Project description:Klf4 is the zinc finger transcription factor indispensable for terminal maturation of epithelial tissues and part of group of proteins for the generation of pluripotent embryonic stem cells from differentiated tissues. No definitive in-vivo-function of Klf4 in the hematopoietic system has been established yet. We show here that Klf4 directly guides the transcriptional program for terminal differentiation of Ly-6Chigh monocytes. Klf4 suppresses transcripts of the interferon immune response and induces a proinflammatory cytokine transcriptional program. Concordantly, Klf4 hematopoietic deficiency in mice results in an enhanced Th1 but impaired Th17 immune response, and, consequently, those mice are resistant towards the induction of experimental autoimmune encephalitis (EAE). We here identify Klf4 is a transcription factor required for ontogenesis and function of Ly-6Chigh monocytes at the interface of innate and acquired immunity. The following constructs were used: Mus musculus Klf4 full length, (ref|NM_010637.1) cDNA, Mus musculus cyclin-dependent kinase inhibitor 1A (P21) (Cdkn1a), cDNA (ref|NM_007669.3) were fused in frame to the modified oestrogen receptor ERT2 (Feil et al., 1997) and cloned into a retroviral vector (Mieg3) expressing the constructs together with eGFP after an internal ribosomal entry site . (Williams et al., 2000). For Klf4, a mutant comprinsing the zinc- finger domains only AA 393 M-bM-^@M-^S 474 (Klf4 dN) as well as a mutant comprising the entire protein except the zinc fingers AA 1-393 (Klf4-dC) were constructed and fused in frame to the modified oestrogen receptor. BM cells from 5-FU treated mice were infected with viral constructs, sorted for GFP expression (day8), plated in IMDM , 10% FCS, 2mM Gln, 1% P/S, supplemented with 50ng/ml rSCF, 100ng/ml TPO and 100ng/ml G-CSF and cultured for 72h (day8-day10). On day10 cells were stimulated with 4-OHT (1uM) for 16h. After that time, total RNA was isolated by Trizol method. Reference List: Feil,R., Wagner,J., Metzger,D., and Chambon,P. (1997). Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237, 752-757. Williams,D.A., Tao,W., Yang,F., Kim,C., Gu,Y., Mansfield,P., Levine,J.E., Petryniak,B., Derrow,C.W., Harris,C., Jia,B., Zheng,Y., Ambruso,D.R., Lowe,J.B., Atkinson,S.J., Dinauer,M.C., and Boxer,L. (2000). Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency. Blood 96, 1646-1654.
2012-07-30 | E-GEOD-11572 | biostudies-arrayexpress
