c-Jun Targets Cockayne Syndrome protein B to Specific Genomic Region for Transcription Regulation. Homo sapiens

Cockayne syndrome is an inherited premature aging syndrome associated with developmental and neurological disorders. Mutations in the genomic locus encoding CSB are associated with 80% Cockayne syndrome cases. Transcription profiling assays reveal the association of mis-regulation of gene expression with Cockayne syndrome, highlighting the importance of CSB in transcription regulation. However, many questions remain unanswered as how CSB regulates transcription. In this study, we dissect the mechanisms by which CSB regulates transcription during normal growth. By anti-CSB chromatin immunoprecipitation followed by deep sequencing, we found CSB is enriched at genomic regions containing TGASTCA motifs, to which the immediate early gene product C-Jun binds specifically. We further demonstrate that c-Jun co-immunoprecipitates with CSB. In addition, the targeting of CSB to genomic region containing TGASTCA motifs was drastically reduced in cells treated c-Jun shRNA. Reverse transcription followed by quantitative PCR indicates that CSB can regulate gene expression nearby its binding sites, both in activation and repression. The remodeling defective CSB∆N1 mutant is also targeted to TGASTCA motifs, but cannot always substitute CSB function in transcription regulating, suggesting the importance of remodeling by CSB in transcription regulation. Notably, the Cockayne syndrome related mutation encoding protein CSBR670W, which is defective in ATP hydrolysis but is targeted to TGASTCA motifs efficiently, indicating that ATP hydrolysis is dispensable for c-Jun mediated CSB targeting, in sharp contrast to the ATP-dependent targeting mechanism by which CSB is relocated to DNA lesion stalled transcription. Together, these results reveal a second CSB targeting mechanism in which DNA sequence specific transcription factor c-Jun targets CSB to specific genomic region and regulate gene expression. Overall design: Genomic localization of CSB and remodeling deficient CSB∆N1

科凯恩综合征（Cockayne syndrome）是一种遗传性早衰综合征，伴随发育异常与神经系统功能障碍。编码CSB的基因组位点发生突变与80%的科凯恩综合征病例相关。转录谱分析实验揭示了基因表达失调与科凯恩综合征的关联，凸显了CSB在转录调控中的重要作用。然而，关于CSB如何调控转录仍有诸多未解之谜。本研究解析了正常生长状态下CSB调控转录的分子机制。通过抗CSB染色质免疫沉淀联合深度测序实验，我们发现CSB富集于含有TGASTCA基序的基因组区域，而即刻早期基因产物C-Jun可特异性结合该基序。我们进一步证实，C-Jun可与CSB发生共免疫沉淀反应。此外，在用c-Jun短发夹RNA（shRNA）处理的细胞中，CSB向含有TGASTCA基序的基因组区域的靶向募集显著降低。逆转录联合定量PCR实验表明，CSB可调控其结合位点附近的基因表达，兼具激活与抑制双重功能。重塑缺陷型CSBΔN1突变体同样可被招募至TGASTCA基序区域，但无法完全替代CSB在转录调控中的功能，这提示CSB的重塑活性在转录调控中具有重要意义。值得注意的是，与科凯恩综合征相关的CSBR670W突变蛋白——其ATP水解功能存在缺陷，但可被高效招募至TGASTCA基序区域——表明ATP水解并非C-Jun介导的CSB靶向募集所必需，这与CSB被招募至DNA损伤阻滞转录位点的ATP依赖型靶向机制形成鲜明对比。综上，本研究揭示了第二种CSB靶向募集机制：即DNA序列特异性转录因子C-Jun将CSB靶向招募至特定基因组区域，进而调控基因表达。实验整体设计：CSB与重塑缺陷型CSBΔN1的基因组定位分析