Whole Genome Expression Profiling Shows that BRG1 Transcriptionally Regulates UV Inducible Genes and Other Novel Targets in Human Cells

<div><p>UV irradiation is known to cause cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone photoproducts (6-4PPs), and plays a large role in the development of cancer. Tumor suppression, through DNA repair and proper cell cycle regulation, is an integral factor in maintaining healthy cells and preventing development of cancer. Transcriptional regulation of the genes involved in the various tumor suppression pathways is essential for them to be expressed when needed and to function properly. BRG1, an ATPase catalytic subunit of the SWI/SNF chromatin remodeling complex, has been identified as a tumor suppressor protein, as it has been shown to play a role in Nucleotide Excision Repair (NER) of CPDs, suppress apoptosis, and restore checkpoint deficiency, in response to UV exposure. Although BRG1 has been shown to regulate transcription of some genes that are instrumental in proper DNA damage repair and cell cycle maintenance in response to UV, its role in transcriptional regulation of the whole genome in response to UV has not yet been elucidated. With whole genome expression profiling in SW13 cells, we show that upon UV induction, BRG1 regulates transcriptional expression of many genes involved in cell stress response. Additionally, our results also highlight BRG1's general role as a master regulator of the genome, as it transcriptionally regulates approximately 4.8% of the human genome, including expression of genes involved in many pathways. RT-PCR and ChIP were used to validate our genome expression analysis. Importantly, our study identifies several novel transcriptional targets of BRG1, such as <i>ATF3</i>. Thus, BRG1 has a larger impact on human genome expression than previously thought, and our studies will provide inroads for future analysis of BRG1's role in gene regulation.</p></div

BRG1 controls ATF3 expression under UV conditions.

<p>(<b>A</b>) RT-PCR analysis of ATF3 gene expression in SW13+vector and SW13+BRG1 cells. Both cells were collected 6 hours after UV treatment. RNA was purified and RT-PCR products were analyzed on agarose gel. (<b>B</b>) Binding of BRG1 to ATF3 gene promoter region. 293T cells were UV irradiated and incubated for 6 hours followed by Chromatin Immunoprecipitation (ChIP) assay with anti-BRG1 antibody.</p

Whole genome view of BRG1 regulatory gene targets (in the absence of UV irradiation) showing the locations of genes on each human chromosome.

<p>The vertical bar above the horizontal line indicates genes on the forward strand, and vertical lines below the horizontal line indicate genes on the reverse strand. (A) Genes upregulated by BRG1. (B) Genes downregulated by BRG1.</p

Comparison of BRG1 regulated gene expression with UV treatment and without.

<p>Comparison of BRG1 regulated gene expression with UV treatment and without.</p

34 Genes Induced by UV Radiation in a BRG1-Dependent Manner.

<p>34 Genes Induced by UV Radiation in a BRG1-Dependent Manner.</p

Microarray analysis of gene expression in response to UV irradiation in SW13 cells with or without BRG1.

<p>(<b>A</b>) Morphological changes of SW13 cells after BRG1 re-expression and a schematic representation of sample preparation for Microarray analysis. SW13 cells with and without reintroduction of BRG1 were irradiated with UV or mock-treated. Three independent biological samples of these treatments were incubated for 6 hours in warm media to allow for recovery, and then collected for RNA extraction. RNA preparations were then used for Microarray analysis. Interestingly, this figure also shows the morphological changes associated with reintroduction of BRG1 in SW13 cells. (<b>B</b>) Western blotting confirming BRG1 protein expression. (<b>C</b>) Venn Diagram showing that BRG1 regulates the transcriptional response to UV irradiation in human cells. Shown here are the number of genes induced by UV in SW13 cells and SW13+BRG1 cells. 34 of these genes are BRG1-dependent genes, and are not expressed without BRG1 reintroduction. Out of the 87 total genes induced by UV, 61 of them are only induced in cells which include expression of BRG1. Thus, more than 70% of UV induced genes are regulated by BRG1.</p

Validation of the Microarray data by RT-PCR.

<p>(<b>A</b>) Florescence intensity of RT-PCR products as a measure of mRNA expression confirming microarray data. Upon reintroduction of BRG1 into SW13 cells, regulation of gene expression differs from those cells without BRG1. GAPDH was loaded as a control. (<b>B</b>) Real time RT-PCR quantitation of CD44 and DLC1 mRNA levels.</p

Plot of the density function of beta distributions parameterized by mean and heterogeneity factor .

<p>On each subplot the density is shown for two choices of namely  = 0.2 (solid line) and  = 0.25 (dotted lines).</p

Plot of non-centrality parameter for the Bourgain test for a case-control study using two incompletely admixed populations as sources of cases and controls respectively.

<p>The parameters chosen refer to a test of a variant associated with disease which has 40 percent overall allele frequency and which is associated with a 10 percent difference in frequency between cases and controls (OR = 1.5 per copy). Cases are assumed to have average admixture percentage of 20 percent and controls 25 percent. The within population heterogeneity is specified by a single common heterogeneity parameter as used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001096#pgen-1001096-g001" target="_blank">Figure 1</a>.</p

Principal components plots of AABC and selected HapMap samples.

<p>Principal components plots of AABC and selected HapMap samples.</p