induce EGR2 expression for SC maturation11–13. EGR2 takes a center stage for myelinogenesis by activating myelin genes, such as Mpz, Pmp22, and Mbp14,15. The negative regulatory cues that inhibit SC myelination include NOTCH, WNT, and SOX2 pathways12,16,17. How chromatin reorganizes to promote expression of promyelin cues while preventing the differentiation-inhibitory events during SC myelination has not been determined. CCCTC-binding factor (CTCF) is one of the most critical organizers for the high-order chromatin structure that enables long-range chromatin interactions3,18,19. Accumulating evidence suggests that CTCF mediates extensive crosstalk between promoters and distant regulatory elements and regulates local balance between active and repressive chromatin marks, therefore ensuring proper transcription levels during various biological processes3,18,19. CTCF can not only mediate long-range chromatin looping and modulate three-dimensional genomic architecture to regulate cell-type-specific transcriptional programs20–22, but also define boundaries between chromosomal topological associating domains (TADs)23,24. To date, the function of CTCF-dependent long-range chromatin interactions and looping in peripheral myelination and regeneration has not been defined. In addition, given its ubiquitous role in gene regulation by CTCF, whether CTCF has a temporally specific role during SC myelination remains elusive. The specific local genomic architecture also depends on histone modifications, DNA modification patterns, and nucleosome positioning or accessibility to maintain the proper conformation for transcription25. Histone modifying enzymes such as the deacetylases HDAC1/2 and polycomb-repressive complex 2 (PRC2) modulate chromatin states to regulate the transcriptional program necessary for myelination and remyelination in the PNS26–28. At present, however, it is unknown how chromatin dynamics coordinate histone modifications to control SC myelination programs. Here, we demonstrate a critical role of CTCF-dependent chromatin reorganization during SC differentiation from their immature precursors and in remyelination after peripheral nerve injury. We show that CTCF interacts with and recruits SUZ12 to 2 suppress SC differentiation-inhibitory pathways. Furthermore, we find that CTCF regulates promyelination transcriptional programs at least in part by establishing an interaction between promoter and enhancer elements of the locus of Egr2, a key regulatory gene for SC myelination12. Thus, our data demonstrate a temporally specific function of the chromatin organizer CTCF for SC differentiation by modulating chromatin organization and epigenetic programs to control peripheral myelination. Results Upregulation of CTCF expression during SC differentiation. To investigate the expression pattern of CTCF in proliferative and differentiated SCs, we treated rat SCs with cAMP to promote their differentiation in vitro. Expression of mature SC markers such as EGR2, MBP, and MPZ increased during differentiation. Strikingly, CTCF protein and mRNA expression levels w (...truncated)