Detection of the glucocorticoid receptors in brain protein extracts by SDS-PAGE

Uncorrected proofGlucocorticoids are steroid hormones vital for organ system homeostasis and for the maintenance of essential biological processes. A significant part of these actions are mediated through glucocorticoid receptor (GR) that belongs to the nuclear receptor superfamily. To cover such variety of processes the different glucocorticoids act through different GR isoforms that are originated due to posttranscriptional and posttranslational mechanisms. For this reason when evaluating the levels of GRs we should preferentially determine protein levels instead of gene expression. Here, we describe the detection by Western blotting of the GR (a and ß isoforms) protein, using macrodissected brain tissue

Genome Res

Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells

DNA Specificity Determinants Associate with Distinct Transcription Factor Functions

To elucidate how genomic sequences build transcriptional control networks, we need to understand the connection between DNA sequence and transcription factor binding and function. Binding predictions based solely on consensus predictions are limited, because a single factor can use degenerate sequence motifs and because related transcription factors often prefer identical sequences. The ETS family transcription factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 and other ETS proteins is observed at promoters of housekeeping genes in T cells due to common sequence preferences and the presence of strong consensus motifs. However, ETS1 exhibits a specific function in T cell activation; thus, unique transcriptional targets are predicted. To uncover the sequence motifs that mediate specific functions of ETS1, a genome-wide approach, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), identified both promoter and enhancer binding events in Jurkat T cells. A comparison with DNase I sensitivity both validated the dataset and also improved accuracy. Redundant occupancy of ETS1 with the ETS protein GABPA occurred primarily in promoters of housekeeping genes, whereas ETS1 specific occupancy occurred in the enhancers of T cell–specific genes. Two routes to ETS1 specificity were identified: an intrinsic preference of ETS1 for a variant of the ETS family consensus sequence and the presence of a composite sequence that can support cooperative binding with a RUNX transcription factor. Genome-wide occupancy of RUNX factors corroborated the importance of this partnership. Furthermore, genome-wide occupancy of co-activator CBP indicated tight co-localization with ETS1 at specific enhancers, but not redundant promoters. The distinct sequences associated with redundant versus specific ETS1 occupancy were predictive of promoter or enhancer location and the ontology of nearby genes. These findings demonstrate that diversity of DNA binding motifs may enable variable transcription factor function at different genomic sites

The Elongator Complex Interacts with PCNA and Modulates Transcriptional Silencing and Sensitivity to DNA Damage Agents

Histone chaperones CAF-1 and Asf1 function to deposit newly synthesized histones onto replicating DNA to promote nucleosome formation in a proliferating cell nuclear antigen (PCNA) dependent process. The DNA replication- or DNA repair-coupled nucleosome assembly pathways are important for maintenance of transcriptional gene silencing and genome stability. However, how these pathways are regulated is not well understood. Here we report an interaction between the Elongator histone acetyltransferase and the proliferating cell nuclear antigen. Cells lacking Elp3 (K-acetyltransferase Kat9), the catalytic subunit of the six-subunit Elongator complex, partially lose silencing of reporter genes at the chromosome VIIL telomere and at the HMR locus, and are sensitive to the DNA replication inhibitor hydroxyurea (HU) and the damaging agent methyl methanesulfonate (MMS). Like deletion of the ELP3, mutation of each of the four other subunits of the Elongator complex as well as mutations in Elp3 that compromise the formation of the Elongator complex also result in loss of silencing and increased HU sensitivity. Moreover, Elp3 is required for S-phase progression in the presence of HU. Epistasis analysis indicates that the elp3Δ mutant, which itself is sensitive to MMS, exacerbates the MMS sensitivity of cells lacking histone chaperones Asf1, CAF-1 and the H3 lysine 56 acetyltransferase Rtt109. The elp3Δ mutant has allele specific genetic interactions with mutations in POL30 that encodes PCNA and PCNA binds to the Elongator complex both in vivo and in vitro. Together, these results uncover a novel role for the intact Elongator complex in transcriptional silencing and maintenance of genome stability, and it does so in a pathway linked to the DNA replication and DNA repair protein PCNA

Dosimetry for the MRI accelerator:the impact of a magnetic field on the response of a Farmer NE2571 ionization chamber

\u3cp\u3eThe UMC Utrecht is constructing a 1.5 T MRI scanner integrated with a linear accelerator (Lagendijk et al 2008 Radiother. Oncol. 86 25-9). The goal of this device is to facilitate soft-tissue contrast based image-guided radiotherapy, in order to escalate the dose to the tumour while sparing surrounding normal tissues. Dosimetry for the MRI accelerator has to be performed in the presence of a magnetic field. This paper investigates the feasibility of using a Farmer NE2571 ionization chamber for absolute dosimetry. The impact of the mcagnetic field on the response of this ionization chamber has been measured and simulated using GEANT4 Monte Carlo simulations. Two orientations of the ionization chamber with respect to the incident beam and the magnetic field which are feasible in the MRI accelerator configuration are taken into account. Measurements are performed using a laboratory magnet ranging from 0 to 1.2 T. In the simulations a range from 0 to 2 T is used. For both orientations, the measurements and simulations agreed within the uncertainty of the measurements and simulations. In conclusion, the response of the ionization chamber as a function of the magnetic field is understood and can be simulated using GEANT4 Monte Carlo simulations.\u3c/p\u3