GRO-seq reported in "Phosphorylated Lamin A/C in the nuclear interior binds active enhancers associated with abnormal transcription in progeria"

LMNA encodes nuclear Lamin A/C that tethers lamina-associated domains (LADs) to the nuclear periphery. Mutations in LMNA cause degenerative disorders including the premature aging disorder Hutchinson-Gilford progeria, but the mechanisms are unknown. We report that Ser22-phosphorylated (pS22) Lamin A/C was localized to the nuclear interior in human fibroblasts throughout the cell cycle. pS22-Lamin A/C interacted with a subset of putative active enhancers, not LADs, at locations co-bound by the transcriptional activator c-Jun. In progeria-patient fibroblasts, a subset of pS22-Lamin A/C-binding sites were lost whereas new pS22-Lamin A/C-binding sites emerged in normally quiescent loci. New pS22-Lamin A/C binding was accompanied by increased histone acetylation, increased c-Jun binding, and upregulation of nearby genes implicated in progeria pathophysiology. These results suggest that Lamin A/C regulates gene expression by enhancer binding. Disruption of the gene regulatory rather than LAD tethering function of Lamin A/C presents a novel mechanism for disorders caused by LMNA mutations. The aims of this project are to 1) identify the locations of the human genome associated with Ser22-phosphorylated LMNA by ChIP-seq in normal and progeria-patient cells; 2) define the chromatin characteristics of Ser22-phosphorylated LMNA-associated sites by histone ChIP-seq and ATAC-seq; and 3) identify genes dysregulated in progeria-patient cells by RNA-seq.

LMNA基因编码核纤层蛋白A/C（Lamin A/C），该蛋白可将核纤层结合结构域（lamina-associated domains，LADs）锚定至核膜边缘。LMNA基因突变可引发多种退行性疾病，其中包括早老化疾病哈钦森-吉尔福德早衰症（Hutchinson-Gilford progeria），但其致病机制迄今尚未阐明。本研究发现，在人类成纤维细胞中，丝氨酸22位磷酸化核纤层蛋白A/C（Ser22-phosphorylated (pS22) Lamin A/C）在整个细胞周期中均定位于细胞核内部。pS22-核纤层蛋白A/C可与一系列潜在活性增强子结合，而非LADs，其结合位点均为转录激活因子c-Jun的共结合区域。在早衰症患者成纤维细胞中，部分pS22-核纤层蛋白A/C结合位点消失，同时在原本处于静息状态的染色质区域出现了新的pS22-核纤层蛋白A/C结合位点。新出现的pS22-核纤层蛋白A/C结合区域伴随组蛋白乙酰化水平升高、c-Jun结合增强，以及与早衰症病理生理过程相关的邻近基因表达上调。上述结果表明，核纤层蛋白A/C可通过结合增强子调控基因表达。LMNA基因突变所致疾病的致病新机制，可能源于核纤层蛋白A/C的基因调控功能受损，而非其LAD锚定功能异常。本项目的研究目标为：1）通过染色质免疫共沉淀测序（ChIP-seq），在正常细胞与早衰症患者细胞中鉴定出与丝氨酸22位磷酸化核纤层蛋白A/C相结合的人类基因组区域；2）通过组蛋白ChIP-seq与转座酶可及性测序（ATAC-seq），阐明pS22-核纤层蛋白A/C结合位点的染色质特征；3）通过RNA测序（RNA-seq），鉴定早衰症患者细胞中表达失调的基因。