SWI/SNF enzymes promote SOX10- mediated activation of myelin gene expression

Abstract

SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to directly activate genes that encode components of peripheral myelin.

Figure 1. Dominant negative BRM and BRG1 inhibit SOX10-mediated activation of myelin genes.

Cell lines that express dominant negative BRM or BRG1 were either infected with a pBabe control vector or pBabe-SOX10 in the presence or absence of tetracycline and then cultured in low serum media to promote differentiation. (A) Western Blot showing expression of SOX10 in cells that were cultured in the presence and absence of tetracycline and the expression of FLAG-tagged dominant negative BRM (left) in the H17 cell line and BRG1 (right) in the B22 cell line, when cells were cultured in the absence of tetracycline. Protein expression was detected from cell extracts and ERK1/2 was used as a loading control (B) Quantitative RT-PCR (qRT-PCR) of SOX10 target genes from pBabe or pBabe-SOX10 infected H17 (left) and B22 (right) cells. KROX20, MPZ, and MBP expression was normalized to expression of RPL7. The data are representative of at least four experiments and are the average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (**p<0.01).

Figure 2. Dominant negative BRG1 inhibits synergistic activation of myelin genes by SOX10 and KROX20.

Cell lines that express dominant negative BRG1 were infected with an empty vector, pBabe-SOX10, pBabe-KROX20, or pBabe-SOX10 together with pBabe-KROX20 in the presence or absence of tetracycline and then cultured in low serum media to promote differentiation. (A) Western Blot showing expression of SOX10 and KROX20 in the presence and absence of tetracycline and the expression of FLAG-tagged dominant negative BRG1 in the B22 cell line when cells were cultured in the absence of tetracycline. Protein expression was detected from nuclear extracts and a non-specific band was used as a loading control. (B) Quantitative RT-PCR (qRT-PCR) of SOX10 target genes from pBabe or pBabe-SOX10, pBabe-SOX10, pBabe-KROX20, or pBabe-SOX10 together with pBabe-Krox20 infected cells. Expression of MPZ and MBP was normalized to expression of RPL7. The data are representative of greater than three experiments and are the average of two independent experiments performed in triplicate. Standard error bars and statistical significance are shown (**p<0.01).

Figure 3. FACS analysis of differentiated B22 cells.

B22 cells were infected with an empty vector, pBabe-SOX10, pBabe-KROX20, or pBabe-SOX10 together with pBabe-KROX20 in the presence or absence of tetracycline and differentiated in low serum media. Propidium iodine stained samples were FACS sorted and analyzed using Cell Quest Pro (BD Biosciences). The data are the average of three independent experiments. Standard error bars are shown.

Figure 4. BRG1 is recruited to the MPZ promoter as a result of SOX10 mediated differentiation.

Chromatin immunoprecipitations (ChIPs) were performed with control IgG, antisera to BRG1, or an antibody to SOX10 on chromatin from B22 cells that were infected with empty vector (EV), KROX20, or SOX10 retrovirus and cultured as described in Figure 1. Enrichment at the MPZ promoter and intron is relative to control IgG and normalized to a control region, SCN2A1. There was minimal variation in the ChIP signal at the SCN2A1 region. (A) Detection of BRG1 interactions with the MPZ promoter and intron. (B) Detection of SOX10 interactions with the MPZ promoter and intron. This data are representative of at least three experiments and are the average of two independent experiments that were assayed four or more times, each in triplicate. Standard errors bars and statistical significance are shown (**p<0.01, * p<0.05).

Figure 5. SOX10 is required to recruit BRG1 to the MPZ promoter in S16 Schwann cells.

A. Cell extracts were prepared from S16 cells that were transfected with siControl or siSOX10 and subjected to Western analysis with antibodies to SOX10, KROX20, and antisera to BRG1 (The starred top band (*) in the KROX20 Western may be a non-specific band. It was not used in the quantitation of KROX20 expression shown in Figure 5B. ERK1/2 is shown as a loading control. B. Quantitation of KROX20 protein levels relative to ERK1/2 protein levels by ImageJ software. The data are the average of three independent experiments. Standard error bars and statistical significance are shown (**p<0.01). C–E. Quantitative RT-PCR (qRT-PCR) of KROX20, MPZ, and MBP mRNA levels. F–H. ChIPs were performed with control IgG, antibodies to SOX10 or KROX20, or antisera to BRG1 on chromatin from S16 cells that were transfected with siControl or siSOX10. Enrichment on the MPZ promoter and intron is relative to control IgG and normalized to a control region, Ig2a enhancer. There was minimal variation in the ChIP signal at the Ig2A enhancer. (F) Detection of SOX10 interactions with the MPZ promoter. (G) Detection of KROX20 interactions with the MPZ promoter. (H) Detection of BRG1 interactions with the MPZ promoter. The data are representative of three independent experiments that were assayed three times. Standard errors bars and statistical significance are shown (**p<0.01, *p<0.05).

Figure 6. Dominant negative BRG1 inhibits expression of SOX10 target genes in S16 Schwann cells.

S16 cells were transfected with GFP (not shown), empty vector (EV), or pBabe-dominant negative BRG1 (dnBRG1). Transfection efficiency was monitored by GFP and was determined to be approximately 30%. (A) Cell extracts were prepared and subjected to Western analysis with antibodies to the Flag epitope, SOX10, or KROX20. Tubulin is shown as a loading control. Quantitative RT-PCR (qRT-PCR) of SOX10 (B), KROX20 (C), MPZ (D), and MBP (E) from empty vector (EV) or pBabe-dnBRG1 transfected cells. Expression of each gene was normalized to that of 18S rRNA. The data are the average of three independent experiments performed in triplicate. Standard error bars are shown (**p<0.01).

Figure 7. The requirement for BRG1 during Schwann cell differentiation and myelination.

A model illustrating the stepwise requirement for BRG1 during Schwann cell differentiation and myelination based on the current study as well as two recent studies [11,30]..