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Identification of primary transcriptional regulation of cell cycle-regulated genes upon DNA damage.

ABSTRACT: The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization.

SUBMITTER: Zhou T

PROVIDER: S-EPMC2117899 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Identification of primary transcriptional regulation of cell cycle-regulated genes upon DNA damage.

Zhou Tong T Chou Jeff J Mullen Thomas E TE Elkon Rani R Zhou Yingchun Y Simpson Dennis A DA Bushel Pierre R PR Paules Richard S RS Lobenhofer Edward K EK Hurban Patrick P Kaufmann William K WK

Cell cycle (Georgetown, Tex.) 20070419 8

The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with ...[more]

PMID: 17404513

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Project description:The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell-cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. Experiment Overall Design: Normal human fibroblasts were synchronized and harvested at G0, G1, S G2 and M cell cycle phase, and logarithmically growing cells were treated with 1.5 Gy IR or sham, and harvested at 2, 6 or 24 h after the treatment. Total RNA was isolated using Qiagen RNeasy kit. The quality of all RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 Âµg of sample RNA and global reference RNA (Stratagene) were converted to cDNA with reverse transcriptase then amplified using T7 RNA polymerase while labeling with either Cy3-dUTP or Cy5-dUTP (Agilentâ??s Low RNA Input Linear Amplification Kit). The quality of each labeled cRNA was evaluated using an Agilent 2100 Bioanalyzer prior to hybridization. 750ng of Cy3 and Cy5-labeled cRNA were used in the hybridization. The labeled cRNA from sham- or IR-treated samples was hybridized with the labeled global reference cRNA on an Agilent 22k human 1A array in a hybridization oven (Robbins Scientific Model 400, 1040-60-1AG) at 60oC for 17 h. Hybridization of sample RNA against reference RNA was done twice with dye swap. Following hybridization, the arrays were scanned using the Agilent DNA Microarray Scanner with SureScanÂ® Technology and microarray images were analyzed using Agilent Feature Extraction Software (v7.1). The gene expression level was presented as ratio of sample intensity against reference intensity.

Project description:DNA mismatch repair during replication is a conserved process essential for maintaining genomic stability. Mismatch repair is also implicated in cell-cycle arrest and apoptosis after DNA damage. Because yeast and human mismatch repair systems are well conserved, we have employed the budding yeast Saccharomyces cerevisiae to understand the regulation and function of the mismatch repair gene MSH2. Using a luciferase-based transcriptional reporter, we defined a 218-bp region upstream of MSH2 that contains cell-cycle and DNA damage responsive elements. The 5' end of the MSH2 transcript was mapped by primer extension and was found to encode a small upstream open reading frame (uORF). Mutagenesis of the uORF start codon or of the uORF stop codon, which creates a continuous reading frame with MSH2, increased Msh2 steady-state protein levels ∼2-fold. Furthermore, we found that the cell-cycle transcription factors Swi6, Swi4, and Mbp1-along with SCB/MCB cell-cycle binding sites upstream of MSH2-are all required for full basal expression of MSH2. Mutagenesis of the cell-cycle boxes resulted in a minor reduction in basal Msh2 levels and a 3-fold defect in mismatch repair. Disruption of the cell-cycle boxes also affected growth in a DNA polymerase-defective strain background where mismatch repair is essential, particularly in the presence of the DNA damaging agent methyl methane sulfonate (MMS). Promoter replacements conferring constitutive expression of MSH2 revealed that the transcriptional induction in response to MMS is required to maintain induced levels of Msh2. Turnover experiments confirmed an elevated rate of degradation in the presence of MMS. Taken together, the data show that the DNA damage regulation of Msh2 occurs at the transcriptional and post-transcriptional levels. The transcriptional and translational control elements identified are conserved in mammalian cells, underscoring the use of yeast as a model system to examine the regulation of MSH2.

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Identification of primary transcriptional regulation of cell cycle-regulated genes upon DNA damage.

Publications

Identification of primary transcriptional regulation of cell cycle-regulated genes upon DNA damage.

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