Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting

Chromatin organization and transcriptional regulation are interrelated processes. A shortcoming of current experimental approaches to these complex events is the lack of methods that can capture the activation process on single promoters. We have recently described a method that combines methyltransferase M.SssI treatment of intact nuclei and bisulfite sequencing allowing the representation of replicas of single promoters in terms of protected and unprotected footprint modules. Here we combine this method with computational analysis to study single molecule dynamics of transcriptional activation in the stress inducible GRP78 promoter. We show that a 350–base pair region upstream of the transcription initiation site is constitutively depleted of nucleosomes, regardless of the induction state of the promoter, providing one of the first examples for such a promoter in mammals. The 350–base pair nucleosome-free region can be dissected into modules, identifying transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress. The interaction of the transcriptional machinery with the GRP78 core promoter is highly organized, represented by six major combinatorial states. We show that the TATA box is frequently occupied in the noninduced state, that stress induction results in sequential loading of the endoplasmic reticulum stress response elements, and that a substantial portion of these elements is no longer occupied following recruitment of factors to the transcription initiation site. Studying the positioning of nucleosomes and transcription factors at the single promoter level provides a powerful tool to gain novel insights into the transcriptional process in eukaryotes

Genomic and Transcriptomic Analyses of Breast Cancer Primaries and Matched Metastases in AURORA, the Breast International Group (BIG) Molecular Screening Initiative

Càncer de mama; Genòmica i transcriptòmicaCáncer de mama; Genómica y transcriptómicaBreast Cancer; Genomic and TranscriptomicAURORA aims to study the processes of relapse in metastatic breast cancer (MBC) by performing multi-omics profiling on paired primary tumors and early-course metastases. Among 381 patients (primary tumor and metastasis pairs: 252 targeted gene sequencing, 152 RNA sequencing, 67 single nucleotide polymorphism arrays), we found a driver role for GATA1 and MEN1 somatic mutations. Metastases were enriched in ESR1, PTEN, CDH1, PIK3CA, and RB1 mutations; MDM4 and MYC amplifications; and ARID1A deletions. An increase in clonality was observed in driver genes such as ERBB2 and RB1. Intrinsic subtype switching occurred in 36% of cases. Luminal A/B to HER2-enriched switching was associated with TP53 and/or PIK3CA mutations. Metastases had lower immune score and increased immune-permissive cells. High tumor mutational burden correlated to shorter time to relapse in HR+/HER2− cancers. ESCAT tier I/II alterations were detected in 51% of patients and matched therapy was used in 7%. Integration of multi-omics analyses in clinical practice could affect treatment strategies in MBC

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

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

<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