The Molecular Mechanisms Of TRIM28 Promoting P53 Ubiquitination And Assembling Transcriptional Repression Complex

TRIM28 is a member of TRIM family,TRIM28 complexes play an important role in regulating the formation and development of tumors at the transcriptional level,epigenetic modification,and post-translational modification level of eukaryotes.Malignant tumor is a major disease threatening human life and health.As a tumor suppressor,p53 is essential to maintain genomic stability and prevent malignant transformation.p53 level is mainly regulated by protein stability.MDM2 is a major E3 ligase that ubiquitinates p53,and TRIM28 could enhance the activity of MDM2 and accelerate p53 degradation through the proteasome pathway.Besides,TRIM28 could bind to melanoma-associated antigen protein MAGE-C2,activate E3 ligase activity and promote p53 ubiquitination independently of MDM2.Gene transcription repression is important for negative regulation of gene expression,and it is a key step to ensure the correct expression of genetic information.Transcriptional inhibition also plays a critical role in cell differentiation,morphogenesis and ontogenesis.In the process of regulating gene transcriptional,TRIM28 induces the assembly of epigenetic silencing complex by recruiting zinc finger protein KRAB-ZNF that specifically recognizes DNA retrotransposon sequence.Heterochromatin protein HP1 is also recruited by TRIM28,leading to chromatin remodeling and targeting to supress gene transcription.MAGE-C2 is highly expressed in various cancers,the molecular mechanism of MAGE-C2 participating in MDM2-mediated p53 ubiquitination is still unclear.The relationship between the two p53 ubiquitination pathways regulated by TRIM28 remains to be further studied.In this paper,the molecular mechanism of p53 protein level regulation,cell proliferation and migration was studied by protein expression,purification,ubiquitination assay,cell proliferation,migration and ultracentrifugation analysis.We proved that the conserved MHD domain of the MAGE-C2 protein is essential for direct binding to MDM2 and inhibiting its E3 ligase activity.In TRIM28 knockdown cells,overexpression of MAGE-C2 could inhibit p53 ubiquitination and slow down cell proliferation.However,the restoration of TRIM28 protein level antagonizes the inhibitory effect of MAGE-C2 and accelerates p53 inactivation by cooperating with TRIM28-MDM2 and TRIM28-MAGE-C2 to promote cell proliferation and migration.This study clarified the relationship between the two p53 ubiquitination pathways participated by TRIM28 and revealed the role of MAGE-C2 and TRIM28 in p53 ubiquitination and cell proliferation,which settled a foundation for further understanding the regulatory mechanism of tumor suppressor p53 and related anti-tumor drugs.In addition,the structure of TRIM28-mediated transcriptional repression complex is still unclear,and the mechanism of how KRAB-ZNF performs its DNA sequencespecific recognition function remains unknown.In order to study the molecular mechanism of TRIM28 as a transcriptional co-repressor regulating gene expression,the KRAB-TRIM28-HP1 complex was recombinated in vitro,the molecular proportion and assembly mode of the complex were analyzed,and we preliminarily obtained the cryoelectron microscopic data of the complex structure.For the partial domain of TRIM28 and HP1 related complex,we utilized crystal screening and diffraction to explore the molecular mechanism of HP1-mediated heterochromatin formation.In addition,a liquid-liquid phase separation model of KRAB-TRIM28-HP1 complexmediated heterochromatin formation was established.Through the study on the structure of the transcriptional repression complex,we revealed the assembly mode of the KRAB-TRIM28-HP1 complex and the structural basis for its role in inhibiting gene transcription and explored the molecular mechanism of KRAB-TRIM28-HP1 mediated liquid-liquid phase separation for DNA sequence-specific transcriptional repression.In this paper,through the study of TRIM28 regulating gene expression at the transcriptional level and the regulation of tumor suppressor p53 ubiquitination at the post-translational modification level,we demonstrated that TRIM28 is essential for promoting cell proliferation and regulating gene transcription,and further revealed the molecular mechanism of tumor formation and development in terms of structure and function.