| Cancer metastasis-associated gene1(MTA1) was first cloned in1993, in a highly metastatic breast cancer cell line. Afterwards, in addition to metastasis, MTA1was proved to promote multiple malignant behaviors of cancer cells, such as proliferation, angiogenesis, resistance to radiation and chemotherapy, and so on, resulting in a high recurrence and poor prognosis of cancer. However, despite two decades of research on MTA1, the mechanism underlying its function is still obscure. Up to now, only nucleosome remodeling and histone deacetylation (NuRD) has been identified as a complex through which MTA1plays its role in transcriptional repression of genes, especially cancer suppressor genes. However, other MTA1-involved processes independent of NuRD have been indicated. There are also questions concerning how MTA1is involved in activating transcriptions of some genes, such as Pax5, BCAS3, FosB and so on. So, we proposed that there may be other unknown underlying mechanisms urgent to be discovered. As for subcellular localization, the only confirmed fact is that MTA1has an obvious localization in nucleus, but no more other details were revealed. There are still debates on whether MTAl possesses cytoplasmic distribution.The exact subcellular localization and co-partners of a protein provide good clues to trace its functions and mechanisms. To further explore the mechanisms through which MTA1promotes cancer development, we initiated a performance to give a comprehensive overview on MTA1distribution by multiple molecular technologies, including immunohistochemistory, cell immuno fluorescence, GFP flag tracking, Western Blot, immunoprecipitation and in situ co-IP. Both endogenous and exogenous MTAl have been examined to confirm its existance in both nucleus and cytoplasm. Moreover, we also found an obvious localization of MTA1on nuclear envelope. A vivid image captured in a stably MTA1-transfected cell line indicated a shuttling model of MTA1between nucleus and cytoplasm through nuclear envelop.MTA1was detected in all samples examined, including mouse and human normal adult tissues, mouse embryonic tissues, colon cancer tissues, normal and cancer cell lines indicating a role of MTA1in both normal and cancer cells. In adult normal tissues, MTA1displayed a relativly higher expression in brain, liver, kidney and cardiac muscle, and was localized mainly in nucleus except cardiac and skeletal muscle fibers. In skeletal muscle, MTA1was co-localized with SMN on Z-line in cytoplasm. At the embryonic stage of development, MTA1was higher in nerve tissues such as brain, eye and spinal cord, and mainly expressed in cytoplasm of most tissues. In the colon cancer tissues evaluated by tissue array staining, both nuclear and cytoplasm MTAl were positively correlated with cancer progression. But only nuclear MTAl was associated with cancer cell differentiation.MTAl works as a higher-order chromatin structure regulator in nucleus. Chromatin structure plays a critical role in determining gene expression pattern and hence cellular biological processes. In this section, we, for the first time, found that Metastasis-associated gene1(MTA1), which has been reported to be a cancer-promoting gene, was a higher-order chromatin structure organizer to decondense the interphase chromatin and mitotic chromosomes. MTAl interacts dynamically with DNA during cell cycle progression, prominently contributing to the mitotic chromatin/chromosome transitions at both prophase and telophase. We showed that the decondensation of interphase chromatin by MTAl is independent of the chromatin remodeling activity of Mi-2with which MTAl interacts to assemble the nucleosome remodeling and histone deacetylation complex (NuRD). H1was reported to stabilize the compact higher-order chromatin structure through its interaction with DNA. Our data showed that MTAl caused a reduced H1-DNA interaction in vivo. Moreover, the dynamic MTA1-DNA interaction in the cell cycle contributed to the periodical H1-DNA interaction, which in turn modulated chromatin structure transitions. Although MTAl drove a global decondensation of chromatin structure, it changed the expression of only a small proportion of genes. After MTAl overexpression, the up-regulated genes were distributed in clusters with down-regulated genes on chromosomes at parallel frequencies. This fact indicates that MTA1induced a structure-dependent, but not only a function-dependent gene set as presumed. Putting the data together, we discovered a novel roleof MTA1as a potent modulator of chromatin higher-order structure to regulate the mitotic chromatin/chromosome structure transitions and interphase chromatin transcriptionMTAl inhibits spindle assembly checkpoint (SAC) activation through binding to spindle microtubules in cytoplasm. At interphase, MTAl was confirmed to bind to microtubules in the cytoplasm though at a relatively low level. During mitosis, most of MTA1moved along with the mitotic spindle apparatus in cytoplasm and showed a cell-cycle-dependent distribution pattern. By nocodazol treatment, we showed that MTA1powerfully inhibited spindle assembly checkpoint activation. When overexpressed in HCT116cells, MTAl caused an obviously larger proportion of micronucleated and multinucleated cells due to defectives in chromosomes alignment and separation, which are the typical characteristics of SAC inactivation. Translocated promoter region (TPR) is a member protein of nuclear pore complex (NPC) which functions in nuclear export of mRNAs and proteins at interphase and SAC regulation during mitosis. By immunoprecipitation and co-localization visualization, we confirmed that MTA1collaborates with TPR both at interphase NPC and mitotic spindle apparatus. By further investigation, we proposed that MTA1inhibits SAC activation by direct binding to TPR and competing with MAD1-MAD2complex for TPR, resulting in a reduced MAD1-MAD2interaction, which is essential in SAC activation.Analysis on subcellular localization and interactome of MTA1indicates a potential role in RNA regulation. MTA1has been reported to be negative in nucleolus. However, by high magnification observation under fluorescence microscope for both endogenous and exogenous MTA1, we found a low but general expression of MTA1in nucleolus. It was showed that MTA1was localized in both DFC and GC regions of nucleolus by high-resolution immunoelectron microscopy. Both the subcellular localization and co-partner identification supported the role of MTA1in rRNA processing and ribosome subunit assembly. We also for the first time located MTA1at cytoplasmic RNP granules which functions in RNA transport, stability maintenance and temporary storage on stress. GO analysis of MTA1interactome also yielded74RNA binding proteins out of228of the interactome (32.5%), involving RNA-regulating processes, such as RNA processing, splicing, translation, stability maintenance, ribosome biogenesis and so on. Transcriptome sequencing revealed significant influence of MTA1depletion by specific siRNA on966alternative splicing events. Interestingly, MTA1itself is under the alternative splicing regulation by MTA1overexpression.To sum up, we displayed a shuttling distribution model of MTA1among nucleolus, nucleus, nuclear envelope and cytoplasm, confirmed a dynamic distribution pattern of MTA1during the cell cycle, and constructed an interactom network in the whole cell, all of which were integrated to establish the three fundamental roles of MTA1in chromatin higher order structure, cytoskeleton and RNA regulation. Dysregulation of these physiological roles in normal cells by MTA1overexpression may lead to its pathological roles in cancer promotion. |
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