Role of Topoisomerase IIβ in DNA Damage Response following IR and Etoposide

The role of topoisomerase IIβ was investigated in cell lines exposed to two DNA damaging agents, ionising radiation (IR) or etoposide, a drug which acts on topoisomerase II. The appearance and resolution of γH2AX foci in murine embryonic fibroblast cell lines, wild type and null for DNA topoisomerase IIβ, was measured after exposure to ionising radiation (IR) or etoposide. Topoisomerase II-DNA adduct levels were also measured. IR rapidly triggered phosphorylation of histone H2AX, less phosphorylation was seen in TOP2β−/− cells, but the difference was not statistically significant. IR did not produce topoisomerase II-DNA adducts above control levels. Etoposide triggered the formation of topoisomerase II-DNA adducts and the phosphorylation of histone H2AX, the γH2AX foci appeared more slowly with etoposide than with IR. Topoisomerase II-DNA complexes in WT cells but not TOP2β−/− cells increased significantly at 24 hours with the proteasome inhibitor MG132, suggesting topoisomerase IIβ adducts are removed by the proteasome

γH2AX Foci Form Preferentially in Euchromatin after Ionising-Radiation

BACKGROUND: The histone variant histone H2A.X comprises up to 25% of the H2A complement in mammalian cells. It is rapidly phosphorylated following exposure of cells to double-strand break (DSB) inducing agents such as ionising radiation. Within minutes of DSB generation, H2AX molecules are phosphorylated in large chromatin domains flanking DNA double-strand breaks (DSBs); these domains can be observed by immunofluorescence microscopy and are termed gammaH2AX foci. H2AX phosphorylation is believed to have a role mounting an efficient cellular response to DNA damage. Theoretical considerations suggest an essentially random chromosomal distribution of X-ray induced DSBs, and experimental evidence does not consistently indicate otherwise. However, we observed an apparently uneven distribution of gammaH2AX foci following X-irradiation with regions of the nucleus devoid of foci. METHODOLOGY/PRINCIPLE FINDINGS: Using immunofluorescence microscopy, we show that focal phosphorylation of histone H2AX occurs preferentially in euchromatic regions of the genome following X-irradiation. H2AX phosphorylation has also been demonstrated previously to occur at stalled replication forks induced by UV radiation or exposure to agents such as hydroxyurea. In this study, treatment of S-phase cells with hydroxyurea lead to efficient H2AX phosphorylation in both euchromatin and heterochromatin at times when these chromatin compartments were undergoing replication. This suggests a block to H2AX phosphorylation in heterochromatin that is at least partially relieved by ongoing DNA replication. CONCLUSIONS/SIGNIFICANCE: We discuss a number of possible mechanisms that could account for the observed pattern of H2AX phosphorylation. Since gammaH2AX is regarded as forming a platform for the recruitment or retention of other DNA repair and signaling molecules, these findings imply that the processing of DSBs in heterochromatin differs from that in euchromatic regions. The differential responses of heterochromatic and euchromatic compartments of the genome to DSBs will have implications for understanding the processes of DNA repair in relation to nuclear and chromatin organization

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons

Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2β have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous—but not individual—depletion of Tra2α and Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α–Tra2β control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability

Mutations in topoisomerase IIβ result in a B cell immunodeficiency.

B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization

TOP2B Is Required to Maintain the Adrenergic Neural Phenotype and for ATRA-Induced Differentiation of SH-SY5Y Neuroblastoma Cells.

The neuroblastoma cell line SH-SY5Y is widely used to study retinoic acid (RA)-induced gene expression and differentiation and as a tool to study neurodegenerative disorders. SH-SY5Y cells predominantly exhibit adrenergic neuronal properties, but they can also exist in an epigenetically interconvertible alternative state with more mesenchymal characteristics; as a result, these cells can be used to study gene regulation circuitry controlling neuroblastoma phenotype. Using a combination of pharmacological inhibition and targeted gene inactivation, we have probed the requirement for DNA topoisomerase IIB (TOP2B) in RA-induced gene expression and differentiation and in the balance between adrenergic neuronal versus mesenchymal transcription programmes. We found that expression of many, but not all genes that are rapidly induced by ATRA in SH-SY5Y cells was significantly reduced in the TOP2B null cells; these genes include BCL2, CYP26A1, CRABP2, and NTRK2. Comparing gene expression profiles in wild-type versus TOP2B null cells, we found that long genes and genes expressed at a high level in WT SH-SY5Y cells were disproportionately dependent on TOP2B. Notably, TOP2B null SH-SY5Y cells upregulated mesenchymal markers vimentin (VIM) and fibronectin (FN1) and components of the NOTCH signalling pathway. Enrichment analysis and comparison with the transcription profiles of other neuroblastoma-derived cell lines supported the conclusion that TOP2B is required to fully maintain the adrenergic neural-like transcriptional signature of SH-SY5Y cells and to suppress the alternative mesenchymal epithelial-like epigenetic state

Bedform migration in a mixed sand and cohesive clay intertidal environment and implications for bed material transport predictions

Many coastal and estuarine environments are dominated by mixtures of non-cohesive sand and cohesive mud. The migration rate of bedforms, such as ripples and dunes, in these environments is important in determining bed material transport rates to inform and assess numerical models of sediment transport and geomorphology. However, these models tend to ignore parameters describing the physical and biological cohesion (resulting from clay and extracellular polymeric substances, EPS) in natural mixed sediment, largely because of a scarcity of relevant laboratory and field data. To address this gap in knowledge, data were collected on intertidal flats over a spring-neap cycle to determine the bed material transport rates of bedforms in biologically-active mixed sand-mud. Bed cohesive composition changed from below 2 vol% up to 5.4 vol% cohesive clay, as the tide progressed from spring towards neap. The amount of EPS in the bed sediment was found to vary linearly with the clay content. Using multiple linear regression, the transport rate was found to depend on the Shields stress parameter and the bed cohesive clay content. The transport rates decreased with increasing cohesive clay and EPS content, when these contents were below 2.8 vol% and 0.05 wt%, respectively. Above these limits, bedform migration and bed material transport was not detectable by the instruments in the study area. These limits are consistent with recently conducted sand-clay and sand-EPS laboratory experiments on bedform development. This work has important implications for the circumstances under which existing sand-only bedform migration transport formulae may be applied in a mixed sand-clay environment, particularly as 2.8 vol% cohesive clay is well within the commonly adopted definition of “clean sand”

Anastrozole versus tamoxifen for the prevention of locoregional and contralateral breast cancer in postmenopausal women with locally excised ductal carcinoma in situ (IBIS-II DCIS): a double-blind, randomised controlled trial

Background Third-generation aromatase inhibitors are more effective than tamoxifen for preventing recurrence in postmenopausal women with hormone-receptor-positive invasive breast cancer. However, it is not known whether anastrozole is more effective than tamoxifen for women with hormone-receptor-positive ductal carcinoma in situ (DCIS). Here, we compare the efficacy of anastrozole with that of tamoxifen in postmenopausal women with hormone-receptor-positive DCIS. Methods In a double-blind, multicentre, randomised placebo-controlled trial, we recruited women who had been diagnosed with locally excised, hormone-receptor-positive DCIS. Eligible women were randomly assigned in a 1:1 ratio by central computer allocation to receive 1 mg oral anastrozole or 20 mg oral tamoxifen every day for 5 years. Randomisation was stratified by major centre or hub and was done in blocks (six, eight, or ten). All trial personnel, participants, and clinicians were masked to treatment allocation and only the trial statistician had access to treatment allocation. The primary endpoint was all recurrence, including recurrent DCIS and new contralateral tumours. All analyses were done on a modified intention-to-treat basis (in all women who were randomised and did not revoke consent for their data to be included) and proportional hazard models were used to compute hazard ratios and corresponding confidence intervals. This trial is registered at the ISRCTN registry, number ISRCTN37546358. Results Between March 3, 2003, and Feb 8, 2012, we enrolled 2980 postmenopausal women from 236 centres in 14 countries and randomly assigned them to receive anastrozole (1449 analysed) or tamoxifen (1489 analysed). Median follow-up was 7·2 years (IQR 5·6–8·9), and 144 breast cancer recurrences were recorded. We noted no statistically significant difference in overall recurrence (67 recurrences for anastrozole vs 77 for tamoxifen; HR 0·89 [95% CI 0·64–1·23]). The non-inferiority of anastrozole was established (upper 95% CI <1·25), but its superiority to tamoxifen was not (p=0·49). A total of 69 deaths were recorded (33 for anastrozole vs 36 for tamoxifen; HR 0·93 [95% CI 0·58–1·50], p=0·78), and no specific cause was more common in one group than the other. The number of women reporting any adverse event was similar between anastrozole (1323 women, 91%) and tamoxifen (1379 women, 93%); the side-effect profiles of the two drugs differed, with more fractures, musculoskeletal events, hypercholesterolaemia, and strokes with anastrozole and more muscle spasm, gynaecological cancers and symptoms, vasomotor symptoms, and deep vein thromboses with tamoxifen. Conclusions No clear efficacy differences were seen between the two treatments. Anastrozole offers another treatment option for postmenopausal women with hormone-receptor-positive DCIS, which may be be more appropriate for some women with contraindications for tamoxifen. Longer follow-up will be necessary to fully evaluate treatment differences

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant