Modified chromosome structure caused by phosphomimetic H2A modulates the DNA damage response by increasing chromatin mobility in yeast.

In budding yeast and mammals, double-strand breaks (DSBs) trigger global chromatin mobility together with rapid phosphorylation of histone H2A over an extensive region of the chromatin. To assess the role of H2A phosphorylation in this response to DNA damage, we have constructed strains where H2A has been mutated to the phosphomimetic H2A-S129E. We show that mimicking H2A phosphorylation leads to an increase in global chromatin mobility in the absence of DNA damage. The intrinsic chromatin mobility of H2A-S129E is not due to downstream checkpoint activation, histone degradation or kinetochore anchoring. Rather, the increased intrachromosomal distances observed in the H2A-S129E mutant are consistent with chromatin structural changes. Strikingly, in this context the Rad9-dependent checkpoint becomes dispensable. Moreover, increased chromatin dynamics in the H2A-S129E mutant correlates with improved DSB repair by non-homologous end joining and a sharp decrease in interchromosomal translocation rate. We propose that changes in chromosomal conformation due to H2A phosphorylation are sufficient to modulate the DNA damage response and maintain genome integrity.This article has an associated First Person interview with the first author of the paper.

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

A pathway of targeted autophagy is induced by DNA damage in budding yeast.

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

The budding yeast Polo-like kinase localizes to distinct populations at centrosomes during mitosis.

The budding yeast Polo-like kinase Cdc5 is a key regulator of many mitotic events. Cdc5 coordinates its functions spatially and temporally by changing its localization during the cell cycle: Cdc5 is imported into the nucleus in G2 phase and released to the cytoplasm in anaphase, where it accumulates at the bud neck. Cdc5 also localizes to the spindle pole bodies (SPBs) from S phase until the end of mitosis. Whether Cdc5 changes its SPB population during the cell cycle is not known. We find that Cdc5 localizes to distinct SPB subpopulations, depending on the mitotic stage. Cdc5 localizes to the nuclear side of the SPBs during metaphase and early anaphase and to the cytoplasmic surface of the SPBs during late anaphase. Cdc14 is necessary to relocalize Cdc5 from the nuclear SPB plaque. Accumulation of Cdc5 at the daughter SPB in late anaphase is controlled by Bfa1. We also show that Cdc5 and Bfa1 are found in spatially distinct locations at the SPBs during G2/M arrest after DNA damage. Collectively our data reveal that Cdc5 is a dynamic component of the SPBs during mitosis and provide new insight into its regulation during both late mitotic events and DNA damage-induced G2/M arrest.

AVX-470, an Orally Delivered Anti-Tumour Necrosis Factor Antibody for Treatment of Active Ulcerative Colitis: Results of a First-in-Human Trial.

BACKGROUND AND AIMS: AVX-470 is an oral, polyclonal bovine-derived anti-tumour necrosis factor (TNF) antibody in development for treatment of inflammatory bowel disease (IBD). AVX-470 neutralizes TNF locally in the gastrointestinal tract, minimizing systemic exposure. This was a double-blind, placebo-controlled, first-in-human trial designed to assess the safety, pharmacokinetics, immunogenicity and preliminary efficacy of 4 weeks of AVX-470 in patients with active ulcerative colitis (UC). METHODS: Thirty-seven patients with active UC were randomized and 36 received AVX-470 (0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Endoscopic activity was assessed by colonoscopy pre- and post-treatment. The primary endpoint was safety. Secondary endpoints included pharmacokinetics and immunogenicity. Clinical and endoscopic response and remission were assessed as exploratory endpoints. RESULTS: Thirty-three (92%) patients completed treatment and follow-up. The incidence of adverse events was similar across treatment groups and no allergic reactions or opportunistic infections were reported. AVX-470 therapy did not induce human anti-bovine antibodies (HABA). Bovine immunoglobulin (Ig) with TNF binding capacity was detected in stool, while bovine Ig levels in serum were low. Across all AVX-470 doses, 25.9% of patients achieved clinical response compared with 11.1% on placebo, with greatest improvements in the 3.5g/day group associated with proximal colon endoscopic improvement and reductions in serum CRP and IL-6. CONCLUSIONS: AVX-470 was safe and well tolerated in this first-in-human trial in UC, with efficacy trends for clinical, endoscopic and biomarker endpoints in the highest dose group (3.5g/day). Results suggest benefit of an orally delivered locally active agent in moderate to severe UC. CLINICAL TRIAL REGISTRATION NUMBER: This trial was registered with Clinicaltrials.gov as study NCT01759056 and with EudraCT as study 2012-004859-27.

Effects of AVX-470, an Oral, Locally Acting Anti-Tumour Necrosis Factor Antibody, on Tissue Biomarkers in Patients with Active Ulcerative Colitis.

BACKGROUND AND AIMS: AVX-470 is an orally administered, bovine-derived, anti-tumour necrosis factor (TNF) antibody with local activity in the gastrointestinal tract. In the first-in-human clinical trial of AVX-470 in active ulcerative colitis, we evaluated inflammatory biomarkers in colon tissue as measures of disease activity and early response to treatment. METHODS: Thirty-six patients received active drug (AVX-470 at 0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Colon biopsy samples were collected from 5 regions of colon at baseline and week 4. Tissue inflammatory biomarkers were evaluated by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), epithelial cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and bovine immunoglobulin by immunohistochemistry and mass spectrometry. Endoscopic activity (Ulcerative Colitis Endoscopic Index of Severity [UCEIS]) at colonoscopy was assessed in each colonic region by a central reader. RESULTS: Bovine immunoglobulin was observed in mucosal tissue before and after dosing in lamina propria and submucosal layers of biopsy tissue. Baseline levels of TNF, myeloperoxidase (MPO), CD68 and interleukin (IL)-1ß and, to a lesser extent, IL-6 mRNA were 2- to 3-fold higher in distal vs proximal colon tissue, corresponding to the 2- to 3-fold differences in baseline severities of endoscopic scores. Reductions of >10-fold in TNF and, to lesser extents, in MPO and epithelial cell apoptosis were observed in proximal and distal colon biopsies after 4 weeks of AVX-470 3.5g/day treatment. Reductions in TNF scores were correlated with changes in MPO and CD3 immunohistochemistry scores. CONCLUSIONS: These results are consistent with anti-TNF activity of orally administered AVX-470 in colon mucosal tissue in ulcerative colitis patients and demonstrate the utility of tissue biomarkers in assessing disease and treatment response in early clinical studies. CLINICAL TRIAL REGISTRATION NUMBER: This trial was registered with Clinicaltrials.gov as study NCT01759056 and with EudraCT as study 2012-004859-27.

AVX-470: a novel oral anti-TNF antibody with therapeutic potential in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, which is currently treated with injected monoclonal antibodies specific for tumor necrosis factor (TNF). We developed and characterized AVX-470, a novel polyclonal antibody specific for human TNF. We evaluated the oral activity of AVX-470m, a surrogate antibody specific for murine TNF, in several well-accepted mouse models of IBD. METHODS: AVX-470 and AVX-470m were isolated from the colostrum of dairy cows that had been immunized with TNF. The potency, specificity, and affinity of both AVX-470 and AVX-470m were evaluated in vitro and compared with infliximab. AVX-470m was orally administered to mice either before or after induction of colitis, and activity was measured by endoscopy, histopathology, immunohistochemistry, and quantitative measurement of messenger RNA levels. Colitis was induced using either 2,4,6-trinitrobenzene sulfonate or dextran sodium sulfate. RESULTS: AVX-470 and AVX-470m were shown to be functionally comparable in vitro. Moreover, the specificity, neutralizing potency, and affinity of AVX-470 were comparable with infliximab. Orally administered AVX-470m effectively reduced disease severity in several mouse models of IBD. Activity was comparable with that of oral prednisolone or parenteral etanercept. The antibody penetrated the colonic mucosa and inhibited TNF-driven mucosal inflammation with minimal systemic exposure. CONCLUSIONS: AVX-470 is a novel polyclonal anti-TNF antibody with an in vitro activity profile comparable to that of infliximab. Oral administration of a surrogate antibody specific for mouse TNF is effective in treating mouse models of IBD, delivering the anti-TNF to the site of inflammation with minimal systemic exposure.