Michael W. Van Dyke

Single-step purification of bacterially expressed polypeptides containing an oligo-histidine domain.

Plasmid expression vectors have been constructed that direct the synthesis in Escherichia coli of fusion proteins containing a stretch of six histidine residues at either the N or C terminus. This oligo-histidine domain allows the single-step purification of the fusion proteins, under nondenaturing conditions, by immobilized metal affinity chromatography on Ni2+ bound to iminodiacetic acid-agarose. Several eukaryotic transcription factors (e.g., the upstream stimulatory factor for the adenovirus major late promoter) have been successfully purified, in an active state, by this method.

Parthenolide promotes the ubiquitination of MDM2 and activates p53 cellular functions.

MDM2 belongs to a class of ring-finger domain–containing ubiquitin ligases that mediate the proteasomal degradation of numerous proteins, including themselves. Arguably, the most important substrate of MDM2 is p53, which controls cell cycle progression and apoptosis. MDM2 and p53 are parts of a feedback regulatory loop whose perturbations are often present in cancer and are targets for anticancer drug development. We found that the natural product, small-molecule anti-inflammatory agent parthenolide (PN), which is actively being investigated as a potential therapeutic for many human cancers, induces ubiquitination of MDM2 in treated cells, resulting in the activation of p53 and other MDM2-regulated tumor-suppressor proteins. Using cells with functional gene deletions and small interfering RNA knockdown studies, we found that these effects required the DNA damage transducer ataxia telangiectasia mutated. The effects of PN on tumor suppressor activation were comparable with that of nutlin-3a, a recently developed small molecule that was designed to interfere with the interaction between MDM2 and p53 but does not promote MDM2 ubiquitination. Our study illustrates an alternative approach for controlling MDM2 and p53 activities and identifies an additional critically important cancer pathway affected by PN. [Mol Cancer Ther 2009;8(3):552–62]

Tumour necrosis factor‐α depletes histone deacetylase 1 protein through IKK2

Class I histone deacetylases (HDACs) are ubiquitous enzymes that repress gene expression by deacetylating histone tails and promoting chromatin compaction. Pro‐inflammatory agents activate programmes of gene expression through transcription factors such as nuclear factor‐κB (NF‐κB), even in the context of ubiquitous HDAC activity. How this is accomplished remains unknown. We found that cells treated with the pro‐inflammatory cytokine tumour necrosis factor‐α rapidly and substantially reduced HDAC1 protein levels without affecting other class I HDACs. In addition, HDAC1 depletion occurred through protein degradation, required IKK2 activity and resulted in increased transcription from both NF‐κB‐associated and unassociated gene promoters. Our study suggests that the activation of programmes of gene expression by pro‐inflammatory agents requires global changes in specific critical epigenetic regulators such as HDAC1.

Initiator sequences direct downstream promoter binding by human transcription factor IID

Whereas the human transcription factor IID generally interacts with only the TATA box element on most class II gene promoters, on certain promoters (e.g., the adenovirus 2 major late promoter) TFIID protects DNA sequences up to +35 base pairs downstream of the start site of transcription from DNase I cleavage. In this study, we show that Ad2 MLP sequences from -3 to +5 were sufficient to direct downstream promoter binding by TFIID when introduced into the human hsp70 gene promoter. These sequences correspond to the initiator transcription control element. Initiator mutations resulting in a loss of downstream binding demonstrated a diminished transcription efficiency in vitro. Likewise, initiator-dependent transcription stimulation required TFIID fractions capable of downstream promoter binding. Given the recent findings that immunopurified, human TFIID exhibited downstream promoter binding on the Ad2 MLP (Zhou, Q., Lieberman, P.M., Boyer, T.G. and Berk, A.J. (1992) Genes Dev. 6, 1964-1974), our data suggest that human TFIID can recognize the initiator element and that this interaction is required for maximally efficient transcription initiation.

In vitro inhibition of c-myc transcription by mithramycin

Mithramycin is a DNA-binding antibiotic that has been reported to selectively affect c-myc expression [Snyder, R. C. et al., (1991) Biochemistry 30, 4290-4297]. We used in vitro transcription to investigate the specificity of mithramycin action. We found that mithramycin inhibited transcription from the human c-myc P1 and P2 promoters, as well as from a minimal adenovirus-2 major late promoter, with equal efficiencies. Mithramycin also inhibited transcription elongation by creating kinetic blockades to the passage of RNA polymerase II. These data suggest that mithramycin may inhibit transcription non-specifically by affecting general processes such as transcription elongation.

Depletion of histone deacetylase protein: A common consequence of inflammatory cytokine signaling?

The dynamics of histone acetylation and deacetylation have long been known to influence gene expression by cellular signaling pathways. However, the mechanisms that regulate histone acetyl transferases (HATs) and histone deacetylases (HDACs) by these pathways have only recently become the focus of scientific investigation, spurred by increasing knowledge that HDACs can promote cancer growth. We recently reported that pro-inflammatory signals such as tumor necrosis factor α (TNFα) induce HDAC1 ubiquitination and proteasomal degradation through the IκB kinase IKKβ. The resulting depletion of cellular HDAC1 levels lead to a consequent depletion of HDAC1 associated with the CDKN1A gene promoter and increased expression of its protein product, p21WAF1/CIP1. This phenomenon heralds a unique mechanism of HDAC regulation that modulates the pro-inflammatory activity of TNFα and other cytokines at the level of gene expression. Here we discuss the current knowledge of pro-inflammatory cytokine-induced regulation of gene expression, emphasizing the involvement of HDAC1, and its possible implications in inflammation, cancer, and their therapy.

Stm1p alters the ribosome association of eukaryotic elongation factor 3 and affects translation elongation

Stm1p is a Saccharomyces cerevisiae protein that is primarily associated with cytosolic 80S ribosomes and polysomes. Several lines of evidence suggest that Stm1p plays a role in translation under nutrient stress conditions, although its mechanism of action is not yet known. In this study, we show that yeast lacking Stm1p (stm1Δ) are hypersensitive to the translation inhibitor anisomycin, which affects the peptidyl transferase reaction in translation elongation, but show little hypersensitivity to other translation inhibitors such as paromomycin and hygromycin B, which affect translation fidelity. Ribosomes isolated from stm1Δ yeast have intrinsically elevated levels of eukaryotic elongation factor 3 (eEF3) associated with them. Overexpression of eEF3 in cells lacking Stm1p results in a growth defect phenotype and increased anisomycin sensitivity. In addition, ribosomes with increased levels of Stm1p exhibit decreased association with eEF3. Taken together, our data indicate that Stm1p plays a complementary role to eEF3 in translation.

Plasmids for the in vitro analysis of RNA polymerase II-dependent transcription based on a G-free template

Described are a series of plasmids containing combinations of adenovirus-2 major late promoter elements, including consensus TATA box and initiator, upstream of G-free transcription cassettes of various lengths. These provide an assortment of tools for investigating both basal and regulated transcription mechanisms by in vitro transcription methods.

Do DNA Triple Helices or Quadruplexes Have a Role in Transcription

Certain DNA sequences preferentially adopt multistranded, non-B-form structures under physiological conditions. These include three-stranded DNA triplexes and four-stranded DNA quadruplexes. Several lines of evidence suggest that multiplex structures can form in vivo, either from the addition of oligonucleotides or through the transient formation of single-stranded regions. The consequences of multiplex structures on many DNA-dependent biological processes have been described. In this chapter I will review the effects of different DNA multiplexes on the process of transcription. The influence of parameters such as multiplex type and multiplex formation conditions on different transcription mechanistic steps in organisms spanning from prokaryotes to Xenopus oocytes and mammalian cells will be discussed.

Sp1, USF, and GAL4 activate transcription independently of TFIID-downstream promoter interactions.

One way specific transcription factors are thought to activate transcription initiation is by facilitating interactions between the general transcription factor TFIID and DNA sequences downstream of the TATA element. Examples supporting this model include transcription activation from the core adenovirus E4 promoter by either the human gene-specific transcription factor ATF or the acidic-domain fusion protein GAL4-AH. In these cases, appearance of downstream promoter binding by TFIID correlated directly with transcription activation by these proteins. Previously we had shown that downstream promoter binding by TFIID involved recognition of the initiator DNA control element and that the extent of this binding correlated directly with initiator-dependent transcription activation in vitro. We now report our use of DNase I footprinting and in vitro transcription to investigate the effects of various stimulatory transcription factors on TFIID binding and transcription efficiency from different initiator-containing promoters. Transcription factors investigated included Sp1, USF, and several GAL4-acidic domain fusion proteins. We found that none of these transcription factors appreciably affected downstream promoter binding by TFIID, whether qualitatively or quantitatively. In fact, all of these transcription factors stimulated transcription in vitro regardless of the strength of the initiator element present. When both elements were present, transcription stimulation mediated by proximally bound transcription factors and by TFIID-initiator interaction seemed to be synergistic. Taken together, our data would suggest that transcription activation by these two means occurs through different steps within the transcriptional process.