MOESM1 of Dynamic bimodal changes in CpG and non-CpG methylation genome-wide upon CGGBP1 loss-of-function

Additional file 1. A total of Tables S1 to S11 and Figures S1 to S7 with legends, details of methods and additional references are contained in the combined additional data file

Growth signals employ CGGBP1 to suppress transcription of Alu-SINEs

<p>CGGBP1 (CGG triplet repeat-binding protein 1) regulates cell proliferation, stress response, cytokinesis, telomeric integrity and transcription. It could affect these processes by modulating target gene expression under different conditions. Identification of CGGBP1-target genes and their regulation could reveal how a transcription regulator affects such diverse cellular processes. Here we describe the mechanisms of differential gene expression regulation by CGGBP1 in quiescent or growing cells. By studying global gene expression patterns and genome-wide DNA-binding patterns of CGGBP1, we show that a possible mechanism through which it affects the expression of RNA Pol II-transcribed genes in trans depends on Alu RNA. We also show that it regulates Alu transcription in cis by binding to Alu promoter. Our results also indicate that potential phosphorylation of CGGBP1 upon growth stimulation facilitates its nuclear retention, Alu-binding and dislodging of RNA Pol III therefrom. These findings provide insights into how Alu transcription is regulated in response to growth signals.</p

Summary of patient characteristics and treatment received.

<p>OII = oligodendroglioma grade II, AII = astrocytoma grade II, Primary GBM = primary glioblastoma, F = female, M = male.</p

MC distribution in the mouse RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced brain tumors.

<p>(<b>A</b>) Immunofluorescence staining for endothelial cell marker CD31 and MC tryptase mMCP-6 in <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse brain tumors revealed perivascular localization of MCs. Lower panel: quantification of perivascular MCs in mouse brain tumors revealed about 50% in the corresponding objective fields with no difference between <i>Ntv-a Arf−/−</i> (n = 5) and <i>Gtv-a Arf−/−</i> (n = 5). Error bars show SD. Scale bar = 50 µM. (<b>B</b>) Immunohistochemical analysis for SCF expression revealing marked expression of SCF in the glioma vascular structures in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mice (indicated by blue arrows). Expression of SCF was also observed in MC granules (indicated by red arrows). Lower panel: quantification of total absolute intensity signal for SCF revealed statistically significant difference between tumor and nontumor areas of the objective fields in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse brain tumors (* p<0.05). Error bars show SD. Scale bar = 50 µM.</p

CXCL12 and CXCR4 expression in mouse RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced gliomas.

<p>Immunofluorescence staining for CXCL12 and CXCR4 was performed in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas. The quantification of intensity signal for CXCL12 (lower left panel) and CXCR4 (lower right panel) revealed statistically significant difference between tumor and nontumor areas of the objective fields in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse brain tumors (* p<0.05). Error bars show SD. Scale bar = 50 µM.</p

Accumulation of MCs in RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced tumors from <i>Ntv-a Ink4a−/−</i>, <i>Ntv-a Arf−/−</i>, <i>Gtv-a Ink4a−/−</i> and <i>Gtv-a Arf−/−</i> mice.

<p>(<b>A</b>) Chloroacetate esterase (CE) and H&E-stained <i>Ntv-a Ink4a−/−</i> and <i>Ntv-a Arf−/−</i> tumors. Arrows in left panels indicate MCs. Scale bar = 100 µM. (<b>B</b>) Chloroacetate esterase (CE)- and H&E-stained <i>Gtv-a Ink4a−/−</i> and <i>Gtv-a Arf−/−</i> tumors. Arrows indicate MCs. (<b>C</b>) Quantification of MCs in both non-tumor MHb (MHb, − tumor), cancerous MHb (MHb, + tumor) and in the tumor area (tumor) of mouse glioma samples. <i>Ntv-a Ink4a−/−</i> (MHb − tumor, n = 6; MHb + tumor, n = 12; tumor, n = 12), <i>Ntv-a Arf−/−</i> (MHb − tumor, n = 6; MHb + tumor, n = 12; tumor, n = 12), <i>Gtv-a Ink4a−/−</i> (MHb − tumor, n = 6; MHb + tumor, n = 2; tumor, n = 2), <i>Gtv-a Arf−/−</i> (MHb − tumor, n = 6; MHb + tumor, n = 12; tumor, n = 12) revealing statistically significant difference between all compared groups for <i>Arf</i> deficient mice (** p<0.01, *** p<0.001). Error bars show SD. (<b>D</b>) MC carboxypeptidase A (MC-CPA)- and mMCP-6 positive MCs from the <i>Ntv-a Arf−/−</i> tumor.</p

MC infiltration of human gliomas.

<p>(<b>A</b>) Immunohistochemical analysis of human MC tryptase (hTRS) in human low-grade gliomas (grade II, n = 8) and glioblastomas multiforme (GBM) (grade IV, n = 10). Right panel: quantification of MCs. Error bars show SD, *** p<0.001. Scale bar = 50 µM. (<b>B</b>) Immunofluorescence staining for CXCL12 and CXCR4 in human GBMs. Scale bar = 50 µM. (<b>C</b>) Immunofluorescence staining for CXCR4 and hTPS in human GBMs displayed co-expression of CXCR4 and hTPS. Scale bar = 25 µM. The inset represents a MC with co-localization of CXCR4 and hTPS at the single-cell level where maximum intensity projection of z-stack confocal images was applied.</p

Demonstration of MC migration toward glioma-conditioned medium.

<p>Co-expression of CXCL12 and CD31 in mouse RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced gliomas. (<b>A</b>) Trans-well assay using CXCL12-neutralizing antibodies revealed statistically significant decreased migration of BMMCs towards glioma-conditioned medium. (<b>B</b>) Trans-well assay using antibodies to block CXCR4 receptor expressed on the BMMC surface demonstrated statistically significant decrease in BMMC migration towards glioma-conditioned medium. Appropriate isotype controls were used (* p<0.05, ** p<0.01). (<b>C</b>) Immunofluorescence staining demonstrated co-localization of CXCL12 and CD31 in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas. Image analysis revealed an average of 16% and 14% of total CXCL12-positive cells in <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas respectively were co-localized with CD31-positive endothelial cells. However, almost 94% and 82% of total CD31-positive cells in <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas correspondingly were co-localized with CXCL12-positive staining. No statistical difference between <i>Ntv-a-</i> and <i>Gtv-a</i> lines was found. Scale bar = 50 µM. Error bars show SD.</p

Co-expression of CXCR4 and mMCP-6 by MCs in mouse RCAS-<i>KRas</i>+RCAS-<i>Akt</i> induced gliomas.

<p>(<b>A</b>) Immunofluorescence staining for CXCR4 and mMCP-6 was performed in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas, demonstrating co-expression of CXCR4 and mMCP-6. Scale bar = 50 µM. (<b>B</b>) Immunofluorescence staining, demonstrated co-localization of CXCR4 and mMCP-6 at the single-cell level in both <i>Ntv-a Arf−/−</i> and <i>Gtv-a Arf−/−</i> mouse gliomas. Maximum intensity projection of z-stack confocal images was applied. Scale bar = 20 µM. (<b>C</b>) The quantification of MCs in mouse brain tumors revealed about 90% to be CXCR4-positive in the corresponding objective fields with no difference between <i>Ntv-a Arf−/−</i> (n = 5) and <i>Gtv-a Arf−/−</i> (n = 5). Error bars show SD.</p

Expression of genes involved in Ca²⁺ signaling in GICs correlating with a NSC-associated transcriptome.

<p>(A) GIC lines rank ordered in relation to NSC lines (second component in a principle component analysis of microarray based mRNA expression data from Pollard et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115698#pone.0115698-Pollard1" target="_blank">[11]</a>, where the first component segregates NSCs and GICs from normal brain tissue). GliNS1 is derived from the G144ED line in the Pollard et al study. (B) Re-analysis of transcriptome profiles in Pollard et al comparing GICs to NSCs indicating a NSC-proximal cluster of stem-like GICs with high similarity to NSCs, sharing e.g. SOX2 and BLBP expression. NSC-distal GIC lines in contrast expressed microglia markers, such as CXCL2, CXCL5 and CCL20. (C) De novo RNA sequencing analysis and pairwise comparisons of NSCs and three individual GIC lines (GliNS1, G179NS and G166NS) showed that NSCs expressed a larger number of genes with 10-fold higher gene expression compared to all GIC lines. (D) Pairwise comparisons of NSCs to the GIC lines GliNS1, G179NS and G166NS, individually. Gene enrichment and gene ontology analysis of sequencing based transcriptome profiles, identified an enrichment of Ca²⁺ signaling genes in NSCs, which increased with rank order distal to NSC in pairwise comparisons. (E) Pairwise comparisons of the NSC-proximal (GliNS1) and NSC-distal (G166NS) GICs. Gene enrichment and gene ontology analysis suggested a switch in Ca²⁺ permeable channels to Ca²⁺ binding genes in the NSC-distal GIC line (upper boxes). In volcano plot, gene names in green denote ion channel/pump/transporter related genes, whereas gene names in purple denote Ca²⁺ binding proteins genes. The volcano plot of the comparison of NSC-proximal and NSC-distal GICs revealed a larger number of ion channels expressed in the NSC-proximal GIC (GliNS1).</p