Howard A. Chansky

An ERG (ets-related gene)-associated histone methyltransferase interacts with histone deacetylases 1/2 and transcription co-repressors mSin3A/B.

Covalent modifications of histone tails play important roles in gene transcription and silencing. We recently identified an ERG ( ets -related gene)-associated protein with a SET (suppressor of variegation, enhancer of zest and trithorax) domain (ESET) that was found to have the activity of a histone H3-specific methyltransferase. In the present study, we investigated the interaction of ESET with other chromatin remodelling factors. We show that ESET histone methyltransferase associates with histone deacetylase 1 (HDAC1) and HDAC2, and that ESET also interacts with the transcription co-repressors mSin3A and mSin3B. Deletion analysis of ESET reveals that an N-terminal region containing a tudor domain is responsible for interaction with mSin3A/B and association with HDAC1/2, and that truncation of ESET enhances its binding to mSin3. When bound to a promoter, ESET represses the transcription of a downstream luciferase reporter gene. This repression by ESET is independent of its histone methyltransferase activity, but correlates with its binding to the mSin3 co-repressors. In addition, the repression can be partially reversed by treatment with the HDAC inhibitor trichostatin A. Taken together, these data suggest that ESET histone methyltransferase can form a large, multi-protein complex(es) with mSin3A/B co-repressors and HDAC1/2 that participates in multiple pathways of transcriptional repression.

Preoperative Supraphysiological Testosterone in Older Men Undergoing Knee Replacement Surgery

OBJECTIVES: Older patients undergoing knee replacement surgery can recover more slowly than younger patients and require extended rehabilitation. Because administration of supraphysiological testosterone (T) dramatically increases strength, we hypothesized that preoperative T therapy would improve functional recovery and reduce hospital stay in older men undergoing knee replacement surgery.

Articular cartilage adjacent to experimental defects is subject to atypical strains.

We tested the hypothesis that articular cartilage adjacent to experimental osteochondral defects is not subject to unusual strains under load. A 2.5-mm drill hole was made in the medial femoral condyle of 15 knees from 10 adult rabbits. Experimental joints were loaded with simulated quadriceps force, then frozen under load and preserved by freeze-substitution fixation. Deformation in the region of the defect was evaluated by scanning electron and light microscopy and compared with nondrilled and nonloaded control knees. To simulate blood clot, alginate was placed into some defects before loading. In loaded knees, articular cartilage at the edge of the drill hole was abnormally flattened and folded into the defect. Opposing tibial cartilage or meniscus intruded into the femoral defect beyond the cement line. Alginate did not prevent incursion of opposing cartilage. In this standard drill-hole model, the articular cartilage defect is occupied by the opposing surface when a joint is loaded. Any tissue growing or surgically implanted in the defect is subject to loading and displacement, therefore complicating attempts to characterize the healing or regenerative potential in similar drill-hole models. Deformation of cartilage at the defect edge suggests load concentration or increased compliance. Either phenomenon would contribute to subsequent degeneration of the cartilage adjacent to defects.

Preoperative opioid use is associated with early revision after total knee arthroplasty a study of male patients treated in the veterans affairs system

Background: Opioid use is endemic in the U.S. and is associated with morbidity and mortality. The impact of long-term opioid use on joint-replacement outcomes remains unknown. We tested the hypothesis that use of opioids is associated with adverse outcomes after total knee arthroplasty (TKA). Methods: We performed a retrospective analysis of patients who had had TKA within the U.S. Veterans Affairs (VA) system over a 6-year period and had been followed for 1 year postoperatively. The length of time for which an opioid had been prescribed and the morphine equivalent dose were calculated for each patient. Patients for whom opioids had been prescribed for >3 months in the year prior to the TKA were assigned to the long-term opioid group. A natural language processing-based machine-learning classifier was developed to classify revisions due to infectious and non-infectious causes on the basis of the postoperative note. Survival curves for the time to knee revision or manipulation were used to compare the long-term opioid group with the patients who did not take opioids long-term. Hazard and odds ratios for knee revision and manipulation were obtained as well. Results: Of 32,636 patients (94.4% male; mean age [and standard deviation], 64.45 ± 9.41 years) who underwent TKA, 12,772 (39.1%) were in the long-term opioid group and 734 (2.2%) had a revision within a year after the TKA. Chronic kidney disease, diabetes, and long-term opioid use were associated with revision within 1 year—with odds ratios (95% confidence intervals [CIs]) of 1.76 (1.37 to 2.22), 1.11 (0.93 to 1.31), and 1.40 (1.19 to 1.64), respectively—and were also the leading factors associated with a revision at any time after the index TKA—with odds ratios (95% CIs) of 1.61 (1.34 to 1.92), 1.21 (1.08 to 1.36), and 1.28 (1.15 to 1.43), respectively. Long-term opioid use had a hazard ratio of 1.19 (95% CI = 1.10 to 0.24) in the analysis of its relationship with knee revision, but the hazard was not significant in the analysis of its association with knee manipulation. The accuracy of the text classifier was 0.94, with the area under the receiver operating characteristic curve being 0.99. There was no association between long-term use of opioids and the specific cause for knee revision. Conclusions: Long-term opioid use prior to TKA was associated with an increased risk of knee revision during the first year after TKA among predominantly male patients treated in the VA system. Level of Evidence: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

The oncogenic TLS-ERG fusion protein exerts different effects in hematopoietic cells and fibroblasts

ABSTRACT The oncogenic TLS-ERG fusion protein is found in human myeloid leukemia and Ewings sarcoma as a result of specific chromosomal translocation. To unveil the potential mechanism(s) underlying cellular transformation, we have investigated the effects of TLS-ERG on both gene transcription and RNA splicing. Here we show that the TLS protein forms complexes with RNA polymerase II (Pol II) and the serine-arginine family of splicing factors in vivo. Deletion analysis of TLS-ERG in both mouse L-G myeloid progenitor cells and NIH 3T3 fibroblasts revealed that the RNA Pol II-interacting domain of TLS-ERG resides within the first 173 amino acids. While TLS-ERG repressed expression of the luciferase reporter gene driven by glycoprotein IX promoter in L-G cells but not in NIH 3T3 cells, the fusion protein was able to affect splicing of the E1A reporter in NIH 3T3 cells but not in L-G cells. To identify potential target genes of TLS-ERG, the fusion protein and its mutants were stably expressed in both L-G and NIH 3T3 cells through retroviral transduction. Microarray analysis of RNA samples from these cells showed that TLS-ERG activates two different sets of genes sharing little similarity in the two cell lines. Taken together, these results suggest that the oncogenic TLS-ERG fusion protein transforms hematopoietic cells and fibroblasts via different pathways.

RNA splicing mediated by YB-1 is inhibited by TLS/CHOP in human myxoid liposarcoma cells

Human myxoid liposarcoma contains a characteristic t(12;16) chromosomal translocation that results in fusion of the N‐terminal domain of the translocated in liposarcoma (TLS) protein to the C/EBP homologous protein (CHOP). TLS possesses structural motifs that suggest it may participate in RNA processing. We demonstrate that in human myxoid liposarcoma cells, wild‐type TLS binds to RNA polymerase II (Pol II) via its N‐terminal domain and to the transcription and translation factor Y‐box binding protein‐1 (YB‐1) through its C‐terminal domain. The liposarcoma fusion protein TLS/CHOP retains the ability to bind RNA Pol II but lacks the ability to recruit YB‐1 due to replacement of the C‐terminal domain of TLS by CHOP. In an in vivo splicing assay, YB‐1 promotes splicing of adenovirus E1A pre‐mRNA predominantly to the 13S isoform. The oncogenic TLS/CHOP fusion protein inhibits this splicing function of YB‐1 in a dominant negative manner. When considered in conjunction with studies on other sarcoma fusion proteins, these data suggest that aberrant RNA splicing may be a common feature of human sarcomas.

ESET histone methyltransferase is essential to hypertrophic differentiation of growth plate chondrocytes and formation of epiphyseal plates

The ESET (also called SETDB1) protein contains an N-terminal tudor domain that mediates protein-protein interactions and a C-terminal SET domain that catalyzes methylation of histone H3 at lysine 9. We report here that ESET protein is transiently upregulated in prehypertrophic chondrocytes in newborn mice. To investigate the in vivo effects of ESET on chondrocyte differentiation, we generated conditional knockout mice to specifically eliminate the catalytic SET domain of ESET protein only in mesenchymal cells. Such deletion of the ESET gene caused acceleration of chondrocyte hypertrophy in both embryos and young animals, depleting chondrocytes that are otherwise available to form epiphyseal plates for endochondral bone growth. ESET-deficient mice are thus characterized by defective long bone growth and trabecular bone formation. To understand the underlying mechanism for ESET regulation of chondrocytes, we carried out co-expression experiments and found that ESET associates with histone deacetylase 4 to bind and inhibit the activity of Runx2, a hypertrophy-promoting transcription factor. Repression of Runx2-mediated gene transactivation by ESET is dependent on its H3-K9 methyltransferase activity as well as its associated histone deacetylase activity. In addition, knockout of ESET is associated with repression of Indian hedgehog gene in pre- and early hypertrophic chondrocytes. Together, these results provide clear evidence that ESET controls hypertrophic differentiation of growth plate chondrocytes and endochondral ossification during embryogenesis and postnatal development.

Prolonged opioid use after knee arthroscopy in military veterans

BACKGROUND:Chronic postoperative pain occurs with an appreciable incidence after elective surgery. Known risk factors include perioperative pain and posttraumatic stress disorder (PTSD). Military veterans are a population at particular risk for PTSD and hence may be at increased risk for chronic pain after surgery. Our goal was to identify risk factors for chronic postoperative pain in young veterans after minor elective surgery, including the contribution of PTSD. METHODS:We reviewed the medical and pharmacy records of veterans (18–50 years old), undergoing elective knee arthroscopy from 2007 to 2010 at the Veteran’s Administration Puget Sound Health Care System. The data included demographics, ASA physical status class, comorbidities, anesthesia medications, and opioid prescriptions starting 3.5 months before surgery and ending 3.5 months after surgery. We documented the presence of PTSD based on either the patient’s problem list or the clinical notes. We used prolonged postoperative opioid prescription longer than 3 months after surgery as a surrogate for chronic postoperative pain. RESULTS:We identified 145 patients who met inclusion criteria. The median age was 39 ± 8 years old. Eighty-seven percent of the patients were men. The prevalence of PTSD was 32% (95% confidence interval, 25%–41%). PTSD was associated with increased incidence of smoking (P = 0.009) and preoperative opioid use (P = 0.0006). Preoperative opioids were prescribed in 44% (63 of 145) of the patients: in 64% (30 of 47) of patients with PTSD, compared with 34% (33 of 98) in patients without PTSD (P = .0006). Chronic postoperative pain was identified in 30% (43 of 145) of patients. The strongest independent predictor of chronic postoperative pain was an opioid prescription before surgery (odds ratio = 65.3; 95% confidence interval, 014.5–293.0). In patients older than 27.5 years who did not receive opioids before surgery, PTSD may also have been a risk factor for chronic postoperative pain. CONCLUSIONS:This single-center retrospective study suggests that the most important predictor of chronic postoperative pain is preoperative opioid use. For patients not taking opioids preoperatively, PTSD may increase the risk of prolonged postoperative opioid prescriptions and chronic postoperative pain, potentially related to patient age.

EWS/FLI1 suppresses retinoblastoma protein function and senescence in Ewing's sarcoma cells

Ewings Family Tumors (EFTs) most commonly harbor a specific t(11;22) translocation that generates the EWS/FLI1 fusion protein responsible for malignant transformation. Many potential downstream targets of EWS/FLI1 have been identified but a detailed mechanism by which the fusion protein brings about transformation remains unknown. In this report, we show that depletion of EWS/FLI1 in Ewings cell lines results in a senescence phenotype, a marked increase in expression of the G1/S regulatory proteins p27kip1 and p57kip2, and a significant decrease in cyclin D1 and CDK2. We also demonstrate for the first time, to our knowledge, that knockdown of EWS/FLI1 leads to hypophosphorylation and functional activation of the retinoblastoma (pRb) family of proteins. Consistent with activation of the pRb proteins, E2F‐responsive genes such as cyclin A are repressed in EWS/FLI1‐depleted cells. Together, these results support the role of EWS/LI1 as an inhibitor of cellular senescence and implicate the retinoblastoma family of proteins as key mediators of this inhibition.

COL11A2 Collagen Gene Transcription Is Differentially Regulated by EWS/ERG Sarcoma Fusion Protein and Wild-type ERG

A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewings sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG andCOL11A2 genes are expressed in the Ewings sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG inCOL11A2 gene expression, we characterized theCOL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction betweenCOL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.