High-Affinity Quasi-Specific Sites in the Genome: How the DNA-Binding Proteins Cope with Them

AbstractMany prokaryotic transcription factors home in on one or a few target sites in the presence of a huge number of nonspecific sites. Our analysis of λ-repressor in the Escherichia coli genome based on single basepair substitution experiments shows the presence of hundreds of sites having binding energy within 3 Kcal/mole of the OR1 binding energy, and thousands of sites with binding energy above the nonspecific binding energy. The effect of such sites on DNA-based processes has not been fully explored. The presence of such sites dramatically lowers the occupation probability of the specific site far more than if the genome were composed of nonspecific sites only. Our Brownian dynamics studies show that the presence of quasi-specific sites results in very significant kinetic effects as well. In contrast to λ-repressor, the E. coli genome has orders of magnitude lower quasi-specific sites for GalR, an integral transcription factor, thus causing little competition for the specific site. We propose that GalR and perhaps repressors of the same family have evolved binding modes that lead to much smaller numbers of quasi-specific sites to remove the untoward effects of genomic DNA

Study of Conformation and DNA Binding Specificity of Transcription Factors

The regulation of gene expression in bacteria primarily occurs at the level of transcription initiation. In bacterial chromosomes, a transcription unit (TU) is the ordered assembly of: a regulatory region, a transcription start site, one or more open reading frames (ORFs) and a transcription termination site. When a TU comprises more than one ORF, the transcribed mRNA is called polycistronic; otherwise, it is called monocistronic. The collection of overlapping TUs constitutes an operon. Even in a polycistronic TU, it has been observed that operons always contain a single promoter that transcribes the whole set of genes conforming its TUs. The regulatory region contains cis elements such as the promoter – where the RNA polymerase initially binds – and transcription factor-binding sites (TFBS) – where transcription factors (TFs) bind to modulate the binding of the RNA polymerase (Browning, D.F. et al., 2004). It is now becoming clear that long non-coding RNA genes can have numerous molecular functions, like modulating transcriptional patterns, regulating protein activities, serving structural or organizational roles, altering RNA processing events, and serving as precursors to small RNAs

Utilization of Cancer Cell Line Screening to Elucidate the Anticancer Activity and Biological Pathways Related to the Ruthenium-Based Therapeutic BOLD-100

BOLD-100 (sodium trans-[tetrachlorobis(1H indazole)ruthenate(III)]) is a ruthenium-based anticancer compound currently in clinical development. The identification of cancer types that show increased sensitivity towards BOLD-100 can lead to improved developmental strategies. Sensitivity profiling can also identify mechanisms of action that are pertinent for the bioactivity of complex therapeutics. Sensitivity to BOLD-100 was measured in a 319-cancer-cell line panel spanning 24 tissues. BOLD-100’s sensitivity profile showed variation across the tissue lineages, including increased response in esophageal, bladder, and hematologic cancers. Multiple cancers, including esophageal, bile duct and colon cancer, had higher relative response to BOLD-100 than to cisplatin. Response to BOLD-100 showed only moderate correlation to anticancer compounds in the Genomics of Drug Sensitivity in Cancer (GDSC) database, as well as no clear theme in bioactivity of correlated hits, suggesting that BOLD-100 may have a differentiated therapeutic profile. The genomic modalities of cancer cell lines were modeled against the BOLD-100 sensitivity profile, which revealed that genes related to ribosomal processes were associated with sensitivity to BOLD-100. Machine learning modeling of the sensitivity profile to BOLD-100 and gene expression data provided moderative predictive value. These findings provide further mechanistic understanding around BOLD-100 and support its development for additional cancer types

Efficacy of histone deacetylase and estrogen receptor inhibition in breast cancer cells due to concerted down regulation of Akt.

Hormonal therapy resistance remains a considerable barrier in the treatment of breast cancer. Activation of the Akt-PI3K-mTOR pathway plays an important role in hormonal therapy resistance. Our recent preclinical and clinical studies showed that the addition of a histone deacetylase inhibitor re-sensitized hormonal therapy resistant breast cancer to tamoxifen. As histone deacetylases are key regulators of Akt, we evaluated the effect of combined treatment with the histone deacetylase inhibitor PCI-24781 and tamoxifen on Akt in breast cancer cells. We demonstrate that while both histone deacetylase and estrogen receptor inhibition down regulate AKT mRNA and protein, their concerted effort results in down regulation of AKT activity with induction of cell death. Histone deacetylase inhibition exerts its effect on AKT mRNA through an estrogen receptor-dependent mechanism, primarily down regulating the most abundant isoform AKT1. Although siRNA depletion of AKT modestly induces cell death, when combined with an anti-estrogen, cytotoxicity is significantly enhanced. Thus, histone deacetylase regulation of AKT mRNA is a key mediator of this therapeutic combination and may represent a novel biomarker for predicting response to this regimen

Alternative Sigma Factors in the Free State Are Equilibrium Mixtures of Open and Compact Conformations

Conformational switching upon core RNA polymerase binding is an integral part of functioning of bacterial sigma factors. Here, we have studied dynamical features of two alternative sigma factors. A study of fluorescence resonance energy transfer and hydrodynamic measurements in Escherichia coli σ32 suggest a compact shape like those found in complex with anti-sigma factors. On the other hand, the fluorescence anisotropy of probes attached to different regions of the protein and previous hydrogen exchange measurements suggest significant internal flexibility, particularly in the C-terminal half and region 1. In a homologous sigma factor, σF of Mycobacterium tuberculosis, emission spectra and fluorescence resonance energy transfer between the single tryptophan (W112) and probes placed in different regions suggest a compact conformation for a major part of the N-terminal half encompassing region 2 and the flexible C-terminal half. Fluorescence anisotropy measurements suggest significant flexibility in the C-terminal half and region 1, as well. Thus, free alternative sigma factors may be in equilibrium between two conformations: a compact one in which the promoter interacting motifs are trapped in the wrong conformation and another less abundant one with a more open and flexible conformation. Such flexibility may be important for promoter recognition and interaction with many partner proteins

Histone Deacetylase Regulation of ATM-Mediated DNA Damage Signaling

Ataxia-telangiectasia mutated (ATM) is a major regulator of the DNA damage response. ATM promotes the activation of BRCA1, CHK2, and p53 leading to the induction of response genes such as CDKN1A (p21), GADD45A and RRM2B that promote cell cycle arrest and DNA repair. The up-regulation of these response genes may contribute to resistance of cancer cells to genotoxic therapies. Here we show that histone deacetylases (HDACs) play a major role in mitigating the response of the ATM pathway to DNA damage. HDAC inhibition decreased ATM activation and expression, and attenuated the activation of p53 in vitro and in vivo. Select depletion of HDAC1 and HDAC2 was sufficient to modulate ATM activation, reduce GADD45A and RRM2B induction, and increase sensitivity to DNA strand breaks. The regulation of ATM by HDAC enzymes therefore suggests a vital role for HDAC1 and HDAC2 in the DNA damage response, and the potential use of the ATM pathway as a pharmacodynamic marker for combination therapies involving HDAC inhibitors

HDACs regulate AKT mRNA expression by an estrogen receptor-(α) dependent mechanism.

<p>(<b>A</b>) MCF7 cells were treated with vehicle (V), 0.1 µM PCI-24781 (P), 10 µM OH-tamoxifen (T), or the combination (PT) for 72 hours and western blotted. (<b>B</b>) MCF7 cells were treated with 0.1 µM PCI-24781 and ESR1 mRNA levels were measured at the indicated times and presented relative to untreated ESR1 levels. (<b>C</b>) The indicated cell lines were treated with vehicle (V) or 0.1 µM PCI-24781 (P) for 6 hours and ESR1 mRNA levels were measured and presented relative to vehicle treated MCF7 cells. (<b>D</b>) MCF7 cells were treated with vehicle (Veh), 0.1 µM PCI-24781 (PCI), 10 µM OH-tamoxifen (Tam), 0.1 µM PCI-24781 and 10 µM OH-tamoxifen (PCI+Tam), 0.1 µM fulvestrant (Ful), or 0.1 µM PCI-24781 and 0.1 µM fulvestrant (PCI-Ful) for 24 hours and AKT1 mRNA levels were measured and presented relative to vehicle treated MCF7 cells. (<b>E</b>) MCF7 cells were transfected with scramble or increasing concentrations ESR1 directed siRNA for 72 hours and assayed for AKT1 and AKT2 mRNA and western blotted for ER and Akt pathway components. AKT1 and AKT2 expression are normalized to individual scramble treatments and not to each other. For both AKT1 and 2, all ESR1 siRNA concentrations resulted in significant reductions (P-value < 0.05) compared to scramble transfection. (<b>F</b>) MCF7 cells were transfected with scramble or 1 µM ESR1 directed siRNA for 24 hours, divided and then treated with vehicle (V) or 0.1 µM PCI-24781 (P) for 72 hours and evaluated by western blot. For all mRNA measurements, experiments were conducted in triplicate and results expressed as the average with the error bars indicating the standard error of the mean. An (*) indicates a significant difference (P-value < 0.05) and a (#) an insignificant difference (P-value > 0.05) compared to vehicle or zero time treatment. A (@) indicates a significant (P-value < 0.05) difference compared to PCI-24781 treatment.</p

PCI-24781 and <b>tamoxifen concertedly down regulate Akt protein</b> and <b>activity in MCF7 cells.</b>

<p>(<b>A</b>) MCF7 cells were treated with increasing concentrations of PCI-24781 for 72 hours and western blotted. MCF7 cells were treated for 72 hours with vehicle (V), 0.1 µM PCI-24781 (P), 10 µM OH-tamoxifen (T), or the combination (PT) and cells extracts were evaluated by western blot (<b>B</b>) for Pan-Akt and active Akt (P^S473) expression or (<b>E</b>) Akt activity <i>in vitro</i> by immunoprecipitating and then incubating phospho-Akt with a GSK-3 substrate and measuring levels of phosphorylated GSK-3. (<b>C</b>) The indicated cell lines were treated with vehicle (V) or 0.1 µM PCI-24781 and 10 µM OH-tamoxifen (PT) for 72 hours and western blotted for active Akt. (<b>D</b>) In cell extracts, levels of Akt isoforms, active Akt (P ^S473and P^S308) and down stream indicators of Akt activity following 48 and 72 hour treatment with 0.1 µM PCI-24781 and 10 µM OH-tamoxifen (PT) are compared to vehicle treated cells (V).</p

The HDAC inhibitor PCI-24781 potentiates tamoxifen by inducing mitochondrial-mediated apoptosis.

<p>(<b>A</b>) MCF-7 cells were treated with increasing concentrations of PCI-24781 and with or without 10 µM OH-tamoxifen for 72 hours and assayed for viability. The dotted line indicates maximal achievable serum levels in patients [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068973#B26" target="_blank">26</a>]. MCF-7 cells were treated with vehicle (V), 0.1 µM PCI-24781 (P), 10 µM OH-tamoxifen (T), or the combination (PT) for 72 h. Cells were evaluated by western blotting for expression of apoptotic BH3 family member proteins, cytoplasmic cytochrome C, and PARP cleavage (<b>C</b>) and by microscopy for TUNEL staining (<b>B</b>). (<b>D</b>) The indicated cell lines were treated with vehicle (V), 0.1 µM PCI-24781 (P), 10 µM OH-tamoxifen (T), or the combination (PT) for 72 hours assayed for viability. Vehicle and combination treated cells were further evaluated by western blot for PARP cleavage. For (<b>A</b>), (<b>B</b>), and (<b>D</b>) the average from three independent experiments is presented, with the error bars indicating the standard error of the mean. An (*) indicates a significant difference compared to vehicle treatment (P-value < 0.05), while an (#) indicates an insignificant difference (P-value > 0.05).</p

PCI-24781 regulates AKT mRNA levels.

<p>(<b>A</b>) Basal levels of AKT1, 2, and 3 mRNA in MCF7 cells are presented relative to AKT1 expression. (<b>B</b>) AKT1 and 2 mRNA expression was determined in MCF7 cells following treatment with increasing concentrations of PCI-24781 for 24 hours. (<b>C</b>) MCF7 cells were treated with 0.1 µM PCI-24781 (P), 5 µg/mL actinomycin D (Actino D), or the combination (P+Actino D) and evaluated for AKT1 mRNA expression after 0, 12, 24, and 48 hours treatment. (<b>D</b>) MCF7 cells were pretreated with vehicle or 10 µg/mL cycloheximide for 1 hour. Vehicle and cycloheximide pretreated cells were then each divided and treated with vehicle (V) or 0.1 µM PCI-24781 (P) with or without 10 µg/mL cycloheximide (CycloH) for 24 hours and evaluated for AKT1 mRNA expression. (<b>E</b>) MCF7 cells were treated with vehicle, 0.1 µM PCI-24781, 3 mM valproic acid (VPA), or 30 nM trichostatin A (TSA) for 24 hours and evaluated for AKT1 mRNA expression. All treatments were conducted in triplicate and expressed as the average with the error bars indicating the standard error of the mean. An (*) indicates a significant difference (P-value < 0.05) and a (#) an insignificant difference (P-value > 0.05) compared to vehicle or zero time treatment.</p