Fig. 1.TIF

Lamin A/C intermediate filament proteins are constituents of the nuclear lamina contributing to the mechanical stability of the nucleus and can be found throughout the nucleoplasm. Examples of lamin A involvement in transcriptional regulation include laminopathies causing lipodystrophies and progeria. Here, we studied the effect of lamin A KO in murine adult fibroblasts on the organization of chromatin and the DNA binding of peroxisome proliferator‐activated receptor gamma (PPARγ), a transcription factor also implicated in adipogenesis and lipodystrophies. We used confocal microscopy to analyze the distribution of different chromatin marks and fluorescence correlation spectroscopy to measure PPARγ mobility and DNA binding. Conspicuous overall changes were detected in response to both lamin A depletion and treatment with the PPARγ agonist rosiglitazone (RSG). The diameter of the nucleus decreased remarkably in the absence of Lamin A and also upon RSG treatment. Heterochromatin (hc.) was enriched in a distinct peripheral rim, and the thickness of the constitutive hc. rim diminished in the absence of lamin A. The overlap between euchromatin (euc.) and hc. decreased in lamin A KO cells. PPARγ was colocalized with euc. whereas its localization was anti-correlated with constitutive hc., even more so in the absence of lamin A. PPARγ had a fast diffusing fraction transiently bound with short residence times on the DNA and a slow, more stably bound fraction throughout the nucleus including the periphery. RSG increased PPARγ’s colocalization with the euc. and its DNA binding, which was more pronounced in lamin A KO cells. Our results suggest that lamin A plays a primary role in determining nuclear volume and overall chromatin architecture with likely functional consequences exemplified by regulating ligand-induced DNA binding of PPARγ, which may have epigenomic and transcriptional consequences relevant in the context of lipodystrophies.

核纤层蛋白A/C（Lamin A/C）中间丝蛋白是核纤层的组成成分，有助于维持细胞核的机械稳定性，且可分布于整个核质中。核纤层蛋白A参与转录调控的典型案例包括引发脂肪营养不良与早老症的核纤层蛋白病。本研究探讨了小鼠成纤维细胞中核纤层蛋白A敲除（KO）对染色质组织以及过氧化物酶体增殖物激活受体γ（PPARγ）DNA结合能力的影响；PPARγ是一种同时参与脂肪生成与脂肪营养不良过程的转录因子。我们采用共聚焦显微镜分析不同染色质标记物的分布情况，并通过荧光相关光谱术测定PPARγ的迁移率与DNA结合能力。本研究检测到，核纤层蛋白A缺失与PPARγ激动剂罗格列酮（RSG）处理均可引发显著的整体细胞变化。在缺失核纤层蛋白A以及经RSG处理后，细胞核直径均出现明显减小。异染色质（hc.）以独特的外周环带形式富集，而在缺失核纤层蛋白A时，组成型异染色质环带的厚度显著降低。核纤层蛋白A敲除细胞中，常染色质（euc.）与异染色质的重叠区域明显减少。PPARγ与常染色质共定位，但其定位与组成型异染色质呈负相关，且在核纤层蛋白A缺失的细胞中这种负相关更为显著。PPARγ存在两类扩散组分：一个是在DNA上瞬时结合、滞留时间较短的快速扩散组分，另一个是在包括核外周在内的整个细胞核中稳定结合的慢速扩散组分。RSG可增强PPARγ与常染色质的共定位及其DNA结合能力，且该效应在核纤层蛋白A敲除细胞中更为明显。本研究结果表明，核纤层蛋白A在决定细胞核体积与整体染色质结构方面发挥核心作用，其功能后果可通过调控配体诱导的PPARγ DNA结合能力得以体现，这一过程可能在脂肪营养不良相关病理场景下产生表观基因组与转录层面的影响。