Transition from Transient DNA Rereplication to Inherited Gene Amplification Following Prolonged Environmental Stress.

Cells require the ability to adapt to changing environmental conditions, however, it is unclear how these changes elicit stable permanent changes in genomes. We demonstrate that, in response to environmental metal exposure, the metallothionein (MT) locus undergoes DNA rereplication generating transient site-specific gene amplifications (TSSGs). Chronic metal exposure allows transition from MT TSSG to inherited MT gene amplification through homologous recombination within and outside of the MT locus. DNA rereplication of the MT locus is suppressed by H3K27me3 and EZH2. Long-term ablation of EZH2 activity eventually leads to integration and inheritance of MT gene amplifications without the selective pressure of metal exposure. The rereplication and inheritance of MT gene amplification is an evolutionarily conserved response to environmental metal from yeast to human. Our results describe a new paradigm for adaptation to environmental stress where targeted, transient DNA rereplication precedes stable inherited gene amplification.

Isolation and analysis of rereplicated DNA by Rerep-Seq.

Changes in gene copy number contribute to genomic instability, the onset and progression of cancer, developmental abnormalities and adaptive potential. The origins of gene amplifications have remained elusive; however, DNA rereplication has been implicated as a source of gene amplifications. The inability to determine which sequences are rereplicated and under what conditions have made it difficult to determine the validity of the proposed models. Here we present Rerep-Seq, a technique that selectively enriches for rereplicated DNA in preparation for analysis by DNA sequencing that can be applied to any species. We validated Rerep-Seq by simulating DNA rereplication in yeast and human cells. Using Rerep-Seq, we demonstrate that rereplication induced in Saccharomyces cerevisiae by deregulated origin licensing is non-random and defined by broad domains that span multiple replication origins and topological boundaries.

Epigenetic Modulation of the Tumor Immune Microenvironment to Potentiate Immune Checkpoint Blockade Therapy.

Response rates to immune checkpoint blockade (ICB) in KRAS-mutant lung adenocarcinoma remain poor. In this issue of Cancer Discovery, Li and colleagues report an in vivo CRISPR screen of epigenetic regulators of the tumor immune microenvironment that uncovers Asf1a as a tumor-intrinsic suppressor of ICB through suppression of GM-CSF expression.See related article by Li et al., p. 270.

The conserved and divergent roles of Prdm3 and Prdm16 in zebrafish and mouse craniofacial development.

The formation of the craniofacial skeleton is a highly dynamic process that requires proper orchestration of various cellular processes in cranial neural crest cell (cNCC) development, including cell migration, proliferation, differentiation, polarity and cell death. Alterations that occur during cNCC development result in congenital birth defects and craniofacial abnormalities such as cleft lip with or without cleft palate. While the gene regulatory networks facilitating neural crest development have been extensively studied, the epigenetic mechanisms by which these pathways are activated or repressed in a temporal and spatially regulated manner remain largely unknown. Chromatin modifiers can precisely modify gene expression through a variety of mechanisms including histone modifications such as methylation. Here, we investigated the role of two members of the PRDM (Positive regulatory domain) histone methyltransferase family, Prdm3 and Prdm16 in craniofacial development using genetic models in zebrafish and mice. Loss of prdm3 or prdm16 in zebrafish causes craniofacial defects including hypoplasia of the craniofacial cartilage elements, undefined posterior ceratobranchials, and decreased mineralization of the parasphenoid. In mice, while conditional loss of Prdm3 in the early embryo proper causes mid-gestation lethality, loss of Prdm16 caused craniofacial defects including anterior mandibular hypoplasia, clefting in the secondary palate and severe middle ear defects. In zebrafish, prdm3 and prdm16 compensate for each other as well as a third Prdm family member, prdm1a. Combinatorial loss of prdm1a, prdm3, and prdm16 alleles results in severe hypoplasia of the anterior cartilage elements, abnormal formation of the jaw joint, complete loss of the posterior ceratobranchials, and clefting of the ethmoid plate. We further determined that loss of prdm3 and prdm16 reduces methylation of histone 3 lysine 9 (repression) and histone 3 lysine 4 (activation) in zebrafish. In mice, loss of Prdm16 significantly decreased histone 3 lysine 9 methylation in the palatal shelves but surprisingly did not change histone 3 lysine 4 methylation. Taken together, Prdm3 and Prdm16 play an important role in craniofacial development by maintaining temporal and spatial regulation of gene regulatory networks necessary for proper cNCC development and these functions are both conserved and divergent across vertebrates.

Preclinical evaluation of (99m)Tc labeled chondroitin sulfate for monitoring of cartilage degeneration in osteoarthritis.

PURPOSE: In previous in-vitro and ex-vivo studies we proved the specific uptake of (99m)Tc radiolabeled chondroitin sulfate (CS) in human articular cartilage. As a logical next step for the clinical use for imaging osteoarthritis we investigated in-vivo uptake of (99m)TcCS in dogs. PROCEDURES: The radiolabeling of CS Condrosulf (IBSA, Lugano, Switzerland) was performed using 25mg of CS and 20-40MBq/kg body weight of (99m)Tc by means of the tin method. In-vivo uptake of (99m)TcCS was evaluated in dogs (n=12, castrated males, 4-9years, with 15-51kg body weight). 6 healthy dogs served as controls and 6 with clinical and radiological signs of osteoarthritis in the carpal, elbow, and tarsal joint were examined. The tracer was i.v. injected into the external cephalic vein. The uptake was monitored after 2, 4, 6 and 24h in healthy and osteoarthritic dogs using a planar gamma camera by regional planar or whole body ventral and dorsal acquisition. For whole body scintigraphy animals were under general anesthesia, for planar under sedation only. RESULTS: In healthy control dogs we did not detect any specific uptake of (99m)TcCS in the cartilage. In contrast, in the diseased dogs suffering from osteoarthritis a significant, specific, persistent uptake between 4 and 6h in tarsal, carpal and cubital joints was documented. Median target (joint) to background (mid antebrachium) ratio (T/B) in the OA joints after 4, 6, and 24h was significantly higher than in healthy controls. Target to background ratio using soft tissue as a background (T/S) a similar significantly higher than in healthy controls. In all osteoarthritic joints we found a significant positive correlation (r=0.8, n=20) between grade of disease (I-III) and T/B. When matching radiographic (X ray) changes in osteoarthritic joints (grade II and III) we found also a maximal uptake of (99m)TcCS at the specific anatomical site of highest cartilage degeneration. None of the dogs experienced any side effects. CONCLUSION: These results suggest that (99m)TcCS might become a promising diagnostic tool for imaging osteoarthritis. More extensive and detailed examinations are required, however, before extending this methodology for application in humans.

The anaphase promoting complex regulates yeast lifespan and rDNA stability by targeting Fob1 for degradation.

Genomic stability, stress response, and nutrient signaling all play critical, evolutionarily conserved roles in lifespan determination. However, the molecular mechanisms coordinating these processes with longevity remain unresolved. Here we investigate the involvement of the yeast anaphase promoting complex (APC) in longevity. The APC governs passage through M and G1 via ubiquitin-dependent targeting of substrate proteins and is associated with cancer and premature aging when defective. Our two-hybrid screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as prey. Fob1 is unstable specifically in G1, cycles throughout the cell cycle in a manner similar to Clb2 (an APC target), and is stabilized in APC (apc5(CA)) and proteasome (rpn10) mutants. Deletion of FOB1 increased replicative lifespan (RLS) in wild type (WT), apc5(CA), and apc10 cells, and suppressed apc5(CA) cell cycle progression and rDNA recombination defects. Alternatively, increased FOB1 expression decreased RLS in WT cells, but did not reduce the already short apc5(CA) RLS, suggesting an epistatic interaction between apc5(CA) and fob1. Mutation to a putative L-Box (Fob1(E420V)), a Destruction Box-like motif, abolished Fob1 modifications, stabilized the protein, and increased rDNA recombination. Our work provides a mechanistic role played by the APC to promote replicative longevity and genomic stability in yeast.

Uptake studies with chondrotropic 99mTc-chondroitin sulfate in articular cartilage. Implications for imaging osteoarthritis in the knee.

Chondroitin sulfate (CS) is an endogenous component of extracellular matrix in the cartilage and can be valuable for imaging of cartilage degeneration after radiolabeling. Data monitoring the uptake of (99m)TcCS by human cartilage are rare. Radiolabeling was performed by (99m)TcO4(-)/tin method at pH5.0 in 0.5M sodium acetate. For uptake studies human articular cartilage (n = 4, 65-79a) derived from individuals undergoing knee replacement (pieces of 3-5mg wet weight), or frozen tissue sections (5 µ) for autoradiography (10 µCi) were used. The uptake was monitored from 10 min up to 96 h to achieve saturation. As the commercially available drug Condrosulf (IBSA, Lugano) contains Mg-stearate (0.25%) as additive (to improve its gastrointestinal resorption), we investigated the uptake ± additive. The washout of the tracer was examined by tissue incubation after uptake experiments (3h and 24h) with PBS-buffer for 10 min to 3h. Using human articular cartilage the maximal uptake of (99m)TcCS (specific activity of 4.1-6.1 Ci/mmol) was continuously increasing with time amounting to a maximum of 53.2% ± 3.2% with additive, versus 39.4% ± 2.3%, without additive, at saturation. Additive increased the resorption of the drug and consecutively its uptake. The washout of the tracer from cartilage after 3h uptake amounted to 1.5% ± 0.2% with additive, versus 2.6% ± 0.5%, without. After 24h washout was lower amounting to 1.1% ± 0.1% versus 1.75% ± 0.15%, respectively. Autoradiography revealed also a continuous increase in uptake of (99m)TcCS with time. After 10 min of incubation the uptake increase was proportional to the incubation time, reaching the maximum at 48-72 h. Enhanced uptake at the surface (superficial zone) as compared to the subchondral part (deep zone) of slices, was observed. The non-specific uptake in the presence of 50-fold excess of cold CS was time-dependent up to a maximum of 15% (tissue) and 10% (autoradiography), at saturation. The uptake studies indicate, that (99m)TcCS accumulates in articular cartilage and prove its chondrotropic effects.

Antagonistic Gcn5-Hda1 interactions revealed by mutations to the Anaphase Promoting Complex in yeast.

BACKGROUND: Histone post-translational modifications are critical for gene expression and cell viability. A broad spectrum of histone lysine residues have been identified in yeast that are targeted by a variety of modifying enzymes. However, the regulation and interaction of these enzymes remains relatively uncharacterized. Previously we demonstrated that deletion of either the histone acetyltransferase (HAT) GCN5 or the histone deacetylase (HDAC) HDA1 exacerbated the temperature sensitive (ts) mutant phenotype of the Anaphase Promoting Complex (APC) apc5CA allele. Here, the apc5CA mutant background is used to study a previously uncharacterized functional antagonistic genetic interaction between Gcn5 and Hda1 that is not detected in APC5 cells. RESULTS: Using Northerns, Westerns, reverse transcriptase PCR (rtPCR), chromatin immunoprecipitation (ChIP), and mutant phenotype suppression analysis, we observed that Hda1 and Gcn5 appear to compete for recruitment to promoters. We observed that the presence of Hda1 can partially occlude the binding of Gcn5 to the same promoter. Occlusion of Gcn5 recruitment to these promoters involved Hda1 and Tup1. Using sequential ChIP we show that Hda1 and Tup1 likely form complexes at these promoters, and that complex formation can be increased by deleting GCN5. CONCLUSIONS: Our data suggests large Gcn5 and Hda1 containing complexes may compete for space on promoters that utilize the Ssn6/Tup1 repressor complex. We predict that in apc5CA cells the accumulation of an APC target may compensate for the loss of both GCN5 and HDA1.

Optimal 99mTc radiolabeling and uptake of glucosamine sulfate by cartilage. A potential tracer for scintigraphic detection of osteoarthritis.

Glucosamine sulfate (GS) is used in treatment of human osteoarthritis, but no data for(99m)TcGS scintigraphy are available. Radiolabeling of GS was performed using the (99m)TcO(4)(-)/tin method. We applied two procedures for separation of free (99m)Tc using PD10 and G10 columns. In each eluted fraction, GS content was estimated by the Elson/Morgan method. For optimal radiolabeling, we varied the amount of GS, tin, (99m)Tc activity, and pH. For uptake age matched human rib cartilage (males, 78 and 63 years old) and 5-10 µCi/well of (99m)TcGS were used. Uptake was monitored up to 72 h. Also, washout of the tracer 3 h and 24 h after uptake was investigated. At pH 2, using PD10 column, the uptake of (99m)TcGS amounted to 100.8 ± 2.9%, n = 6 at saturation time of 72 h. Uptake was age-dependent; at pH 5, it amounted to 99.8% as compared to 66.1% at 78 vs 63 years old. When the amount of tin was varied at pH 2, the tracer uptake amounted to 21.37% (1 mg) vs 45.99% (2.5 mg) at saturation. At pH 7, the amount of needed tin was much lower and amounted to 42.50 ± 2.50% using 0.1 mg vs 25.11 ± 1.90% using 0.05 mg. Although the uptake at pH 7 (0.1 mg tin) is comparable with that at pH 2 (2.5 mg tin), the washout of the tracer amounted only to 4.10 ± 1.25% and 2.05 ± 0.65% after 3 h and 24 h, respectively. During degeneration of cartilage, incorporation of (99m)TcGS is high and could therefore be a promising tracer not only to target osteoarthritis but also to monitor the effects of therapy.

Uptake of 99mTc-labeled chondroitin sulfate by chondrocytes and cartilage: a promising agent for imaging of cartilage degeneration?

Chondroitin sulfate (CS) is used in the treatment of human osteoarthritis as a slow-acting symptomatic drug. For this reason, we performed uptake studies with (99m)TcCS using different chondrocyte cultures, as well as cartilage tissue in vitro. For uptake studies, adherent monolayer cultures of human chondrocytes (2.7 x 10(4) cells/well) and (99m)TcCS (1 microCi) were used. In parallel, we also performed uptake studies with cell suspensions of human chondrocytes at 1 x 10(6) cells/well incubated with (99m)TcCS (5 microCi) under identical conditions. Uptake was studied also in cartilage tissue samples and frozen tissue sections for autoradiography. The uptake was monitored for 10-240 min, every 10-30 min for cell cultures and for cartilage tissue up to 72 h. As the commercially available drug Condrosulf (IBSA, Lugano, Switzerland) contains magnesium (Mg) stearate as additive, we investigated the uptake with and without this additive. The washout of the tracer was assessed after the uptake experiments with PBS buffer for different time intervals (10 min-3 h). Tracer uptake in monolayer+/-additives with low number of cells was low. With the use of chondrocytes in culture suspensions with higher number of cells, a higher uptake of 5.9+/-0.65% and 1.0+/-0.1% (n=6) was found, with and without additive, respectively. The saturation was achieved after 100 min. With the use of human rib cartilage, the uptake of (99m)TcCS was continuously increasing with time and was very high with additive amounting to 101.8+/-5.2% vs. 53.0+/-8.3% (n=6) without after 72 h and showing delayed saturation up to 30 h. Thus, not only the resorption of the drug is enhanced by Mg-stearate, but also the uptake. The washout of the tracer from cartilage after 3 h of uptake amounted to 3.75+/-1.5% with additive vs. 13.1+/-2.1% without. After 24 h, washout was lower amounting to 1.75+/-0.15% vs. 3.25+/-0.25%, respectively. The autoradiographic studies paralleled the results of in vitro cartilage tissue uptake. These data show that (99m)TcCS accumulates in cartilage tissue, either by acting as a substrate for proteoglycan synthesis or by adsorption to cartilage. (99m)TcCS could therefore be a possible agent to target and radioimage osteoarthritis.