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Flow CytometryPublications

Cell type-specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP.
Bonn, S., Zinzen, R.P., Perez-Gonzalez, A., Riddell, A., Gavin, A.C. & Furlong, E.E.
Nat Protoc. 2012 Apr 26;7(5):978-94. doi: 10.1038/nprot.2012.049.
This protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. Embryos expressing a cell type-specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using FACS on the basis of the cell type-specific nuclear marker. Tissue-specific chromatin is extracted, sheared by sonication and used for ChIP-seq or other analyses. The key advantages of this method are the covalent cross-linking before embryo dissociation, which preserves the transcriptional context, and the use of FACS of nuclei, yielding very high purity. The protocol has been optimized for Drosophila, but with minor modifications should be applicable to any model system. The full protocol, including sorting, immunoprecipitation and generation of sequencing libraries, can be completed within 5 d.
Europe PMC

Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development.
Bonn, S., Zinzen, R.P., Girardot, C., Gustafson, E.H., Perez-Gonzalez, A., Delhomme, N., Ghavi-Helm, Y., Wilczynski, B., Riddell, A. & Furlong, E.E.
Nat Genet. 2012 Jan 8;44(2):148-56. doi: 10.1038/ng.1064.
Chromatin modifications are associated with many aspects of gene expression, yet their role in cellular transitions during development remains elusive. Here, we use a new approach to obtain cell type-specific information on chromatin state and RNA polymerase II (Pol II) occupancy within the multicellular Drosophila melanogaster embryo. We directly assessed the relationship between chromatin modifications and the spatio-temporal activity of enhancers. Rather than having a unique chromatin state, active developmental enhancers show heterogeneous histone modifications and Pol II occupancy. Despite this complexity, combined chromatin signatures and Pol II presence are sufficient to predict enhancer activity de novo. Pol II recruitment is highly predictive of the timing of enhancer activity and seems dependent on the timing and location of transcription factor binding. Chromatin modifications typically demarcate large regulatory regions encompassing multiple enhancers, whereas local changes in nucleosome positioning and Pol II occupancy delineate single active enhancers. This cell type-specific view identifies dynamic enhancer usage, an essential step in deciphering developmental networks.
Europe PMC

Regulatory T cells contribute to diabetes protection in lipopolysaccharide-treated non-obese diabetic mice.
Caramalho, I., Rodrigues-Duarte, L., Perez, A., Zelenay, S., Penha-Goncalves, C. & Demengeot, J.
Scand J Immunol. 2011 Dec;74(6):585-95. doi: 10.1111/j.1365-3083.2011.02627.x.
It is well established that viral, parasitic or bacterial infections can prevent type 1 diabetes (T1D) occurrence in non-obese diabetic (NOD) mice. On the other hand, defects in CD4(+) Regulatory T cell (Treg) numbers and/or function contribute to T1D aetiology in NOD mice and in humans. In this work, we formally tested whether the protective role of the bacterial product lipopolysaccharide (LPS) on diabetes incidence results from enhanced Treg activity. We first report that weekly administration of LPS to young prediabetic NOD mice, presenting or not insulitis at the time of treatment, afforded full protection from diabetes. Taking advantage from the high but incomplete penetrance of diabetes in NOD mice raised in specific pathogen free (SPF) conditions we compared untreated disease-free old animals with gender- and age-matched LPS-treated mice. Histological and flow cytometry analysis indicated that LPS treatment did not prevent islet infiltration or priming of diabetogenic T cells but increased Foxp3(+) and CD103(+) Treg frequency and numbers. By performing adoptive transfer experiments into alymphoid NOD/SCID recipients, we further demonstrated that CD25(+) cells from LPS-treated NOD mice, but not from naturally protected animals, maintained diabetogenic cells at check. Our study suggests that T cell regulation represents a cellular mechanism to explain the 'hygiene hypothesis' and reinforces the notion that immune activity consolidates dominant tolerance.
Europe PMC

Sphingosine kinase-1 (SphK-1) regulates Mycobacterium smegmatis infection in macrophages.
Prakash, H., Luth, A., Grinkina, N., Holzer, D., Wadgaonkar, R., Gonzalez, A.P., Anes, E. & Kleuser, B.
PLoS One. 2010 May 17;5(5):e10657. doi: 10.1371/journal.pone.0010657.
Sphingosine kinase-1 is known to mediate Mycobacterium smegmatis induced inflammatory responses in macrophages, but its role in controlling infection has not been reported to date. We aimed to unravel the significance of SphK-1 in controlling M. smegmatis infection in RAW 264.7 macrophages. Our results demonstrated for the first time that selective inhibition of SphK-1 by either D, L threo dihydrosphingosine (DHS; a competitive inhibitor of Sphk-1) or Sphk-1 siRNA rendered RAW macrophages sensitive to M. smegmatis infection. This was due to the reduction in the expression of iNOs, p38, pp-38, late phagosomal marker, LAMP-2 and stabilization of the RelA (pp-65) subunit of NF-kappaB. This led to a reduction in the generation of NO and secretion of TNF-alpha in infected macrophages. Congruently, overexpression of SphK-1 conferred resistance in macrophages to infection which was due to enhancement in the generation of NO and expression of iNOs, pp38 and LAMP-2. In addition, our results also unraveled a novel regulation of p38MAPK by SphK-1 during M. smegmatis infection and generation of NO in macrophages. Enhanced NO generation and expression of iNOs in SphK-1++ infected macrophages demonstrated their M-1(bright) phenotype of these macrophages. These findings thus suggested a novel antimycobacterial role of SphK-1 in macrophages.
Europe PMC