Role of the Pbrm1 subunit and the PBAF complex in Schwann cell development

www.nature.com/scientificreports OPEN Role of the Pbrm1 subunit and the PBAF complex in Schwann cell development Vanessa Polanetzki1,3, Franziska Fröb1,3, Tina Baroti1, Margit Schimmel2, Ernst R. Tamm2 & Michael Wegner1* Myelin sheath formation in the peripheral nervous system and the ensuing saltatory conduction rely on differentiated Schwann cells. We have previously shown that transition of Schwann cells from an immature into a differentiated state requires Brg1 that serves as the central energy generating subunit in two related SWI/SNF-type chromatin remodelers, the BAF and the PBAF complex. Here we used conditional deletion of Pbrm1 to selectively interfere with the PBAF complex in Schwann cells. Despite efficient loss of Pbrm1 early during lineage progression, we failed to detect any substantial alterations in the number, proliferation or survival of immature Schwann cells as well as in their rate and timing of terminal differentiation. As a consequence, postnatal myelin formation in peripheral nerves appeared normal. There were no inflammatory alterations in the nerve or other signs of a peripheral neuropathy. We conclude from our study that Pbrm1 and very likely the PBAF complex are dispensable for proper Schwann cell development and that Schwann cell defects previously observed upon Brg1 deletion are mostly attributable to altered or absent function of the BAF complex. Schwann cells represent the main population of glial cells in the peripheral nervous system (PNS) and line axons in the peripheral nerves as myelinating or non-myelinating Schwann cells. Their development from the neural crest is coordinated by combinatorially acting, continuously present or stage-specific transcription factors that form an evolving gene regulatory network in cooperation with regulatory RNAs and various classes of chromatin-modifying proteins or complexes, such as histone modifying enzymes, DNA methyltransferases and chromatin remodeling c omplexes1–4. Among chromatin remodeling complexes, several have been studied in Schwann cells. This includes the Chd4-containing NuRD complex, the Ep400/Tip60 complex and Brg1-containing complexes of the SWI/SNFtype5–7. The in vivo impact of chromatin remodelers in higher organisms is usually studied by deleting a central component of the complex, frequently the ATP-hydrolyzing, energy generating subunit. In case of the SWI/ SNF-type remodelers, we previously deleted the ATP-hydrolyzing Brg1 subunit in the late Schwann cell precursor stage and observed a radial sorting defect. Schwann cells did not activate the late stage transcription factors Oct6 and Egr2 and failed to transit from the immature into the pro-myelinating and further on into the myelinating Schwann cell s tages7. Our findings thus established an essential role of SWI/SNF-type chromatin remodelers in late stage Schwann cell development. SWI/SNF-type remodelers are intrinsically heterogeneous in their subunit composition and are frequently grouped into BAF and PBAF complexes in v ertebrates8. BAF complexes contain either Brg1 or its close relative Brm as the ATP-hydrolyzing subunit, PBAF complexes exclusively Brg1. Better than by their ATP-hydrolyzing component, both complexes can be distinguished by signature subunits that occur only in one of the complexes. As an example, Arid1a/b subunits are specific for BAF complexes. Pbrm1 (also known as Polybromo-1, BAF180 and PB1) and Arid2 are regarded as signature subunits of PBAF complexes. Pbrm1 contains six consecutive bromodomains that serve as readers for various acetylated histones, followed by a DNA-binding high-mobilitygroup domain9. Pbrm1 is thus likely a major determinant of the DNA-binding specificity and thus of fundamental properties of PBAF complexes. Its essential role is documented by frequent loss-of-function mutations in several tumors, most prominently in clear renal cell carcinomas, its function as a tumor suppressor, its essential role in the potentiation of transcriptional activation by nuclear receptors as well as severe placental and cardiac defects upon Pbrm1 loss in the e mbryo10–12. 1 Institut für Biochemie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany. 2Institut für Humananatomie und Embryologie, Universität Regensburg, Regensburg, Germany. 3These authors contributed equally: Vanessa Polanetzki and Franziska Fröb. *email: Scientific Reports | (2022) 12:2651 | https://doi.org/10.1038/s41598-022-06588-8 1 Vol.:(0123456789) www.nature.com/scientificreports/ ◂ Figure 1.  Pbrm1 occurrence in peripheral nerves and Schwann cells. (A) Co-immunohistochemistry with antibodies directed against Pbrm1 (red, left and middle panels) and Sox10 (green, middle panels) in embryonic spinal nerves (boundaries depicted by dotted lines) at E12.5, E14.5, E16.5 and E18.5 as well as postnatal sciatic nerves at P0, P7, P14, P21 and P60. Nuclei were counterstained with DAPI (blue, right panels). (B) Co-immunohistochemistry with antibodies directed against Pbrm1 (red, left and right panels) and Sox2, Oct6 and Egr2 as stage-specific Schwann cell markers (green, middle and right panels) at E14.5 and E18.5 as indicated. (C, D) Analysis of Pbrm1 expression in Schwann cells cultured under proliferating (hatched bars) and differentiation (black bars) conditions according to published RNA-Seq data (GSE101153, presented as absolute number of counts for n = 3, in C) and quantitative RT-PCR (with average normalized expression levels in proliferating cells set to 1 and shown as mean ± SEM for n = 4, in D). Statistical significance was determined by unpaired two-tailed Student’s t-test (**P ≤ 0.01). (E) Co-immunohistochemistry with antibodies directed against Pbrm1 (red) and various markers for other nerve-associated cell types (green), including fibronectin (FN), α-smooth muscle actin (Sma), Desmin, Pecam, Iba and CD3 in sciatic nerve tissue at P14 Scale bars: 10 µm (A, B, E). Considering that all essential and constitutive subunits of BAF and PBAF complexes occur in developing Schwann cells1,13,14 it is currently not clear, to what extent the previously observed defects in Schwann cell development after Brg1 deletion are due to impaired function of the Brg1-containing BAF complex as opposed to the equally Brg1-containing PBAF complex. To shed light onto the role of the PBAF complex, we here chose to delete the essential PBAF subunit Pbrm1 from Schwann cells and study their development. From the absence of major alterations in lineage progression, precursor cell survival and expansion as well as terminal differentiation and myelination, we conclude that Pbrm1 is of minor relevance in developing Schwann cells. In comparison to PBAF complexes, the Brg1-containing BAF complexes therefore appear to be the major SWI/SNF-type chromatin remodelers in Schwann cells. Results Pbrm1 expression in Schwann cells. To be able to study Pbrm1 on the protein level, we generated an antibody against amino a (...truncated)