Processes that Regulate the Ubiquitination of Chromatin and Chromatin-Associated Proteins

Ubiquitin is a post-translational modification important for many different processes in the cell, including antigen presentation and proteosomal degradation of proteins. It is heavily involved in the regulation of chromatin and the proteins that control chromatin-related processes. In this review, we will focus on ubiquitin-based chromatin regulation involved in four different processes. The first is DNA double strand break (DSB) repair and the role that ubiquitin plays in not just recruiting and stimulating DSB repair, but also the choice of pathway. The second is the PAF1 complex, which is involved in transcriptional elongation and interacts with RNAPII. The third is polycomb repressive complexes, specifically polycomb repressive complex 1, which utilizes ubiquitin to repress constitutively inactive genes. The last role of ubiquitin discussed is ubiquitin as a mitotic bookmark, which serves to provide a record of -active genes as cells transit mitosis. Each of these processes has independent pathways, but each is necessary for proper cellular function and organismal health

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively

MICA: microRNA integration for active module discovery

A successful method to address disease-specific module discovery is the integration of the gene expression data with the protein-protein interaction~(PPI) network. Although many algorithms have been developed for this purpose, they focus only on the network genes~(mostly on the well-connected ones); totally neglecting the genes whose interactions are partially or totally not known. In addition, they only make use of the gene expression data which does not give the complete picture about the actual protein expression levels. The cell uses different mechanisms, such as microRNAs, to post-transcriptionally regulate the proteins without affecting the corresponding genes' expressions. Due to this complexity, using a single data type is definitely not the correct way to find the correct module(s). Today, the unprecedented amount of publicly available disease-related heterogeneous data encourages the development of new methodologies to better understand complex diseases. In this work, we propose a novel workflow Mica, which, to the best of our knowledge, is the first study integrating miRNA, mRNA, and PPI information to identify disease-specific gene modules. The novelty of the Mica lies in many directions, such as the early modification of mRNA expression with microRNA to better highlight the indirect dependencies between the genes. We applied Mica on microRNA-Seq and mRNA-Seq data sets of $699$ invasive ductal carcinoma samples and $150$ invasive lobular carcinoma samples from the Cancer Genome Atlas Project~(TCGA). The Mica modules are shown to unravel new and interesting dependencies between the genes. Additionally, the modules accurately differentiate between the case and control samples while being highly enriched with disease-specific pathways and genes

Functional analysis of BARD1 missense variants in homology-directed repair and damage sensitivity

The BARD1 protein, which heterodimerizes with BRCA1, is encoded by a known breast cancer susceptibility gene. While several BARD1 variants have been identified as pathogenic, many more missense variants exist that do not occur frequently enough to assign a clinical risk. In this paper, whole exome sequencing of over 10,000 cancer samples from 33 cancer types identified from somatic mutations and loss of heterozygosity in tumors 76 potentially cancer-associated BARD1 missense and truncation variants. These variants were tested in a functional assay for homology-directed repair (HDR), as HDR deficiencies have been shown to correlate with clinical pathogenicity for BRCA1 variants. From these 76 variants, 4 in the ankyrin repeat domain and 5 in the BRCT domain were found to be non-functional in HDR. Two known benign variants were found to be functional in HDR, and three known pathogenic variants were non-functional, supporting the notion that the HDR assay can be used to predict the clinical risk of BARD1 variants. The identification of HDR-deficient variants in the ankyrin repeat domain indicates there are DNA repair functions associated with this domain that have not been closely examined. In order to examine whether BARD1-associated loss of HDR function results in DNA damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity. These findings improve the understanding of BARD1 functional domains in DNA repair and support that this functional assay is useful for predicting the cancer association of BARD1 variants.</div

Fanconi anemia protein FANCD2 inhibits TRF1 polyADP-ribosylation through tankyrase1-dependent manner

Background: Fanconi anemia (FA) is a rare autosomal recessive syndrome characterized by developmental abnormalities, progressive bone marrow failure, and predisposition to cancer. The key FA protein FANCD2 crosstalks with members of DNA damage and repair pathways that also play a role at telomeres. Therefore, we investigated whether FANCD2 has a similar involvement at telomeres. Results: We reveal that FANCD2 may perform a novel function separate to the FANCD2/BRCA pathway. This function includes FANCD2 interaction with one of the telomere components, the PARP family member tankyrase-1. Moreover, FANCD2 inhibits tankyrase-1 activity in vitro. In turn, FANCD2 deficiency increases the polyADP-ribosylation of telomere binding factor TRF1. Conclusions: FANCD2 binding and inhibiting tankyrase-1PARsylation at telomeres may provide an additional step within the FA pathway for the regulation of genomic integrity

Identification of the Chromophores in Prussian blue

Prussian blue was the world's first synthetic dye. Its structural, optical and magnetic properties have led to many applications in technology and medicine, and provide paradigms for understanding coordination polymers, framework materials and mixed-valence compounds. The intense red absorption of Prussian blue that characterises chemical and physical properties critical to many of these applications is now shown to arise from localised intervalence charge transfer transitions within two chromophoric variants (ligand isomers) of an idealised "dimer" fragment {(NC)5FeII}(mu-CN){FeIII(NC)3(H2O)2}. This fragment is only available in modern interpretations of the material's crystal structure, with the traditional motif {(NC)5FeII}(mu-CN){FeIII(NC)5} shown not to facilitate visible absorption. Essential to the analysis is the demonstration, obtained independently using absorption and magnetic circular dichroism spectroscopies, that spectra of Prussian blues are strongly influenced by particle size and (subsequent) light scattering. These interpretations are guided and supported by density functional theory calculations (CAM-B3LYP), supplemented by coupled cluster and Bethe-Salpeter spectral simulations, as well as electron paramagnetic resonance spectroscopy of Prussian blue and a model molecular dimeric ion [Fe2(CN)11]6-

Inference of hierarchical regulatory network of estrogen-dependent breast cancer through ChIP-based data

<p>Abstract</p> <p>Background</p> <p>Global profiling of in vivo protein-DNA interactions using ChIP-based technologies has evolved rapidly in recent years. Although many genome-wide studies have identified thousands of ERα binding sites and have revealed the associated transcription factor (TF) partners, such as AP1, FOXA1 and CEBP, little is known about ERα associated hierarchical transcriptional regulatory networks.</p> <p>Results</p> <p>In this study, we applied computational approaches to analyze three public available ChIP-based datasets: ChIP-seq, ChIP-PET and ChIP-chip, and to investigate the hierarchical regulatory network for ERα and ERα partner TFs regulation in estrogen-dependent breast cancer MCF7 cells. 16 common TFs and two common new TF partners (RORA and PITX2) were found among ChIP-seq, ChIP-chip and ChIP-PET datasets. The regulatory networks were constructed by scanning the ChIP-peak region with TF specific position weight matrix (PWM). A permutation test was performed to test the reliability of each connection of the network. We then used DREM software to perform gene ontology function analysis on the common genes. We found that FOS, PITX2, RORA and FOXA1 were involved in the up-regulated genes.</p> <p>We also conducted the ERα and Pol-II ChIP-seq experiments in tamoxifen resistance MCF7 cells (denoted as MCF7-T in this study) and compared the difference between MCF7 and MCF7-T cells. The result showed very little overlap between these two cells in terms of targeted genes (21.2% of common genes) and targeted TFs (25% of common TFs). The significant dissimilarity may indicate totally different transcriptional regulatory mechanisms between these two cancer cells.</p> <p>Conclusions</p> <p>Our study uncovers new estrogen-mediated regulatory networks by mining three ChIP-based data in MCF7 cells and ChIP-seq data in MCF7-T cells. We compared the different ChIP-based technologies as well as different breast cancer cells. Our computational analytical approach may guide biologists to further study the underlying mechanisms in breast cancer cells or other human diseases.</p

The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner. © 2013 Wysocka-Kapcinska et al

Camptothecin resistance is determined by the regulation of topoisomerase I degradation mediated by ubiquitin proteasome pathway

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance