Transcriptional profiling of the cell cycle checkpoint gene krüppel-like factor 4 reveals a global inhibitory function in macromolecular biosynthesis

Abstract

Krüppel-like factor 4 (KLF4; also known as gut-enriched Krüppel-like factor or GKLF) is known to exhibit checkpoint function during the G1/S and G2/M transitions of the cell cycle. The mechanism by which KLF4 exerts these effects is not fully established. Here we investigated the expression profile of KLF4 in an inducible system over a time course of 24 h. Using oligonucleotide microarrays, we determined that the fold changes relative to control in expression levels of KLF4 exhibited a time-dependent increase from 3- to 20-fold between 4 and 24 h following KLF4 induction. During this period and among a group of 473 cell cycle regulatory genes examined, 96 were positively correlated and 86 were negatively correlated to KLF4's expression profile. Examples of upregulated cell cycle genes include those encoding tumor suppressors such as MCC and FHIT, and cell cycle inhibitors such as CHES1 and CHEK1. Examples of downregulated genes include those that promote the cell cycle including several cyclins and those required for DNA replication. Unexpectedly, several groups of genes involved in macromolecular synthesis, including protein biosynthesis, transcription, and cholesterol biosynthesis, were also significantly inhibited by KLF4. Thus, KLF4 exerts a global inhibitory effect on macromolecular biosynthesis that is beyond its established role as a cell cycle inhibitor.

Figure 1

Time-dependent increase in the expression levels of KLF4 following induction in EcR-RKO/KLF4 cells. EcR-RKO/KLF4 cells were treated with 5 μM ponasterone A (PA) or vehicle control for the time indicated. RNA was extracted in duplicate at each time point and analyzed using the cDNA microarrays as described in Materials and Methods. The expression levels of KLF4 were calculated as fold changes by comparing the signal intensities between induced and control cells at the corresponding time point (A). Shown is the mean of two independent hybridizations. (B) Proteins were extracted from PA-treated (+) or vehicle-treated (−) cells for the time periods indicated and analyzed for KLF4 by Western blotting.

Figure 2

Expression profiles of cell cycle regulatory genes that correlate with KLF4 between 4 and 24 h of induction. A total of 473 cell cycle regulatory genes were present on the gene chips and selected for analysis. Those with an expression ratio between induced and control cells greater than 2 at the corresponding time point were considered upregulated (solid bars) and those with a ratio below 0.5 were considered downregulated (striped bars). Genes that function in cell cycle arrest (A) and cell cycle promoting (B).

Figure 3

Gene trees of cell cycle-related genes that are positively or negatively correlated with the expression profile of KLF4. The expression profiles of 62 cell cycle-related genes that were positively correlated with KLF4 are shown in (A) and those of 47 genes negatively correlated with KLF4 in (B). The total branch similarities of the upregulated and downregulated genes with KLF4 are 0.329 and 0.344, respectively. The location of KLF4 is indicated in (A).

Figure 4

Comparison of select groups of gene families that are positively or negatively correlated with KLF4. The percentages of genes that are either positively (solid bars) or negatively (striped bars) correlated to KLF4 are shown for three families: protein biosynthesis, transcription, and cell cycle control.

Figure 5

Gene trees of protein biosynthesis and transcription genes that are inversely correlated with KLF4. (A) and (B) contain the protein biosynthesis and transcription genes, respectively, whose expression were inversely correlated with that of KLF4 over the time course of the experiment.

Figure 6

Gene trees of cholesterol biosynthesis genes that are inversely correlated with KLF4. Shown are genes involved in cholesterol biosynthesis whose expression is inversely correlated with that of KLF4. These genes form two branches, one above and one below the expression profile of KLF4, identified by the arrow. The top branch (I) has a branch similarity of 0.378. The bottom branch (II) has a branch similarity of 1.386.

Figure 7

Western blot analysis of HMGCR in response to KLF4 induction. EcR-RKO/KLF4 cells were treated with 5 μM ponasterone A for the time periods indicated followed by Western blot analysis of HMGCR and KLF4 levels. Actin was used as a loading control.