Diverse Class 2 CRISPR Effectors as Active Nucleases with Expanded Targeting Capabilities.

CRISPR-Cas systems have proven effective in a variety of applications due to their ease of use and relatively high editing efficiency. Yet, any individual CRISPR-Cas system has inherent limitations, necessitating a diversity of RNA-guided nucleases to suit applications with distinct needs. We searched through metagenomic sequences to identify RNA-guided nucleases and found enzymes from diverse CRISPR-Cas types and subtypes, the most promising of which we developed into gene-editing platforms. Based on prior annotations of the metagenomic sequences, we establish the likely taxa and sampling locations where Class 2 CRISPR-Cas systems active in eukaryotes may be found. The newly discovered systems show robust capabilities as gene editors and base editors.

Should we screen aging physicians for cognitive decline?

OBJECTIVES: To synthesize evidence relevant for informed decisions concerning cognitive testing of older physicians. METHODS: Relevant literature was systematically searched in Medline, EMBASE, PsycInfo, and ERIC, with key findings abstracted and synthesized. RESULTS: Cognitive abilities of physicians may decline in an age range where they are still practicing. Physician competence and clinical performance may also decline with age. Cognitive scores are lower in physicians referred for assessment because of competency or performance concerns. Many physicians do not accurately self-assess and continue to practice despite declining quality of care; however, perceived cognitive decline, although not an accurate indicator of ability, may accelerate physicians' decision to retire. Physicians are reluctant to report colleagues' cognitive problems. Several issues should be considered in implementing cognitive screening. Most cognitive assessment tools lack normative data for physicians. Scientific evidence linking cognitive test results with physician performance is limited. There is no known level of cognitive decline at which a doctor is no longer fit to practice. Finally, relevant domains of cognitive ability vary across medical specialties. CONCLUSION: Physician cognitive decline may impact clinical performance. If cognitive assessment of older physicians is to be implemented, it should consider challenges of cognitive test result interpretation.

Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

Commercially produced antibodies are essential research tools. Investigators at universities and pharmaceutical companies use them to study human proteins, which carry out all the functions of the cells. Scientists usually buy antibodies from commercial manufacturers who produce more than 6 million antibody products altogether. Yet many commercial antibodies do not work as advertised. They do not recognize their intended protein target or may flag untargeted proteins. Both can skew research results and make it challenging to reproduce scientific studies, which is vital to scientific integrity. Using ineffective commercial antibodies likely wastes $1 billion in research funding each year. Large-scale validation of commercial antibodies by an independent third party could reduce the waste and misinformation associated with using ineffective commercial antibodies. Previous research testing an antibody validation pipeline showed that a commercial antibody widely used in studies to detect a protein involved in amyotrophic lateral sclerosis did not work. Meanwhile, the best-performing commercial antibodies were not used in research. Testing commercial antibodies and making the resulting data available would help scientists identify the best study tools and improve research reliability. Ayoubi et al. collaborated with antibody manufacturers and organizations that produce genetic knock-out cell lines to develop a system validating the effectiveness of commercial antibodies. In the experiments, Ayoubi et al. tested 614 commercial antibodies intended to detect 65 proteins involved in neurologic diseases. An effective antibody was available for about two thirds of the 65 proteins. Yet, hundreds of the antibodies, including many used widely in studies, were ineffective. Manufacturers removed some underperforming antibodies from the market or altered their recommended uses based on these data. Ayoubi et al. shared the resulting data on Zenodo, a publicly available preprint database. The experiments suggest that 20-30% of protein studies use ineffective antibodies, indicating a substantial need for independent assessment of commercial antibodies. Ayoubi et al. demonstrated their side-by-side antibody comparison methods were an effective and efficient way of validating commercial antibodies. Using this approach to test commercial antibodies against all human proteins would cost about $50 million. But it could save much of the $1 billion wasted each year on research involving ineffective antibodies. Independent validation of commercial antibodies could also reduce wasted efforts by scientists using ineffective antibodies and improve the reliability of research results. It would also enable faster, more reliable research that may help scientists understand diseases and develop new therapies to improve patient's lives.

Cooperative regulation of coupled oncoprotein synthesis and stability in triple-negative breast cancer by EGFR and CDK12/13.

Evidence has long suggested that epidermal growth factor receptor (EGFR) may play a prominent role in triple-negative breast cancer (TNBC) pathogenesis, but clinical trials of EGFR inhibitors have yielded disappointing results. Using a candidate drug screen, we identified that inhibition of cyclin-dependent kinases 12 and 13 (CDK12/13) dramatically sensitizes diverse models of TNBC to EGFR blockade. This combination therapy drives cell death through the 4E-BP1-dependent suppression of the translation and translation-linked turnover of driver oncoproteins, including MYC. A genome-wide CRISPR/Cas9 screen identified the CCR4-NOT complex as a major determinant of sensitivity to the combination therapy whose loss renders 4E-BP1 unresponsive to drug-induced dephosphorylation, thereby rescuing MYC translational suppression and promoting MYC stability. The central roles of CCR4-NOT and 4E-BP1 in response to the combination therapy were further underscored by the observation of CNOT1 loss and rescue of 4E-BP1 phosphorylation in TNBC cells that naturally evolved therapy resistance. Thus, pharmacological inhibition of CDK12/13 reveals a long-proposed EGFR dependence in TNBC that functions through the cooperative regulation of translation-coupled oncoprotein stability.

The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence.

Profilin-1, a member of the Profilin family, is a ubiquitously expressed protein that controls actin polymerization in a concentration-dependent manner. As mutations in the Profilin-1 gene have potential implications in neurodegenerative disease progression, well-characterized anti-Profilin-1 antibodies would be beneficial to the scientific community. In this study, we characterized sixteen Profilin-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence applications, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

The identification of high-preforming antibodies for Ubiquilin-2 for use in Western Blot, immunoprecipitation, and immunofluorescence.

Ubiquilin-2, a member of the ubiquilin protein family, plays a role in the regulation of various protein degradation pathways, and is mutated in some neurodegenerative diseases. Well-characterized anti-Ubiquilin-2 antibodies would advance reproducible research for Ubiquilin-2 and in turn, benefit the scientific community. In this study, we characterized ten Ubiquilin-2 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Long-term effects of volanesorsen on triglycerides and pancreatitis in patients with familial chylomicronaemia syndrome (FCS) in the UK Early Access to Medicines Scheme (EAMS).

BACKGROUND AND AIMS: The VOL4002 study assessed the efficacy and safety of volanesorsen in 22 adults with genetically confirmed familial chylomicronaemia syndrome (FCS) treated in the UK Early Access to Medicines Scheme (EAMS), with ("prior exposure") or without ("treatment naive") previous treatment in the APPROACH and/or APPROACH-OLE volanesorsen phase 3 studies. METHODS: Data collection focused on triglyceride (TG) levels, platelet counts and pancreatitis events. Pancreatitis incidence during volanesorsen treatment was compared against the 5-year period preceding volanesorsen exposure. Volanesorsen 285 mg was self-administered subcutaneously once every 2 weeks. RESULTS: Individual patient volanesorsen exposure ranged from 6 to 51 months (total cumulative exposure, 589 months). Among treatment-naive patients (n = 12), volanesorsen treatment resulted in an averaged median 52% reduction (-10.6 mmol/L) from baseline (26.4 mmol/L) in TG levels at 3 months, which were maintained through time points over 15 months of treatment (47%-55% reductions). Similarly, prior-exposure patients (n = 10) experienced a 51% reduction (-17.8 mmol/L) from pre-treatment baseline (28.0 mmol/L), with reductions of 10%-38% over 21 months of treatment. A comparison of pancreatitis event rates found a 74% reduction from the 5-year period before (one event/2.8 years) and during (one event/11.0 years) volanesorsen treatment. Platelet declines were consistent with observations in phase 3 clinical trials. No patient recorded a platelet count <50 × 109/L. CONCLUSIONS: This longitudinal study supports the efficacy of volanesorsen in patients with FCS for lowering TG levels over treatment periods up to 51 months with no apparent safety signals related to increased duration of exposure.

Epigenome editing of the CFTR-locus for treatment of cystic fibrosis.

BACKGROUND: Mechanisms governing the diversity of CFTR gene expression throughout the body are complex. Multiple intronic and distal regulatory elements are responsible for regulating differential CFTR expression across tissues. METHODS: Drawing on published data, 18 high-priority genomic regions were identified and interrogated for CFTR-enhancer function using CRISPR/dCas9-based epigenome editing tools. Each region was evaluated by dCas9p300 and dCas9KRAB for its ability to enhance or repress CFTR expression, respectively. RESULTS: Multiple genomic regions were tested for enhancer activity using CRISPR/dCas9 epigenome editing. dCas9p300 mediates a significant increase in CFTR mRNA levels when targeted to the promoter and a region 44 kb upstream of the transcriptional start site in a CFTR-low expressing cell line. Multiple gRNAs targeting the promoter induced a robust increase in CFTR protein levels. In contrast, dCas9KRAB-mediated repression is much more robust with 10 of the 18 evaluated genomic regions inducing CFTR protein knockdown. To evaluate the therapeutic efficacy of modulating CFTR gene regulation, dCas9p300 was used to induce elevated levels of CFTR from the endogenous locus in ΔF508/ΔF508 human bronchial epithelial cells. Ussing chamber studies demonstrated a synergistic increase in ion transport in response to CRISPR-induced expression of ΔF508 CFTR mRNA along with VX809 treatment. CONCLUSIONS: CRISPR/dCas9-based epigenome-editing provides a previously unexplored tool for interrogating CFTR enhancer function. Here, we demonstrate that therapeutic interventions that increase the expression of CFTR may improve the efficacy of CFTR modulators. A better understanding CFTR regulatory mechanisms could uncover novel therapeutic interventions for the development of cystic fibrosis therapies.

Causal network inference from gene transcriptional time-series response to glucocorticoids.

Gene regulatory network inference is essential to uncover complex relationships among gene pathways and inform downstream experiments, ultimately enabling regulatory network re-engineering. Network inference from transcriptional time-series data requires accurate, interpretable, and efficient determination of causal relationships among thousands of genes. Here, we develop Bootstrap Elastic net regression from Time Series (BETS), a statistical framework based on Granger causality for the recovery of a directed gene network from transcriptional time-series data. BETS uses elastic net regression and stability selection from bootstrapped samples to infer causal relationships among genes. BETS is highly parallelized, enabling efficient analysis of large transcriptional data sets. We show competitive accuracy on a community benchmark, the DREAM4 100-gene network inference challenge, where BETS is one of the fastest among methods of similar performance and additionally infers whether causal effects are activating or inhibitory. We apply BETS to transcriptional time-series data of differentially-expressed genes from A549 cells exposed to glucocorticoids over a period of 12 hours. We identify a network of 2768 genes and 31,945 directed edges (FDR ≤ 0.2). We validate inferred causal network edges using two external data sources: Overexpression experiments on the same glucocorticoid system, and genetic variants associated with inferred edges in primary lung tissue in the Genotype-Tissue Expression (GTEx) v6 project. BETS is available as an open source software package at https://github.com/lujonathanh/BETS.

Sex differences in cardiovascular morbidity associated with familial hypercholesterolaemia: A retrospective cohort study of the UK Simon Broome register linked to national hospital records.

BACKGROUND AND AIMS: The UK Simon Broome (SB) familial hypercholesterolaemia (FH) register previously reported 3-fold higher standardised mortality ratio for cardiovascular disease (CVD) in women compared to men from 2009 to 2015. Here we examined sex differences in CVD morbidity in FH by national linkage of the SB register with Hospital Episode Statistics (HES). METHODS: Of 3553 FH individuals in the SB register (aged 20-79 years at registration), 2988 (52.5% women) had linked HES records. Standardised Morbidity Ratios (SMbR) compared to an age and sex-matched UK general practice population were calculated [95% confidence intervals] for first CVD hospitalisation in HES (a composite of coronary heart disease (CHD), myocardial infarction (MI), stable or unstable angina, stroke, TIA, peripheral vascular disease (PVD), heart failure, coronary revascularisation interventions). RESULTS: At registration, men had significantly (p < 0.001) higher prevalence of previous CHD (24.8% vs 17.6%), previous MI (13.2% vs 6.3%), and were commenced on lipid-lowering treatment at a younger age than women (37.5 years vs 42.3 years). The SMbR for composite CVD was 6.83 (6.33-7.37) in men and 7.55 (6.99-8.15) in women. In individuals aged 30-50 years, SMbR in women was 50% higher than in men (15.04 [12.98-17.42] vs 10.03 [9.01-11.17]). In individuals >50 years, SMbR was 33% higher in women than men (6.11 [5.57-6.70] vs 4.59 [4.08-5.15]). CONCLUSIONS: Excess CVD morbidity due to FH remains markedly elevated in women at all ages, but especially those aged 30-50 years. This highlights the need for earlier diagnosis and optimisation of lipid-lowering risk factor management for all FH patients, with particular attention to young women with FH.

Non-HDL or LDL cholesterol in heterozygous familial hypercholesterolaemia: findings of the Simon Broome Register.

PURPOSE OF REVIEW: The role of non-HDL-C in the identification and management of lipid disorders is not clearly defined, although UK guidelines recommend its wider use in assessing the need for lipid-lowering therapy and as a treatment target. RECENT FINDINGS: We examined the implications of the use of non-HDL-C as opposed to LDL-C in 253 people with hypercholesterolaemia before treatment and 573 after treatment in whom fasting total serum cholesterol, HDL-C and LDL-C had been recorded and the diagnosis of heterozygous familial hypercholesterolemia (heFH) was investigated by genetic testing. The difference and the limits of agreement between non-HDL-C and LDL-C calculated using the Friedewald formula were assessed in those with and without heFH-causing mutations. SUMMARY: There were 147 mutation-positive and 106 mutation-negative pretreatment participants and 395 mutation-positive and 178 mutation-negative patients receiving treatment. The difference between non-HDL-C and LDL-C pretreatment in mutation-positive people (mean LDL-C 7.73 mmol/l) was 0.67 mmol/l (95% CI 0.62-0.73) and posttreatment (mean LDL-C 4.71 mmol/l) was 0.62 mmol/l (95% CI 0.59-0.65) with wide limits of agreement of -0.02 to 1.37 and 0.07-1.18 mmol/l, respectively. Among patients with heterozygous familial hypercholesterolaemia, use of estimated LDL-C derived from non-HDL-C in place of calculated LDL-C may result in diagnostic misclassification and difficulty in assessing the true reduction in LDL-C with treatment, because of the wide inter-individual limits of agreement around the mean difference between non-HDL-C and LDL-C.

Mental health during and after protests, riots and revolutions: A systematic review.

OBJECTIVES: Protests, riots and revolutions have long been a part of human history and are increasing globally, yet their impact on mental health remains largely unknown. We therefore systematically reviewed studies on collective actions and mental health. METHOD: We searched PubMed, Web of Science, PsycINFO and CINAHL Plus for published studies from their inception until 1 January 2018. Study quality was rated using the Newcastle-Ottawa Scale. RESULTS: We identified 52 studies (n = 57,487 participants) from 20 countries/regions. The prevalence of post-traumatic stress disorder ranged from 4% to 41% in riot-affected areas. Following a major protest, the prevalence of probable major depression increased by 7%, regardless of personal involvement in the protests, suggestive of community spillover effects. Risk factors for poorer mental health included female sex, lower socioeconomic status, exposure to violence, interpersonal conflicts, frequent social media use and lower resilience and social support. Nevertheless, two studies suggested that collective actions may reduce depression and suicide, possibly due to a collective cathartic experience and greater social cohesion within subpopulations. CONCLUSION: We present the first systematic review of collective actions and mental health, showing compelling evidence that protests even when nonviolent can be associated with adverse mental health outcomes. Health care professionals therefore need to be vigilant to the mental and psychological sequelae of protests, riots and revolutions. Further research on this emerging sociopolitical determinant of mental health is warranted.

Coronary heart disease mortality in severe vs. non-severe familial hypercholesterolaemia in the Simon Broome Register.

BACKGROUND AND AIMS: The International Atherosclerosis Society (IAS) has proposed that patients with "severe" FH (SFH) would warrant early and more aggressive cholesterol-lowering treatment such as with PCSK9 inhibitors. SFH is diagnosed if LDL-cholesterol (LDLC) > 10 mmol/L, or LDLC >8.0 mmol/L plus one high-risk feature, or LDLC >5 mmol/L plus two high-risk features. Here we compare CHD mortality in SFH and non-SFH (NSFH) patients in the UK prospective Simon Broome Register since 1991, when statin use became routine. METHODS: 2929 definite or possible PFH patients (51% women) aged 20-79 years were recruited from 21 UK lipid clinics and followed prospectively between 1992 and 2016. The excess CHD standardised mortality ratio (SMR) compared to the England and Wales population was calculated (with 95% confidence intervals). RESULTS: 1982 (67.7%) patients met the SFH definition. Compared to the non-SFH, significantly (p < 0.001) more SFH patients had diagnosed CHD at baseline (24.6% vs. 17.5%), were current smokers (21.9% vs 10.2%) and had a BMI > 30 kg/m2 (14.9% vs. 7.8%). The SMR for CHD mortality was significantly (p = 0.007) higher for SFH (220 (184-261) (34,134 person years, 129 deaths observed, vs. 59 expected) compared to NSFH of 144 (98-203) (15,432 person years, 32 observed vs. 22 expected). After adjustment for traditional risk factors, the Hazard Ratio for CHD mortality in SFH vs. NSFH was 1.22 (0.80-1.87) p = 0.36, indicating that the excess risk was largely accounted for by these factors. CONCLUSIONS: CHD mortality remains elevated in treated FH, especially for SFH, emphasising the importance of optimal lipid-lowering and management of other risk factors.

Estimation of the prevalence of cholesteryl ester storage disorder in a cohort of patients with clinical features of familial hypercholesterolaemia.

BACKGROUND AND AIM: Familial hypercholesterolaemia is caused by variants in the low-density lipoprotein cholesterol metabolic pathway involving LDLR, APOB and PCSK9 genes. A national genetic testing service in Wales, UK has observed that no familial hypercholesterolaemia variant is found in almost 80% patients with the familial hypercholesterolaemia phenotype. It has recently been suggested that some adult patients with a familial hypercholesterolaemia phenotype may have cholesteryl ester storage disease which can also present as a mixed hyperlipidaemia. The commonest genetic cause of cholesteryl ester storage disease is an exon 8 splice junction variant in the LIPA gene (rs116928232, c.894G>A; E8SJM) previously found to have an allele frequency of 0.0011 (1 in 450 individuals) in a large European population. This study investigated the prevalence of the E8SJM in patients with a familial hypercholesterolaemia phenotype in Wales, UK. METHOD: A total of 1203 patients with a clinical suspicion of familial hypercholesterolaemia but no familial hypercholesterolaemia variant were invited to participate. Of these, 668 patients provided informed written consent. Stored DNA samples from 663 patients were genotyped for the E8SJM variant. RESULTS: Three heterozygotes were identified (allele frequency 0.0023). Whole gene sequencing of the LIPA gene was undertaken in these three individuals, but no other variants were found. Therefore, there were no cholesteryl ester storage disease patients (homozygote or compound heterozygote) identified in this cohort. CONCLUSION: The allele frequency 0.0023 (1 in 221 individuals) for the E8SJM variant was more prevalent in this cohort than in a European population study; however, no cholesteryl ester storage disease homozygotes were identified. We found no evidence to support routine testing for cholesteryl ester storage disease in adult patients with a familial hypercholesterolaemia phenotype.

Human genome-wide measurement of drug-responsive regulatory activity.

Environmental stimuli commonly act via changes in gene regulation. Human-genome-scale assays to measure such responses are indirect or require knowledge of the transcription factors (TFs) involved. Here, we present the use of human genome-wide high-throughput reporter assays to measure environmentally-responsive regulatory element activity. We focus on responses to glucocorticoids (GCs), an important class of pharmaceuticals and a paradigmatic genomic response model. We assay GC-responsive regulatory activity across >108 unique DNA fragments, covering the human genome at >50×. Those assays directly detected thousands of GC-responsive regulatory elements genome-wide. We then validate those findings with measurements of transcription factor occupancy, histone modifications, chromatin accessibility, and gene expression. We also detect allele-specific environmental responses. Notably, the assays did not require knowledge of GC response mechanisms. Thus, this technology can be used to agnostically quantify genomic responses for which the underlying mechanism remains unknown.

Glucocorticoid receptor recruits to enhancers and drives activation by motif-directed binding.

Glucocorticoids are potent steroid hormones that regulate immunity and metabolism by activating the transcription factor (TF) activity of glucocorticoid receptor (GR). Previous models have proposed that DNA binding motifs and sites of chromatin accessibility predetermine GR binding and activity. However, there are vast excesses of both features relative to the number of GR binding sites. Thus, these features alone are unlikely to account for the specificity of GR binding and activity. To identify genomic and epigenetic contributions to GR binding specificity and the downstream changes resultant from GR binding, we performed hundreds of genome-wide measurements of TF binding, epigenetic state, and gene expression across a 12-h time course of glucocorticoid exposure. We found that glucocorticoid treatment induces GR to bind to nearly all pre-established enhancers within minutes. However, GR binds to only a small fraction of the set of accessible sites that lack enhancer marks. Once GR is bound to enhancers, a combination of enhancer motif composition and interactions between enhancers then determines the strength and persistence of GR binding, which consequently correlates with dramatic shifts in enhancer activation. Over the course of several hours, highly coordinated changes in TF binding and histone modification occupancy occur specifically within enhancers, and these changes correlate with changes in the expression of nearby genes. Following GR binding, changes in the binding of other TFs precede changes in chromatin accessibility, suggesting that other TFs are also sensitive to genomic features beyond that of accessibility.

Pre-established Chromatin Interactions Mediate the Genomic Response to Glucocorticoids.

The glucocorticoid receptor (GR) is a hormone-inducible transcription factor involved in metabolic and anti-inflammatory gene expression responses. To investigate what controls interactions between GR binding sites and their target genes, we used in situ Hi-C to generate high-resolution, genome-wide maps of chromatin interactions before and after glucocorticoid treatment. We found that GR binding to the genome typically does not cause new chromatin interactions to target genes but instead acts through chromatin interactions that already exist prior to hormone treatment. Both glucocorticoid-induced and glucocorticoid-repressed genes increased interactions with distal GR binding sites. In addition, while glucocorticoid-induced genes increased interactions with transcriptionally active chromosome compartments, glucocorticoid-repressed genes increased interactions with transcriptionally silent compartments. Lastly, while the architectural DNA-binding proteins CTCF and RAD21 were bound to most chromatin interactions, we found that glucocorticoid-responsive chromatin interactions were depleted for CTCF binding but enriched for RAD21. Together, these findings offer new insights into the mechanisms underlying GC-mediated gene activation and repression.

Clustering gene expression time series data using an infinite Gaussian process mixture model.

Transcriptome-wide time series expression profiling is used to characterize the cellular response to environmental perturbations. The first step to analyzing transcriptional response data is often to cluster genes with similar responses. Here, we present a nonparametric model-based method, Dirichlet process Gaussian process mixture model (DPGP), which jointly models data clusters with a Dirichlet process and temporal dependencies with Gaussian processes. We demonstrate the accuracy of DPGP in comparison to state-of-the-art approaches using hundreds of simulated data sets. To further test our method, we apply DPGP to published microarray data from a microbial model organism exposed to stress and to novel RNA-seq data from a human cell line exposed to the glucocorticoid dexamethasone. We validate our clusters by examining local transcription factor binding and histone modifications. Our results demonstrate that jointly modeling cluster number and temporal dependencies can reveal shared regulatory mechanisms. DPGP software is freely available online at https://github.com/PrincetonUniversity/DP_GP_cluster.