Adenovirus type 5 E1A proteins interact with cellular regulators of transcription to reprogram gene expression in the infected or transformed cell. Although E1A also interacts with DNA directly in vitro , it is not clear how this relates to its function in vivo . The N-terminal conserved regions 1, 2 and 3 and the C-terminal portions of E1A were prepared as purified recombinant proteins and analyses showed that only the C-terminal region bound DNA in vitro . Deletion of E1A amino acids 201–220 inhibited binding and a minimal fragment encompassing amino acids 201–218 of E1A was sufficient for binding single- and double-stranded DNA. This portion of E1A also bound the cation-exchange resins cellulose phosphate and carboxymethyl Sepharose. As this region contains six basic amino acids, in vitro binding of E1A to DNA probably results from an ionic interaction with the phosphodiester backbone of DNA. Studies in Saccharomyces cerevisiae have shown that expression of a strong transcriptional activation domain fused to a DNA-binding domain can inhibit growth. Although fusion of the C-terminal region of E1A to a strong transcriptional activation domain inhibited growth when expressed in yeast, this was not mediated by the DNA-binding domain identified in vitro . These data suggest that E1A does not bind DNA in vivo .
Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1 - PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression—most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1—implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12 —is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 . Importantly, the specific increased expression of PRC2 targets/ HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.
This investigation reports the use of surfactants and colorless leuco triarylmethane dyes to form a new class of radiochromic micelle hydrogels for three-dimensional (3D) water-equivalent dosimetry. Gelatin gel samples with several surfactants and leuco dyes were prepared and evaluated for optical transparency, dose sensitivity and diffusion rates. The addition of Triton X-100, a non-ionic surfactant, at levels exceeding the critical micelle concentration provides a transparent hydrogel in which the water insoluble leuco Malachite Green (LMG) can dissolve. During irradiation, the LMG dye precursor converts to Malachite Green (MG+). The most sensitive reported LMG gel formulation contains 0.3 mM LMG leuco dye, 16 mM trichloroacetic acid, 7 mM Triton X-100 and 4% w/w gelatin. A diffusion coefficient of 0.14 mm2 h−1 was determined for MG+ in this gel by fitting the time-dependent degradation of the transmission profile after irradiating half of the sample. The diffusion rate was three times lower than the standard radiochromic ferrous xylenol-orange (FX) gel. The primary feature of this 3D hydrogel is that it introduces transparent, radiochromic, micelle hydrogels. The radiochromic response to dose is instantaneous and images are stable for several hours. A dosimetric characterization revealed that the dose response is reproducible to within 10% over five separate batches and independent of both energy and dose rate. Uniform pre-irradiation of samples to 5 Gy provided a subsequent near linear response to greater than 110 Gy. LMG gels when read with a fast optical CT scanner can provide full 3D dose distributions in less than 30 min post-irradiation. LMG micelle gels scanned with a 633 nm light source are a promising system for quantitative water- or tissue-equivalent 3D dose verification in the 5–100 Gy dose range. These gels are useful for the scanning of larger volume dosimeters (i.e. >15 cm diameter) since they are easily prepared with inexpensive ingredients, are transparent and have a low initial optical absorbance prior to irradiation. In this gel, only one colored molecular species is produced. This results in a linear relation between the concentration of the colored dye product and optical absorption independent of the sampled wavelength.
Abstract The NuA4 lysine acetyltransferase complex acetylates histone and nonhistone proteins and functions in transcription regulation, cell cycle progression, and DNA repair. NuA4 harbors an interesting duality in that its catalytic module can function independently and distinctly as picNuA4. At the molecular level, picNuA4 anchors to its bigger brother via physical interactions between the C-terminus of Epl1 and the HSA domain of Eaf1, the NuA4 central scaffolding subunit. This is reflected at the regulatory level, as picNuA4 can be liberated genetically from NuA4 by disrupting the Epl1−Eaf1 interaction. As such, removal of either Eaf1 or the Epl1 C-terminus offers a unique opportunity to elucidate the contributions of Eaf1 and Epl1 to NuA4 biology and in turn their roles in balancing picNuA4 and NuA4 activities. Using high-throughput genetic and gene expression profiling, and targeted functional assays to compare eaf1Δ and epl1-CΔ mutants, we found that EAF1 and EPL1 had both overlapping and distinct roles. Strikingly, loss of EAF1 or its HSA domain led to a significant decrease in the amount of picNuA4, while loss of the Epl1 C-terminus increased picNuA4 levels, suggesting starkly opposing effects on picNuA4 regulation. The eaf1Δ epl1-CΔ double mutants resembled the epl1-CΔ single mutants, indicating that Eaf1’s role in picNuA4 regulation depended on the Epl1 C-terminus. Key aspects of this regulation were evolutionarily conserved, as truncating an Epl1 homolog in human cells increased the levels of other picNuA4 subunits. Our findings suggested a model in which distinct aspects of the Epl1−Eaf1 interaction regulated picNuA4 amount and activity.
