Few Combinatorial Modes of <i>GRP78</i> Promoter Organization

<p>Shown are clustered protection patterns for the 294 sampled promoters (rows, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020160#s4" target="_blank">Materials and Methods</a>). Only few modes of promoter organization are observed, including clusters representing high levels of TATA binding (cluster 1), cassette like loading of the ESREs (clusters 2–4), recruitment of factors to the TIS (cluster 5), and release of the ESRE modules (cluster 6). Statistical enrichment analysis (Materials and Methods) confirms that specific modes of activity (clusters) are overrepresented in specific phases of the ER-stress activation process, enabling us to arrange the clusters in a chronological order. The designation of each row (= protection pattern of one promoter molecule) to the time point from which it originated is marked by the blue boxes on the right. The early induction time points (1, 0.5, and 6 h) are pooled.</p

A Model Proposed for the Activation Process at the <i>GRP78</i> Promoter

<div><p>(A) The TATA box is occupied to a high extent even before induction, probably by TBP but also other factors.</p><p>(B-D) After stress induction the ERSEs are sequentially loaded, from E1 to E3 while each ERSE is occupied at a different level (indicated by the size of the circles).</p><p>(E, F) Recruitment of factors to the TIS is followed by release of the transcription factors from the ERSEs.</p></div

Transcription Factor Binding and Nucleosome Depletion at the Human <i>GRP78</i> Promoter

<div><p>(A) ChIP analysis of the <i>GRP78</i> promoter was performed on noninduced or TG-induced LD419 cells using antibodies against TBP, RNA polII, NF-Y, total H3, and acetylated H3-K9/14. Precipitated DNA was quantified by real-time PCR using primers specific for the indicated four regions of the promoter. The enrichment at each region is plotted as percentage of input. The data are representatives of experiments performed from two or three independent chromatin preparations.</p><p>(B, C) MNase assay. (B) A mixture of oligonucleosomes (lane 2) from LD419 nuclei digested with MNase was fractionated through a sucrose gradient to obtain mononucleosomal, dinucleosomal, and trinucleosomal DNA (lanes 4, 5, and 6). Purified genomic DNA partially digested with MNase was used as naked DNA control (D1 and D2, lanes 7 and 8; M in lanes 1 and 9 indicates size marker). (C) Relative enrichment of nucleosomal and naked DNA at four regions of the <i>GRP78</i> promoter. The values plotted are normalized with respect to the value at the R1 region, arbitrarily defined as 1. The four regions are depicted on the promoter diagram at the top of the figure. The TATA box (T), TIS (bent arrow), and ERSEs (E1–E3) are marked.</p><p>(D) DNase I hypersensitivity assay. Top: EtBr staining. Bottom: Southern blot. The assay was performed on nuclei extracted from noninduced or TG-induced cells. Genomic DNA was included as a control to show a lack of sequence specificity of the enzyme. DNase I–digested samples were resolved by gel electrophoresis, showing varying extents of digestion (EtBr staining). Southern blot performed after RsaI digestion revealed a hypersensitive region in the <i>GRP78</i> promoter similar in size in both noninduced and induced samples. On the left, a map of the GRP78 promoter region is shown indicating the 1,802-bp DNA fragment generated by RsaI digestion, transcription start site (bent arrow), and probe fragment (black box). Numbers next to the marker indicate size in bp.</p></div

<i>GRP78</i> Activation during ER Stress

<div><p>LD419 cells were harvested at 0, 0.5, 1, 2, 6, and 16 h after induction with 300 nM TG. The nuclei were purified and treated with M.SssI followed by bisulfite genomic sequencing.</p><p>(A) Relative <i>GRP78</i> mRNA levels at the different points after TG induction determined by quantitative RT-PCR (normalized to GAPDH).</p><p>(B) Sequencing data for the various time points. The diagrams on top, drawn to scale, represent the region analyzed (core promoter amplicon, short) and indicate the distribution density of the 37 CpG sites included in this region. The TIS (bent arrow), TATA box (T), and ERSE elements (E1–E3) are marked. Each horizontal line with a string of circles represents the methylation profile for one DNA molecule. White circles indicate unmethylated, and black circles, methylated CpG sites. The modules, as defined by the correlation analysis in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020160#pgen-0020160-g003" target="_blank">Figure 3</a>, are color coded: blue, TATA box module; yellow, TIS module; bright green, ERSE1 module; dark green, ERSE2 module; olive, ERSE3 module; dark red, CpGs 49–50 and 51–53 modules; pink, nucleosomal module. The modules were colored if the majority of CpGs comprising the module were protected. The nucleosomal module was colored according to the previous nucleosomal patch definition [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020160#pgen-0020160-b012" target="_blank">12</a>] where more than two consecutive protected CpGs are considered a patch and the occurrence of one unmethylated CpG site does not break the contiguity of the patch.</p><p>(C) Protection levels of nucleosomal modules (left), TATA and TIS (middle), and ERSEs (right) were calculated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020160#s4" target="_blank">Materials and Methods</a>) and are shown at the different time points.</p><p>(D) ATF6 enrichment at the ERSE region shown by ChIP analyses on LD419 cells harvested at 0, 1, 4, and 16 h after TG stress induction. DNA was quantified by real-time PCR using primers specific for the indicated four regions of the promoter (as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020160#pgen-0020160-g001" target="_blank">Figure 1</a>A).</p></div

Definition of Footprint Modules in the Core Promoter

<div><p>(A) Shown is a color-coded representation of the correlations among protection patterns of 37 CpGs in the <i>GRP78</i> core promoter region (CpGs 26–62). Red indicates positive correlation, and green indicates negative correlation. The evident red blocks on the diagonal correspond to contiguous sets of CpGs that are highly co-protected in the pool of 294 promoter sequences. Although discovered in a completely unsupervised fashion, these blocks correspond to distinct and known functional promoter elements.</p><p>(B) A similar correlation analysis performed as a negative control on 40 sequences derived from naked DNA which was methylated to achieve 60% average methylation. No defined modules similar to those as in (A) are found. For both (A) and (B), note that the matrix is symmetric by definition; both upper and lower triangular parts are drawn for visualization purpose only.</p></div