Lamin A upregulation reorganizes the genome during rod photoreceptor degeneration

Neurodegenerative diseases are accompanied by dynamic changes in gene expression, including the upregulation of hallmark stress-responsive genes. While the transcriptional pathways that impart adaptive and maladaptive gene expression signatures have been the focus of intense study, the role of higher order nuclear organization in this process is less clear. Here, we examine the role of the nuclear lamina in genome organization during the degeneration of rod photoreceptors. Two proteins had previously been shown to be necessary and sufficient to tether heterochromatin at the nuclear envelope. The lamin B receptor (Lbr) is expressed during development, but downregulates upon rod differentiation. A second tether is the intermediate filament lamin A (LA), which is not normally expressed in murine rods. Here, we show that in the rd1 model of retinitis pigmentosa, LA ectopically upregulates in rod photoreceptors at the onset of degeneration. LA upregulation correlated with increased heterochromatin tethering at the nuclear periphery in rd1 rods, suggesting that LA reorganizes the nucleus. To determine how heterochromatin tethering affects the genome, we used in vivo electroporation to misexpress LA or Lbr in mature rods in the absence of degeneration, resulting in the restoration of conventional nuclear architecture. Using scRNA-seq, we show that reorganizing the nucleus via LA/Lbr misexpression has relatively minor effects on rod gene expression. Next, using ATAC-seq, we show that LA and Lbr both lead to marked increases in genome accessibility. Novel ATAC-seq peaks tended to be associated with stress-responsive genes. Together, our data reveal that heterochromatin tethers have a global effect on genome accessibility, and suggest that heterochromatin tethering primes the photoreceptor genome to respond to stress. Overall design: After flow cytometric sorting, cells were barcoded with 'anchor' and 'co-anchor' lipid-modified oligonucleotides generously provided by the Zev Gartner lab. Barcode oligonucleotides were purchased from Integrated DNA Technologies as follows. Barcode 1: F: 5'- CCTTGGCACCCGAGAATTCCAGGAGAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3'; Barcode 2: F: 5'- CCTTGGCACCCGAGAATTCCACCACAATGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3'; Barcode 3: F: 5'- CCTTGGCACCCGAGAATTCCATGAGACCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3'; Each replicate was incubated with barcode oligonucleotides for 10 minutes. Cells were pelleted and washed 3 times with PBS. Replicates were pooled and processed in a single 10X Genomics Chromium run.

神经退行性疾病伴随基因表达的动态变化，包括标志性应激响应基因的上调。既往研究多聚焦于介导适应性与适应不良性基因表达特征的转录通路，而高阶核结构（higher order nuclear organization）在该过程中的作用仍不甚明确。
本研究探讨核纤层（nuclear lamina）在视杆感光细胞（rod photoreceptors）变性过程中基因组组织的作用。此前已有两种蛋白被证实可将异染色质（heterochromatin）锚定在核被膜（nuclear envelope）上：核纤层蛋白B受体（lamin B receptor, Lbr）在发育阶段表达，但在视杆细胞分化后下调；第二种锚定蛋白为中间丝蛋白核纤层蛋白A（lamin A, LA），正常情况下在小鼠视杆细胞中不表达。
本研究发现，在视网膜色素变性（retinitis pigmentosa）rd1模型（rd1 model）中，LA在变性启动阶段的视杆感光细胞中异位上调。LA上调与rd1模型视杆细胞中核周异染色质锚定增强相关，提示LA可重塑细胞核架构。
为明确异染色质锚定对基因组的影响，我们利用体内电穿孔（in vivo electroporation）技术在未发生变性的成熟视杆细胞中异位表达LA或Lbr，成功恢复了常规的细胞核结构。通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析，我们发现通过LA/Lbr异位表达重塑细胞核对视杆细胞基因表达的影响相对轻微。随后利用转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）分析显示，LA与Lbr均可显著提升基因组的染色质可及性。新增的ATAC-seq峰多与应激响应基因相关。
综上，我们的研究结果表明异染色质锚定对基因组可及性具有全局性影响，并提示异染色质锚定可使感光细胞基因组做好应激响应的准备。
实验设计概述：经流式细胞分选（flow cytometric sorting）后，细胞使用由Zev Gartner实验室惠赠的带有"anchor"与"co-anchor"标记的脂质修饰寡核苷酸进行条形码标记。条形码寡核苷酸购自Integrated DNA Technologies（IDT），具体序列如下：
条形码1正向引物：5'- CCTTGGCACCCGAGAATTCCAGGAGAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3'；
条形码2正向引物：5'- CCTTGGCACCCGAGAATTCCACCACAATGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3'；
条形码3正向引物：5'- CCTTGGCACCCGAGAATTCCATGAGACCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3'；
每个重复样本与条形码寡核苷酸孵育10分钟。随后收集细胞并以磷酸盐缓冲液（phosphate-buffered saline, PBS）洗涤3次。将所有重复样本混合后，通过单次10X Genomics Chromium平台测序流程进行处理。