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. 2018 Jul 23;218(8):1336–1347. doi: 10.1093/infdis/jiy299

Figure 5.

Figure 5.

Host delivery of Cdg7_FLc_1030 triggers transcription of the Dkk1 gene in epithelial cells. A, Levels of the transcription activity markers, H3K4me1 and H3K36me3, as well as enrichment of Pol II, associated with the Dkk1 gene locus in IEC4.1 cells following Cryptosporidium parvum infection. Cells were exposed to C. parvum infection for 24 hours, followed by chromatin immunoprecipitation (ChIP) analysis using anti-H3K4me1, anti-H3K36me3, or anti-Pol II, respectively, and the PCR primer sets as designed. Increased enrichment of H3K4me1 and H3K36me3, as well as Pol II, was detected in the Dkk1 gene locus in cells following infection. B, Assembly of Cdg7_FLc_1030 to the Pol II complex in cells following C. parvum infection. IEC4.1 were exposed to C. parvum infection for 24 hours or transfected with Full-Cdg7_FLc_1030 for 24 hours, followed by RNA immunoprecipitation (RIP) analysis using anti-Pol II and PCR primers for Cdg7_FLc_1030. Assembly of the U2 RNA was also measured as control (Ctrl). C, Recruitment of Cdg7_FLc_1030 to the Dkk1 gene locus in IEC4.1 cells following C. parvum infection. Cells were exposed to C. parvum infection for 24 hours, following by chromatin isolation by RNA purification (ChIRP) analysis using a pool of probes specific to Cdg7_FLc_1030 and the PCR primer sets as designed. Increased recruitment of Cdg7_FLc_1030 was detected in the Dkk1 gene locus in cells following infection. D, C. parvum infection did not promote the luciferase activity associated with the transcription of the Dkk1-promoter luciferase reporter constructs. Various regions of Dkk1 promoter were cloned into the luciferase reporter constructs and the interleukin 8 luciferase reporter (IL8-Luc) plasmid containing human IL8 promoter was used for the positive control. IEC4.1 cells were transfected with the constructs for 12 hours and followed by exposure to C. parvum infection for 24 hours. Cells transfected with the empty vector were used as the control. The luciferase activities of the cells were then measured and normalized to β-gal. Data represent means ± SEs from 3 independent experiments. *P < .01 ANOVA versus noninfected or empty vector controls.