Synovial Sarcoma Chromatin Dynamics Reveal a Continuum in SS18:SSX Reprograming.

Synovial sarcoma (SyS) is an aggressive soft-tissue malignancy characterized by a pathognomonic chromosomal translocation leading to the formation of the SS18::SSX fusion oncoprotein. SS18::SSX associates with mammalian BAF complexes suggesting deregulation of chromatin architecture as the oncogenic driver in this tumour type. To examine the epigenomic state of SyS we performed comprehensive multi-omics analysis on 52 primary pre-treatment human SyS tumours. Our analysis revealed a continuum of epigenomic states across the cohort at fusion target genes independent of rare somatic genetic lesions. We identify cell-of-origin signatures defined by enhancer states and reveal unexpected relationships between H2AK119Ub1 and active marks. The number of bivalent promoters, dually marked by the repressive H3K27me3 and activating H3K4me3 marks, has strong prognostic value and outperforms tumor grade in predicting patient outcome. Finally, we identify SyS defining epigenomic features including H3K4me3 expansion associated with striking promoter DNA hypomethylation in which SyS displays the lowest mean methylation level of any sarcoma subtype. We explore these distinctive features as potential vulnerabilities in SyS and identify H3K4me3 inhibition as a promising therapeutic strategy.

ATRX guards against aberrant differentiation in mesenchymal progenitor cells.

Alterations in the tumor suppressor ATRX are recurrently observed in mesenchymal neoplasms. ATRX has multiple epigenetic functions including heterochromatin formation and maintenance and regulation of transcription through modulation of chromatin accessibility. Here, we show in murine mesenchymal progenitor cells (MPCs) that Atrx deficiency aberrantly activated mesenchymal differentiation programs. This includes adipogenic pathways where ATRX loss induced expression of adipogenic transcription factors and enhanced adipogenic differentiation in response to differentiation stimuli. These changes are linked to loss of heterochromatin near mesenchymal lineage genes together with increased chromatin accessibility and gains of active chromatin marks. We additionally observed depletion of H3K9me3 at transposable elements, which are derepressed including near mesenchymal genes where they could serve as regulatory elements. Finally, we demonstrated that loss of ATRX in a mesenchymal malignancy, undifferentiated pleomorphic sarcoma, results in similar epigenetic disruption and de-repression of transposable elements. Together, our results reveal a role for ATRX in maintaining epigenetic states and transcriptional repression in mesenchymal progenitors and tumor cells and in preventing aberrant differentiation in the progenitor context.

Tumor-intrinsic PRC2 inactivation drives a context-dependent immune-desert microenvironment and is sensitized by immunogenic viruses.

Immune checkpoint blockade (ICB) has demonstrated clinical success in "inflamed" tumors with substantial T cell infiltrates, but tumors with an immune-desert tumor microenvironment (TME) fail to benefit. The tumor cell-intrinsic molecular mechanisms of the immune-desert phenotype remain poorly understood. Here, we demonstrated that inactivation of the polycomb-repressive complex 2 (PRC2) core components embryonic ectoderm development (EED) or suppressor of zeste 12 homolog (SUZ12), a prevalent genetic event in malignant peripheral nerve sheath tumors (MPNSTs) and sporadically in other cancers, drove a context-dependent immune-desert TME. PRC2 inactivation reprogramed the chromatin landscape that led to a cell-autonomous shift from primed baseline signaling-dependent cellular responses (e.g., IFN-γ signaling) to PRC2-regulated developmental and cellular differentiation transcriptional programs. Further, PRC2 inactivation led to diminished tumor immune infiltrates through reduced chemokine production and impaired antigen presentation and T cell priming, resulting in primary resistance to ICB. Intratumoral delivery of inactivated modified vaccinia virus Ankara (MVA) enhanced tumor immune infiltrates and sensitized PRC2-loss tumors to ICB. Our results identify molecular mechanisms of PRC2 inactivation-mediated, context-dependent epigenetic reprogramming that underline the immune-desert phenotype in cancer. Our studies also point to intratumoral delivery of immunogenic viruses as an initial therapeutic strategy to modulate the immune-desert TME and capitalize on the clinical benefit of ICB.

A Phase Ib/II Randomized Study of RO4929097, a Gamma-Secretase or Notch Inhibitor with or without Vismodegib, a Hedgehog Inhibitor, in Advanced Sarcoma.

PURPOSE: Because the Hedgehog and Notch pathways are often overexpressed in mesenchymal malignancies, we evaluated the efficacy of concurrent inhibition of Notch and Hedgehog signaling using the gamma-secretase inhibitor (GSI) RO4929097 and the smoothened antagonist vismodegib in unresectable or metastatic sarcoma. PATIENTS AND METHODS: In this investigator-initiated trial, phase Ib used standard 3+3 dose escalation in which patients first received vismodegib once daily for 21 days, followed by the combination of RO4929097 concurrently with vismodegib in 21-day cycles. In phase II, patients were randomized to RO4929097 alone or in combination with vismodegib. RESULTS: Nine patients were treated in phase Ib with no dose-limiting toxicities. RO4929097 at 15 mg daily in combination with 150 mg daily of vismodegib was declared the recommended phase II dose. Most adverse events were grade ≤ 2. In phase II (closed early due to discontinuation of RO4929097 evaluation), 34 patients were randomized to RO4929097 alone and 33 to RO4929097 plus vismodegib. RO4929097 did not interfere with the steady-state concentration of vismodegib, while vismodegib reduced the plasma concentration of RO492909. No patients had an objective response. Neither progression-free nor overall survival differed significantly between treatment arms. Paired tumor biopsies from a subset of patients demonstrated inhibition of cleaved Notch. CONCLUSIONS: The combination of RO4929097 plus vismodegib was generally well tolerated. Although accrual to this study was not completed, vismodegib did not meaningfully enhance the clinical efficacy of RO4929097 in an unplanned analysis. GSIs and GSIs plus vismodegib can inhibit intratumoral Notch and downstream phosphorylated Akt signaling.

Outcome of 1000 Patients With Gastrointestinal Stromal Tumor (GIST) Treated by Surgery in the Pre- and Post-imatinib Eras.

OBJECTIVE: To characterize the results of surgery for gastrointestinal stromal tumor (GIST) in the pre and post-imatinib eras at a single institution and to identify current prognostic clinicopathologic factors. BACKGROUND: Imatinib has radically changed the management of GIST, yet the magnitude of impact on outcome across the spectrum of GIST presentation and relevance of historical prognostic factors are not well defined. METHODS: We retrospectively analyzed 1000 patients who underwent surgery for GIST at our institution from 1982 to 2016. Patients were stratified by presentation status as primary tumor only (PRIM), primary with synchronous metastasis (PRIM + MET), or metachronous recurrence/metastases (MET), and also imatinib era (before and after it became available). Cox proportional-hazard models and Kaplan-Meier methods were used to model and estimate overall survival (OS) and recurrence-free survival (RFS). RESULTS: OS was longer in the imatinib era compared with the pre-imatinib era in each presentation group, including in Miettinen high-risk primary tumors. Among PRIM patients from the pre-imatinib era, tumor site, size, and mitotic rate were independently associated with OS and RFS on multivariate analysis. PRIM patients in the imatinib era who received imatinib (neoadjuvant and/or adjuvant) had higher risk tumors, but after adjusting for treatment, only size >10 cm remained independently prognostic of RFS [hazard ratio (HR) 3.85, 95% confidence interval (CI) 2.00-7.40, P < 0.0001) and OS (HR 3.37, 95% CI 1.60-7.13, P = 0.001)]. CONCLUSIONS: Patients treated in the imatinib era had prolonged OS across all presentations. In the imatinib era, among site, size, and mitotic rate, high-risk features were associated with treatment with the drug, but only size >10 cm correlated with outcome. Imatinib should still be prescribed for patients with high-risk features.

Prognostic Factors After Neoadjuvant Imatinib for Newly Diagnosed Primary Gastrointestinal Stromal Tumor.

INTRODUCTION: Neoadjuvant imatinib (Neo-IM) therapy may facilitate R0 resection in primary gastrointestinal stromal tumors (GISTs) that are large or in difficult anatomic locations. While response to preoperative tyrosine kinase inhibitors is associated with better outcome in metastatic GIST, little is known about prognostic factors after Neo-IM in primary GIST. STUDY DESIGN: Patients with primary GIST with or without synchronous metastases who underwent Neo-IM were retrospectively analyzed from a prospective maintained institutional database for Response Evaluation Criteria in Solid Tumors (RECIST), tumor viability, and mitotic rate. Overall survival (OS) was estimated by Kaplan-Meier and compared by log-rank test. Cox proportionate hazard models were used for univariate and multivariate analysis. RESULTS: One hundred and fifty patients were treated for a median of 7.1 months (range 0.2-160). By RECIST, partial response, stable disease, and progressive disease were seen in 40%, 51%, and 9%, respectively. By pathologic analysis, ≤ 50% of the tumor was viable in 72%, and the mitotic rate was ≤ 5/50HPF in 74%. On multivariate analysis, RECIST response and tumor viability were not associated with OS, while post-treatment high mitotic rate (hazard ratio (HR) for death 5.3, CI 2.3-12.4), R2 margins (HR 6.0, CI 2.3-15.5), and adjuvant imatinib (HR 0.4, CI 0.2-0.9) were (p < 0.05). Five-year OS was 81 vs. 38% for low vs. high mitotic rate; 81, 59, and 39% for R0, R1, and R2 margins; and 75 vs 61% for adjuvant vs. no adjuvant imatinib therapy (p < 0.05). CONCLUSIONS: In primary GIST undergoing Neo-IM therapy, progression was uncommon, but substantial down-sizing occurred in the minority. High tumor mitotic rate and incomplete resection following Neo-IM were associated with poor outcome, while adjuvant imatinib was associated with prolonged survival.

Objective Response Rate Among Patients With Locally Advanced or Metastatic Sarcoma Treated With Talimogene Laherparepvec in Combination With Pembrolizumab: A Phase 2 Clinical Trial.

Importance: Patients with advanced sarcoma have limited treatment options. Talimogene laherparepvec (T-VEC) has been shown to increase tumor-specific immune activation via augmenting antigen presentation and T-cell priming. Objective: To examine whether T-VEC in combination with pembrolizumab is associated with increased tumor-infiltrating lymphocyte infiltration and programmed death-ligand 1 expression and thus with increased antitumor activity in patients with locally advanced or metastatic sarcoma. Design, Setting, and Participants: This open-label, single-institution phase 2 interventional trial of T-VEC plus pembrolizumab enrolled 20 patients with locally advanced or metastatic sarcoma between March 16 and December 4, 2017, for whom at least 1 standard systemic therapy had failed. The median duration of therapy was 16 weeks (range, 7-67 weeks). Reported analyses include data through December 14, 2018. Intervention: Patients received pembrolizumab (200-mg flat dose) intravenously and T-VEC (first dose, ≤4 mL × 106 plaque-forming units [PFU]/mL; second and subsequent doses, ≤4 mL × 108 PFU/mL) injected into palpable tumor site(s) on day 1 of each 21-day cycle. Main Outcomes and Measures: The primary end point was objective response rate (ORR; complete response and partial response) at 24 weeks determined by Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1, criteria. Secondary end points included best ORR by immune-related RECIST criteria, progression-free survival rate at 24 weeks, overall survival, and safety. Results: All 20 patients (12 women [60%]; median age, 63.5 years [range, 24-90 years]) were evaluable for response. The study met its primary end point of evaluating the best ORR at 24 weeks determined by RECIST, version 1.1, criteria; the best ORR was 30% (95% CI, 12%-54%; n = 6). The ORR overall was 35% (95% CI, 15%-59%; n = 7). The incidence of grade 3 treatment-related adverse events was low (4 patients [20%]). There were no grade 4 treatment-related adverse events or treatment-related deaths. Conclusions and Relevance: In this phase 2 clinical trial, treatment with T-VEC plus pembrolizumab was associated with antitumor activity in advanced sarcoma across a range of sarcoma histologic subtypes, with a manageable safety profile. This combination therapy met its predefined primary study end point; further evaluation of T-VEC in combination with pembrolizumab for patients with select sarcoma subtypes is planned. Trial Registration: ClinicalTrials.gov identifier: NCT03069378.

A Phase I Study of Binimetinib (MEK162) Combined with Pexidartinib (PLX3397) in Patients with Advanced Gastrointestinal Stromal Tumor.

LESSONS LEARNED: The combination of pexidartinib and binimetinib was safe and tolerable and demonstrated encouraging signs of efficacy in two patients with advanced gastrointestinal stromal tumor (GIST) refractory to tyrosine kinase inhibitors (TKIs).Molecular profiling of GISTs at diagnosis and upon progression may provide insight into the mechanisms of response or resistance to targeted therapies.Additional trials are needed to further explore combined KIT and MEK inhibition in treatment-naïve and TKI-refractory patients with advanced GIST. BACKGROUND: Nearly all patients with advanced gastrointestinal stromal tumor (GIST) develop resistance to imatinib, and subsequent treatments have limited efficacy. Dual inhibition of KIT and MAPK pathways has synergistic antitumor activity in preclinical GIST models. METHODS: This was an investigator-initiated, phase I, dose escalation study of the MEK inhibitor binimetinib combined with pexidartinib, a potent inhibitor of CSF1R, KIT, and FLT3, in patients with advanced or metastatic GIST who progressed on imatinib. The primary endpoint was phase II dose determination; secondary endpoints included safety, tolerability, and efficacy. An expansion cohort to further evaluate safety and efficacy was planned. RESULTS: Two patients were treated at dose level one (binimetinib 30 mg b.i.d. and pexidartinib 400 mg every morning and 200 mg every evening), after which the study was terminated by the manufacturer. No dose-limiting toxicities (DLTs) were reported, and treatment was well tolerated. The only grade ≥3 treatment-emergent adverse event (TEAE) was asymptomatic elevated creatine phosphokinase (CPK). Both patients had a best response of stable disease (SD) by RECIST. Progression-free survival (PFS) and overall survival (OS) were 6.1 and 14.6 months, respectively, in one patient with five prior lines of therapy. The second patient with NF1-mutant GIST had a 27% decrease in tumor burden by RECIST and remains on study after 19 months of treatment. CONCLUSION: Pexidartinib combined with binimetinib was tolerable, and meaningful clinical activity was observed in two imatinib-refractory patients.

Rectal Gastrointestinal Stromal Tumor (GIST) in the Era of Imatinib: Organ Preservation and Improved Oncologic Outcome.

BACKGROUND: Approximately 5% of gastrointestinal stromal tumors (GISTs) originate in the rectum, and historically, radical resection was commonly performed. Little is known about the outcome for rectal GIST in the era of imatinib. METHODS: Using a prospectively maintained database, this study retrospectively analyzed 47 localized primary rectal GISTs treated at our center from 1982 to 2016, stratified by when imatinib became available in 2000. Overall survival (OS), disease-specific survival (DSS), and recurrence-free survival (RFS) were analyzed by the Kaplan-Meier method. RESULTS: Rectal GISTs represented 7.1% of 663 primary GISTs. The findings showed 17 patients in the pre-imatinib era and 30 patients in the imatinib era. The two groups had similar follow-up evaluation, age, gender, Miettinen risk, and distance to the anal verge. In the imatinib era, tumors were smaller at diagnosis (median 4 vs. 5 cm; p = 0.029), and 24 of the 30 patients received perioperative imatinib. In the high-risk patients, organ preservation and negative margins were more common among the 13 patients treated with neoadjuvant imatinib than among the 21 patients treated directly with surgery. High-risk patients who received perioperative imatinib (n = 15) had greater (or nearly significantly greater) 5-year OS, DSS, local RFS, and distant RFS than those who did not (n = 19) (91, 100, 100, and 71% vs. 47, 65, 74, and 41%; p = 0.049, 0.052, 0.077, 0.051, respectively). In the imatinib era, no patient has had a local recurrence or death due to GIST. CONCLUSIONS: The use of imatinib is associated with organ preservation and improved oncologic outcome for patients with rectal GIST.

Transcriptome-wide survey of mouse CNS-derived cells reveals monoallelic expression within novel gene families.

Monoallelic expression is an integral component of regulation of a number of essential genes and gene families. To probe for allele-specific expression in cells of CNS origin, we used next-generation sequencing (RNA-seq) to analyze four clonal neural stem cell (NSC) lines derived from Mus musculus C57BL/6 (B6)×Mus musculus molossinus (JF1) adult female mice. We established a JF1 cSNP library, then ascertained transcriptome-wide expression from B6 vs. JF1 alleles in the NSC lines. Validating the assay, we found that 262 of 268 X-linked genes evaluable in at least one cell line showed monoallelic expression (at least 85% expression of the predominant allele, p-value<0.05). For autosomal genes 170 of 7,198 genes (2.4% of the total) showed monoallelic expression in at least 2 evaluable cell lines. The group included eight known imprinted genes with the expected pattern of allele-specific expression. Among the other autosomal genes with monoallelic expression were five members of the glutathione transferase gene superfamily, which processes xenobiotic compounds as well as carcinogens and cancer therapeutic agents. Monoallelic expression within this superfamily thus may play a functional role in the response to diverse and potentially lethal exogenous factors, as is the case for the immunoglobulin and olfactory receptor superfamilies. Other genes and gene families showing monoallelic expression include the annexin gene family and the Thy1 gene, both linked to inflammation and cancer, as well as genes linked to alcohol dependence (Gabrg1) and epilepsy (Kcnma1). The annotated set of genes will provide a resource for investigation of mechanisms underlying certain cases of these and other major disorders.

Dual DNA methylation patterns in the CNS reveal developmentally poised chromatin and monoallelic expression of critical genes.

As a first step towards discovery of genes expressed from only one allele in the CNS, we used a tiling array assay for DNA sequences that are both methylated and unmethylated (the MAUD assay). We analyzed regulatory regions of the entire mouse brain transcriptome, and found that approximately 10% of the genes assayed showed dual DNA methylation patterns. They include a large subset of genes that display marks of both active and silent, i.e., poised, chromatin during development, consistent with a link between differential DNA methylation and lineage-specific differentiation within the CNS. Sixty-five of the MAUD hits and 57 other genes whose function is of relevance to CNS development and/or disorders were tested for allele-specific expression in F(1) hybrid clonal neural stem cell (NSC) lines. Eight MAUD hits and one additional gene showed such expression. They include Lgi1, which causes a subtype of inherited epilepsy that displays autosomal dominance with incomplete penetrance; Gfra2, a receptor for glial cell line-derived neurotrophic factor GDNF that has been linked to kindling epilepsy; Unc5a, a netrin-1 receptor important in neurodevelopment; and Cspg4, a membrane chondroitin sulfate proteoglycan associated with malignant melanoma and astrocytoma in human. Three of the genes, Camk2a, Kcnc4, and Unc5a, show preferential expression of the same allele in all clonal NSC lines tested. The other six genes show a stochastic pattern of monoallelic expression in some NSC lines and bi-allelic expression in others. These results support the estimate that 1-2% of genes expressed in the CNS may be subject to allelic exclusion, and demonstrate that the group includes genes implicated in major disorders of the CNS as well as neurodevelopment.

Monoallelic expression of multiple genes in the CNS.

The inheritance pattern of a number of major genetic disorders suggests the possible involvement of genes that are expressed from one allele and silent on the other, but such genes are difficult to detect. Since DNA methylation in regulatory regions is often a mark of gene silencing, we modified existing microarray-based assays to detect both methylated and unmethylated DNA sequences in the same sample, a variation we term the MAUD assay. We probed a 65 Mb region of mouse Chr 7 for gene-associated sequences that show two distinct DNA methylation patterns in the mouse CNS. Selected genes were then tested for allele-specific expression in clonal neural stem cell lines derived from reciprocal F(1) (C57BL/6xJF1) hybrid mice. In addition, using a separate approach, we directly analyzed allele-specific expression of a group of genes interspersed within clusters of OlfR genes, since the latter are subject to allelic exclusion. Altogether, of the 500 known genes in the chromosomal region surveyed, five show monoallelic expression, four identified by the MAUD assay (Agc1, p (pink-eyed dilution), P4ha3 and Thrsp), and one by its proximity to OlfR genes (Trim12). Thrsp (thyroid hormone responsive SPOT14 homolog) is expressed in hippocampus, but the human protein homolog, S14, has also been implicated in aggressive breast cancer. Monoallelic expression of the five genes is not coordinated at a chromosome-wide level, but rather regulated at individual loci. Taken together, our results suggest that at least 1% of previously untested genes are subject to allelic exclusion, and demonstrate a dual approach to expedite their identification.

Arterial grafting results in reduced operative mortality and enhanced long-term quality of life in octogenarians.

BACKGROUND: Despite well-established benefits of arterial (ART) grafting, surgeons have been reluctant to use this conduit in octogenarians. This study explores the influence of arterial revascularization on operative and long-term outcomes of coronary artery bypass grafting surgery. METHODS: A retrospective analysis was conducted of 987 consecutive patients 80 years of age or older who underwent isolated coronary artery bypass grafting between January 1989 and November 2000. Patients with saphenous vein graft only (SVG; n = 574) were compared with those receiving arterial and saphenous vein grafts (ART+SVG; n = 413). Mean follow-up for SVG patients was 3.8 years (range, 4 months to 12.6 years) and 98.6% complete, and mean follow-up was 3.1 years for ART+SVG patients (range, 2 months to 11.2 years) and 97.3% complete. RESULTS: Patients with SVG had a significantly higher (p = 0.009) operative mortality (11.1% versus 6.3%) and significantly longer postoperative length of stay (12.9 versus 10.7 days; p = 0.002) than ART+SVG recipients. More ART+SVG than SVG patients were free of all postoperative complications (290 of 413; 70.2% versus 372 of 574; 64.8%; p = 0.086). Multivariable analysis identified SVG as an independent predictor of operative mortality (p = 0.014) and late mortality (p = 0.040). When patients were matched by equivalent propensity scores to receive SVG only, operative mortality was higher for SVG patients in four of the five quintiles. At 10 years, 97.0% +/- 1.2% of SVG and 92.9% +/- 3.7% of ART+SVG current survivors were free of all late major adverse cardiac events (p = 0.565), and 95.5% of SVG patients and 97.5% of ART+SVG patients were in Canadian class 1 or 2 (p = 0.162). On the SF-36 quality-of-life assessment, ART+SVG patients scored significantly higher than both SVG patients and age-adjusted normal subjects. Physical health summary component scores were 36.8 +/- 11.0 for SVG and 41.0 +/- 10.3 for ART+SVG (p = 0.001). Mental health summary scores were comparable for the two groups. CONCLUSIONS: Arterial grafting confers an operative survival benefit, and an enhanced long-term quality of life in elderly patients.

Use of a reverse transcriptase-polymerase chain reaction assay to analyze allele-specific expression in individual hippocampal neurons.

We report here a single-cell RT-PCR assay for allele-specific gene expression that can be used to probe for somatic variability within the CNS. Such variability, arising from epigenetic (nonmutational) events or somatic mutation early in development, may give clues as to clonal origin and may also affect the inheritance pattern of some CNS disorders. As a model system, we used reciprocal F1 hybrids of the cross Mus musculus C57BL/6J x Mus musculus castaneus. RNA was isolated from individual dissociated pyramidal neurons from hippocampi of F1 pups. For each gene of interest, single base polymorphisms were identified between the two parental strains by automated sequencing of RT-PCR products. Allele-specific expression was then analyzed by means of the previously described quantitative RT-PCR single nucleotide primer extension (SNuPE) assay (Singer-Sam et al., PCR Methods Appl. 1:160-163, 1992). Individual neurons showed monoallelic expression of the two control genes, X-linked Rps4, and the imprinted gene Snrpn; in contrast expression of Ncam and F3cam, coding for neural cell adhesion molecules, was found to be biallelic.

Methylation analysis by genomic sequencing of 5' region of mouse Pgk-1 gene and a cautionary note concerning the method.

We have used genomic sequencing aided by ligation-mediated PCR (LMPCR) to assay for 5-methylcytosine in the CpG-rich promoter region of the mouse X-linked phosphoglycerate kinase gene (Pgk-1). Earlier studies showed that there was very heavy methylation of CpG dinucleotides in the CpG-rich promoter of the human PGK1 gene on the inactive X chromosome (the Xi), but that these same sites were completely unmethylated on the active X chromosome (the Xa). For mouse Pgk-1, previous restriction enzyme analysis had shown apparently complete methylation of only one cytosine in the promoter region on the Xi, at HpaII site H7, which is located in the untranslated region, 28 nucleotides upstream of the translation start site. We analyzed this potentially critical region by combining the use of HpaII with LMPCR, and find that the CpG dinucleotides near H7 are either unmethylated or only partially methylated on the Xi. LMPCR analysis of male and female DNA over a 490-bp sequence including the promoter and enhancer extend the finding of relative hypomethylation on the mouse Xi to include all CpG dinucleotides in this region. These results are relevant to the role of DNA methylation in stabilizing the inactive state of chromatin. In addition, we find that caution must be exercised in using LMPCR for methylation analysis of some sequences. A DNA concentration-dependent band-suppression artifact can incorrectly suggest methylation of both CpG and nonCpG dinucleotides.

Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation.

A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days; about the time of X-chromosome inactivation of the inner cell mass.

Evolutionary conservation of the substrate-binding cleft of phosphoglycerate kinases.

The primary structures of six phosphoglycerate kinases (PGKs) are known: three from mammals, one from yeast, and two from trypanosomes. Comparison of the amino acid sequence of these enzymes reveals 154 invariant positions out of 392 positions in the aligned sequences. Most of the conserved positions fall into the twelve beta-sheets and adjacent peptide regions that form the inner loops surrounding the ATP and 3-phosphoglycerate-binding cleft. The homology between mammalian and yeast PGKs is greater than 94% for the inner-loop region, even though the overall homology is less than 65%. Trypanosome PGK has only 44% overall homology with the mammalian enzyme, but shows 74% homology in the inner-loop region. Trypanosome PGK contains a polypeptide segment in its N-terminal domain that is transposed in comparison with the other species.

The nucleotide sequence of a cDNA clone containing the entire coding region for mouse X-chromosome-linked phosphoglycerate kinase.

A clone containing cDNA for X chromosome-linked phosphoglycerate kinase (PGK-1) was isolated from a mouse myeloma cDNA library. The nucleotide (nt) sequence of the cDNA has been determined, and the amino acid (aa) sequence of the enzyme thereby deduced. At the nt level, the coding region of mouse PGK cDNA has 93% homology with human X-linked cDNA and 60% homology with the yeast gene. Mouse PGK-1 protein contains 416 aa and is 98%, 96% and 64% homologous with human, horse, and yeast enzyme sequences, respectively.

Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe.

We have obtained a cDNA clone encoding most of human X-linked 3-phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3). Total mRNA was prepared from human adenocarcinoma-derived cell line LS174T and used for cDNA preparation. Double-stranded cDNA was inserted, after tailing with oligo(dC), into the plasmid vector pBR327 and cloned in Escherichia coli K-12. Transformants were screened by colony hybridization with a mixture of 32P-labeled oligodeoxyribonucleotides. A pool of hexadecamers complementary to all 32 possible sequences encoding amino acids 291-296 of X-linked PGK was used for the initial screen. One clone among 2,500 gave a strong positive signal. Plasmid DNA from this clone was purified and characterized by hybridization first to the hexadecamer probe mixture and then to an undecamer probe consisting of a mixture of four sequences. The cloned fragment hybridizes preferentially to DNA from human cells with five X chromosomes. DNA sequence analysis has established that the 1.2-kilobase-pair fragment encodes PGK from amino acid 121 through the COOH terminus.