Yin Yang 1 promotes mTORC2-mediated AKT phosphorylation.

Yin Yang 1 (YY1) regulates both gene expression and protein modifications, and has shown a proliferative role in cancers. In this study, we demonstrate that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. YY1 expression positively correlated with AKT(S473) phosphorylation in a tissue microarray and cultured cells of breast cancer, but negatively associated with the distant metastasis-free survival of 166 breast cancer patients. YY1 promotes AKT phosphorylation at S473 through direct interaction with AKT, and the AKT-binding site is mapped to the residues G201-S226 on YY1. These residues are also involved in YY1 interaction with Mdm2, Ezh2, and E1A, and thus are designated as the oncogene protein binding (OPB) domain. YY1-promoted AKT phosphorylation relies on the OPB domain but is independent of either transcriptional activity of YY1 or the activity of phosphoinositide-3-kinases. We also determine that YY1-promoted mTORC2 access to AKT leads to its phosphorylation at S473. Importantly, a peptide based on the OPB domain blocks YY1 interaction with AKT and reduces AKT phosphorylation and cell proliferation. Thus, we demonstrate for the first time that YY1 promotes mTORC2-mediated AKT activation and disrupting YY1-AKT interaction by OPB domain-based peptide may represent a potential strategy for cancer therapy.

DNA vector-based RNA interference to study gene function in cancer.

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

Yin Yang 1 plays an essential role in breast cancer and negatively regulates p27.

Yin Yang 1 (YY1) is highly expressed in various types of cancers and regulates tumorigenesis through multiple pathways. In the present study, we evaluated YY1 expression levels in breast cancer cell lines, a breast cancer TMA, and two gene arrays. We observed that, compared with normal samples, YY1 is generally overexpressed in breast cancer cells and tissues. In functional studies, depletion of YY1 inhibited the clonogenicity, migration, invasion, and tumor formation of breast cancer cells, but did not affect the clonogenicity of nontumorigenic cells. Conversely, ectopically expressed YY1 enhanced the migration and invasion of nontumorigenic MCF-10A breast cells. In both a monolayer culture condition and a three-dimensional Matrigel system, silenced YY1 expression changed the architecture of breast cancer MCF-7 cells to that resembling MCF-10A cells, whereas ectopically expressed YY1 in MCF-10A cells had the opposite effect. Furthermore, we detected an inverse correlation between YY1 and p27 expression in both breast cancer cells and xenograft tumors with manipulated YY1 expression. Counteracting the changes in p27 expression attenuated the effects of YY1 alterations on these cells. In addition, YY1 promoted p27 ubiquitination and physically interacted with p27. In conclusion, our data suggest that YY1 is an oncogene and identify p27 as a new target of YY1.

Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1.

Yin Yang 1 (YY1) is a multifunctional protein with regulatory potential in tumorigenesis. Ample studies demonstrated the activities of YY1 in regulating gene expression and mediating differential protein modifications. However, the mechanisms underlying YY1 gene expression are relatively understudied. G-quadruplexes (G4s) are four-stranded structures or motifs formed by guanine-rich DNA or RNA domains. The presence of G4 structures in a gene promoter or the 5'-UTR of its mRNA can markedly affect its expression. In this report, we provide strong evidence showing the presence of G4 structures in the promoter and the 5'-UTR of YY1. In reporter assays, mutations in these G4 structure forming sequences increased the expression of Gaussia luciferase (Gluc) downstream of either YY1 promoter or 5'-UTR. We also discovered that G4 Resolvase 1 (G4R1) enhanced the Gluc expression mediated by the YY1 promoter, but not the YY1 5'-UTR. Consistently, G4R1 binds the G4 motif of the YY1 promoter in vitro and ectopically expressed G4R1 increased endogenous YY1 levels. In addition, the analysis of a gene array data consisting of the breast cancer samples of 258 patients also indicates a significant, positive correlation between G4R1 and YY1 expression.

PIASy-mediated sumoylation of Yin Yang 1 depends on their interaction but not the RING finger.

As a multifunctional protein, Yin Yang 1 (YY1) has been demonstrated to regulate both gene expression and protein posttranslational modifications. However, gaps still exist in our knowledge of how YY1 can be modified and what the consequences of its modifications are. Here we report that YY1 protein can be sumoylated both in vivo and in vitro. We have identified lysine 288 as the major sumoylation site of YY1. We also discovered that PIASy, a SUMO E3 ligase, is a novel YY1-interacting protein and can stimulate the sumoylation of YY1 both in vitro and in vivo. Importantly, the effects of PIASy mutants on in vivo YY1 sumoylation correlate with the YY1-PIASy interaction but do not depend on the RING finger domain of PIASy. This regulation is unique to YY1 sumoylation because PIASy-mediated p53 sumoylation still relies on the integrity of PIASy, which is also true of all of the previously identified substrates of PIASy. In addition, PIASy colocalizes with YY1 in the nucleus, stabilizes YY1 in vivo, and differentially regulates YY1 transcriptional activity on different target promoters. This study demonstrates that YY1 is a target of SUMOs and reveals a novel feature of a SUMO E3 ligase in the PIAS family that selectively stimulates protein sumoylation independent of the RING finger domain.