Posttranscriptional regulation of transformation by human papillomavirus type 16 E7 and expression of this oncogene

Many epidemiological and experimental studies have strongly implicated human papillomavirus type 16 (IIPV-16) in cervical neoplasia. The oncogenic potential of this virus can be demonstrated by transformation of primary baby rat kidney (BRK) cells by cotransfection of the viral genome with the activated EJ-ras oncogene. I performed a mutational analysis of the viral genome to map the regions essential for its transforming activity. For the SV40-bascd early region expression plasmids, the disruption of the E6, E2, E5 and the 3' region of the El open reading frames (ORFs) did not affect the transforming activity of mutated plasmids, whereas the insertion of translation termination linkers within the E7 ORE abolished transformation. Additional sequences present in the 3'-flanking region of the E7 ORF were also required for efficient transformation. -- The 3' flanking region sequences were analyzed in detail in SV40-based E7 expression plasmids by progressive deletion analysis and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within this 3'-flanking region abolished transformation. Regeneration of the wild- type sequence in a previously transformation incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in higher levels of transformation, whereas mutating the + 2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the transformation-deficient splice site mutants accumulated lower levels of E7 RNA, primarily because of rapid destabilization of E7 RNA. -- The splice sites present within the E6 ORF were examined for their ability to substitute for the loss of nt 880 splice donor site function. The wild-type E6 splice sites, as well as the heterologous splice sites of the SV40 intron, were able to partially substitute for the nt 880 splice site function. These results indicated that the efficient accumulation of HPV-16 E7 RNA and transformation of BRK cells depend on the presence of an intron in the transcription unit. -- Recent studies have indicated the presence of naturally occurring HPV-16 antisense (AS) RNA in cervical carcinomas. I have detected AS E7 and E7/E1 RNA in Cos-1 cells transiently transfected with SV40-based HPV-16 early region expression plasmids. By deletion mutation analysis, the AS promoter was localized to nt 4031-4338 of HPV-16. Primer extension analysis and RNase protection assays indicated that the AS RNA was initiated from multiple sites in an AT-rich region around nt 4100. The AS promoter was active when cloned downstream of the chloramphenicol acetyl transferase (Cat) gene, giving rise to AS Cat RNA. -- AS E7 RNA was detected in both the nucleus and cytoplasm. The AS RNA formed a duplex with the sense (S) E7 RNA. The presence of AS RNA was correlated with reduced splicing of E7 RNA from the nt 880 splice site and the synthesis of E7 protein

GATA6 Promotes Colon Cancer Cell Invasion by Regulating Urokinase Plasminogen Activator Gene Expression12

GATA6 is a zinc finger transcription factor expressed in the colorectal epithelium. We have examined the expression of GATA6 in colon cancers and investigated the mechanisms by which GATA6 regulates colon cancer cell invasion. GATA6 was overexpressed in colorectal polyps and primary and metastatic tumors. GATA6 was strongly expressed in both the nuclear and cytoplasmic compartments of the colon cancer cells. GATA6 expression was upregulated in invasive HT29 and KM12L4 cells compared with the parental HT29 and KM12 cells and positively correlated with urokinase-type plasminogen activator (uPA) gene expression. Small interfering RNA (siRNA) knockdown of GATA6 resulted in reduced uPA gene expression and cell invasion. GATA6 bound to the uPA gene regulatory sequences in vivo and activated uPA promoter activity in vitro. uPA promoter deletion analysis indicated that the promoter proximal Sp1 sites were required for GATA6 activation of the uPA promoter. Accordingly, GATA6 physically associated with Sp1 and siRNA knockdown of Sp1 decreased GATA6 activation of the uPA promoter activity suggesting that Sp1 recruits GATA6 to the uPA promoter and mediates GATA6 induced activation of the uPA promoter activity. On the basis of our results, we conclude that GATA6 is an important regulator of uPA gene expression, and the dysregulated expression of GATA6 contributes to colorectal tumorigenesis and tumor invasion

Nuclear localization of FOXA2 is independent of sumoylation.

<p>INS-1E cells plated on cover slips were not transfected (panels L,M,N) or transfected with pCGN empty vector (panels A,B,C) or HA epitope tagged FOXA2 (panels D,E,F,G) or FOXA2K6R mutant (panels H,I,J,K,O). Cells were fixed 36 hours post-transfection, permeabilized and stained with mouse HA antibody and fluorescent Alexa Fluor 594 donkey anti-mouse secondary antibody. No primary antibody was used for panels L,M,N. Cover slips were mounted in DAPI containing mounting media. Panels B,E,I and M are DAPI images. Panel C is superimposed A and B images. Panel F is superimposed D and E images. Panel J is superimposed H and I images. Panel N is superimposed L and M images. All images are at 400× magnification. Cells in the marked boxes in images F and J are digitally magnified and shown in panels G,K and O. Four times longer exposure were used for panels A, H and L. Two times lesser exposure was used for panel I.</p

Forkhead Box Protein A2 (FOXA2) Protein Stability and Activity Are Regulated by Sumoylation

<div><p>The forkhead box protein A2 (FOXA2) is an important regulator of glucose and lipid metabolism and organismal energy balance. Little is known about how FOXA2 protein expression and activity are regulated by post-translational modifications. We have identified that FOXA2 is post-translationally modified by covalent attachment of a small ubiquitin related modifier-1 (SUMO-1) and mapped the sumoylation site to the amino acid lysine 6 (K6). Preventing sumoylation by mutating the SUMO acceptor K6 to arginine resulted in downregulation of FOXA2 protein but not RNA expression in INS-1E insulinoma cells. K6R mutation also downregulated FOXA2 protein levels in HepG2 hepatocellular carcinoma cells, HCT116 colon cancer cells and LNCaP and DU145 prostate cancer cells. Further, interfering with FOXA2 sumoylation through siRNA mediated knockdown of UBC9, an essential SUMO E2 conjugase, resulted in downregulation of FOXA2 protein levels. Stability of sumoylation deficient FOXA2K6R mutant protein was restored when SUMO-1 was fused in-frame. FOXA2 sumoylation and FOXA2 protein levels were increased by PIAS1 SUMO ligase but not a SUMO ligase activity deficient PIAS1 mutant. Although expressed at lower levels, sumoylation deficient FOXA2K6R mutant protein was detectable in the nucleus indicating that FOXA2 nuclear localization is independent of sumoylation. Sumoylation increased the transcriptional activity of FOXA2 on <em>Pdx-1</em> area I enhancer. Together, our results show that sumoylation regulates FOXA2 protein expression and activity.</p> </div