Csf2 and Ptgs2 Epigenetic Dysregulation in Diabetes-prone Bicongenic B6.NODC11bxC1tb Mice.

In Type 1 diabetic (T1D) human monocytes, STAT5 aberrantly binds to epigenetic regulatory sites of two proinflammatory genes, CSF2 (encoding granulocyte-macrophage colony-stimulating factor) and PTGS2 (encoding prostaglandin synthase 2/cyclooxygenase 2). Bicongenic B6.NOD C11bxC1tb mice re-create this phenotype of T1D monocytes with only two nonobese diabetic (NOD) Idd subloci (130.8 Mb-149.7 Mb, of Idd5 on Chr 1 and 32.08-53.85 Mb of Idd4.3 on Chr11) on C57BL/6 genetic background. These two Idd loci interact through STAT5 binding at upstream regulatory regions affecting Csf2 (Chr 11) and Ptgs2 (Chr 1) expression. B6.NODC11bxC1tb mice exhibited hyperglycemia and immune destruction of pancreatic islets between 8 and 30 weeks of age, with 12%-22% penetrance. Thus, B6.NODC11bxC1tb mice embody NOD epigenetic dysregulation of gene expression in myeloid cells, and this defect appears to be sufficient to impart genetic susceptibility to diabetes in an otherwise genetically nonautoimmune mouse.

Persistent STAT5 phosphorylation and epigenetic dysregulation of GM-CSF and PGS2/COX2 expression in Type 1 diabetic human monocytes.

STAT5 proteins are adaptor proteins for histone acetylation enzymes. Histone acetylation at promoter and enhancer chromosomal regions opens the chromatin and allows access of transcription enzymes to specific genes in rapid response cell signals, such as in inflammation. Histone acetylation-mediated gene regulation is involved in expression of 2 key inflammatory response genes: CSF2, encoding granulocyte-macrophage colony stimulating factor (GM-CSF), and PTGS2, encoding prostaglandin synthase 2/cyclooxygenase 2 (PGS2/COX2). Prolonged CSF2 expression, high GM-CSF production, and GM-CSF activation of PTGS2 gene expression all are seen in type 1 diabetes (T1D) monocytes. Persistent phosphorylation activation of monocyte STAT5 (STAT5Ptyr) is also found in individuals with or at-risk for T1D. To examine whether elevated T1D monocyte STAT5Ptyr may be associated with aberrant inflammatory gene expression in T1D, blood monocytes from non-autoimmune controls and T1D patients were analyzed by flow cytometry for STAT5Ptyr activation, and by chromatin immuno-precipitation (ChIP) analyses for STAT5Ptyr's ability to bind at CSF2 and PTGS2 regulatory sites in association with histone acetylation. In unstimulated monocytes, STAT5Ptyr was elevated in 59.65% of T1D, but only 2.44% of control subjects (p<0.0001). Increased STAT5Ptyr correlated with T1D disease duration (p = 0.0030, r(2) = 0.0784). Unstimulated (p = 0.140) and GM-CSF-stimulated (p = 0.0485) T1D monocytes, had greater STAT5Ptyr binding to epigenetic regulatory sites upstream of CSF2 than control monocytes. Increased STAT5Ptyr binding in T1D monocytes was concurrent with binding at these sites of STAT6Ptyr (p = 0.0283), CBP/P300 histone acetylase, acetylated histones H3, SMRT/NCoR histone deacetylase (p = 0.0040), and RNA Polymerase II (p = 0.0040). Our study indicates that in T1D monocytes, STAT5Ptyr activation is significantly higher and that STAT5Ptyr is found bound to CSF2 promoter and PTGS2 enhancer regions coincident with histone acetylation and RNA polymerase II. These findings suggest that the persistent activation of STAT5 by GM-CSF may be involved in altering the epigenetic regulation of these inflammatory response genes in T1D monocytes.

An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier.

Specific proteins are concentrated within primary cilia, whereas others remain excluded. To understand the mechanistic basis of entry into cilia, we developed an in vitro assay using cells in which the plasma membrane was permeabilized, but the ciliary membrane was left intact. Using a diffusion-to-capture system and quantitative analysis, we find that proteins >9 nm in diameter (∼100 kD) are restricted from entering cilia, and we confirm these findings in vivo. Interference with the nuclear pore complex (NPC) or the actin cytoskeleton in permeabilized cells demonstrated that the ciliary diffusion barrier is mechanistically distinct from those of the NPC or the axon initial segment. Moreover, applying a mass transport model to this system revealed diffusion coefficients for soluble and membrane proteins within cilia that are compatible with rapid exploration of the ciliary space in the absence of active transport. Our results indicate that large proteins require active transport for entry into cilia but not necessarily for movement inside cilia.