GM-CSF induces STAT5 binding at epigenetic regulatory sites within the Csf2 promoter of non-obese diabetic (NOD) mouse myeloid cells

Abstract

Myeloid cells from non-obese diabetic (NOD) mouse and human type 1 diabetic (T1D) patients overexpress granulocyte-macrophage colony stimulation factor (GM-CSF). This overproduction prolongs the activation of signal transduction and activator of transcription 5 (STAT5) proteins, involved in GM-CSF-induced control of myeloid cell gene expression. We found that GM-CSF can regulate the binding of STAT5 on the promoter of its own gene, Csf2, within regions previously identified as sites of chromatin epigenetic modification important to the regulation of GM-CSF during myeloid differentiation and inflammation. We found multiple sequence polymorphisms within NOD mouse chromosome 11 Idd4.3 diabetes susceptibility region that alter STAT5 GAS binding sequences within the Csf2 promoter. STAT5 binding at these sites in vivo is increased significantly in GM-CSF-stimulated-bone marrow cells and in unactivated, high GM-CSF-producing macrophages from NOD mice as compared to non-autoimmune C57BL/6 mouse myeloid cells. Thus, GM-CSF overproduction by NOD myeloid cells may be perpetuating a positive epigenetic regulatory feedback on its own gene expression through its induction of STAT5 binding to its promoter. These findings suggest that aberrant STAT5 binding at epigenetic regulatory sites may contribute directly to immunopathology through cytokine-induced gene expression dysregulation that can derail myeloid differentiation and increase inflammatory responsiveness.

Figure 1. GM-CSF Production and STAT5 Phosphorylation are Aberrantly High in NOD Mouse Myeloid Cells

A. Four to five million bone marrow cells and adherence-isolated peritoneal macrophages were cultured without supplementation for 24hr at 37C/5%CO₂. Cell-free culture supernatants were then analyzed by ELISA and/or Luminex for the presence of GM-CSF. GM-CSF concentrations were normalized to pg/million plated cells for comparison. The p values listed were obtained from Mann-Whitney U test analysis of the data. Patterned bars indicate the mean GM-CSF production from NOD samples and open bars the mean of C57BL/6 samples. Error bars represent SEM. B. Ex vivo myeloid cells from NOD and C57BL/6 mice (peritoneal macrophages, peripheral blood, and bone marrow cells) were collected and fixed within 4hr of collection and then analyzed for phosphorylated STAT5 by intracellular flow cytometry [–14]. Data shown represents the percentage of gated CD11b+ cells that are also positive for phosphorylated STAT5. In macrophage and bone marrow cell analyses, 10000 events were counted, while 5000 events were collected for each run of the peripheral blood analysis. The p values listed were obtained from Mann-Whitney U test analysis of the data. Patterned bars indicate the mean %STAT5Ptyr+/CD11b+ cells detected in NOD samples and open bars the mean of C57BL/6 samples. Error bars represent SEM. The p values listed are from pair wise (Student t or Mann Whitney U) or group wise ANOVA analyses.

Figure 2. Sequence Comparisons for Csf2 Promoter Region Reveals Polymorphism in NOD

A. Genomic DNA from NOD and C57BL/6 mice were amplified in PCR using primers specific for the −181 to +10bp region upstream of the Csf2 gene [9]. PCR products run on a 5% agarose gel show a small but reproducible size variation between the two mouse strains. Data are representative of 12 runs of the analysis. B. Sequence analysis of the −3 to −969bp region upstream of the Csf2 gene in NOD and C57BL/6 mouse genomic DNA samples revealed differences in STAT5 binding sites (yellow, boxed), many half or imperfect binding sites (yellow), and a microsatellite DNA insertion (blue) in the region that has a length polymorphism between the two strains. Putative STAT5 binding site NOD polymorphisms are indicated by underlined bold, boxed sequences. * indicate sequence homology between the two strains.

Figure 3. Macrophage Chromatin Immunoprecipitation (ChIP) Analysis shows STAT5 binding within the promoter region upstream of the gene which encodes for GM-CSF, Csf2

Four million peritoneal macrophages (PMAC) from NOD and non-autoimmune C57BL/6 mice were incubated for 24hr without exogenous GM-CSF. Cells were fixed in situ, and extracted for protein-chromatin complexes as described in the Materials & Methods. Extracts divided in 4 parts and immunoprecipitated with anti-STAT5Ptyr antibodies. One hundred nanograms of the ChIP-isolated STAT5Ptyr-associated DNA were analyzed by conventional PCR/Ethidium bromide agarose gel (insert) and by SybrGreen Real Time PCR for the presence of Csf2 promoter (−181 to +10bp) DNA sequences. Key: 5P= ChIP anti-STAT5 precipitated DNA, T= total cellular DNA from unprecipitated fixed cell extracts, Ig= ChIP non-specific mouse IgG precipitated DNA, W= DNA-free water control. Patterned bars (log of mean R values) and gel represent data obtained from 3 independent runs of each strain. Error bars represent SEM.

Figure 4. ChIP Analysis of GM-CSF-Induced STAT5 Binding Upstream at Multiple sites within the Csf2 Promoter involves DNA Secondary Structure

Four million cell cultures of NOD and C56BL/6 mouse bone marrow cells (BM) were grown for 24hr in the presence (GM or G) or absence (0) of 1000U/ml GM-CSF before being fixed and extracted for ChIP analysis. Aliquots of 100ng of total DNA extracted from ChIP protein-chromatin complexes precipitated with anti-STAT5 antibodies were amplified using primers to potential epigenetic modification sites within the Csf2 promoter region [8,9]. A. Real time PCR analysis using specific primers to amplify and identify Csf2 promoter regions previously identified as epigenetic control sites for Csf2 gene expression. Unpatterned bars indicate cells without treatment (0) and hatched bars indicate cells treated with GM-CSF (G). Data representative of 2–3 sample sets. B. Real time PCR analysis using the hot start and DMSO protocol described in the Materials and Methods to remove secondary structure and the primers depicted in A to amplify and identify Csf2 promoter regions previously identified as epigenetic control sites for Csf2 gene expression. Unpatterned bars indicate cells without treatment (0) and hatched bars indicate cells treated with GM-CSF (G). Data representative of 2 (combined A–I) and 3 (Promoter −3 to −969bp) sample sets. The p values indicate one-way ANOVA analysis (above graphs) or Mann-Whitney U tests (on graph) of pairwise comparisons.