Transcriptional profiling of Krüppel-like factor 4 reveals a function in cell cycle regulation and epithelial differentiation

Abstract

Krüppel-like factor 4 (KLF4) is an epithelially enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Constitutive expression of KLF4 inhibits G1/S transition of the cell cycle but the manner by which it accomplishes this effect is unclear. To better understand the biochemical function of KLF4, we identified its target genes using cDNA microarray analysis in an established human cell line containing inducible KLF4. RNA extracted from induced and control cells were hybridized differentially to microarray chips containing 9600 human cDNAs. In all, 84 genes with significantly increased expression and 107 genes with significantly reduced expression due to KLF4 induction were identified. The affected genes are sorted to several clusters on the basis of functional relatedness. A major cluster belongs to genes involved in cell-cycle control. Within this cluster, many up-regulated genes are inhibitors of the cell cycle and down-regulated genes are promoters of the cell cycle. Another up-regulated gene cluster includes nine keratin genes, of which seven are located in a specific region on chromosome 12. The results indicate that KLF4 is involved in the control of cell proliferation and does so by eliciting changes in expression of numerous cell-cycle regulatory genes in a concerted manner. Furthermore, KLF4 regulates expression of a group of epithelial-specific keratin genes in a manner consistent with a potential locus control region function.

Figure 1

Results of cDNA micro-array hybridization. EcR-RKO/KLF4 cells were treated with 5 μM ponasterone A (PA) or vehicle alone for 24 hours before subject to mRNA isolation. In experiment A, mRNAs from induced and control cells were reverse-transcribed and labeled with Cy3 and Cy5 fluorescence dye, respectively. The dyes were reversed in experiment B. Labeled cDNAs from induced and control cells were then combined and hybridized to the human cDNA microarray chip as described in Experimental Procedures. The signal value for each cDNA in the treated and control cells was measured and plotted in the Figure for both experiments A and B.

Figure 2

Scatter plot analysis of microarray hybridization results from experiments A and B. The signal value for each cDNA from induced cells was compared to that from control cells and expressed as a balanced differential expression value. Shown is a scatter plot of balanced differential expression values for all the genes in experiments A and B. Genes in the center of the plot have a value of 1 and are unchanged in their expression by treatment with ponasterone A. Genes in the right upper quadrants are up-regulated and those in the lower left quadrants are down-regulated in both experiments.

Figure 3

Verification of expression levels of up-regulated genes identified by microarrays. (A) Total RNA was isolated from EcR-RKO/KLF4 cells treated for 24 hours with ponasterone A (PA) (+) or vehicle alone (−) treated and analyzed by Northern blot analysis using cDNA probes encoding some of the up-regulated genes identified by microarray analysis. ALPI, intestinal alkaline phosphatase; KRT18, keratin 18; IGFBP6, insulin-like growth factor binding protein 6; SFN, stratifin or 14-3-3σ. β-Actin was used as a loading control. (B) Proteins were extracted from treated or control cells and analyzed by Western blot analysis using antibodies directed against p21^WAF1/Cip1, p57^Kip2, β-actin, and KLF4.

Figure 4

Verification of expression levels of down-regulated genes identified by microarrays. Total RNA was isolated from EcR-RKO/KLF4 cells treated for 24 hours with ponasterone A (PA) (+) or vehicle alone (−) treated and analyzed by Northern blot analysis using cDNA probes encoding some of the down-regulated genes identified by microarray analysis. See Table 2 for abbreviations. β-Actin was used as a loading control.

Figure 5

Time + course of expression of up-regulated gene during induction of KLF4RNA was isolated from EcR-RKO/KLF4 cells treated with ponasterone A for the time indicated and probed by Northern blot analysis using cDNA encoding the genes in the Figure. β-Actin was used as a loading control.

Figure 6

A cluster of keratin and keratin-related genes is up-regulated by KLF4. (A) The sequence-relatedness of the group of keratin and keratin-related genes that are up-regulated by KLF4. Shown are also the chromosomal locations for each of the genes. KRT, keratin; VIM, vimentin; VIL2/p81, villin 2 or eczrin; DSG2, desmoglein 2; SNL/p55, singed-like. (B) The up-regulated keratin genes are sorted on the basis of their chromosomal localization.