NuMA function in nonmalignant and malignant breast cells

描述(申请人提供)：染色质结构和基因表达的改变是癌症的标志。染色质重塑复合体(CRCs)参与的蛋白质通过修饰DMA和组蛋白直接影响染色质结构，在癌症中CRCs靶向性改变。像NUMA这样的结构蛋白被认为是核结构的组织者，也与染色质相互作用，它们在癌症中的分布发生了变化。然而，NUMA在基因表达调控中的作用尚不清楚。NUMA与依赖于ATP的CRCs的成分共分离。使用针对其C末端(CT)的抗体或通过表达其CT的远端部分来改变NUMA可以改变染色质结构和乳腺上皮细胞的表型。CT截短的NUMA和突变的NUMA分别与白血病和乳腺癌的发生有关。我们建议研究NUMA控制乳腺上皮细胞染色质结构和基因表达的机制。假设NUMA通过与CRCs的成分相互作用来参与基因转录的调控，以便将这些复合体靶向特定的基因。我们提出了三个目标：(1)通过亲和层析和免疫共沉淀检测NUMA与依赖于ATP的CRCs的Snf2和BAF型成分的相互作用，以及抑制NUMA表达对CRCs组装和功能的影响；(2)通过比较干扰NUMA的类HPC2、GAS41结合和CH结合区的功能对染色质组织、CRC功能和乳腺上皮分化的影响，分析NUMA参与染色质调节的初级序列；(3)通过干扰NUMA的功能，评估控制细胞增殖的基因的表达状态(开或关)与它们的定位和启动子上是否存在含NUMA的CRC，以确定NUMA在通过位置效应调控基因表达中的作用。意义：当前的科学和医学挑战是揭开癌症发展过程中基因表达机制或调控变化背后的机制。了解这些机制将有助于制定预防癌症进展和控制肿瘤行为的策略。因此，现在应该强调结构核蛋白在基因转录中的作用，以获得基因表达调控的完整图景。

DESCRIPTION (provided by applicant): Alterations in chromatin structure and gene expression are a hallmark of cancer. Proteins involved in chromatin remodeling complexes (CRCs) directly affect chromatin structure by modifying DMA and histones, and CRCs targeting is altered in cancer. Structural proteins like NuMA, considered as organizers of nuclear architecture, also interact with chromatin, and their distribution is altered in cancer. However, the role of NuMA in the control of gene expression is unknown. NuMA co-isolates with components of ATP-dependent CRCs. Altering NuMA using an antibody against its C-terminus (CT) or by expressing the distal portion of its CT modifies chromatin structure and the phenotype of breast epithelial cells. CT-truncated NuMA and mutated NuMA are implicated in leukemia and breast cancer development, respectively. We propose to investigate the mechanisms by which NuMA controls chromatin structure and gene expression in breast epithelial cells. The hypothesis is that NuMA participates in the control of gene transcription by interacting with components of CRCs, in order to target these complexes to specific genes. Three aims are proposed: (1) To identify the specific CRCs in which NuMA is involved by assessing the interaction of NuMA with components of SNF2h and BAF types of ATP-dependent CRCs by affinity chromatography and co- immunoprecipitation, and assessing the effect of inhibiting NuMA expression on the assembly and function of CRCs; (2) To analyze the primary sequence of NuMA with regards to its involvement in chromatin regulation by comparing the effects of interrupting the function of HPC2-like, GAS41-binding, and CH-binding regions of NuMA on chromatin organization, CRC function, and mammary epithelial differentiation; (3) To define the role of NuMA in gene expression control by position effect, by interfering with NuMA function and evaluating the expression status (on or off) of genes that control cell proliferation in relation to their localization and the presence of NuMA-containing CRCs at their promoter. Significance: A current scientific and medical challenge is to unravel the mechanisms that underlie changes in the machinery or regulation of gene expression during cancer development. Understanding these mechanisms will help develop strategies to prevent cancer progression and control tumor behavior. Thus, it is time to emphasize the role of structural nuclear proteins in gene transcription in order to get a complete picture of gene expression control.