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. ? ? ?
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