Regulation Of Era Mediated Estrogen Signaling Pathway By Sumoylated CLOCK

There is a link between the disruption of circadian rhythm and the generation of a variety of hormone-related cancers, such as breast cancer, in which aberrant ERa signaling is a major contributor. However, the molecular mechanisms associated with the disruption of circadian rhythm and the development and progression of cancers remain largely undefined. The core clock protein CLOCK belongs to the bHLH superfamily having a bHLH-PAS domain. CLOCK usually heterodimerizes with BMAL1, forming a transcriptional complex that regulates the transcription of down-stream target genes, and thus plays important roles in maintaining the circadian rhythm. At present, there are few reports focusing on the issue of whether CLOCK directly participates in the pathway related to cancer development and progression.Regulation of transcriptional factors usually involves post-translational modification of proteins, such as phosphorylation, acetylation, sumoylation and ubiquitination. Currently, few studies have focused on the post-translational modification of CLOCK other than its phosphorylation by PKC (protein kinase C) and GSK-3(glycogen synthase kinase-3). The research of this thesis focused on the regulation of CLOCK by sumoylation, and the role such modification plays in its function, especially in the estrogen signaling pathway. The main results of the findings are as follow:1. CLOCK was shown to coimmunoprecipitate with ERa in MCF-7cell extract, suggesting that these two proteins could interact with each other. This interaction was enhanced by treatment of the cells with E2. Luciferase reporter assay showed that CLOCK promoted the transcriptional activity of ERa in the presence of E2, suggesting that CLOCK may function as a coactivator for ERa and participates in estrogen signaling.2. Structure and bioinformatic analysis of the amino acid sequence of CLOCK revealed two putative SUMOylation sites, K67and K851, suggesting that it may be subjected to sumoylation. CLOCK sumoylation was subsequently demonstrated in MCF-7cells and treatment of the cells with E2appeared to enhance the sumoylation of CLOCK. Site-directed mutagenesis was performed in which K67and K851were replaced with arginine to give K67R and K851R mutants or a double mutant2K/2R. Single mutation (K67R or K851R) reduced the level of sumoylation relative to wild type, whereas double mutation (2K/2R) abolished sumoylation, resulting in reduced transcriptional activity. Desumoylation analysis using SENP proteins showed that SENP1was a desumoylation enzyme of CLOCK, and desumoylation decreased its transcriptional activity.3. Nucleo-cytoplasmic separation and immunofluorescence analyses showed that sumoylation of CLOCK enhanced its localization in the nucleus, while desumoylation of CLOCK attenuated its localization in the nucleus. Further study revealed that the double mutant CLOCK2K/2R had reduced interaction with BMAL1, which led to reduced level of transcriptional activity and protein localization in the nucleus.4. Sumoylation usually changes the structure of a protein, therefore affecting its interaction with other proteins. Desumoylation of CLOCK was shown to attenuate its interaction with ERa, as determined by immunoprecipitation assay. Sumoylation of CLOCK exerted a positive effect on the transcriptional activity of ERa. Overexpression of wild type CLOCK clearly promoted the transcriptional activity of ERa, while overpression of its mutant2K/2R had no effect.5. Cyclin D1is a classical ERa target gene, and it is closely linked to cell proliferation. Through realtime-PCR and reporter assays, wild type CLOCK was shown to promote the expression of Cyclin D1, while the mutant CLOCK2K/2R had no effect on Cyclin Dl expression. The status of cell proliferation was investigated by MTT assay, and the result showed that wild type CLOCK can promote the proliferation of MCF-7cell. Flow cytometry analysis of MCF-7cells transfected with wild type CLOCK revealed a reduction in the percentage of cells in G0/G1phase with a corresponding increasing in the percentage of cells in the S phase compared to control cells. MCF-7cells transfected with the double mutant CLOCK2K/2R showed little change in cell cycle compared to control cells.Taken together, these results clearly showed a link between the circadian protein CLOCK and the estrogen-signaling pathway mediated by ERa, and identified sumoylation as an additional form of post-translational modification of CLOCK. Revelation that sumoylation of CLOCK plays a crucial role in regulating the estrogen signaling pathway may provide a theoretical basis to uncover the molecular mechanism of the relationship between circadian rhythm disruption and the generation and development of breast cancer.