Cell synchronization helps identify phase-specific genes3,12,15
but does not provide quantitatively accurate expression levels of cell cycle-specific genes because of the lack of perfect synchrony. By applying simultaneous equations, the expression levels of genes in theoretically pure G1
, S, G2
or M cell cycle phases were estimated. These gene expression levels were thereafter used to identify cell cycle-specific genes and to estimate the expression levels of cell cycle-regulated genes in an asynchronous cell population based on the proportions of G1
, S, G2
and M cells. The results provide another image of the regulation of gene expression as human cells synchronously pass through the cell division cycle, and suggest that many cell cycle-regulated genes are actively repressed in response to IR-induced DNA damage.
It must be noted that artificially synchronized cell cycle phases do not biologically equal the phases existing in physiologically cycling cells. Although a total of 2410 genes or ESTs showing cell cycle-specific expression were identified based on nine extracted patterns by using EPIG method, they were not necessarily cycle-regulated genes. Many of these genes may have been selected because of serum stimulation after release from G0
or by a direct response to aphidicolin or colcemid treatment.27
Only genes whose expression levels could be correctly estimated in asynchronous cell populations based on the proportions of cells in the cycle compartments were accepted as cell cycle-regulated. A total of 406 among the 2410 genes were identified as cell cycle-regulated genes.
One of the purposes of the present study was to identify the transcriptional targets of a genotoxin among the cell cycle-regulated genes. It has been well-documented that IR induces DNA double-strand-breaks and activates checkpoints to arrest cells in G1
Hundreds of cell-cycle-regulated genes were observed to change in response to DNA damage.9,16,35,36
Genes that were not cell-cycle- regulated were easy to identify as transcriptional targets because expression of these genes was not affected by the DNA damage-induced synchronization effect. However, it is impossible in these studies to distinguish genes that were changed by primary transcriptional regulation from genes changed resulting from cell synchrony caused by the checkpoint response to DNA damage. Our model was developed to address this question. The methodology used here enabled estimation of the changes of gene expression resulting from the cell synchrony effect and identification of transcriptional targets of IR whose changes in gene expression were greater than that from cell synchrony alone.
Although the G2
checkpoint was clearly activated at 2 hr post-IR irradiation, the expression of genes that are important for the G2
/M transition, such as CDC2
, did not show any difference between the measured and the estimated values, indicating that post-translational modifications account for the G2
checkpoint function at this time point and changes in expression of these genes were due to accumulation of the G2
population (, ). Starting at 6 hr post-IR, the time when G1
arrest was clearly observed, the expression of some S phase genes, like MCM2
, showed greater repression than estimated, suggesting a direct transcriptional repression mechanism in G1
arrest. Further at 24 h post-IR, the expression of many important cell cycle-regulated genes, including CCNB1
and 2, CDC2, CDC20, CDC7L1, CDK2, MCM2, 3
and 7, RFC4, TIMELESS
α showed greater repression in the observed values also suggesting direct inhibition by IR in addition to the cell synchrony effects. These results suggest that expression of some cell-cycle-regulated genes is actively changed in response to DNA damage to contribute to cell cycle arrest. Many other cell-cycle-regulated genes were just down-regulated passively in response to DNA damage secondary to cell cycle arrest. The mechanisms of primary down-regulation of the target genes are still not clear. p53 has been reported to play an important role in trans-repression of cell-cycle-regulatory genes.35,37,38
Well-known p53-responsive genes include CDC2
, and TOP2
Despite the transcription factors E2F and NF-Y being enriched in both groups, cell cycle-dependent element (CDE) was significantly enriched in the target group and upstream stimulating factor (USF) was enriched in the non-target group. Previous analyses of CDC2
promoters suggested that p53 interacted with NF-Y to mediate trans-repression,42
although a subsequent study suggested that p53 trans-repressed through interaction with SP1.43
Conversely several groups have reported that genes containing CDE/CHR in their promoters, such as CCNB1
, can be directly trans-repressed by IR-induced DNA damage through a p53-dependent signaling pathway.44–46
Two independent mechanisms, direct binding or via CDE/CHR element, are involved in p53-dependent transcriptional repression of some cell cycle-regulated genes.44
Six of the above-mentioned target genes appeared in our target gene list (). The results presented here suggest that a large set of as many as 150 cell-cycle-regulated genes may be subject to p53-dependent trans-repression in response to DNA damage.
Analysis of gene ontology in the 150 potential target genes showed that although they distributed in various categories of biological process, cellular component and molecular functions, almost all of the over-represented categories were related to cell cycle regulation, DNA metabolism and cell proliferation (). Beside the post-translational modifications of checkpoint sensors, transducers, mediators and effectors, the repression of cell cycle-regulated genes may play an important role in cell cycle checkpoint function in response to IR-induced DNA damage.
In our previous publication, 1811 IR-responding genes or ESTs were identified using the EPIG method with the same criteria for significant gene extraction.16
Among the 1811 genes, 328 of the 406 cell cycle-regulated genes were identified, and all of the 150 IR-target genes were on the list. The other 1483 genes that were IR-responsive but not cell cycle-regulated included early DNA damage response genes, such as CDKN1A
that initiate or regulate cell cycle checkpoint functions. Such genes may work together with targeted repression in transcription of cell cycle-regulated genes to cause and maintain cell cycle arrest, and with genes passively responding to G0
-like growth quiescence.16
Identification of transcriptional targets of the DNA damage response helps us understand the mechanisms of cell cycle arrest caused by direct regulation of cell cycle-regulated genes at the level of transcription, in addition to those checkpoint functions regulated by post-translational modification of protein. Both responses appear to play important roles in maintaining cell cycle arrest. By accounting for the changes in gene expression that are indirect manifestations of cell synchronization, this model may improve elucidation of the mechanisms of genotoxicity by environmental chemicals or therapeutic drugs that directly induce or repress transcription of target genes.