Repertoire and evolution of miRNA genes in four divergent nematode species.

miRNAs are approximately 22-nt RNA molecules that play important roles in post-transcriptional regulation. We have performed small RNA sequencing in the nematodes Caenorhabditis elegans, C. briggsae, C.

Candidate biomarkers of response to an experimental cancer drug identified through a large-scale RNA interference genetic screen.

Purpose: A major impediment in the optimal selection of cancer patients for the most effective therapy is the lack of suitable biomarkers that foretell the response of a patient to a given drug. In the present study, we have used large-scale RNA interference-based genetic screens to find candidate biomarkers of resistance to a new acyl sulfonamide derivative, R3200. This compound inhibits the proliferation of tumor cells in vitro and in vivo, but its mechanism of action is unknown.

Wip1 confers G2 checkpoint recovery competence by counteracting p53-dependent transcriptional repression.

Activation of the DNA damage checkpoint causes a cell-cycle arrest through inhibition of cyclin-dependent kinases (cdks). To successfully recover from the arrest, a cell should somehow be maintained in its proper cell-cycle phase. This problem is particularly eminent when a cell arrests in G2, as cdk activity is important to establish a G2 state.

RAMA1 is a novel kinetochore protein involved in kinetochore-microtubule attachment.

During mitosis, kinetochores need to attach to microtubules emanating from spindle poles. Several protein complexes have been shown to mediate the kinetochore-microtubule interaction. However, with the continually growing number of newly identified kinetochore proteins, it is unclear whether all major components of the kinetochore-microtubule interface have been identified.

Aurora-A and hBora join the game of Polo.

Overactivation of both Polo-like kinase-1 (Plk1) and Aurora-A is linked to cancer development, and small-molecule inhibitors that target these kinases are currently tested as anticancer drugs. Here, we discuss recent advances in the understanding of the functional crosstalk between Plk1 and Aurora-A before and during mitosis. Several recent findings have led to a better appreciation of how the activities of these distinct mitotic kinases are intertwined.

Next-generation sequencing approaches in genetic rodent model systems to study functional effects of human genetic variation.

Rapid advances in DNA sequencing improve existing techniques and enable new approaches in genetics and functional genomics, bringing about unprecedented coverage, resolution and sensitivity. Enhanced toolsets can facilitate the untangling of connections between genomic variation, environmental factors and phenotypic effects, providing novel opportunities, but may also pose challenges in data interpretation, especially in highly heterogeneous human populations. Laboratory rodent strains, however, offer a variety of tailored model systems with controlled genetic backgrounds, facilitating complex genotype/phenotype relationship studies.

ZNF423 is critically required for retinoic acid-induced differentiation and is a marker of neuroblastoma outcome.

Retinoids play key roles in differentiation, growth arrest, and apoptosis and are increasingly being used in the clinic for the treatment of a variety of cancers, including neuroblastoma. Here, using a large-scale RNA interference-based genetic screen, we identify ZNF423 (also known as Ebfaz, OAZ, or Zfp423) as a component critically required for retinoic acid (RA)-induced differentiation. ZNF423 associates with the RARalpha/RXRalpha nuclear receptor complex and is essential for transactivation in response to retinoids.

Relaying the checkpoint signal from kinetochore to APC/C.

The mitotic checkpoint delays chromosome segregation until the last chromosome has correctly attached to the spindle. Exactly how this unattached chromosome can generate a checkpoint signal and inhibit the anaphase promoting complex/cyclosome (APC/C) is unknown. Two Developmental Cell papers in this issue by Kulukian et al.

RAD50 and NBS1 form a stable complex functional in DNA binding and tethering.

The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function.

An Xpb mouse model for combined xeroderma pigmentosum and cockayne syndrome reveals progeroid features upon further attenuation of DNA repair.

Patients carrying mutations in the XPB helicase subunit of the basal transcription and nucleotide excision repair (NER) factor TFIIH display the combined cancer and developmental-progeroid disorder xeroderma pigmentosum/Cockayne syndrome (XPCS). Due to the dual transcription repair role of XPB and the absence of animal models, the underlying molecular mechanisms of XPB(XPCS) are largely uncharacterized. Here we show that severe alterations in Xpb cause embryonic lethality and that knock-in mice closely mimicking an XPCS patient-derived XPB mutation recapitulate the UV sensitivity typical for XP but fail to show overt CS features unless the DNA repair capacity is further challenged by crossings to the NER-deficient Xpa background.

Relevance of breast cancer antiestrogen resistance genes in human breast cancer progression and tamoxifen resistance.

Purpose: We have previously identified a set of breast cancer antiestrogen resistance (BCAR) genes causing estrogen independence and tamoxifen resistance in vitro using a functional genetic screen. Here, we explored whether these BCAR genes provide predictive value for tamoxifen resistance and prognostic information for tumor aggressiveness in breast cancer patients.

Patients And Methods: mRNA levels of 10 BCAR genes (AKT1, AKT2, BCAR1, BCAR3, EGFR, ERBB2, GRB7, SRC, TLE3, and TRERF1) were measured in estrogen receptor-positive breast tumors using quantitative reverse-transcriptase polymerase chain reaction.

RNAi Delivers Insights into Liver Cancer.

Of the myriad alterations in gene copy number found in cancer cells, which alterations are critical for the cancer phenotype? In this issue of Cell, Zender et al. (2008) describe an integrative genomics approach to identify new tumor suppressor genes involved in hepatocellular carcinoma.

Mechanisms of dealing with DNA damage-induced replication problems.

During every S phase, cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. Given the large size of mammalian genomes and the required precision of DNA replication, genome duplication requires highly fine-tuned corrective and quality control processes. A major threat to the accuracy and efficiency of DNA synthesis is the presence of DNA lesions, caused by both endogenous and exogenous damaging agents.

DNA repair by the MRN complex: break it to make it.

Genomic instability in ataxia telangiectasia-like disorder and Nijmegen breakage syndrome is due to disruption of the Mre11-Rad50-Nbs1 complex. Buis et al. (2008) and Williams et al.

Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals.

Background: The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive.

Results: As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background.

Zebrafish with mutations in mismatch repair genes develop neurofibromas and other tumors.

Defective mismatch repair (MMR) in humans causes hereditary nonpolyposis colorectal cancer. This genetic predisposition to colon cancer is linked to heterozygous familial mutations, and loss-of-heterozygosity is necessary for tumor development. In contrast, the rare cases with biallelic MMR mutations are juvenile patients with brain tumors, skin neurofibromas, and café-au-lait spots, resembling the neurofibromatosis syndrome.

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos.

S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos.

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches.

Lack of DNA mismatch repair protein MSH6 in the rat results in hereditary non-polyposis colorectal cancer-like tumorigenesis.

To understand genetic instability in relation to tumorigenesis, experimental animal models have proven very useful. The DNA mismatch repair (MMR) machinery safeguards genomic integrity by repairing mismatches, insertion or deletion loops and responding to genotoxic agents. Here, we describe the functional characterization of a novel rat mutant model in which the MMR gene Msh6 has been genetically inactivated by N-ethyl-N-nitrosourea-driven target-selected mutagenesis.

From DNA end chemistry to cell-cycle response: the importance of structure, even when it's broken.

In this issue of Molecular Cell, Barlow et al. (2008) show that not all DNA double-strand breaks are processed equally and that the chemical nature of DNA ends guides different paths to DNA repair.

Functional genomic analysis reveals cross-talk between peroxisome proliferator-activated receptor gamma and calcium signaling in human colorectal cancer cells.

Activation of PPARgamma in MOSER cells inhibits anchorage-dependent and anchorage-independent growth and invasion through Matrigel-coated transwell membranes. We carried out a longitudinal two-class microarray analysis in which mRNA abundance was measured as a function of time in cells treated with a thiazolidinedione PPARgamma agonist or vehicle. A statistical machine learning algorithm that employs an empirical Bayesian implementation of the multivariate HotellingT2 score was used to identify differentially regulated genes.

An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria.

Inborn defects in nucleotide excision DNA repair (NER) can paradoxically result in elevated cancer incidence (xeroderma pigmentosum [XP]) or segmental progeria without cancer predisposition (Cockayne syndrome [CS] and trichothiodystrophy [TTD]). We report generation of a knockin mouse model for the combined disorder XPCS with a G602D-encoding mutation in the Xpd helicase gene. XPCS mice are the most skin cancer-prone NER model to date, and we postulate an unusual NER dysfunction that is likely responsible for this susceptibility.

Gene expression profiles associated with treatment response in oligodendrogliomas.

Oligodendrogliomas are a specific subtype of brain tumor of which the majority responds favorably to chemotherapy. In this study, we made use of expression profiling to identify chemosensitive oligodendroglial tumors. Correlation of expression profiles to loss of heterozygosity on 1p and 19q, common chromosomal aberrations associated with response to treatment, identified 376, 64, and 60 differentially expressed probe sets associated with loss of 1p, 19q or 1p, and 19q, respectively.

DNA amplification method tolerant to sample degradation.

Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in an unbiased fashion remains a serious challenge for genetic diagnosis. We describe a new whole genome amplification procedure, RCA-RCA (Restriction and Circularization-Aided Rolling Circle Amplification), which retains the allelic differences among degraded amplified genomes while achieving almost complete genome coverage. RCA-RCA utilizes restriction digestion and whole genome circularization to generate genomic sequences amenable to rolling circle amplification.