|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) regulate transcription and recombination via alteration of local chromatin configuration. The ade6-M26 allele of Schizasaccharomyces pombe creates a meiotic recombination hotpot that requires a cAMP-responsive element (CRE)-like sequence MX the Atf1/Pcr1 heterodimeric ATF/CREB transcription factor, the Gcn5 HAT, and the Snf22 SWI2/SNF2 family ADCR. Chromatin alteration occurs meiotically around M26 leading to the activation of meiotic recombination. We newly report the roles of other chromatin remodeling factors that function positively and negatively in chromatin alteration at M26 two CHD-1 family ADCRs (Hrp1 and Hrp3), a SAGA component (Ada2), and a member of MYST family (Mst2). Ada2, Mst2, and Hrp3 are required for the full activation of chromatin changes around M26 and meiotic recombination. Acetylation of histone H3 around M26 is remarkably reduced in gcn5Δ ada2Δ and sni22Δ, suggesting coopera
tive functions of these HAT complexes and Snf22. Conversely, Hrp1, another CHD-1 family ADCR, maintains repressive chromatin configuration at ade6-M26. Interestingly, transcriptional initiation site is shifted to a site around M26 from the original initiation sites, in couple with the histone acetylation and meiotic chromatin alteration induced around 3' region of M26, suggesting a collaboration between these chromatin modulators and the transcriptional machinery to form accessible chromatin. These HATs and ADCRs are also required for the regulation of transcription and chromatin structure around M26 in response to osmotic stress. Thus, multiple chromatin modulators regulate chromatin structure reversibly and participate in the regulation of both meiotic recombination and stress-induced transcription around CRE-lika sequences.
The fission yeast global co-repressors Tup11 and Tup12, orthologs of budding yeast Tup1, are involved in the glucose-dependent transcriptional repression and chromatin alteration of the fbp1^+ gene. The fbp1^+ promoter contains two regulatory elements, UAS1 and UAS2, of which UAS2 serves as a binding site for two antagonizing C_2H_2 Zn-finger transcription factors, the Rst2 activator and the Scr1 repressor. We analyzed the role of Tup proteins and Scr1 in chromatin remodeling at fbp1^+ during glucose repression. We found that Scr1, co-operating with Tup11 and Tup12, functions to maintain the chromatin of the fbp1^+ promoter in a transcriptionally inactive state under glucose-rich conditions. Consistent with this notion, Scr1 is quickly exported from the nucleus to the cytoplasm at the initial stage of derepression, immediately after the glucose starvation, at which time Rst2 is imported into the nucleus. In addition, chromatin immunoprecipitation assays reveal a switching of Scr1 to Rst2 bound at UAS2 during glucose derepression. On the other hand, Tup11 and Tup12 persist in the nucleus and bind to the fbp1^+ promoter in both derepressed and repressed conditions. These observations suggest taht Tup1-like are recruited to the fbp1^+ promoter by either of two antagonizing C_2H_2Zn-finger proteins. Tup11 and Tup12 are likely regulated by reciprocal nuclear shuttling of the two antagonizing Zn-finger proteins in response to extracellular glucose concentration.
In addition, we have examined whether or not such alterations in chromatin occur at natural meiotic DNA double-strand break (DSB) sites in S. pombe. At one of the prominent DSB sites, mbs1 (meiotic break site1), the chromatin structure has constitutively accessible configuration at or near the DSB sites. The establishment of the chromatin state and DSB formation are independent of the CRE-binding transcription factor, Atf1. On the other hand, analysis on several naturally occurring CRE-dependent DSB sites reveal that meiotic chromatin alteration occurs at the tdh1^+ locus, which harbors a CRE-consensus sequence near the DSB site. Another example is cds1^+ locus, in which chromatin constitutively exhibits an open configuration. Importantly, Atf1 is required for the open chromatin in both tdh1^+ and cds1^+. These result suggest that CRE-dependent meiotic chromatin changes are intrinsic processes related to DSB formation in fission yeast meiosis. In addition, the results suggest that chromatin configuration in natural meiotic recombination hotspots can be classified into at least three distinct categories: (1) Atf1・CRE-independent constitutively open chromatin configration, (2) Atf1・CRE-dependent meiotically induced open chromatin configuration, and (3) Atf1・CRE-dependent constitutively accessible chromatin configuration. Less