ASH1L guards cis-regulatory elements against cyclobutane pyrimidine dimer induction [ChIP-seq]. ASH1L guards cis-regulatory elements against cyclobutane pyrimidine dimer induction [ChIP-seq]

The histone methyltransferase ASH1L, first discovered for its role in transcription, has been shown to accelerate the removal of ultraviolet (UV) light-induced cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair. Previous reports demonstrated that CPD excision is most efficient at transcriptional regulatory elements, including enhancers, relative to other genomic sites. Therefore, we analyzed DNA damage maps in ASH1L-proficient and ASH1L-deficient cells to understand how ASH1L controls enhancer stability. This comparison showed that ASH1L protects enhancer sequences against the induction of CPDs besides stimulating repair activity. ASH1L reduces CPD formation at C-containing but not at TT dinucleotides, and no protection occurs against pyrimidine-(6,4)-pyrimidone photoproducts or cisplatin crosslinks. The diminished CPD induction extends to gene promoters but excludes retrotransposons. This guardian role against CPDs in regulatory elements is associated with the presence of H3K4me3 and H3K27ac histone marks, which are known to interact with the PHD and BRD motifs of ASH1L, respectively. Molecular dynamics simulations identified a DNA-binding AT hook of ASH1L that alters the distance and dihedral angle between neighboring C nucleotides to disfavor dimerization. The loss of this protection results in a higher frequency of C–>T transitions at enhancers of skin cancers carrying ASH1L mutations compared to ASH1L-intact counterparts. Overall design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for histone modifications H3K4me1 and H3K27ac in ASH1L-proficient and ASH1L-deficient U2OS cells not exposed to UV or 3 hours after UV-C exposure (20 J/m2). ChIP-seq samples and input controls were performed in biological triplicates. Please note that each processed data file was generated from both ChIP and Input samples, and is linked to the corresponding ChIP sample records.

组蛋白甲基转移酶ASH1L（histone methyltransferase ASH1L）最初因参与转录调控而被发现，后续研究证实其可通过核苷酸切除修复（nucleotide excision repair）加速清除紫外线（ultraviolet, UV）诱导的环丁烷嘧啶二聚体（cyclobutane pyrimidine dimers, CPDs）。既往研究显示，相较于其他基因组位点，CPD的切除效率在包括增强子（enhancers）在内的转录调控元件（transcriptional regulatory elements）中显著更高。因此，本研究通过分析ASH1L功能正常（ASH1L-proficient）与功能缺陷（ASH1L-deficient）细胞中的DNA损伤图谱（DNA damage maps），以探究ASH1L如何调控增强子稳定性。对比分析结果显示，除促进修复活性外，ASH1L还可保护增强子序列免受CPDs的诱导形成。ASH1L可降低含C二核苷酸位点的CPD形成水平，但对TT二核苷酸位点无此调控效应，且对嘧啶(6,4)-嘧啶酮光产物（pyrimidine-(6,4)-pyrimidone photoproducts）和顺铂交联（cisplatin crosslinks）无保护作用。这种受抑制的CPD诱导效应可延伸至基因启动子区域，但不涵盖逆转录转座子（retrotransposons）。该保护作用与H3K4me3和H3K27ac两种组蛋白修饰的存在密切相关，已知这两种修饰可分别与ASH1L的PHD结构域（PHD motif）和BRD结构域（BRD motif）发生相互作用。分子动力学模拟（molecular dynamics simulations）鉴定发现，ASH1L存在一个DNA结合AT钩模体，该模体可改变相邻C核苷酸之间的距离与二面角，从而抑制二聚体的形成。相较于ASH1L功能完整的对照细胞，携带ASH1L突变的皮肤癌细胞的增强子区域中C→T转换突变的频率显著升高，这正是该保护作用丧失所导致的结果。实验整体设计：对未接受紫外线照射或经20 J/m² 紫外线C（UV-C）照射3小时后的ASH1L功能正常与功能缺陷U2OS细胞（U2OS cells），开展针对组蛋白修饰H3K4me1和H3K27ac的染色质免疫共沉淀测序（Chromatin immunoprecipitation DNA-sequencing, ChIP-seq）。所有ChIP-seq样本及输入对照均设置3次生物学重复。请注意：每份处理后的数据文件均由ChIP样本与Input对照样本共同生成，并与对应的ChIP样本记录相关联。