A compendium of chromatin contact maps reflecting regulation by chromatin remodelers in budding yeast. A compendium of chromatin contact maps reflecting regulation by chromatin remodelers in budding yeast

We employed in situ Hi-C with an auxin-inducible degron (AID) system to demonstrate the effect of chromatin remodeling on 3D genome organization in yeast. Overall design: IAA (Sigma, I2886) was added to a final concentration of 0.5 mM for degradation of target proteins and the same volume of 100% ethanol was used as a control. For G1 arrest, alpha-factor was added at a final concentration of 50 ng/ml to bar1Δ strains; after 2 h, 0.5 mM IAA was added and the cells were incubated for an additional 3 h. For S or G2/M arrest, alpha-factor was added at a final concentration of 50 ng/ml to bar1Δ strains; after 1.5 h, the yeast cells were transferred to fresh YPD medium containing 200 mM hydroxyurea (HU; Sigma, H8627) for S arrest or 15 ug/ml nocodazole (Sigma, M1404) for G2 arrest. At 1.5 h after HU/nocodazole treatment, 0.5 mM IAA was added and cells were incubated for an additional 3 h. For G2 arrest, an additional 10 ug/ml of nocodazole was applied along with the IAA. Yeast cells (50 O.D.600) were cultured, fixed with 3% formaldehyde (Wako, 064-00406), quenched, pelleted, pre-incubated with β-ME buffer (20 mM EDTA and 0.7 M β-ME) for 10 min at 30℃, and then lysed with 2 mg of zymolase (US Biological, Z1004) in 2 ml lyticase buffer (1 M sorbitol, 50 mM Tris-Cl (pH 8.0), 5 mM β-ME) for 20 min at 30℃. The obtained spheroplasts were resuspended in 2 ml of ice-cold PBS. The isolated nuclear DNA was resuspended in 50 ul of 0.5% SDS, incubated for 10 min at 62℃, mixed with 170 ul of 1.47% TritonX-100 and incubated for 15 min at 37℃. The generated libraries were sequenced using 150-bp paired-end reads on an Illumina Novaseq6000 and/or HiSeqX.

我们借助生长素诱导降解（auxin-inducible degron, AID）系统结合原位Hi-C（in situ Hi-C）技术，阐明了染色质重塑对酵母三维基因组组织的调控作用。整体实验设计如下：向体系中添加终浓度为0.5 mM的IAA（Sigma, I2886）以实现靶蛋白的降解，同时以等体积的100%乙醇作为空白对照。针对G1期阻滞实验：向bar1Δ缺失菌株中添加终浓度为50 ng/ml的α因子；2小时后加入0.5 mM IAA，继续孵育3小时。针对S期或G2/M期阻滞实验：先向bar1Δ缺失菌株中添加终浓度为50 ng/ml的α因子；1.5小时后，将酵母细胞转移至含200 mM羟基脲（hydroxyurea, HU; Sigma, H8627）的新鲜YPD培养基中以实现S期阻滞，或转移至含15 μg/ml诺考达唑（nocodazole; Sigma, M1404）的新鲜YPD培养基中以实现G2期阻滞。在HU/诺考达唑处理1.5小时后，加入0.5 mM IAA并继续孵育3小时；针对G2期阻滞组，需同时额外添加10 μg/ml诺考达唑。培养50个OD600单位的酵母细胞，采用3%甲醛（Wako, 064-00406）进行固定，随后淬灭固定反应、收集细胞沉淀；将沉淀重悬于β-巯基乙醇（β-ME）缓冲液（含20 mM EDTA与0.7 M β-ME）中，于30℃预孵育10分钟；随后使用2 mg溶细胞酶（zymolase; US Biological, Z1004）在2 ml溶菌酶缓冲液（含1 M山梨醇、50 mM Tris-Cl（pH 8.0）、5 mM β-ME）中于30℃裂解20分钟。将得到的原生质体重悬于2 ml冰预冷的PBS中；分离得到的核DNA重悬于50 μl 0.5% SDS溶液中，62℃孵育10分钟后，加入170 μl 1.47% TritonX-100并于37℃孵育15分钟。构建完成的文库采用Illumina Novaseq6000和/或HiSeqX测序平台，以150 bp双端读长进行测序。