dna-vector-based-rna-interference-to-study-gene-function-in-cancer

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 ...

Wake Forest University School of Medicine

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

The transcriptional co-repressor CtBP (C-terminal binding protein) is implicated in tumorigenesis because it is targeted by the adenovirus E1A protein during oncogenic transformation. Genetic studies have also identified a crucial function for CtBP in animal development. CtBP is recruited to DNA by transcription factors that contain a PXDLS motif, but the detailed molecular events after the recruitment of CtBP to DNA and the mechanism of CtBP function in tumorigenesis are largely unknown. Here we report the identification of a CtBP complex that contains the essential components for both gene targeting and coordinated histone modifications, allowing for the effective repression of genes targeted by CtBP. Inhibiting the expression of CtBP and its associated histone-modifying activities by RNA-mediated interference resulted in alterations of histone modifications at the promoter of the tumour invasion suppressor gene E-cadherin and increased promoter activity in a reporter assay. These findings identify a molecular mechanism by which CtBP mediates transcriptional repression and provide insight into CtBP participation in oncogenesis.

Coordinated histone modifications mediated by a CtBP co-repressor complex.

Yin Yang 1 (YY1) is a transcription factor that plays an essential role in development. However, the full spectrum of YY1's functions and mechanism of action remains unclear. We find that YY1 ablation results in p53 accumulation due to a reduction of p53 ubiquitination in vivo. Conversely, YY1 overexpression stimulates p53 ubiquitination and degradation. Significantly, recombinant YY1 is sufficient to induce Hdm2-mediated p53 polyubiquitination in vitro, suggesting that this function of YY1 is independent of its transcriptional activity. We identify direct physical interactions of YY1 with Hdm2 and p53 and show that the basis for YY1-regulating p53 ubiquitination is its ability to facilitate Hdm2-p53 interaction. Importantly, the tumor suppressor p14ARF compromises the Hdm2-YY1 interaction, which is important for YY1 regulation of p53. Taken together, these findings identify YY1 as a potential cofactor for Hdm2 in the regulation of p53 homeostasis and suggest a possible role for YY1 in tumorigenesis.

Yin Yang 1 is a negative regulator of p53.

The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assembles multiprotein complexes containing numerous transcription factors and cofactors, including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled-coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic-activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of the cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and the transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.

The ubiquitin carboxyl hydrolase BAP1 forms a ternary complex with YY1 and HCF-1 and is a critical regulator of gene expression.

Host Cell Factor 1 (HCF-1) plays critical roles in regulating gene expression in a plethora of physiological processes. HCF-1 is first synthesized as a precursor, and subsequently specifically proteolytically cleaved within a large middle region termed the proteolytic processing domain (PPD). Although the underlying mechanism remains enigmatic, proteolysis of HCF-1 regulates its transcriptional activity and is important for cell cycle progression. Here we report that HCF-1 proteolysis is a regulated process. We demonstrate that a large proportion of the signaling enzyme O-linked-N-acetylglucosaminyl transferase (OGT) is complexed with HCF-1 and this interaction is essential for HCF-1 cleavage. Moreover, HCF-1 is, in turn, required for stabilizing OGT in the nucleus. We provide evidence indicating that OGT regulates HCF-1 cleavage via interaction with and O-GlcNAcylation of the HCF-1 PPD. In contrast, although OGT also interacts with the basic domain in the HCF-1 amino-terminal subunit, neither the interaction nor the O-GlcNAcylation of this region are required for proteolysis. Moreover, we show that OGT-mediated modulation of HCF-1 impacts the expression of the herpes simplex virus immediate-early genes, targets of HCF-1 during the initiation of viral infection. Together the data indicate that O-GlcNAcylation of HCF-1 is a signal for its proteolytic processing and reveal a unique crosstalk between these posttranslational modifications. Additionally, interactions of OGT with multiple HCF-1 domains may indicate that OGT has several functions in association with HCF-1.

Crosstalk between O-GlcNAcylation and proteolytic cleavage regulates the host cell factor-1 maturation pathway.

Vitamin D(3) is a promising preventative and therapeutic agent for prostate cancer, but its implementation is hampered by a lack of understanding about its mechanism of action. Upon treatment with 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3), vitamin D(3)], the metabolically active form of vitamin D(3), adult prostate progenitor/stem cells (PrP/SC) undergo cell-cycle arrest, senescence, and differentiation to an androgen receptor-positive luminal epithelial cell fate. Microarray analyses of control- and vitamin D(3)-treated PrP/SCs revealed global gene expression signatures consistent with induction of differentiation. Interestingly, one of the most highly upregulated genes by vitamin D(3) was the proinflammatory cytokine interleukin-1α (IL-1α). Systems biology analyses supported a central role for IL-1α in the vitamin D(3) response in PrP/SCs. siRNA-mediated knockdown of IL-1α abrogated vitamin D(3)-induced growth suppression, establishing a requirement for IL-1α in the antiproliferative effects of vitamin D(3) in PrP/SCs. These studies establish a system to study the molecular profile of PrP/SC differentiation, proliferation, and senescence, and they point to an important new role for IL-1α in vitamin D(3) signaling in PrP/SCs.

Interleukin-1α mediates the antiproliferative effects of 1,25-dihydroxyvitamin D3 in prostate progenitor/stem cells.

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.

Article	Year
Free radical biology & medicine\| PubMed ID: 22387176	2012
Yin Yang 1 plays an essential role in breast cancer and negatively regulates p27. The American journal of pathology\| PubMed ID: 22440256	2012
Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 11960009	2002
Coordinated histone modifications mediated by a CtBP co-repressor complex. Nature\| PubMed ID: 12700765	2003
Yin Yang 1 is a negative regulator of p53. Cell\| PubMed ID: 15210108	2004
Molecular and cellular biology\| PubMed ID: 15899857	2005
Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 15990402	2005
Cancer research\| PubMed ID: 16585168	2006
The Journal of biological chemistry\| PubMed ID: 16963455	2006
Molecular and cellular biology\| PubMed ID: 17353273	2007
The EMBO journal\| PubMed ID: 17581637	2007
Molecular cancer\| PubMed ID: 20478051	2010
The Prostate\| PubMed ID: 20583132	2010
Molecular cell\| PubMed ID: 20670891	2010
Science translational medicine\| PubMed ID: 20686179	2010
The ubiquitin carboxyl hydrolase BAP1 forms a ternary complex with YY1 and HCF-1 and is a critical regulator of gene expression. Molecular and cellular biology\| PubMed ID: 20805357	2010
Crosstalk between O-GlcNAcylation and proteolytic cleavage regulates the host cell factor-1 maturation pathway. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21285374	2011
Transcription\| PubMed ID: 21326896	2010
Interleukin-1α mediates the antiproliferative effects of 1,25-dihydroxyvitamin D3 in prostate progenitor/stem cells. Cancer research\| PubMed ID: 21653679	2011
Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1. Nucleic acids research\| PubMed ID: 21993297	2012
Carcinogenesis\| PubMed ID: 22159221	2012
Critical reviews in oncogenesis\| PubMed ID: 22248053	2011
Cancer research\| PubMed ID: 22331460	2012
Cancer research\| PubMed ID: 22467172	2012
PloS one\| PubMed ID: 22536409	2012
PloS one\| PubMed ID: 22606284	2012

Guangchao Sui

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