Interplay between promoter occupancy and chromatin remodeling requirements in transactivation of the S.cerevisiae PHO5 gene

In higher eukaryotes, DNA is packaged with histones and other proteins into chromatin. While this is important in the control of unwanted gene expression, chromatin also serves as a barrier to many vital functions in the cell. Therefore, cells have evolved many different types of chromatin remodeling enzymes to contend with this inhibitory structure and enable gene expression and other functions. The Saccharomyces cerevisiae PHO5 gene is triggered in response to phosphate starvation. In this study, I evaluate the chromatin remodeling requirements of this gene with respect to the multisubunit complexes SWI/SNF and SAGA. I show, for the first time, physical recruitment of SWI/SNF to the PHO5 promoter. I also demonstrate the role of promoter occupancy in influencing requirements for chromatin remodeling enzymes. Further, I describe various interactions between these two complexes at the PHO5 promoter. This study presents evidence for the first instance of excess recruitment of an ATP-dependent remodeler potentially compensating for the lack of a histone acetyltransferase

Working a second job: Cell adhesion proteins that moonlight in the nucleus

Cells are adept at sensing changes in their environment, transmitting signals internally to coordinate responses to external stimuli, and thereby influencing adaptive changes in cell states and behavior. Often, this response involves modulation of gene expression in the nucleus, which is seen largely as a physically separated process from the rest of the cell. Mechanosensing, whereby a cell senses physical stimuli, and integrates and converts these inputs into downstream responses including signaling cascades and gene regulatory changes, involves the participation of several macromolecular structures. Of note, the extracellular matrix (ECM) and its constituent macromolecules comprise an essential part of the cellular microenvironment, allowing cells to interact with each other, and providing both structural and biochemical stimuli sensed by adhesion transmembrane receptors. This highway of information between the ECM, cell adhesion proteins, and the cytoskeleton regulates cellular behavior, the disruption of which results in disease. Emerging evidence suggests a more direct role for some of these adhesion proteins in chromatin structure and gene regulation, RNA maturation and other non-canonical functions. While many of these discoveries were previously limited to observations of cytoplasmic-nuclear transport, recent advances in microscopy, and biochemical, proteomic and genomic technologies have begun to significantly enhance our understanding of the impact of nuclear localization of these proteins. This review will briefly cover known cell adhesion proteins that migrate to the nucleus, and their downstream functions. We will outline recent advances in this very exciting yet still emerging field, with impact ranging from basic biology to disease states like cancer

The Transcription Factors Snail and Slug Activate the Transforming Growth Factor-Beta Signaling Pathway in Breast Cancer

The transcriptional repressors Snail and Slug are situated at the core of several signaling pathways proposed to mediate epithelial to mesenchymal transition or EMT, which has been implicated in tumor metastasis. EMT involves an alteration from an organized, epithelial cell structure to a mesenchymal, invasive and migratory phenotype. In order to obtain a global view of the impact of Snail and Slug expression, we performed a microarray experiment using the MCF-7 breast cancer cell line, which does not express detectable levels of Snail or Slug. MCF-7 cells were infected with Snail, Slug or control adenovirus, and RNA samples isolated at various time points were analyzed across all transcripts. Our analyses indicated that Snail and Slug regulate many genes in common, but also have distinct sets of gene targets. Gene set enrichment analyses indicated that Snail and Slug directed the transcriptome of MCF-7 cells from a luminal towards a more complex pattern that includes many features of the claudin-low breast cancer signature. Of particular interest, genes involved in the TGF-beta signaling pathway are upregulated, while genes responsible for a differentiated morphology are downregulated following Snail or Slug expression. Further we noticed increased histone acetylation at the promoter region of the transforming growth factor beta-receptor II (TGFBR2) gene following Snail or Slug expression. Inhibition of the TGF-beta signaling pathway using selective small-molecule inhibitors following Snail or Slug addition resulted in decreased cell migration with no impact on the repression of cell junction molecules by Snail and Slug. We propose that there are two regulatory modules embedded within EMT: one that involves repression of cell junction molecules, and the other involving cell migration via TGF-beta and/or other pathways

Promoter Occupancy Is a Major Determinant of Chromatin Remodeling Enzyme Requirements

Chromatin creates transcriptional barriers that are overcome by coactivator activities such as histone acetylation by Gcn5 and ATP-dependent chromatin remodeling by SWI/SNF. Factors defining the differential coactivator requirements in the transactivation of various promoters remain elusive. Induction of the Saccharomyces cerevisiae PHO5 promoter does not require Gcn5 or SWI/SNF under fully inducing conditions of no phosphate. We show that PHO5 activation is highly dependent on both coactivators at intermediate phosphate concentrations, conditions that reduce the nuclear concentration of the Pho4 transactivator and severely diminish its association with PHO5 in the absence of Gcn5 or SWI/SNF. Conversely, physiological increases in Pho4 nuclear concentration and binding at PHO5 suppress the need for both Gcn5 and SWI/SNF, suggesting that coactivator redundancy is established at high Pho4 binding site occupancy. Consistent with this, we demonstrate, using chromatin immunoprecipitation, that Gcn5 and SWI/SNF are directly recruited to PHO5 and other strongly transcribed promoters, including GAL1-10, RPL19B, RPS22B, PYK1, and EFT2, which do not require either coactivator for expression. These results show that activator concentration and binding site occupancy play crucial roles in defining the extent to which transcription requires individual chromatin remodeling enzymes. In addition, Gcn5 and SWI/SNF associate with many more genomic targets than previously appreciated

PARP-1/2 Inhibitor Olaparib Prevents or Partially Reverts EMT Induced by TGF-β in NMuMG Cells

Poly- adenosine diphosphate (ADP)-ribose (PAR) is a polymer synthesized as a posttranslational modification by some poly (ADP-ribose) polymerases (PARPs), namely PARP-1, PARP-2, tankyrase-1, and tankyrase-2 (TNKS-1/2). PARP-1 is nuclear and has also been detected in extracellular vesicles. PARP-2 and TNKS-1/2 are distributed in nuclei and cytoplasm. PARP or PAR alterations have been described in tumors, and in particular by influencing the Epithelial- Mesenchymal Transition (EMT), which influences cell migration and drug resistance in cancer cells. Pro-EMT and anti-EMT effects of PARP-1 have been reported while whether PAR changes occur specifically during EMT is currently unknown. The PARP-1/2 inhibitor Olaparib (OLA) is approved by FDA to treat certain patients harboring cancers with impaired homologous recombination. Here, we studied PAR changes and OLA effects on EMT. Total and nuclear PAR increased in EMT while PAR belts were disassembled. OLA prevented EMT, according to: (i) molecular markers evaluated by immuno-cytofluorescence/image quantification, Western blots, and RNA quantitation, (ii) morphological changes expressed as anisotropy, and (iii) migration capacity in the scratch assay. OLA also partially reversed EMT. OLA might work through unconventional mechanisms of action (different from synthetic lethality), even in non-BRCA (breast cancer 1 gene) mutated cancers

Coupling Phosphate Homeostasis to Cell Cycle-Specific Transcription: Mitotic Activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead Proteins ▿

Cells devote considerable resources to nutrient homeostasis, involving nutrient surveillance, acquisition, and storage at physiologically relevant concentrations. Many Saccharomyces cerevisiae transcripts coding for proteins with nutrient uptake functions exhibit peak periodic accumulation during M phase, indicating that an important aspect of nutrient homeostasis involves transcriptional regulation. Inorganic phosphate is a central macronutrient that we have previously shown oscillates inversely with mitotic activation of PHO5. The mechanism of this periodic cell cycle expression remains unknown. To date, only two sequence-specific activators, Pho4 and Pho2, were known to induce PHO5 transcription. We provide here evidence that Mcm1, a MADS-box protein, is essential for PHO5 mitotic activation. In addition, we found that cells simultaneously lacking the forkhead proteins, Fkh1 and Fkh2, exhibited a 2.5-fold decrease in PHO5 expression. The Mcm1-Fkh2 complex, first shown to transactivate genes within the CLB2 cluster that drive G2/M progression, also associated directly at the PHO5 promoter in a cell cycle-dependent manner in chromatin immunoprecipitation assays. Sds3, a component specific to the Rpd3L histone deacetylase complex, was also recruited to PHO5 in G1. These findings provide (i) further mechanistic insight into PHO5 mitotic activation, (ii) demonstrate that Mcm1-Fkh2 can function combinatorially with other activators to yield late M/G1 induction, and (iii) couple the mitotic cell cycle progression machinery to cellular phosphate homeostasis

MBD3 Localizes at Promoters, Gene Bodies and Enhancers of Active Genes

<div><p>The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status.</p></div

MBD3 localizes to active CpG rich promoters independent of cell type.

<p>A. The Venn diagram depicts the overlap of transcripts with an MBD3 peak within 3-7 and 9,310 in MDA-231 were included in the overlap analysis. A total of 4292 transcripts had MBD3 peaks at TSS in both cell lines. B. The heatmap displays MBD3-DamID signal, H3K4me3 signal (MCF-7 ChIP-seq data is from UW, GEO accession number GSM945269, ENCODE Project Consortium 2011; MDA-231 ChIP-seq, this study) and gene expression in MCF-7 and MDA-231 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-Khaitan1" target="_blank">[54]</a>. 8,207 genes were selected for display as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>. Genomic intervals from −7 to +3 kb relative to TSS were binned into 20 equal bins and ranked by MBD3-DamID signal. H3K4me3 and gene expression were displayed in the same order. The color scale for interpretation of signal intensity is located at the bottom of the heatmap. C. Refseq transcript 5′ ends were selected as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>. The plot depicts average DamID signal for all transcripts across the interval from −3 to +3 kb relative to TSS. MCF-7 and MDA-231 plots are in green and tan, respectively. D. For each cell type, promoters were subdivided as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-Weber1" target="_blank">[16]</a>. The plot displays the average DamID signal for each promoter class by cell type across the indicated genomic interval. Promoter class is indicated by color coding as indicated in the figure. E. A composite gene model for genes selected as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#s4" target="_blank">Methods</a>. DamID signal intensity was averaged for genes in the two cell lines and displayed in the plot. The location of TSS and TES is indicated. Cell lines are distinguished by color coding as indicated. F. The column graph depicts the enrichment percentage derived from Functional Analysis. Genes were selected as MBD3 targets if there was an MBD3 peak within 3 kb of TSS. The luminal and basal discriminatory genes were as described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004028#pgen.1004028-CharafeJauffret1" target="_blank">[47]</a>.</p