Three-dimensional folding and functional organization principles of the Drosophila genome.

Chromosomes are the physical realization of genetic information and thus form the basis for its readout and propagation. Here we present a high-resolution chromosomal contact map derived from a modified genome-wide chromosome conformation capture approach applied to Drosophila embryonic nuclei. The data show that the entire genome is linearly partitioned into well-demarcated physical domains that overlap extensively with active and repressive epigenetic marks. Chromosomal contacts are hierarchically organized between domains. Global modeling of contact density and clustering of domains show that inactive domains are condensed and confined to their chromosomal territories, whereas active domains reach out of the territory to form remote intra- and interchromosomal contacts. Moreover, we systematically identify specific long-range intrachromosomal contacts between Polycomb-repressed domains. Together, these observations allow for quantitative prediction of the Drosophila chromosomal contact map, laying the foundation for detailed studies of chromosome structure and function in a genetically tractable system.

Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila.

In Drosophila melanogaster, Hox genes are organized in an anterior and a posterior cluster, called Antennapedia complex and bithorax complex, located on the same chromosome arm and separated by 10 Mb of DNA. Both clusters are repressed by Polycomb group (PcG) proteins. Here, we show that genes of the two Hox complexes can interact within nuclear PcG bodies in tissues where they are corepressed. This colocalization increases during development and depends on PcG proteins. Hox gene contacts are conserved in the distantly related Drosophila virilis species and they are part of a large gene interaction network that includes other PcG target genes. Importantly, mutations on one of the loci weaken silencing of genes in the other locus, resulting in the exacerbation of homeotic phenotypes in sensitized genetic backgrounds. Thus, the three-dimensional organization of Polycomb target genes in the cell nucleus stabilizes the maintenance of epigenetic gene silencing.

Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide.

Polycomb group (PcG) proteins are required for normal differentiation and development and are frequently deregulated in cancer. PcG proteins are involved in gene silencing; however, their role in initiation and maintenance of transcriptional repression is not well defined. Here, we show that knockout of the Polycomb repressive complex 2 (PRC2) does not lead to significant gene expression changes in mouse embryonic stem cells (mESCs) and that it is dispensable for initiating silencing of target genes during differentiation. Transcriptional inhibition in mESCs is sufficient to induce genome-wide ectopic PRC2 recruitment to endogenous PcG target genes found in other tissues. PRC2 binding analysis shows that it is restricted to nucleosome-free CpG islands (CGIs) of untranscribed genes. Our results show that it is the transcriptional state that governs PRC2 binding, and we propose that it binds by default to nontranscribed CGI genes to maintain their silenced state and to protect cell identity.

In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites.

In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. These sites include gene promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset reveals DSB-independent interactions between PRDM9 and genomic sites, such as the binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot-bound PRDM9 and genomic sequences located on the chromosome axis.

Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos.

Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. Here, we used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, we mapped histone modifications associated with PcG-dependent silencing and TRX-mediated activation. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. Our results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N-bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation.

Continuous estimation of ground reaction force during long distance running within a fatigue monitoring framework: A Kalman filter-based model-data fusion approach.

Estimation of ground reaction forces in runners has been limited to laboratory environments by means of instrumented treadmills, in-ground force plates and optoelectronic systems. Recent advances in estimation techniques using wearable sensors for kinematic analysis and sports performance could enable estimation outside the laboratory. This paper proposes a state-input-parameter estimation framework to continuously estimate the vertical ground reaction force waveform during running. By modeling a runner as a single degree of freedom mass-spring-damper with acceleration measurements at the sacrum a state-space formulation can be applied using Newtonian methods. A dual-Kalman filter is employed to estimate the unmeasured system input which feeds through to an unscented Kalman filter to estimate system dynamics and unknown model parameters (e.g. spring stiffness). For validation, 14 subjects performed three one-minute running trials at three different speeds (self-selected slow, comfortable, and fast) on a pressure-sensor-instrumented treadmill. The estimated vertical ground reaction force waveform parameters; peak vertical ground reaction force (RMSE=6.1-7.2%,ρ=0.95-0.97), vertical impulse (RMSE=8.5-13.0%,ρ=0.50-0.60), loading rate (RMSE=24.6-39.4%,ρ=0.85-0.93), and cadence RMSE<1%,ρ=1.00 were compared against the instrumented treadmill measurements. The proposed state-input-parameter estimation framework could monitor personalized vertical ground reaction force metrics for potential biofeedback applications. The feedback mechanism could provide information about the vertical ground reaction force characteristics to the runner as they are running to provide knowledge of both desirable and undesirable loading characteristics experienced.

The impact of a physician-directed health information technology system on diabetes outcomes in primary care: a pre- and post-implementation study.

PURPOSE: To determine the impact of a physician-directed, multifaceted health information technology (HIT) system on diabetes outcomes. METHODS: A pre/post-interventional study. SETTING AND PARTICIPANTS: The setting was Providence Primary Care Research Network in Oregon, with approximately 71 physicians caring for 117 369 patients in 13 clinic locations. The study covered Network patients with diabetes age 18 years and older. INTERVENTION: The study intervention included implementation of the CareManager HIT system which augments an electronic medical record (EMR) by automating physician driven quality improvement interventions, including point-of-care decision support and care reminders, diabetes registry with care prompts, performance feedback with benchmarking and access to published evidence and patient educational materials. MEASURES: The primary clinical measures included the change in mean value for low density lipoprotein (LDL) target <100 mg/dL or 2.6 mmol/l, blood pressure (BP) target <130/80 mmHg and glycated haemoglobin (HbA1c) target <7%, and the proportion of patients meeting guideline-recommended targets for those measures. All measures were analysed using closed and open cohort approaches. RESULTS: A total of 6072 patients were identified at baseline, 70% of whom were continuously enrolled during the 24-month study. Significant improvements were observed in all diabetes related outcomes except mean HbA1c. LDL goal attainment improved from 32% to 56% (P=0.002), while mean LDL decreased by 13 mg/dL (0.33 mmol/l, P=0.002). BP goal attainment increased significantly from 30% to 52%, with significant decreases in both mean systolic and diastolic BP. The proportion of patients with an HbA1c below 7% was higher at the end of the study (P=0.008). Mean patient satisfaction remained high, with no significant difference between baseline and follow-up. Total Relative Value Units per patient per year significantly increased as a result of an increase in the number of visits in year one and the coding complexity throughout. CONCLUSION: Implementation of a physician-directed, multifaceted HIT system in primary care was associated with significantly improved diabetes process and outcome measures.

A randomized controlled trial of team-based care: impact of physician-pharmacist collaboration on uncontrolled hypertension.

OBJECTIVE: Evaluate the effectiveness of collaborative management of hypertension by primary care-pharmacist teams in community-based clinics. STUDY DESIGN: A 12-month prospective, single-blind, randomized, controlled trial in the Providence Primary Care Research Network of patients with hypertension and uncontrolled blood pressure. METHODS: As compared to usual primary care, intervention consisted of pharmacy practitioners participating in the active management of hypertension in the primary care office according to established collaborative treatment protocols. At baseline, there was no significant difference in blood pressure between groups. Primary outcome measures were the differences in mean systolic and diastolic blood pressures between arms at study end. Secondary measures included blood pressure goal attainment (<140/90 mmHg), hypertension-related knowledge, medication adherence, home blood pressure monitoring, resource utilization, quality of life, and satisfaction. RESULTS: A total of 463 subjects were enrolled (n = 233 control, n = 230 intervention). Subjects receiving the intervention achieved significantly lower systolic (p = 0.007) and diastolic (p = 0.002) blood pressures compared to control (137/75 mmHg vs. 143/78 mmHg). In addition, 62% of intervention subjects achieved target blood pressure compared to 44% of control subjects (p = 0.003). The intervention group received more total office visits (7.2 vs. 4.9, p < 0.0001), however had fewer physician visits (3.2 vs. 4.7, p < 0.0001) compared to control. Intervention subjects were prescribed more antihypertensive medications (2.7 vs. 2.4, p = 0.02), but did not take more antihypertensive pills per day (2.4 vs. 2.5, p = 0.87). There were minimal differences between groups in hypertension-related knowledge, medication adherence, quality of life, or satisfaction. CONCLUSIONS: Patients randomized to collaborative primary care-pharmacist hypertension management achieved significantly better blood pressure control compared to usual care with no difference in quality of life or satisfaction.

EZH2 is a potential therapeutic target for H3K27M-mutant pediatric gliomas.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brain tumor that is located in the pons and primarily affects children. Nearly 80% of DIPGs harbor mutations in histone H3 genes, wherein lysine 27 is substituted with methionine (H3K27M). H3K27M has been shown to inhibit polycomb repressive complex 2 (PRC2), a multiprotein complex responsible for the methylation of H3 at lysine 27 (H3K27me), by binding to its catalytic subunit EZH2. Although DIPGs with the H3K27M mutation show global loss of H3K27me3, several genes retain H3K27me3. Here we describe a mouse model of DIPG in which H3K27M potentiates tumorigenesis. Using this model and primary patient-derived DIPG cell lines, we show that H3K27M-expressing tumors require PRC2 for proliferation. Furthermore, we demonstrate that small-molecule EZH2 inhibitors abolish tumor cell growth through a mechanism that is dependent on the induction of the tumor-suppressor protein p16INK4A. Genome-wide enrichment analyses show that the genes that retain H3K27me3 in H3K27M cells are strong polycomb targets. Furthermore, we find a highly significant overlap between genes that retain H3K27me3 in the DIPG mouse model and in human primary DIPGs expressing H3K27M. Taken together, these results show that residual PRC2 activity is required for the proliferation of H3K27M-expressing DIPGs, and that inhibition of EZH2 is a potential therapeutic strategy for the treatment of these tumors.

Depression screening interfaced with an electronic health record: a feasibility study in a primary care clinic using optical mark reader technology.

OBJECTIVE: Strategies to effectively and efficiently screen for depression remain elusive in the primary care setting. The purpose of this study was to assess the feasibility of a depression screening program in which patients completed a validated questionnaire in the waiting room. Using Optical Mark Reader technology (PatientLink), patient responses were interfaced into the electronic health record (EHR), where the responses and score were available to practitioners at the time of the visit. METHOD: This was a prospective, observational study that enrolled all consenting patients, aged 18 years and older, who spoke English and arrived for any type of visit during a 1-week period at a family practice clinic (the first week of August 2004). Patient feedback was sought using a standardized survey. Feedback from practitioners and staff was sought using focus group methodology. The primary outcome measure was the proportion of patients successfully screened for depression by the front desk personnel using PatientLink. RESULTS: A total of 189 patients met eligibility criteria. Of those, 169 (89.4%) were successfully screened for depression. Of the patients who completed PHQ-9 questionnaires, 30 (17.8%) met DSM-IV criteria for moderate to severe depression. Four (2.4%) of these subjects with major depressive disorder were found not to have preexisting documentation of a depressive disorder in the medical record. In no case was the lack of successful screening due to technology error. Patients, staff, and practitioners supported this new screening strategy. No additional staff were needed to conduct the screening program. CONCLUSION: This study demonstrates that depression screening using a Scantron-based PHQ-9 questionnaire completed by patients in the waiting room and uploaded into an EHR is technically feasible and resource efficient.

Chromosome Conformation Capture on Chip (4C): Data Processing.

4C methods are useful to investigate dependencies between regulatory mechanisms and chromatin structures by revealing the frequency of chromatin contacts between a locus of interest and remote sequences on the chromosome. In this chapter we describe a protocol for the data analysis of microarray-based 4C experiments, presenting updated versions of the methods we used in a previous study of the large-scale chromatin interaction profile of a Polycomb response element in Drosophila. The protocol covers data preparation, normalization, microarray probe selection, and the multi-resolution detection of regions with enriched chromatin contacts. A reanalysis of two independent mouse datasets illustrates the versatility of this protocol and the importance of data processing in 4C. Methods were implemented in the R package MRA.TA (Multi-Resolution Analyses on Tiling Array data), and they can be used to analyze ChIP-on-chip data on broadly distributed chromatin components such as histone marks.

Maintaining cell identity: PRC2-mediated regulation of transcription and cancer.

Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb repressive complex 2 (PRC2), has attracted broad research attention in the past few years because of its involvement in the development and maintenance of many types of cancer and the use of specific EZH2 inhibitors in clinical trials. Several observations show that PRC2 can have both oncogenic and tumour-suppressive functions. We propose that these apparently opposing roles of PRC2 in cancer are a consequence of the molecular function of the complex in maintaining, rather than specifying, the transcriptional repression state of its several thousand target genes.

Data processing and exchange tools to facilitate chemical genetic screening processes.

Due to the development of chemical genomics, the screening of chemical libraries is used more and more by research laboratories to identify small molecule inhibitors or activators of cell functions. To facilitate the treatment and archiving of screening data, we developed a multiuser web application called Elisa Data Exchanger (EDE). The program is able to automatically identify which chemical compounds were tested. Several data exchange formats can be generated for visualization, printing, charting, or exporting to chemical analysis software. These data exchange functions allow for a comparison of results obtained from screening several targets in order to select the most specific compounds. EDE is freely available online at https://ibph.pharma.univ-montpl.fr/ede/ (login: evalede, password: loginede).

Team-based care approach to cholesterol management in diabetes mellitus: two-year cluster randomized controlled trial.

BACKGROUND: Creative, cost-effective interventions to improve the quality of care of chronic illnesses are needed. This study was designed to evaluate the impact of remote physician-pharmacist team-based care on cholesterol levels in patients with diabetes mellitus (DM). METHODS: This 2-year prospective, cluster randomized controlled trial was conducted within the Providence Primary Care Research Network in Oregon. Participants at least 18 years of age were identified by a diagnosis of DM. The intervention included remote physician-pharmacist team-based care focused on cholesterol management in DM. All clinicians in the study had access to the health information technology tool CareManager, which provided automated DM-related point-of-care prompts, a Web-based registry, and performance feedback with benchmarking. Study outcomes included the difference in low-density lipoprotein cholesterol (LDL-C) goal attainment, mean LDL-C, prescribed lipid-lowering therapy, and patient satisfaction between the intervention and control arms. RESULTS: A total of 6963 patients with DM cared for by 68 physicians in 9 clinics were evaluated. Patients in the intervention arm were more likely to achieve their target LDL-C levels compared with controls (78% vs 50%; P = .003). The mean LDL-C level was 12 mg/dL lower in the intervention arm compared with the control arm (P < .001). The rate of LDL-C testing was significantly higher in the intervention arm compared with the control arm. Patients in the intervention arm were also 15% more likely to receive a prescription for a lipid-lowering medication (P = .008). There was no significant difference in patient satisfaction between study arms (P = .15). CONCLUSION: Remotely located physician-pharmacist team-based care resulted in significantly improved LDL-C levels and goal attainment among patients with DM.

Genome regulation by polycomb and trithorax proteins.

Polycomb group (PcG) and trithorax group (trxG) proteins are critical regulators of numerous developmental genes. To silence or activate gene expression, respectively, PcG and trxG proteins bind to specific regions of DNA and direct the posttranslational modification of histones. Recent work suggests that PcG proteins regulate the nuclear organization of their target genes and that PcG-mediated gene silencing involves noncoding RNAs and the RNAi machinery.

Provider feedback to improve 5A's tobacco cessation in primary care: a cluster randomized clinical trial.

The electronic health record (EHR) may be an effective tool to help clinicians address tobacco use more consistently. To evaluate the impact of EHR-generated practice feedback on rates of referral to a state-level tobacco quitline, we conducted a cluster randomized clinical trial (feedback versus no feedback) within 19 primary care clinics in Oregon. Intervention clinics received provider-specific monthly feedback reports generated from EHR data. The reports rated provider performance in asking, advising, assessing, and assisting with tobacco cessation compared with a clinic average and an achievable benchmark of care. During 12 months of follow-up, EHR-documented rates of advising, assessing, and assisting were significantly improved in the intervention clinics compared with the control clinics (p<.001). A higher case-mix index and presence of a clinic champion were associated with higher rates of referral to a state-level quitline. EHR-generated provider feedback improved documentation of assistance with tobacco cessation. Connecting physician offices to a state-level quitline was feasible and well accepted.

Gene expression signature in advanced colorectal cancer patients select drugs and response for the use of leucovorin, fluorouracil, and irinotecan.

PURPOSE: In patients with advanced colorectal cancer, leucovorin, fluorouracil, and irinotecan (FOLFIRI) is considered as one of the reference first-line treatments. However, only about half of treated patients respond to this regimen, and there is no clinically useful marker that predicts response. A major clinical challenge is to identify the subset of patients who could benefit from this chemotherapy. We aimed to identify a gene expression profile in primary colon cancer tissue that could predict chemotherapy response. PATIENTS AND METHODS: Tumor colon samples from 21 patients with advanced colorectal cancer were analyzed for gene expression profiling using Human Genome GeneChip arrays U133. At the end of the first-line treatment, the best observed response, according to WHO criteria, was used to define the responders and nonresponders. Discriminatory genes were first selected by the significance analysis of microarrays algorithm and the area under the receiver operating characteristic curve. A predictor classifier was then constructed using support vector machines. Finally, leave-one-out cross validation was used to estimate the performance and the accuracy of the output class prediction rule. RESULTS: We determined a set of 14 predictor genes of response to FOLFIRI. Nine of nine responders (100% specificity) and 11 of 12 nonresponders (92% sensitivity) were classified correctly, for an overall accuracy of 95%. CONCLUSION: After validation in an independent cohort of patients, our gene signature could be used as a decision tool to assist oncologists in selecting colorectal cancer patients who could benefit from FOLFIRI chemotherapy, both in the adjuvant and the first-line metastatic setting.