Molecular Genetic Analysis of Non-Catalytic Pol IV and V Subunits

Among eukaryotes, plants have the distinction of encoding multisubunit RNA polymerases used exclusively for RNA directed DNA Methylation: RdDM) in addition to Pol I, II, and III. In Arabidopsis thaliana, Pol IV is required for the biogenesis of 24nt siRNAs whereas Pol V transcription is needed for cytosine methylation of the DNA sequences corresponding to these siRNAs. The ancestry of Pol IV and V can be traced back to Pol II, and Pol II, IV and V still utilize multiple non-catalytic subunits encoded by the same genes. Genetic analysis of non-catalytic subunits that are highly similar reveals that these subunits are not necessarily redundant. For instance, NRPB9b but not its 97% similar paralog, NRPB9a is required for RdDM. Likewise, Pol IV and Pol V-specific 7th largest subunits are very similar yet have different involvements in RdDM. In some of the non-catalytic subunit mutants of Pol IV, 24nt siRNA accumulation is not dramatically reduced, yet RNA silencing is disrupted. This contrasts with Pol IV catalytic subunit mutants in which siRNA biogenesis and RdDM are coordinately disrupted. Taken together, these results suggest that Pol IV might possess functions in RdDM that are in addition to, and separable from siRNA biogenesis. Differences in Pol V subunit composition based on the use of alternative non-catalytic subunit variants might also have functional consequences for RdDM. The evidence suggests that alternative non-catalytic subunits in Pol IV and V are likely to influence interactions with other proteins needed for RdDM

Point Mutations in Centromeric Histone Induce Post-zygotic Incompatibility and Uniparental Inheritance.

The centromeric histone 3 variant (CENH3, aka CENP-A) is essential for the segregation of sister chromatids during mitosis and meiosis. To better define CENH3 functional constraints, we complemented a null allele in Arabidopsis with a variety of mutant alleles, each inducing a single amino acid change in conserved residues of the histone fold domain. Many of these transgenic missense lines displayed wild-type growth and fertility on self-pollination, but exhibited frequent post-zygotic death and uniparental inheritance when crossed with wild-type plants. The failure of centromeres marked by these missense mutation in the histone fold domain of CENH3 reproduces the genome elimination syndromes described with chimeric CENH3 and CENH3 from diverged species. Additionally, evidence that a single point mutation is sufficient to generate a haploid inducer provide a simple one-step method for the identification of non-transgenic haploid inducers in existing mutagenized collections of crop species. As proof of the extreme simplicity of this approach to create haploid-inducing lines, we performed an in silico search for previously identified point mutations in CENH3 and identified an Arabidopsis line carrying the A86V substitution within the histone fold domain. This A87V non-transgenic line, while fully fertile on self-pollination, produced postzygotic death and uniparental haploids when crossed to wild type

Functional consequences of subunit diversity in RNA polymerases II and V

SummaryMultisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V

Catastrophic chromosomal restructuring during genome elimination in plants.

Genome instability is associated with mitotic errors and cancer. This phenomenon can lead to deleterious rearrangements, but also genetic novelty, and many questions regarding its genesis, fate and evolutionary role remain unanswered. Here, we describe extreme chromosomal restructuring during genome elimination, a process resulting from hybridization of Arabidopsis plants expressing different centromere histones H3. Shattered chromosomes are formed from the genome of the haploid inducer, consistent with genomic catastrophes affecting a single, laggard chromosome compartmentalized within a micronucleus. Analysis of breakpoint junctions implicates breaks followed by repair through non-homologous end joining (NHEJ) or stalled fork repair. Furthermore, mutation of required NHEJ factor DNA Ligase 4 results in enhanced haploid recovery. Lastly, heritability and stability of a rearranged chromosome suggest a potential for enduring genomic novelty. These findings provide a tractable, natural system towards investigating the causes and mechanisms of complex genomic rearrangements similar to those associated with several human disorders

Naturallyoccurring differences in cenh3 affect chromosome segregation in zygotic mitosis of hybrids

The point of attachment of spindle microtubules to metaphase chromosomes is known as the centromere. Plant and animal centromeres are epigenetically specified by a centromere-specific variant of Histone H3, CENH3 (a.k.a. CENP-A). Unlike canonical histones that are invariant, CENH3 proteins are accumulating substitutions at an accelerated rate. This diversification of CENH3 is a conundrum since its role as the key determinant of centromere identity remains a constant across species. Here, we ask whether naturally occurring divergence in CENH3 has functional consequences. We performed functional complementation assays on cenh3-1, a null mutation in Arabidopsis thaliana, using untagged CENH3s from increasingly distant relatives. Contrary to previous results using GFP-tagged CENH3, we find that the essential functions of CENH3 are conserved across a broad evolutionary landscape. CENH3 from a species as distant as the monocot Zea mays can functionally replace A. thaliana CENH3. Plants expressing variant CENH3s that are fertile when selfed show dramatic segregation errors when crossed to a wild-type individual. The progeny of this cross include hybrid diploids, aneuploids with novel genetic rearrangements and haploids that inherit only the genome of the wild-type parent. Importantly, it is always chromosomes from the plant expressing the divergent CENH3 that missegregate. Using chimeras, we show that it is divergence in the fast-evolving N-terminal tail of CENH3 that is causing segregation errors and genome elimination. Furthermore, we analyzed N-terminal tail sequences from plant CENH3s and discovered a modular pattern of sequence conservation. From this we hypothesize that while the essential functions of CENH3 are largely conserved, the N-terminal tail is evolving to adapt to lineage-specific centromeric constraints. Our results demonstrate that this lineage-specific evolution of CENH3 causes inviability and sterility of progeny in crosses, at the same time producing karyotypic variation. Thus, CENH3 evolution can contribute to postzygotic reproductive barriers

Analysis of cell proliferation and tissue remodelling uncovers a KLF4 activity score associated with poor prognosis in colorectal cancer

Human cancers can be classified based on gene signatures quantifying the degree of cell proliferation and tissue remodelling (PR). However, the specific factors that drive the increased tissue remodelling in tumours are not fully understood. Here we address this question using colorectal cancer as a case study. We reanalysed a reported cohort of colorectal cancer patients. The patients were stratified based on gene signatures of cell proliferation and tissue remodelling. Putative transcription factors activity was inferred using gene expression profiles and annotations of transcription factor targets as input. We demonstrate that the PR classification performs better than the currently adopted consensus molecular subtyping (CMS). Although CMS classification differentiates patients with a mesenchymal signature, it cannot distinguish the remaining patients based on survival. We demonstrate that the missing factor is cell proliferation, which is indicative of good prognosis. We also uncover a KLF4 transcription factor activity score associated with the tissue remodelling gene signature. We further show that the KLF4 activity score is significantly higher in colorectal tumours with predicted infiltration of cells from the myeloid lineage. The KLF4 activity score is associated with tissue remodelling, myeloid cell infiltration and poor prognosis in colorectal cancer

Cover to Volume 3

The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth

Light smoking at base-line predicts a higher mortality risk to women than to men; evidence from a cohort with long follow-up

BACKGROUND: There is conflicting evidence as to whether smoking is more harmful to women than to men. The UK Cotton Workers’ Cohort was recruited in the 1960s and contained a high proportion of men and women smokers who were well matched in terms of age, job and length of time in job. The cohort has been followed up for 42 years. METHODS: Mortality in the cohort was analysed using an individual relative survival method and Cox regression. Whether smoking, ascertained at baseline in the 1960s, was more hazardous to women than to men was examined by estimating the relative risk ratio women to men, smokers to never smoked, for light (1–14), medium (15–24), heavy (25+ cigarettes per day) and former smoking. RESULTS: For all-cause mortality relative risk ratios were 1.35 for light smoking at baseline (95% CI 1.07-1.70), 1.15 for medium smoking (95% CI 0.89-1.49) and 1.00 for heavy smoking (95% CI 0.63-1.61). Relative risk ratios for light smoking at baseline for circulatory system disease was 1.42 (95% CI 1.01 to 1.98) and for respiratory disease was 1.89 (95% CI 0.99 to 3.63). Heights of participants provided no explanation for the gender difference. CONCLUSIONS: Light smoking at baseline was shown to be significantly more hazardous to women than to men but the effect decreased as consumption increased indicating a dose response relationship. Heavy smoking was equally hazardous to both genders. This result may help explain the conflicting evidence seen elsewhere. However gender differences in smoking cessation may provide an alternative explanation

Review: ‘Gimme five’: future challenges in multiple sclerosis. ECTRIMS Lecture 2009

This article is based on the ECTRIMS lecture given at the 25th ECTRIMS meeting which was held in Düsseldorf, Germany, from 9 to 12 September 2009. Five challenges have been identified: (1) safeguarding the principles of medical ethics; (2) optimizing the risk/benefit ratio; (3) bridging the gap between multiple sclerosis and experimental autoimmune encephalitis; (4) promoting neuroprotection and repair; and (5) tailoring multiple sclerosis therapy to the individual patient. Each of these challenges will be discussed and placed in the context of current research into the pathogenesis and treatment of multiple sclerosis

Longitudinal residual strain and stress-strain relationship in rat small intestine

BACKGROUND: To obtain a more detailed description of the stress-free state of the intestinal wall, longitudinal residual strain measurements are needed. Furthermore, data on longitudinal stress-strain relations in visceral organs are scarce. The present study aims to investigate the longitudinal residual strain and the longitudinal stress-strain relationship in the rat small intestine. METHODS: The longitudinal zero-stress state was obtained by cutting tissue strips parallel to the longitudinal axis of the intestine. The longitudinal residual stress was characterized by a bending angle (unit: degrees per unit length and positive when bending outwards). Residual strain was computed from the change in dimensions between the zero-stress state and the no-load state. Longitudinal stresses and strains were computed from stretch experiments in the distal ileum at luminal pressures ranging from 0–4 cmH(2)O. RESULTS: Large morphometric variations were found between the duodenum and ileum with the largest wall thickness and wall area in the duodenum and the largest inner circumference and luminal area in the distal ileum (p < 0.001). The bending angle did not differ between the duodenum and ileum (p > 0.5). The longitudinal residual strain was tensile at the serosal surface and compressive at the mucosal surface. Hence, the neutral axis was approximately in the mid-wall. The longitudinal residual strain and the bending angle was not uniform around the intestinal circumference and had the highest values on the mesenteric sides (p < 0.001). The stress-strain curves fitted well to the mono-exponential function with determination coefficients above 0.96. The α constant increased with the pressure, indicating the intestinal wall became stiffer in longitudinal direction when pressurized. CONCLUSION: Large longitudinal residual strains reside in the small intestine and showed circumferential variation. This indicates that the tissue is not uniform and cannot be treated as a homogenous material. The longitudinal stiffness of the intestinal wall increased with luminal pressure. Longitudinal residual strains must be taken into account in studies of gastrointestinal biomechanical properties