Epigenetic regulation of the extrinsic oncosuppressor PTX3 gene in inflammation and cancer
Marcello Rubino1, Paolo Kunderfranco1, Gianluca Basso1, Carolina Magdalen Greco1, Fabio Pasqualini1, Simone Serio1, Massimo Roncalli1, Luigi Laghi1, Alberto Mantovani1,2, Roberto Papait1,3,a, Cecilia Garlanda1,a 1
Humanitas Clinical and Research Center, Rozzano (Milan), 20089, Italy;
2
Humanitas University of Milan, Rozzano, 20089, Italy.
3
Institute of Genetics and Biomedical Research, National Research Council, Rozzano (Milan),
20089, Italy.
Running Title: PTX3 enhancers in inflammation and cancer
a
Contact information:
Cecilia Garlanda Humanitas Clinical and Research Center Via Manzoni 113, 20089 Rozzano (Milan), Italy
[email protected] or Roberto Papait Humanitas Clinical and Research Center Via Manzoni 113, 20089 Rozzano (Milan), Italy
[email protected]
1
Supplementary figures
Supplementary Figure S1 - PTX3 regulatory regions are conserved among species A) Top: schematic view of the PTX3 locus and regions identified as enhancers; bottom: ChIP-Seq profiles for the histone markers H3K4me1 and H3K27Ac in human normal mucosa and in colorectal cancer (CRC). The peaks show the enrichment of the histone modifications compared to input DNA. Data were downloaded from GEO archive (GSE36401). B) Analysis of genome conservation among species of the PTX3 gene and putative enhancers through ECR browser. The conservation rate is calculated by the browser comparing the sequence of the PTX3 human gene with PTX3 gene in other species: fugu, frog, chicken, opossum, dog, mouse, rat and Rhesus macaque.
2
Supplementary Figure S2 - Histone modifications of PTX3 regulatory regions in 8387 and HCoEpiC cells Analysis of H3K4me1, H3K4me3, H3K9Ac, H3K27Ac and H3K27me3 histone modifications at enhancer 1, promoter and enhancer 2 by ChIP in the human 8387 cell line (A) and in human colonic epithelial cells (HCoEpiC) (B) in basal and inflammatory conditions. Regions analysed are reported in the upper part of the panels. Results are expressed as fold change relative to IgG (N = 2 experiments). *: p ≤ 0.05, **: p ≤ 0.01; Student’s t-test.
3
Supplementary Figure S3 - PTX3 enhancers and promoter are activated and silenced by specific transcription factors and complex in 8387 cells A) ChIP assay for Suz12 and EZH2 and for TAF1 and RNA Pol II in the 8387 cell line in basal and inflammatory conditions. Regions analysed are reported in the upper part of the panels. Results are expressed as fold change relative to IgG and as mean ± SEM (N = 2 experiments). *: p ≤ 0.05, **: p ≤ 0.01; Student’s t-test.
4
Supplementary Figure S4 - Effect of PTX3 SNPs on PTX3 RNA expression and enhancer 2 activation A) Analysis by RT-qPCR of PTX3 mRNA expression in human macrophages from healthy donors with ACA or GAG PTX3 haplotypes (rs2305619, rs3816527, rs1840680), associated to PTX3 enhancer 2, in basal conditions and after TNFα treatment (20 ng/ml, 4h). PTX3 mRNA expression is expressed as mean ± SEM (N = 4-5 donors for haplotypes). *: p ≤ 0.05; Student’s t-test. B) Analysis of H3K27Ac histone modifications and TAF1 peaks in enhancer 2 by ChIP in human macrophages from healthy donors with ACA or GAG PTX3 haplotypes, in basal and inflammatory conditions (TNFα 20 ng/ml, 4h). Results are expressed as fold change relative to IgG (N = 4 experiments). IgG are shown as mean percentage of enrichment relative to input *: p ≤ 0.05; Student’s t-test.
5
Supplementary Figure S5 - 5’AZA-dC treatment restores the activity of PTX3 enhancers in HCT116 cells ChIP assay for H3K4me1, H3K4me3, H3K9Ac, H3K27Ac, H3K27me3 in HCT116 cells upon treatment with TNFα and 5’AZA-dC. Regions analysed are reported in the upper part of the panels. Results are expressed as fold change relative to IgG and as mean (N = 2 experiments). *, ^, $: p ≤ 0.05, **, $$: p ≤ 0.01; ***, $$, p
