Study On The Mechanism Of EWSR1 In Mitosis

Human Ewing sarcoma breakpoint region 1 protein(EWSR1), which belongs to the TET family, is a RNA binding protein. This family could directly bind RNA and DNA, and could also bind transcription factor as a transcription cofactor. They are involved in gene expression regulation, mRNA splicing, signaling pathways and DNA repair.Initially, researchers found chromosomal translocation(t(ll; 22)(q24; q12) to form a fusion protein in Ewing sarcoma and primitive neuroectodermal tumor related subtypes. Ewing sarcoma is the second most common osteosarcoma occurred in children and young adults. Ewing sarcoma can occur at any age, but the peak incidence is before the age of 30. About 5% of adults and 10-15% of children occur such sarcoma, and there are no gender differences. The treatment of Ewing sarcoma includes chemotherapy, radiotherapy and surgery, but unfortunately, the prognosis is poor, and numbers of cases follow primary tumor and tumor metastasis. Translocations between EWSR1 and ETS genes FLI1 or ERG are found in more than 90% of the Ewing sarcoma. These translocation fusion proteins target cell growth, proliferation, senescence and tumor related genes, change the fate of cells, and thus lead to abnormal proliferation and viruous tumors. Therefore, the study on the function of EWSR1 is mainly focused on the process of the development of the fusion protein with multiple transcription factors, which is involved in the occurrence and development of malignant tumors.EWSR1 gene locates on the chromosome 22q12.2 and encodes 656 amino acid, including an N-terminal transcriptional activation domain and a C-terminal RNA binding domain. The last 18 amino acids in C-terminal domain is its nuclear localization sequence. There are 6 different transcription isoform of EWSR1, of which only two have been confirmed by experiments. EWSR1 is conserved in evolution and widely expressed in most tissues and cell lines. EWSR1 is mainly localized in the nucleus, but may also be localized in the cytoplasm and cell surface. Recent studies indicate that EWSR1 is dynamic subcellular localization in different cell types and different cell stages.In the nucleus, EWSR1 is involved with transcriptional regulation process by interacting with transcription factor TFIID, RNA polymerase II(RNAP II), and transcriptional activator or repressor. EWSR1 could also combine with RNA and DNA, transcription splicer and non encoding RNA. Recent years, researchers begin to pay attention to other features of EWSR1. EWSR1 may inhibit microtubule depolymerization and affect the process of cell cycle. EWSR1 deletion results in mitotic spindle defects and cell apoptosis. Further studies showed that EWS/FLi1 inhibits the function of EWSR1 by associated with EWSR1, EWSR1 depletion alters the localization of Aurora B in midzone during anaphase, resulting in cytokinesis failure and aneuploid. However, the molecular mechanism of EWSR1 involved in mitotic process is still not clear.This study focused on the expression patterns of EWSR1 in specific temporal and spatial, examined the dynamic location of EWSR1 during cell cycle, and explained the molecular mechanism of dynamic regulation of acetylation of spindle microtubules by EWSR1.Our study showed that the expression level of EWSR1 in the M phase was high. With the increasing proportion of M phase, the proportion of cells in which EWSR1 spread in whole cell also increased, suggesting that EWSR1 plays an important role in the M period. The flow cytometry detection showed that EWSR1 depletion prevented cells from exiting G2/M phase after the cells being synchronized to the M release by nocodazole. The indirect immunofluorescence experiments showed that EWSR1 depletion prevented cells leaving M phase. We further counted the proportion of different phase, and found that the depletion of EWSR1 mainly inhibits the cells from leaving the prometaphase and metaphase. Then the time-lapse experiments showed that after EWSR1 depletion, the time during prometaphase and metaphase was delayed, but the time during metaphase and anaphase did not change. Overexpression of siRNA insensitive GFP-EWSR1 after EWSR1 knockdown could accelerate the cells to leave the G2/M phase, and rescue the delay of prometaphase caused by EWSR1 knockdown. To better understand the mechanism of EWSR1 in regulation of mitosis, we used immunofluorescence assay to determine the location of EWSR1 in each phase of the cell cycle. In interphase, EWSR1 mainly localized in the nucleus. When the nuclear membrane was ruptured and the cells entered mitosis prometaphase, EWSR1 was whole cell distribution; during metaphase, anaphase and telophase, EWSR1 localized on the spindle. Treatment with PEM containing Taxol comfirmed the co-localization of EWSR1 and spindle. Exogenous GFP-EWSR1 also located on the spindle. Aftr konckdown of EWSR1 by special siRNA, the fluorescence of EWSR1 in spindle position was disappeared, which confirmed the specificity localization of EWSR1 on spindle. Subsequently, immune coprecipitation assay showed that EWSR1 interacted with α-Tubulin in the synchronized cells to M phase but not in asynchronized cells. Pull-down GST experiment showed that EWSR1 interacted with α-Tubulin directly. Overexpression of EWSR1ΔNLS mutant which is lack of nuclear lacalization sequence(NLS) rescued the prolonged time from NEB to metaphase induced by EWSR1 knockdown. After treatment with α-amanitin, the RNA polymerase II inhibitor, no obvious changes were found in the inhibition effects of EWSR1-siRNA on mitosis. These results suggest that the role of EWSR1 in mitosis is independent on its functionin in nuclear.Microtubule regrowth experiments indicated that depletion of EWSR1 inhibited the formation of spindle microtubules and cold treatment experiments indicated that knockdown of EWSR1 led to spindle microtubules unstable. BubR1 and Mad2 are mitotic checkpoint SAC protein. When microtubules combine with kinetochores and all chromosomes are arranged to the metaphase plate, the signals of BubR1 and Mad2 are inactivate and the APC/C is actived, followed with chromosome segregation. Our results indicate that knockdown of EWSR1 did not affect the localization of BubR1 and Mad2 on the kinetochores. And knockdown of EWSR1 did not change the location of of Aurora B in prometaphase and metaphase, which indicates that Aurora B does not participate in the regulation of mitosis by EWSR1.There is a close relationship between the acetylation of the K40-Tubulin and the stability of microtubules. Western blot experiments showed that in synchronized cells of M phase, overexpression of EWSR1 promoted the acetylation of α-Tubulin, depletion of EWSR1 inhibited the acetylation of α-Tubulin. But when the cells were asynchronous, depletion of EWSR1 did not affect the acetylation of α-Tubulin. Immunofluorescence assay showed that EWSR1 depeltion inhibited the acetylation of α-Tubulin in the prometaphase and metaphase, but did not affect other periods of cell cycle. Further we ruled out the effects of chromosome arrangement abnormalities and MG132 on acetylation. Overexpression of Myc-EWSR1ΔNLS could promote the acetylation of α-Tubulin as overexpression of Myc-EWSR1. After treatment with α-amanitin, no obvious changes were found in the inhibition effects of EWSR1-siRNA on Ac-tubulin in mitotic cells, which suggests that EWSR1 regulates α-Tubulin acetylation in a transcription-independent manner. EWSR1 knockdown did not affect the detyrosination and polyglutamylation of α-Tubulin. HDAC6 and SIRT2 are known to be the deacetylases of α-Tubulin. The inhibition of Ac-tubulin induced by EWSR1 knockdown was obviously weakened in cells treated with HDAC6 specific inhibitor tubacin or HDAC6 siRNA, which suggests that EWSR1 affects the acetylation of α-Tubulin through HDAC6. EWSR1 knockdown could still inhibit the acetylation of α-Tubulin in cells after SIRT2 depletion, which indicates that SIRT2 does not participate in the acetylation of α-Tubulin by EWSR1. Furthermore, tubacin can reverse the effect EWSR1 knockdown on abnormal chromosome arrangement and multipolar spindle. The immunoprecipitation results indicates that EWSR1 might competitively bind with spindle MTs, leading to the decreased binding of HDAC6 with spindle.In conclusion, our study suggests that EWSR1 plays a crucial role in the mitotic progression and spindle dynamics and promotes MTs acetylation in the mitotic spindle by inhibiting the activity of HDAC6. Our study might benefit to extend our understanding about the mechanism of EWSR1 in mitotic arrest and supply some new ideas for developing drugs targeting mitosis disorder for tumor treatment.