YY2 Suppresses Tumorigenesis By Inducing Ferroptosis

Yin Yang(YY)family consists two members with high homology,Yin yang 1(YY1)and Yin yang 2(YY2),that belong to the GLI-Krüppel zinc finger transcription factor.YY1,the first-identified YY family member,could act as both positive and negative regulators of target genes depend on the contexts.YY1 plays an important role in various biological processes.YY2 is another member of YY family identified more recently.YY2 originated from YY1 m RNA retrotransposed and inserted into the region between exons 5 and 6 of the Mbtps2 gene after the divergence of placental mammals from other mammals.YY2 is highly homologous with YY1 in their nucleotide and amino acid sequences.While YY1 has been known to be expressed ubiquitously in many tissues and is involved in the regulation of various biological pathways,our knowledge regarding the functions of YY2 is still very limited.Initially,YY2 was assumed to simply aid YY1 in regulating its target genes;however,increasing evidences indicated that YY2 exerts its own specific function in maintaining embryonic stem cells,as well as in cardiovascular and nervous system development.While YY1 is overexpressed in various cancers and functions as an oncogene,YY2 mutations and aberrantly low expression has been found in various tumors.Furthermore,our previous study showed that YY2 could inhibit cell cycle progression and proliferation by inducing p53/p21 pathway.These results suggest that YY2 might be a tumor suppressor;however,the role of YY2 in tumor progression as well as its molecular mechanism have not been totally elucidated.Furthermore,the molecular mechanism underlying the opposite function of these homologous genes also need to be elucidated.In this study,we found that YY2 could induce ferroptosis and thereby suppress tumorigenesis.Using transcriptomic analysis,molecular biology,cellular biology,and biochemistry as well as tumor xenograft model,we systematically analysed YY2 regulation on tumor cell ferroptosis and ferroptosis-mediated tumor suppressive effect,as well as the molecular mechanism underlying it.We also analysed the molecular mechanism underlying the opposite effect of YY1 and YY2.We first found that YY2 could significantly suppress the proliferation potential of several colorectal cancer cell lines and the tumorigenesis potential in xenograft model.Furthermore,its expression level showed a negative correlation with the poor prognosis of clinical colorectal cancer patients.Using colorectal cancer cell line HCT116 and YY2-overexpression HCT116 cells,we performed RNA-seq transcriptomic analysis.By analyzing differentially-expressed gene and performing Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,we found that YY2 plausibly involved in ferroptosis regulation through regulating cystine/glutamate transporter SLC7A11.SLC7A11 is a transporter protein localizes at cell membrane,its suppression leads to the reduction of cellular cystine,and in turn,the reduction of glutathione synthesis.Glutathione is an important factor in maintaining cellular homeostasis,its downregulation enhances the formation of lipid peroxide,and subsequently induces ferroptosis.We found that the expression levels of YY2 and SLC7A11 showed a negative correlation in clinical colorectal cancer lesion.Furthermore,using cellular and xenograft experiments,we demonstrated that YY2 downregulated the GSH level in tumor cells and xenografted tumor tissue by suppressing SLC7A11,and that YY2/SLC7A11 signaling pathway plays critical role in regulating ferroptosis and tumorigenesis.To further reveal the molecular regulatory mechanism of YY2 on SLC7A11,we performed dual luciferase reporter assay,chromatin immunoprecipitation assay,and point mutation analysis,and proved that SLC7A11 could directly bind to the CCAT sequence at-667 to-664 of the SLC7A11 promoter and suppressed its transcriptional activity.Furthermore,we found that mutations at the first and third zinc finger domains of YY2 diminished its ability to suppress SLC7A11 transcription.YY2 and YY1 are highly homologous,and the homology at their zinc finger domains are especially high(86%).Furthermore,the core binding site of YY2 on SLC7A11 promoter,5?-CCAT-3?,is a common DNA binding site of both YY1 and YY2.Thus,we next further examined whether YY1 could also regulate SLC7A11.Our results showed that in contrast to YY2,YY1 significantly upregulated the transcriptional activity of SLC7A11 promoter and promoted SLC7A11 expression level.We further elucidated the relation of YY2 and YY1 regulation on SLC7A11 Using DNA affinity precipitation assay(DAPA),we revealed that YY2 and YY1 competed to bind to the same site of the SLC7A11 promoter.Furthermore,while YY1 suppressed lipid peroxidation and the percentage of cell death,YY2 cancelled this effect.These results indicated that YY2 and YY1 could competitively bind to SLC7A11 promoter,thereby exerting opposite regulations on SLC7A11 expression and ferroptosis,and subsequently,their opposite effect on tumorigenesis.Given that YY1 and YY2 have high homology and share common binding sites,we examined the effect of YY1 overexpression on SLC7A11.In contrast to YY2,YY1 significantly promoted SLC7A11 expression.These results,together with YY1 overexpression blocking the downregulation of SLC7A11 induced by YY2 overexpression,hinted at a competitive regulation by YY1 and YY2 of the same site on the SLC7A11 promoter.To further explore the potential competitive transcriptional regulation of SLC7A11 by YY1 and YY2,we performed a DNA affinity precipitation assay suggested that YY1 and YY2 acted antagonistically in regulating ferroptosis in tumor cells.This effect was achieved through competitive binding and regulation of the same site on the SLC7A11 promoter,thereby leading to opposite regulation of ferroptosis.In summary,our findings describe a competitive binding between YY2 and YY1 on the promoter of cystine/glutamate transporter SLC7A11 that results in the opposite regulation on its transcriptional activity.This in turn regulates ferroptosis in an opposite manner,and furthermore,highlighting their different roles in tumorigenesis.Furthermore,our results also show,for the first time,that the balanced YY1 and YY2 expression is crucial for determining cell fate,and the loss of this balance might lead to the progression of tumors and other diseases.