CHK2 activation contributes to the development of oxaliplatin resistance in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC), the most common cancer type, causes high morbidity and mortality. Patients who develop drug resistance to oxaliplatin-based regimens have short overall survival. Thus, identifying molecules involved in the development of oxaliplatin resistance is critical for designing therapeutic strategies. METHODS: A proteomic screen was performed to reveal altered protein kinase phosphorylation in oxaliplatin-resistant (OR) CRC tumour spheroids. The function of CHK2 was characterised using several biochemical techniques and evident using in vitro cell and in vivo tumour models. RESULTS: We revealed that the level of phospho-CHK2(Thr68) was elevated in OR CRC cells and in ~30% of tumour samples from patients with OR CRC. We demonstrated that oxaliplatin activated several phosphatidylinositol 3-kinase-related kinases (PIKKs) and CHK2 downstream effectors and enhanced CHK2/PARP1 interaction to facilitate DNA repair. A phosphorylation mimicking CHK2 mutant, CHK2T68D, but not a kinase-dead CHK2 mutant, CHK2D347A, promoted DNA repair, the CHK2/PARP1 interaction, and cell growth in the presence of oxaliplatin. Finally, we showed that a CHK2 inhibitor, BML-277, reduced protein poly(ADP-ribosyl)ation (PARylation), FANCD2 monoubiquitination, homologous recombination and OR CRC cell growth in vitro and in vivo. CONCLUSION: Our findings suggest that CHK2 activity is critical for modulating oxaliplatin response and that CHK2 is a potential therapeutic target for OR CRC.

YHMI: a web tool to identify histone modifications and histone/chromatin regulators from a gene list in yeast.

Post-translational modifications of histones (e.g. acetylation, methylation, phosphorylation and ubiquitination) play crucial roles in regulating gene expression by altering chromatin structures and creating docking sites for histone/chromatin regulators. However, the combination patterns of histone modifications, regulatory proteins and their corresponding target genes remain incompletely understood. Therefore, it is advantageous to have a tool for the enrichment/depletion analysis of histone modifications and histone/chromatin regulators from a gene list. Many ChIP-chip/ChIP-seq datasets of histone modifications and histone/chromatin regulators in yeast can be found in the literature. Knowing the needs and having the data motivate us to develop a web tool, called Yeast Histone Modifications Identifier (YHMI), which can identify the enriched/depleted histone modifications and the enriched histone/chromatin regulators from a list of yeast genes. Both tables and figures are provided to visualize the identification results. Finally, the high-quality and biological insight of the identification results are demonstrated by two case studies. We believe that YHMI is a valuable tool for yeast biologists to do epigenetics research.

Ecological genomics in Xanthomonas: the nature of genetic adaptation with homologous recombination and host shifts.

BACKGROUND: Comparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas pathogenic to various plants. RESULTS: Using whole-genome sequences, we examined the genetic divergence in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting. CONCLUSION: Altogether, mediated with lasting gene flow, species formation in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification.

The Yeast Nucleosome Atlas (YNA) database: an integrative gene mining platform for studying chromatin structure and its regulation in yeast.

BACKGROUND: Histone modification and remodeling play crucial roles in regulating gene transcription. These post-translational modifications of histones function in a combinatorial fashion and can be recognized by specific histone-binding proteins, thus regulating gene transcription. Therefore, understanding the combinatorial patterns of the histone code is vital to understanding the associated biological processes. However, most of the datasets regarding histone modification and chromatin regulation are scattered across various studies, and no comprehensive search and query tool has yet been made available to retrieve genes bearing specific histone modification patterns and regulatory proteins. DESCRIPTION: For this reason, we developed the Yeast Nucleosome Atlas database, or the YNA database, which integrates the available experimental data on nucleosome occupancy, histone modifications, the binding occupancy of regulatory proteins, and gene expression data, and provides the genome-wide gene miner to retrieve genes with a specific combination of these chromatin-related datasets. Moreover, the biological significance analyzer, which analyzes the enrichments of histone modifications, binding occupancy, transcription rate, and functionality of the retrieved genes, was constructed to help researchers to gain insight into the correlation among chromatin regulation and transcription. CONCLUSIONS: Compared to previously established genome browsing databases, YNA provides a powerful gene mining and retrieval interface, and is an investigation tool that can assist users to generate testable hypotheses for studying chromatin regulation during transcription. YNA is available online at http://cosbi3.ee.ncku.edu.tw/yna/.

Downregulation of BRCA1-BRCA2-containing complex subunit 3 sensitizes glioma cells to temozolomide.

We previously found that BRCA1-BRCA2-containing complex subunit 3 (BRCC3) was highly expressed in tumorigenic rat glioma cells. However, the functional role of BRCC3 in human glioma cells remains to be characterized. This study indicated that the upregulation of BRCC3 expression was induced in two human malignant glioblastoma U251 and A172 cell lines following exposure to the alkylating agent, temozolomide (TMZ). Homologous recombination (HR)-dependent DNA repair-associated genes (i.e. BRCA1, BRCA2, RAD51 and FANCD2) were also increased in U251 and A172 cells after treatment with TMZ. BRCC3 gene knockdown through lentivirus-mediated gene knockdown approach not only significantly reduced the clonogenic and migratory abilities of U251 and A172 cells, but also enhanced their sensitization to TMZ. The increase in phosphorylated H2AX foci (γH2AX) formation, an indicator of DNA damage, persisted in TMZ-treated glioma cells with stable knockdown BRCC3 expression, suggesting that BRCC3 gene deficiency is associated with DNA repair impairment. In summary, we demonstrate that by inducing DNA repair, BRCC3 renders glioma cells resistant to TMZ. The findings point to BRCC3 as a potential target for treatment of alkylating drug-resistant glioma.

Retinoic acid mediates the expression of glutamate transporter-1 in rat astrocytes through genomic RXR action and non-genomic protein kinase C signaling pathway.

Astrocytic glutamate transporter-1 (GLT-1) is responsible for 90% of forebrain glutamate uptake in the adult CNS. Retinoic acid (RA) is a potent regulator of neural cell differentiation and neuronal maturation in the developing CNS through activation of RA receptors/retinoic X receptors (RXRs) or non-genomic mechanisms. Although rat GLT-1 contains several RXR binding regions, RA-triggered RXR mechanisms regulating GLT-1 expression remain unknown. RA applied at submicromolar concentrations for 24 h significantly reduced GLT-1 mRNA and membrane levels in astrocytes and dibutyryl cAMP (dbcAMP)-primed astrocytes. An RXR agonist reduced astrocytic GLT-1 mRNA expression, whereas an RXR antagonist blocked the effects of RA on the reduction of astrocytic GLT-1 mRNA expression. Electrophoresis motility shift assay indicated that RA-treatment increased astrocytic RXR-DNA binding activity. RA-induced reduction in GLT-1 mRNA expression was also observed in dbcAMP-primed astrocytes. Through lentivirus-mediated astrocytic over-expression of rat GLT-1, levels of GLT-1 in the processes of dbcAMP-treated astrocytes were attenuated by exposure to RA. The protein kinase C inhibitor, Bis I, restored GLT-1 distribution in the processes of RA-treated dbcAMP-primed astrocytes. These results suggest that RA reduces astrocytic GLT-1 levels through both RXR-mediated inhibition at the transcriptional level and triggering activation of protein kinase C which reduces cell surface GLT-1 levels.

The exon junction complex component Magoh controls brain size by regulating neural stem cell division.

Brain structure and size require precise division of neural stem cells (NSCs), which self-renew and generate intermediate neural progenitors (INPs) and neurons. The factors that regulate NSCs remain poorly understood, and mechanistic explanations of how aberrant NSC division causes the reduced brain size seen in microcephaly are lacking. Here we show that Magoh, a component of the exon junction complex (EJC) that binds RNA, controls mouse cerebral cortical size by regulating NSC division. Magoh haploinsufficiency causes microcephaly because of INP depletion and neuronal apoptosis. Defective mitosis underlies these phenotypes, as depletion of EJC components disrupts mitotic spindle orientation and integrity, chromosome number and genomic stability. In utero rescue experiments showed that a key function of Magoh is to control levels of the microcephaly-associated protein Lis1 during neurogenesis. Our results uncover requirements for the EJC in brain development, NSC maintenance and mitosis, thereby implicating this complex in the pathogenesis of microcephaly.

Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks.

Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability.

Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination.

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3'-5' DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Delta mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.

Genomic organization of the chicken CD8 locus reveals a novel family of immunoreceptor genes.

The genomic organization of the chicken CD8alpha gene was investigated to determine the basis of its polymorphism. Contiguous to the CD8alpha gene we identified multiple DNA blocks possessing sequences homologous to CD8alpha. Gene conversions and recombination over evolutionary time among CD8alpha and these CD8alpha homologous genes seem to account for the observed polymorphism. Furthermore, these CD8alpha-like DNAs encode a distinct multigene family of immunoreceptors that have a charged or polar residue in place of the interspecies-conserved CD8alpha transmembrane proline residue and a short cytoplasmic tail nonhomologous to CD8alpha. The identification of this novel multigene family with an organization reminiscent of human killer Ig-like receptors raises compelling questions on their evolutionary relationship among immunoreceptors.