Cell-type specific regulation of gene expression by simian virus 40 T antigens

Abstract

SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examined global gene expression in SV40-transformed mouse embryo fibroblasts, and in enterocytes obtained from transgenic mice. SV40 transformation alters the expression of approximately 800 cellular genes in both systems. Much of this regulation is observed in both MEFs and enterocytes and is consistent with T antigen action on the Rb-E2F pathway. However, the regulation of many genes is cell-type specific, suggesting that unique signaling pathways are activated in different cell types upon transformation, and that the consequences of SV40 transformation depends on the type of cell targeted.

Fig 1

Design of the mouse microarray experiments. Biological replicates of the mouse intestine and MEF arrays were implemented as a block design and reference design, respectively. Total RNA was isolated from control (C) and SV40-transformed (T) mouse small intestine and MEFs. Total RNA was converted to cDNA and labeled with both Cy3 and Cy5 in independent reactions. Control and SV40-transformed cDNA was mixed and hybridized to the Agilent Mouse cDNA microarray in dye-swap fashion. The cDNA microarray contains two duplicate arrays on one glass slide.

Fig 2

Analysis of SV40 regulated genes in MEFs and enterocytes. The intersection of the genes that were 2-fold up or 2-fold down in the MEF and enterocyte (E) microarrays were overlapped and visualized by Venn Diagrams. The number of genes in each set is indicated. The number above the purple circle represent genes that were regulated by array (1.2- to 2-fold) in the other class but did not meet the 2-fold cutoff. Uniquely regulated genes are indicated by the purple circle. These genes show either no regulation in the other class or are oppositely regulated. For instance, the 128 uniquely upregulated genes in transformed MEFs are less than 1.2-fold upregulated in transformed enterocytes.

Fig 3

Cell-type specific gene regulation is confirmed by RT-PCR. Genes were selected to represent different classes (“Class”) of cell-type specific genes that were observed by microarray. cDNA was synthesized from 1 µg of total RNA from control (C) or SV40-transformed (T) MEF or fractionated villi cells. Each gene was amplified with specific primers by PCR and the products were resolved on a 2% agarose gel and stained with GelStar. Amplification with primers for the Rpl5 transcript was used as a normalizing control. Up arrow, transcript is upregulated in SV40-transformed cells; down arrow, downregulated; dash, transcript level not changed.

Fig 4

Microarray confirmation by immunoblot. 50 µg of control (C) or SV40-transformed (T) cell lysate from two-day post-confluent MEFs and fractionated villi were resolved by SDS-PAGE and transferred to PVDF. An appropriate primary antibody was added (protein symbols shown on the left) and ECL Plus (Amersham) was used to visualize protein expression. Gapdh was used as a loading control. The microarray average log ratio from the MEF and enterocyte (E) arrays is shown on the right for each gene.

Fig 5

Mouse intestine microarrays show similar patterns of gene expression. (A) The average log ratio of E2F responsive genes that were upregulated 3-fold or more in the whole intestine (W), fractionated villi (V) and LCM extracted enterocyte (E) arrays is shown in the top panel. Similarly, the middle panel shows DNA replication genes upregulated at least 3-fold. In the bottom panel, detoxification genes that are downregulated 1.4-fold or more are shown. (B) The biological replicates of the whole intestine, fractionated villi and enterocyte microarray data were filtered by ANOVA (p=0.01) which resulted in 284 clones. These clones were hierarchically clustered using the Euclidean distance metric and average linkage. Clones are colored red and green for clones that are up and downregulated in SV40-transformed tissue, respectively. Grey clones represent missing data. Four expression patterns were revealed from the cluster diagram and the log ratio values of these clones are plotted on the right. The pink line represents the average log ratio for the set of clones in each biological replicate.

Fig 6

Model for SV40 regulation of gene expression. The cell-type specific gene regulation by SV40 is indicated by classes of genes that are uniquely or commonly regulated in each cell type. For example, SV40 upregulates the interferon pathway in MEFs and downregulates the detoxification pathway in enterocytes.