Expression profiling by array Genome binding/occupancy profiling by genome tiling array
Summary
Expression profiling, ChiP-CHIP and phenotypic analysis were used to investigate the functional relationships of class III NAD+-dependent HDACs (Sirtuins) in fission yeast. We detected significant histone acetylation increases in Sirtuin mutants at their specific genomic binding targets and were thus able to identify an in vivo substrate preference for each Sirtuin. At heterochromatic loci, we demonstrate that although Hst2 is mainly cytoplasmic, a nuclear pool of Hst2 co-localises with the other Sirtuins at silent regions (cen, mat, tel, rDNA) and that like the other Sirtuins, Hst2 is required for rDNA and centromeric silencing. Interestingly we found specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation. Hst2 directly represses genes involved in transport and membrane function whereas Hst4 represses amino acid biosynthesis genes and Tf2 retrotransposons. A specific role for Hst4 in Tf2 5’ mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation. Keywords: chromatin; fission yeast; gene silencing; retrotransposon; Sirtuin; HDAC
Overall design
The combined microarray strategy in this study was performed essentially as outlined in (Wiren et al., 2005). cDNA expression profiling was carried out according to (Xue et al., 2004). We used the S. pombe ORF and combined IGR+ORF spotted microarrays (Eurogentec, Belgium custom DNA microarray services). For histone acetylation maps, ChIP-CHIP method was used using according to (Robyr and Grunstein, 2003). Specific acetylation antibodies were used against H3K9ac, H3K14ac, H4K5ac, H4K16ac and H4K12ac (Suka et al., 2002). The histone ‘H3cter’ antibody was purchased from Upstate and used for ChIP according to manufacturers recommendations. For HDAC bindings maps we used we used the ChIP- CHIP procedure described by (Kurdistani et al., 2002). Corrections for nucleosome loss were performed as described in (Wiren et al., 2005). GeneSpring software was used for all data analysis. Expression profiling and histone acetylation mutant vs wt data sets were normalized using Lowess (per spot per chip) intensity-dependent normalization, which corrects nonlinear rates for dye incorporation. Cut off values of 1,5 and 2,0 were used to generate ‘high’ and ‘low’ gene lists for ORF and IGR regions. The HDAC-binding data were normalized using per chip (the 50th percentile). For HDAC binding, Sir2-Myc data were extracted from (Wiren et al., 2005), ProtA-Hst4 binding were carried out …( Fredrik fill in here). Media and spotting assays Standard fission yeast genetic techniques and media were used according to (Moreno et al., 1991). For microarray analysis, cells were grown in YES media. Silencing assays plating and serial dilution experiments were performed as described (Allshire et al., 1994). Fivefold dilutions were plated onto EMM plates as a growth control (+ura), onto EMM plates lacking uracil (-ura) and onto 5-FOA plates (+FOA). Thiabendazole (TBZ; Sigma) was dissolved in DMSO to give stock solution at 20 mg/ml. Construction of strains Haploidization of diploid heterozygote strain h+/h+ ade6-M210/ade6-M216 leu1-32/leu1-32 ura4-D18/ura4-D1 hst2/hst2∆::kanMx4 (Bioneer Corporation) was performed in YES liquid media containing 10µg/ml of TBZ during 2-3 days. Then the cells were plated on YES (–ade) plates containing 150µg/ml G418 (Sigma). G418 resistant haploid colonies with red or pink colony color were then selected, checked by PCR and backcrossed with the wt strain Hu303. 13xMyc epitope tagging of Hst2 was performed according (Bahler et al., 1998). The heterozygous diploid h+/h+ ade6-M210/ade6-M216 hst2::kanMx4/hst2+ leu1-32/leu1-32 ura4-D18/uraD-18 knockout strain was obtained from Bioneer company (South Korea). Diploid cells were haploidized as described above. All strains used in this study are listed in supplementary data Table S1. Northerns and RT PCR… IF microscopy…
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