Augmentation of Myc-Dependent Mitotic Gene Expression by the Pygopus2 Chromatin Effector.

Mitotic segregation of chromosomes requires precise coordination of many factors, yet evidence is lacking as to how genes encoding these elements are transcriptionally controlled. Here, we found that the Pygopus (Pygo)2 chromatin effector is indispensable for expression of the MYC-dependent genes that regulate cancer cell division. Depletion of Pygo2 arrested SKOV-3 cells at metaphase, which resulted from the failure of chromosomes to capture spindle microtubules, a critical step for chromosomal biorientation and segregation. This observation was consistent with global chromatin association findings in HeLa S3 cells, revealing the enrichment of Pygo2 and MYC at promoters of biorientation and segmentation genes, at which Pygo2 maintained histone H3K27 acetylation. Immunoprecipitation and proximity ligation assays demonstrated MYC and Pygo2 interacting in nuclei, corroborated in a heterologous MYC-driven prostate cancer model that was distinct from Wnt/ß-catenin signaling. Our evidence supports a role for Pygo2 as an essential component of MYC oncogenic activity required for mitosis.

Wnt/ß-catenin-dependent acetylation of Pygo2 by CBP/p300 histone acetyltransferase family members.

Pygopus 2 (Pygo2) is a chromatin effector that plays an essential role in canonical Wnt signaling associated with development and stem cell growth. Its function is to facilitate histone acetylation by recruitment of histone acetyltransferases (HATs) at active sites of ß-catenin-mediated transcription. In the present study, we report that Pygo2 itself is transiently acetylated when bound to the activated TCF/ß-catenin transcription complex, which correlated with ß-catenin binding and Axin2 gene activation. The HAT CBP/p300, but not GCN5/PCAF, targeted specific lysine residues of the N-terminal homology domain of Pygo2 for acetylation. Functional analyses revealed that the presence of CBP and p300 increased the association of Pygo2 with GCN5, independent of Pygo2 acetylation status. Finally, while acetylation of Pygo2 had little effect on active ß-catenin complex formation, p300-mediated Pygo2 acetylation resulted in the displacement of Pygo2 from the nucleus to the cytoplasm by targeting specific lysine residues in the Pygo2 nuclear localization sequence. Taken together, these findings are consistent with a model in which acetylation of Pygo2 by CBP/p300 family members in the active TCF/ß-catenin complex occurs coincident with histone acetylation and may be required for the recycling of Pygo2 away from the complex subsequent to target gene activation.

Evidence of a novel role for Pygopus in rRNA transcription.

Increased protein synthesis during cell proliferation is accompanied by a compensatory increase in efficient ribosome production, but the mechanisms by which cells adapt to this requirement are not fully understood. In the present study, we demonstrate evidence that Pygo (Pygopus), a protein originally identified as a core component of the Wnt-ß-catenin transcription complex is also involved in rRNA transcription during cancer cell growth. Pygo was detected in the nucleoli of several transformed cell lines and was associated with treacle and UBF (upstream binding factor), proteins that are essential for ribosome biogenesis in development and cancer. Pygo was also detected at the ribosomal gene promoter along with core components of the rDNA (ribosomal DNA) transcription complex. RNAi (RNA interference)-mediated depletion of hPygo2 (human Pygo 2) reduced histone H4 acetylation at the rDNA promoter, down-regulated rRNA production, and induced growth arrest in both p53-positive and -negative cells. In p53-positive cells, hPygo2 knockdown triggered the ribosomal stress pathway, culminating in p53-dependent growth arrest at G1-phase of the cell cycle. The results of the present study suggest a novel involvement of Pygo in the promotion of rRNA transcription in cancer cells.

Human papilloma virus (HPV) E7-mediated attenuation of retinoblastoma (Rb) induces hPygopus2 expression via Elf-1 in cervical cancer.

The human papillomavirus (HPV) is the etiologic agent of cervical cancer. In this study, we provide evidence for the human Pygopus (hPygo)2 gene as a cellular biomarker for HPV-related disease. In a tumor microarray of cervical cancer progression, hPygo2 levels were greater in high-grade lesions and squamous cell carcinomas than in normal epithelia. Similarly, hPygo2 mRNA and protein levels were greater in HPV-positive cervical cancer cells relative to uninfected primary cells. RNA interference (RNAi)-mediated depletion of HPV-E7 increased whereas E74-like factor (Elf)-1 RNAi decreased association of Retinoblastoma (Rb) tumor suppressor with the hPygo2 promoter in cervical cancer cell lines. Transfection of dominant-active Rb inhibited Elf-1-dependent activation of hPygo2, whereas Elf-1 itself increased hPygo2 expression. Chromatin immunoprecipitation assays showed that Rb repressed hPygo2 by inhibiting Elf-1 at the Ets-binding site in the hPygo2 promoter. These results suggested that abrogation of Rb by E7 resulted in derepression of Elf-1, which in turn stimulated expression of hPygo2. Thus, initiation of hPygo2 expression by Elf-1 was required for proliferation of cervical cancer cells and its expression therefore may act as a surrogate marker for dysplasia.

A co-dependent requirement of xBcl9 and Pygopus for embryonic body axis development in Xenopus.

The Wnt/beta-catenin transcriptional activation complex requires the adapter protein Pygopus (Pygo), which links the basal transcription machinery to beta-catenin, by its association with legless (Lgs)/ B-cell lymphoma-9 (Bcl9). Pygo was shown to be required for development in vertebrates, but the role of Lgs/Bcl9 is unknown. We identified an amphibian orthologue of Lgs/Bcl9, XBcl9, which interacted biochemically with Xbeta-catenin and XPygo2. The body axis promoting ability of Xbeta-catenin was diminished when residues required for its interaction with XBcl9 were mutated. In blastula embryos, XBcl9 was transiently preferentially expressed in nuclei of dorsoanterior cells and ectopically expressed XBcl9 required XPygo2 to localize to nuclei. Furthermore, while neither XBcl9 nor XPygo2 alone affected development when ectopically expressed, both were required to induce supernumerary axis and dorsal gene activation. Like XPygo2, depletion of maternal XBcl9 alone caused dorsal defects. These results indicated an essential role of the Pygo-Bcl9 duet in vertebrate body axis formation.

The transcriptional activity of Pygopus is enhanced by its interaction with cAMP-response-element-binding protein (CREB)-binding protein.

Pygopus is a core component of the beta-catenin/TCF (T-cell factor) transcriptional activation complex required for the expression of canonical Wnt target genes. Recent evidence suggests that Pygopus could interpret histone methylation associated with target genes and it was shown to be required for histone acetylation. The involvement of a specific acetyltransferase, however, was not determined. In this report, we demonstrate that Pygopus can interact with the HAT (histone acetyltransferase) CBP [CREB (cAMP-responsive-element-binding protein)-binding protein]. The interaction is via the NHD (N-terminal homology domain) of Pygopus, which binds to two regions in the vicinity of the HAT domain of CBP. Transfected and endogenous hPygo2 (human Pygopus2) and CBP proteins co-immunoprecipitate in HEK-293 (human embryonic kidney 293) cells and both proteins co-localize in SW480 colorectal cancer cells. The interaction with CBP also enhances both DNA-tethered and TCF/LEF1 (lymphoid enhancing factor 1)-dependent transcriptional activity of Pygopus. Furthermore, immunoprecipitated Pygopus protein complexes displayed CBP-dependent histone acetyltransferase activity. Our data support a model in which the NHD region of Pygopus is required to augment TCF/beta-catenin-mediated transcriptional activation by a mechanism that includes both transcriptional activation and histone acetylation resulting from the recruitment of the CBP histone acetyltransferase.

Oncogenic activation of the human Pygopus2 promoter by E74-like factor-1.

Pygopus is a component of the T-cell factor/beta-catenin transcriptional complex essential for activation of Wnt target genes and is also required for cell regulation in the absence of Wnt signaling. Human Pygopus2 (hPygo2) is overexpressed in a high proportion of breast and epithelial ovarian malignant tumors and is required for the growth of several cell lines derived from these carcinomas. The mechanisms regulating hPygo2 gene activation, however, are unknown. Here, we have determined cis- and trans-interacting factors responsible for hPygo2 expression in cancer. The minimal region required for a maximal 109-fold activation of the hPygo2 promoter in MCF-7 breast cancer cells is 48 bp upstream of the start of transcription. Within 25 bp of the transcriptional start, there are two overlapping tandem Ets transcription factor-binding sites, which are critical for hPygo2 promoter activity. In vitro DNA pull-down assays and proteomic analyses identified the Ets family members Elk-1 and E74-like factor-1 (Elf-1) as potential hPygo2 promoter binding factors, whereas in vivo chromatin immunoprecipitation assays verified that only Elf-1 specifically bound to the hPygo2 promoter in MCF-7 cells. Modulation of elf-1 in MCF-7 cells by silencing via RNA interference or overexpression caused a corresponding decrease or increase, respectively, in hPygo2 promoter activity. Overexpression of Elf-1 in HeLa cells, in which Elf-1 is expressed at a lower level than in MCF-7 cells, caused a 4-fold increase in endogenous hPygo2 mRNA levels. These results provide new evidence that Elf-1 is involved in transcriptional activation of hPygo2. Like hPygo2, previous studies implicated Elf-1 in breast and ovarian cancer and our present findings suggest that the oncogenic requirement of hPygo2 is fulfilled, in part, by Elf-1.

Regulation of the response to Nodal-mediated mesoderm induction by Xrel3.

The Xenopus egg has a yolk-laden vegetal hemisphere juxtaposed to a darkly pigmented animal hemisphere. Mesoderm is derived from the marginal zone, located at the interface between the two hemispheres. The vegetal-most cells become endoderm and release TGF-beta-related factors, including the Xenopus Nodal related (Xnr) proteins, which diffuse to induce the marginal zone to form mesoderm. The remaining animal cells become ectoderm, but our understanding of the mechanisms that limit the response to induction is incomplete. In this study, we provide evidence to suggest that Xrel3, a member of the Rel/NF-kappaB family, plays a role in defining the boundary separating induced from uninduced cells by regulating Xnr-responsive gene transcription. Ectopic Xrel3 expressed in prospective mesoderm caused repression of mesoderm-specific genes resulting in loss-of-function phenotypes that were rescued by co-expression of Xnr2. Depletion of Xrel3 from embryos with antisense morpholinos increased Xnr-dependent transcription, broadened expression of the pan-mesoderm marker Xbra and sensitized animal cells to mesoderm induction by Xnr2. We propose that an additional component to the mechanism that differentiates the ectoderm from the mesoderm involves regulation of nodal-dependent gene transcription by Xrel3.

Requirement of Pygopus 2 in breast cancer.

The development of novel therapeutic strategies for breast cancer requires the identification of molecular targets involved in malignancy. Human Pygopus (Pygo)-1 and -2 are recently discovered components of the Wnt signaling pathway required for beta-Catenin/Tcf dependent transcription in embryos and colorectal cancer cells, but the role of these proteins in malignant cell growth and survival has not yet been determined. We report the expression and requirement for proliferation of hPygo2 in breast cancer cells. hPygo2 protein was overexpressed in malignant breast tumors and in the nuclei of five breast cancer cell lines, but was not expressed in the nuclei of non-malignant breast cells. Phosphorothioated antisense oligonucleotides were used to specifically knockdown expression hPygo2 in Mcf-7 and MDA-MB-468 cell lines. hPygo2 was required for the growth, in tissue culture and anchorage-independent assays, of both cell lines and for the expression of the Wnt target gene Cyclin D1. We conclude that hPygo2 is highly expressed in, and required for the growth of breast carcinoma cells.

Antisense suppression of pygopus2 results in growth arrest of epithelial ovarian cancer.

PURPOSE: The Pygopus proteins are critical elements of the canonical Wnt/beta-catenin transcriptional complex. In epithelial ovarian cancer, constitutively active Wnt signaling is restricted to one (endometrioid) tumor subtype. The purpose of this study was to determine the level of expression and growth requirements of human Pygopus2 (hPygo2) protein in epithelial ovarian cancer. EXPERIMENTAL DESIGN: Expression and subcellular localization of hPygo2 was determined in epithelial ovarian cancer cell lines and tumors using Northern blot, immunoblot, and immunofluorescence. Immunohistochemistry was done on 125 archived patient epithelial ovarian cancer tumors representing all epithelial ovarian cancer subtypes. T-cell factor-dependent transcription levels were determined in epithelial ovarian cancer cells using TOPflash/FOPflash in vivo assays. Phosphorothioated antisense oligonucleotides were transfected into cell lines and growth assayed by cell counting, anchorage-independent colony formation on soft agar, and xenografting into severe combined immunodeficient mice. RESULTS: All six epithelial ovarian cancer cell lines and 82% of the patient samples overexpressed nuclear hPygo2 compared with control cells and benign disease. Depletion of hPygo2 by antisense oligonucleotides in both Wnt-active (TOV-112D) and Wnt-inactive serous (OVCAR-3, SKOV-3) and clear cell (TOV-21G) carcinoma cell lines halted growth, assessed using tissue culture, anchorage-independent, and xenograft assays. CONCLUSIONS: hPygo2 is unexpectedly widely expressed in, and required in the absence of, Wnt signaling for malignant growth of epithelial ovarian cancer, the deadliest gynecologic malignancy. These findings strongly suggest that inhibition of hPygo2 may be of therapeutic benefit for treating this disease.