Molecular Mechanism And Functional Studies Of YY1 Oligomerization In Breast Cancer

Yin Yang 1(YY1)is an important multifunctional transcription factor,and highly expressed in almost all types of solid tumors.It exerts crucial regulatory activities in the onset and development of cancers.In recent years,with the extensive functional studies,the potential of YY1 as a cancer therapeutic target has received increasing attention and recognition.Generally,at the transcriptional level,YY1 can bind to the promoters containing its specific binding motifs,and subsequently recruit various of histone modifying enzymes,such as p300,EZH2 and HDACs,to regulate gene expression through modulating chromatin remodeling.At the protein level,YY1 binds MDM2 through the oncoprotein binding(OPB)domain located in amino acid stretch201-226,and promotes MDM2-mediated p53 ubiquitination and degradation.Using the OPB domain,YY1 also binds and activates AKT through promoting its phosphorylation at the S473.In recent years,YY1 dimerization has been reported in several studies,which can facilitate chromatin looping to pull distant enhancers and promoters close,and subsequently regulate target gene expression.In this study,we carried out innovative exploration on the molecular mechanism underlying YY1 dimerization.The main experimental results of this study include:1.Identification of the binding regions responsible for YY1 intermolecular interactions: The recombinant proteins of wild type(wt)YY1 and its truncated mutants were expressed in bacteria and subsequently purified.Through protein interaction assays,we preliminarily identified that the regions involved in YY1 intermolecular interactions were mainly located in the middle and C terminal regions of the YY1 protein.Then,using the surface plasmon resonance(SPR)technique,we verified that the OPB and zinc finger(ZF)domains of YY1 were involved in the interactions of YY1 monomers.2.Determining the binding model of YY1 intermolecular interactions: We generated eukaryotic expression vectors of YY1(wt)and its truncated mutants with different tags.In co-transfection and co-IP studies,the mutual interactions between YY1 homologous regions were determined as the major binding mode for its intermolecular interaction.The ZF region is responsible for DNA binding,and is generally reputed for promiscuous interactions among proteins.Therefore,in the following studies,we focused on the YY1(ΔZF)to determine YY1 intermolecular interactions.3.Identification of YY1’s ability of oligomerization: In co-IP studies to evaluate how OPB-containing fusion proteins could affect the intermolecular interactions of YY1,we observed that the addition of the OPB peptides promoted the process of YY1(ΔZF)dimerization or oligomerization.Consistent with these results,chemically synthesized OPB peptides could also promote the recombinant YY1(ΔΖF)to form oligomers with high molecular weights visualized in native polypropylene gel electrophoresis.4.Identification of the crucial amino acids that determine YY1 oligomerization:Firstly,we employed molecular model prediction algorithms,including SWISSMODEL,ZDOCK and COOT,to perform bimolecular docking simulations and evaluations of the OPB domains.The results indicated that the interactions between OPB domains were modulated by 8 hydrophobic residues.The docking analyses of the hydrophobic side chains based on the two conformations with the highest simulative binding scores were carried out,and three hydrophobic amino acids,I212,L215 and F219,were determined to most likely affect YY1 intermolecular interactions.Four YY1(ΔΖF)mutants expressing vectors containing mutations of I212 A,L215A,F219 A and 3A(with all three hydrophobic residues mutated to alanine)were generated.Using co-IP studies,we demonstrated that F219 in the OPB domain was the key amino acid playing a decisive role in YY1 oligomer formation.5.Identification of the effects of YY1 oligomerization on breast cancer cells proliferation and migration: In breast cancer MDA-MB-231,MDA-MB-453 and MCF-7 cells,sh RNA targeting the 3’-UTR of the YY1 m RNA was used to knockdown the endogenous YY1.Subsequently,YY1(wt)and its mutants were stably expressed in these cells through lentiviral infection,following by the examination of cell proliferation and migration.The results indicated that the oligomerizationdeficient mutants,YY1(F219A)and YY1(3A),displayed stronger ability than YY1(wt)to promote the proliferation and migration of breast cancer cells.6.Exploring the molecular mechanism underlying the improved proliferative ability of the YY1 oligomerization-deficient mutants compared to YY1(wt):According to previous reports,the OPB domain of the YY1 protein can bind the oncoproteins EZH2,AKT and MDM2,and enhance their activities.In co-IP experiments,compared to YY1(wt),the YY1(F219A)and YY1(3A)mutants exhibited increased binding affinity to EZH2 and AKT,but decreased binding to MDM2.Therefore,we proposed that the amount of monomeric YY1 in cells was increased due to the oligomerizing deficiency of the YY1(F219A)and YY1(3A)mutants,and consequently improved their binding opportunities to EZH2 and AKT.As a result,the two YY1 mutants could better activate EZH2-and AKT-mediated oncogenic signaling pathways than YY1(wt)to enhance the proliferation and migration of breast cancer cells.In summary,this study first identified the OPB domain as the major domain to promote YY1 intermolecular interactions.Furthermore,the hydrophobic residue F219 in the OPB domain was revealed as the crucial amino acid to determine YY1 oligomerization.Additionally,the competitive relationship between YY1 oligomerization and its interactive regulation to oncoproteins EZH2 and AKT was revealed.This study delivers direct experimental evidence for in-depth understanding of the transcriptional regulatory mechanism of YY1 at the chromatin level,and also provides a new theoretical basis for YY1 as a novel target of cancer therapies.