| BackgroundAcute myeloid leukemia(AML)is a heterogeneous hematologic malignancy,which characterized by the accumulation of immature cells and blocked differentiation.AML is the commonest acute leukemia in adults which usually has a poor prognosis.Methyltransferase-like protein 3(METTL3)was identified as an S-adenosine methionine-binding protein.Recent studies have confirmed that METTL3 is abnormally overexpressed in AML and is related to the occurrence and development of AML.Our group’s previous studies have found that METTL3 plays an important role in the chemoresistance of AML.STM2457,the small molecule inhibitor of METTL3,has shown good therapeutic effect in AML animal models.However,METTL3-mediated m6A modification plays a significant role in the central and cardiac systems,so the prospects for systemic treatment of the inhibitor is less clear.Therefore,the establishment of AML-specific targeted intervention mechanisms of METTL3 is of great significance for the application of METTL3 in the clinical treatment of AML.Studies have confirmed that METTL3 expression is regulated by different regulatory mechanisms in different tumors and organs,and the abnormal expression of METTL3 in different tumors suggests that METTL3 expression is finely regulated in different environments.Elucidate the mechanism of abnormal overexpression of METTL3 in AML has become a new choice for targeted intervention.Yin-Yang 1(YY1)is a ubiquitous zinc-finger structure transcription factor.A large number of literatures showed that YY1 was highly expressed in different tumors and acted as an oncogene.Examples include prostate cancer,breast cancer,osteosarcoma,AML and colon cancer.YY1 plays a dual role in transcriptional regulation,and YY1 inhibits or activates gene transcription depending on the cellular environment.Firstly,as a traditional transcription factor,YY1 interacts with a variety of cofactors to promote or inhibit gene transcription.Secondly,YY1 interacts with the chromatin remodeling complexes,affecting chromatin modification and the gene expression.Thirdly,YY1 can activate gene expression by controlling chromatin loops and stabilizing enhance-promoter interactions.The potential mechanism of YY1 to realize its complex regulatory effect has been the focus of research.Liquid-liquid phase separation(LLPS)refers to the process by which key molecules aggregate with other proteins or RNAs into closed liquid-like cavities when threshold concentrations are reached and is involved in the development of various body pathological and physiological processes,such as immunity,transcription,autophagy,cancer and neurodegenerative diseases.LLPS can be regulated by many factors,such as temperature,ubiquitination,acetylation,RNA and DNA.The dual characteristic of YY1,which can both inhibit and activate transcription,are similar to the dynamic balance characteristic of LLPS,suggesting that new explanation can be provided for the "Yin" and "Yang" regulation of YY1 from the perspective of LLPS.To sum up,this study can divided into two parts.The first part aims to explore the mechanism of the abnormal expression of METTL3 in AML and clarify the transcriptional regulation of YY1 on METTL3.The second part aims to clarify the specific mechanism of YY1 regulating METTL3 expression from the perspective of LLPS,so as to provide a therapeutic target for AML.ObjectivesThe first part1.Identify upstream regulatory molecules of METTL3 through website prediction and bioinformatics analysis.2.Molecular biology and cytology experiments were used to verify the regulatory effect of YY1 on METTL3,and clarify that YY1 could promote the proliferation of AML cells by regulating METTL3.The second part1.Explore the potential upstream mechanism of YY1 regulating AML proliferation through METTL3,and analyze the influence of interaction between HD AC 1/3 and YY1 on METTL3.2.Analysis the "Yin" and "Yang" regulatory mechanisms of YY1 from the perspective of LLPS,and clarify the regulation of phase separation of YY1 on METTL3 expression and AML cells proliferation.MethodsThe first part1.Using ChIPBase v2.0,UCSC and GEPIA website to predict the transcription factors of METTL3 and screen out the key molecule YY1.2.Bone marrow samples were collected from AML patients,and the mononuclear cells of bone marrow were isolated by Ficoll lymphocyte isolation solution for RNA extraction.The expressions of YY1 and METTL3 in bone marrow samples of AML patients were detected by qRT-PCR assay,and the correlation between YY1 and METTL3 was analyzed.The correlation between YY1 and METTL3 in AML cell lines was detected by qRT-PCR and Western blot experiments.The binding of YY1 to the promoter region of METTL3 was confirmed by ChIP-qPCR technique and double luciferase reporter assay.3.The regulation of YY1 on AML cells proliferation through METTL3 was verified by EdU,CCK-8,clonal formation assay and other experimental techniques.The second part1.The correlation between YY1 and HD AC 1/3 was analyzed by Co-IP assay,immunofluorescence co-localization assay and other experimental methods,and on this basis,the influence of HDACi on the regulation of YY1 was discussed through basic experiments.2.Laser confocal microscope was used to observe the phase separation characteristics of YY1.Co-IP,CCK-8,dual luciferase reporter analysis,qRT-PCR and other experimental methods were used to clarify the key site of YY1 phase separation and clarify the function of the key sites.ResultsThe first part1.YY1 promoted the expression of METTL3 as a transcription factor.1.1 YY1 is the transcription factor of METTL3:We first used ChIPBase v2.0 website to predict METTL3 transcription factors and found that YY1 was the most important transcription factor.Then,we found that YY1 was obviously enriched in the promoter region of METTL3 gene through UCSC website.1.2 YY1 was significantly positively correlated with METTL3 expression:Firstly,we used the TCGA database of GEPIA website and collected marrow fluid samples of AML patients to analyze the correlation between YY1 and METTL3 expression,and found that they were positively correlated.Then,YY1 was overexpressed/interfered with in AML cell lines,and we found that the expression of METTL3 was consistent with YY1.1.3 YY1 binds to the promoter region of METTL3 to promote its expression:We confirmed the binding of YY1 to the promoter region of METTL3 by ChIP-qPCR.The specific binding sites of YY1 to the promoter region of METTL3 were identified by dual luciferase reporter analysis,and indicated that YY1 positively regulates METTL3 expression.2.YY1 promoted the proliferation of AML cells in a METTL3-dependent manner.2.1 YY1 can promote the multiplication of AML cells:We found that the cellular multiplication was significantly slowed down after YY1 was interfered through EdU,CCK-8 and clonal formation assays.2.2 YY1 promoted the proliferation of AML cells in a METTL3-dependent manner:EdU,CCK-8 and clonal formation assays showed that the propagation of AML cells was accelerated after YY1 was overexpressed.Interference with METTL3 on the basis of YY1 overexpression could reverse the stepped-up proliferation of AML cells caused by YY1 overexpression.2.3 METTL3 is an important downstream functional target of YY1 in AML cells:EdU,CCK-8 and clonal formation tests showed that the proliferation of AML cells was significantly weakened after YY1 interference.Overexpression of METTL3 while interfering with YY1 could save the decreased proliferation of AML cells caused by YY1 interference.The second part1.HDACi treatment can disrupt the binding of YY1 to HD AC 1/3,and inhibit the expression of METTL3 and the proliferation of AML cells.1.1 Interaction between YY1 and HD AC 1/3:We first confirmed the existence of protein interaction between YY1 and HD AC 1/3 through Co-IP experiment.Meanwhile,cell immunofluorescence experiment showed that YY1 was co-located with HD AC 1/3,and the co-location of both was reduced after HDACi treatment.It is suggested that YY1 can interact with HD AC 1/3 to form molecular complex.1.2 HDACi treatment can inhibit the transcriptional regulation of YY1 on METTL3:After HDACi treatment,the acetylation level of YY1 was significantly increased,but the expression of YY1 did not change.In addition,HDACi decreased the expression of METTL3 in AML cells and reversed the increase of METTL3 expression caused by YY1 overexpression.It is suggested that HDACi inhibits the transcriptional regulation of YY1 on METTL3 by regulating YY1.1.3 HDACi regulates the proliferation of AML cells through YY1:EdU,CCK-8 and clonal formation experiments showed that HDACi could inhibit the proliferation of AML cells and reverse the proliferation-promoting effect of YY1.2.The separation of YY1 from HD AC 1/3 resulted in enhanced liquid-liquid phase separation ability.2.1 YY1 can undergo LLPS in/out of cells:EGFP-YY1 protein can form droplets in vitro,and has the two basic characteristics of phase separation,droplet fusion and recovery after fluorescence bleaching.In addition,YY1-EGFP can also undergo LLPS in U20S cells.2.2 The phase separation ability of YY1 were enhanced after YY1 separated from HD AC1/3:After treatment with the HD AC inhibitor,the intracellular particles of AML cells increased significantly,and after interfering with intracellular HD AC 1/3,the intracellular particles also increased significantly.It shows that the degree of the LLPS of YY1 is related to HDAC1/3.3.The mutation of YY1 and HD AC 1/3 binding site affected the regulation of YY1 on METTL3 expression and AML cell proliferation.3.1 The phase separation ability of YY1-6R mutant was enhanced:After the mutation of YY1 and HD AC1/3 binding site,intracellular particles increased significantly.3.2 YY1-6R mutant inhibits the regulation of METTL3 by YY1 and the proliferation of AML cells:Analysis of METTL3 expression and cell proliferation in AML cells showed that the decreased binding of YY1 to HDACs affected the regulation of METTL3,and then,influenced the proliferation of AML cells.ConclusionsHD AC 1/3 dependent YY1 liquid-liquid phase separation promoting METTL3 expression and ultimately promoting the proliferation of AML cells. |
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