Project description:Atf1 was overexpressed in wt S. pombe cells for 24 hours and gene expression changes were analysed all samples were processed in duplicates. Gene expression in cells overexpressing ATf1 was compared with respect to the levels in control cells transformed with the empty vector.
Project description:Clear cell sarcoma (CCS) is an aggressive soft tissue malignant tumor characterized by a unique t(12; 22) translocation, leading to the expression of a chimeric EWS/ATF1 fusion gene. However, little is known about the mechanisms underlying how EWS/ATF1 is involved in the development of CCSs. In addition, the cells of origin for CCSs remain to be determined. We generated EWS/ATF1-inducible mice, and examined the effects of EWS/ATF1 expression in adult cells. We show that the forced expression of EWS/ATF1 results in the development of EWS/ATF1-dependent sarcomas in mice. The histology of EWS/ATF1-induced sarcomas resembles that of CCSs and EWS/ATF1-induced tumor cells express CCS-markers, such as S100, Sox10, and Mitf. A lineage tracing experiment revealed that such sarcomas are derived from neural crest-lineage cells. Finally, we found that EWS/ATF1 directly induces Fos in an ERK-independent manner, and demonstrated that the increased Fos expression is important for the active cell proliferation in not only EWS/ATF1-induced sarcomas, but also in human CCSs. Our results indicate that FOS, as well as EWS/ATF1 itself, could be a promising therapeutic target for the treatment of EWS/ATF1-related sarcomas. Tumor cell lines were exposed to different concentrations of doxycycline, and total RNAs were isolated at 24 hours after the doxycycline exposure. Total RNAs were also isolated directly from a tumor developed in a EWS/ATF1-inducible mouse given doxycycline for 3 months. The doxycycline concentration and time point for each sample is EWS-ATF1_control; 0 microg/ml (no dox), 24 hours after the exposure, EWS-ATF1_24h_highDox; 0.2 microg/ml, 24 hours after the exposure and EWS-ATF1_tumor1; a tumor was resected from a EWS/ATF1-inducible mouse given doxycycline for 3 months.
Project description:In the fission yeast Schizosaccharomyces pombe, heterochromatic silencing is quite stable at pericentromeres but unstable at the mating-type (mat) locus under chronic heat stress. We found that the compromised gene silencing at the mat locus at elevated temperature is linked to the phosphorylation status of Atf1 (a member of the ATF/CREB superfamily) by Sty1, one of the mitogen-activated protein kinases (MAPKs). To gain a full picture of Sty1-dependent phosphosite map of Atf1 under heat stress, we purified HA-Atf1 protein from cultures of wild type and sty1Δ strains shifted from 25℃ to either 30℃ or 37℃ for 5 hr followed by immunoprecipitation. Samples were separated by SDS-PAGE, and Coomassie blue-stained gel slices were excised and submitted for mass spectrometry analyses.
Project description:Gene expression profiling of S. pombe set1/COMPASS/H3K4 mutants, atf1 and set1 atf1 deletion mutants, set1 clr3 deletion mutants; ChIP-chip tiling microarray profiling of S. pombe Set1, Atf1, H3K4me3 in wt/atf1 deletion mutants, H3K9me2 in wt/set1 clr3 deletion mutants Custom Agilent 60mer microarrays were used to assay gene expression in set1/COMPASS mutants in combination with atf1 and clr3 mutants, and to profile genome-wide binding of Set1, Atf1, H3K4me3 and H3K9me2 in S. pombe cells (two-color mutant vs. wildtype, ChIP vs. input experiments).
Project description:ATF1 encodes a sequence-specific activating TF containing a bZIP DNA binding domain, which is overexpressed in CRC tumor tissues compared with their paired normal tissues in two independent cohorts and that ATF1 amplification also frequently occurs across multiple cancer types. Moreover, ATF1 is more highly expressed in advanced stages of CRC. Mechanistically, ATF1 overexpression could provoke cell proliferation and xenograft growth in vitro and in vivo by affecting cell apoptosis, However, the exact mechanism and downstream transcriptional programs by which ATF1 provokes tumor activity are not well understood. We performed ChIP-seq in HCT116cells to investigate the regulatory mechamisms of ATF1.
Project description:RNA sequencing of total RNA purified from wild type S. pombe cells after 0, 5 and 24 hours culture in YES containing 100% D2O (deuterium oxide).