NuMA function in nonmalignant and malignant breast cells

描述（由申请人提供）：染色质结构和基因表达的改变是癌症的标志。参与染色质重塑复合物（CRC）的蛋白质通过修饰DMA和组蛋白直接影响染色质结构，并且CRC靶向在癌症中改变。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，并且它们的分布在癌症中改变。然而，NuMA在基因表达控制中的作用尚不清楚。NuMA与ATP依赖性CRC组分共分离。使用针对NuMA 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展有关。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。该假说是NuMA通过与CRC的组分相互作用参与基因转录的控制，以便将这些复合物靶向特定基因。提出了三个目标：（1）通过亲和层析和免疫共沉淀评估NuMA与SNF 2 h和BAF型ATP依赖性CRCs组分的相互作用，并评估抑制NuMA表达对CRCs组装和功能的影响，以鉴定NuMA参与的特异性CRCs;（2）通过比较阻断HPC 2样、GAS 41结合、和NuMA的CH结合区对染色质组织、CRC功能和乳腺上皮分化的影响;（3）通过干扰NuMA功能并评价控制细胞增殖的基因的表达状态（开或关）（与其定位和在其启动子处存在含NuMA的CRC有关），通过位置效应确定NuMA在基因表达控制中的作用。重要性：当前科学和医学的挑战是揭示癌症发展过程中基因表达机制或调控变化的机制。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，现在是时候强调结构核蛋白在基因转录中的作用，以获得基因表达控制的全貌。

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.