Dynamics and function of the transcription factor SRF inside the nucleus of neurons revealed by single-molecule analysis and super-resolution optical microscopy

单分子分析和超分辨率光学显微镜揭示神经元核内转录因子SRF的动态和功能

• 批准号: 406037611
• 负责人: Professor Dr. Bernd Knöll
• 金额: --
• 依托单位: Institut Physiologische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/406037611?language=en
• 关键词: Dynamics; function; transcription; factor; SRF

Millions of molecules contribute to gene transcription in nuclei of living cells. So far, investigation of gene transcription is mostly restricted to ensemble analysis of all these molecules within one cell. Such ensemble analysis assumes that all individual copies of a particular transcription factor (TF) have identical function and operate as a homogenous population. However, within a protein population, sub-populations of TF molecules with distinct biophysical properties might exist and fulfil different roles in the same process. This requires analysis at the single molecule level, a technique just starting to take-off in cell biological research. In this proposal we analyse such dynamic processes of nuclear gene transcription at the single molecule level. We focus on SRF (serum response factor), a TF regulating physiological processes in the brain such as neuronal growth and synaptic function. In mouse models of neuronal disease including axon injury, neuronal degeneration and e.g. epilepsy, SRF function is impaired. SRF mediates neuronal activity mediated gene transcription through transcription of immediate early genes (IEGs) such as c-Fos and Egr1. In addition, SRF regulates actin cytoskeletal genes thereby impinging on nerve fibre growth and axonal regeneration. We employ a fusion protein of SRF and the Halo tag (SRF-Halo) to analyse single SRF molecules using single particle tracking with light sheet illumination in living neurons. In addition, we will perform single molecule localisation microscopy (SMLM) in fixed cells. These techniques will be used to determine several parameters of nuclear SRF transcriptional dynamics:i) the fraction of unbound to DNA-bound SRFii) the DNA residence time of SRFiii) the sub-nuclear SRF localization, e.g. co-localization with transcription hubsSo far, only few TFs have been analysed at the single molecule level and research focused on immortalised cell culture lines. Here, we will use primary cells, i.e. primary mouse hippocampal neurons stimulated with growth factors such as BDNF (brain derived neurotrophic factor). In a first step, we compare results obtained in neurons with data obtained from NIH3T3 cells, acquired in work building up to this proposal. This provides information as to whether SRF binding dynamics between proliferative and differentiated cells are conserved or divergent. We further analyse how DNA binding dynamics of SRF are altered along the time course of neuronal polarisation, e.g. during protrusion of the first neurites or later on in synapse formation. Finally, we will investigate injured primary neurons generated using laser axotomy to address a potential modulation of SRF activity dynamics by axonal degeneration and regeneration.Since SRF fulfils essential physiological and pathological functions in all organs investigated and not only the brain, our data will provide important insight into regulation of SRF and general TF activity relevant for a wealth of tissues.

数以百万计的分子参与活细胞核中的基因转录。到目前为止，基因转录的研究主要局限于一个细胞内所有这些分子的整体分析。这种集成分析假设特定转录因子 (TF) 的所有单独副本都具有相同的功能并作为同质群体运行。然而，在蛋白质群体中，可能存在具有不同生物物理特性的 TF 分子亚群，并在同一过程中发挥不同的作用。这需要在单分子水平上进行分析，这是一种在细胞生物学研究中刚刚起步的技术。在本提案中，我们在单分子水平上分析了核基因转录的动态过程。我们专注于 SRF（血清反应因子），这是一种调节大脑生理过程（如神经元生长和突触功能）的 TF。在小鼠神经元疾病模型中，包括轴突损伤、神经元变性等。癫痫，SRF功能受损。 SRF 通过立即早期基因 (IEG)（例如 c-Fos 和 Egr1）的转录来介导神经元活动介导的基因转录。此外，SRF 调节肌动蛋白细胞骨架基因，从而影响神经纤维生长和轴突再生。我们采用 SRF 和 Halo 标签的融合蛋白 (SRF-Halo)，通过活体神经元中的单粒子跟踪和光片照明来分析单个 SRF 分子。此外，我们将在固定细胞中进行单分子定位显微镜（SMLM）。这些技术将用于确定核 SRF 转录动力学的几个参数：i) 未结合到 DNA 结合 SRF 的分数ii) SRF 的 DNA 停留时间iii) 亚核 SRF 定位，例如与转录中心的共定位到目前为止，只有少数转录因子在单分子水平上进行了分析，并且研究集中在永生化细胞培养系上。在这里，我们将使用原代细胞，即用 BDNF（脑源性神经营养因子）等生长因子刺激的原代小鼠海马神经元。第一步，我们将神经元中获得的结果与从 NIH3T3 细胞中获得的数据进行比较，这些数据是在构建该提案的工作中获得的。这提供了关于增殖细胞和分化细胞之间的 SRF 结合动力学是否保守或不同的信息。我们进一步分析了 SRF 的 DNA 结合动力学如何随着神经元极化的时间过程而改变。在第一个神经突突出期间或稍后在突触形成期间。最后，我们将研究使用激光轴切术产生的受损原代神经元，以解决轴突变性和再生对 SRF 活动动态的潜在调节。由于 SRF 在所有研究的器官（而不仅仅是大脑）中发挥重要的生理和病理功能，因此我们的数据将为 SRF 和与大量组织相关的一般 TF 活动的调节提供重要的见解。