Probabilistic association of differentially expressed genes with cis-regulatory elements.

Differential gene expression in response to perturbations is mediated at least in part by changes in binding of transcription factors (TFs) and other proteins at specific genomic regions. Association of these cis-regulatory elements (CREs) with their target genes is a challenging task that is essential to address many biological and mechanistic questions. Many current approaches rely on chromatin conformation capture techniques or single-cell correlational methods to establish CRE-to-gene associations. These methods can be effective but have limitations, including resolution, gaps in detectable association distances, and cost. As an alternative, we have developed DegCre, a nonparametric method that evaluates correlations between measurements of perturbation-induced differential gene expression and differential regulatory signal at CREs to score possible CRE-to-gene associations. It has several unique features, including the ability to use any type of CRE activity measurement, yield probabilistic scores for CRE-to-gene pairs, and assess CRE-to-gene pairings across a wide range of sequence distances. We apply DegCre to six data sets, each using different perturbations and containing a variety of regulatory signal measurements, including chromatin openness, histone modifications, and TF occupancy. To test their efficacy, we compare DegCre associations to Hi-C loop calls and CRISPR-validated CRE-to-gene associations, establishing good performance by DegCre that is comparable or superior to competing methods. DegCre is a novel approach to the association of CREs to genes from a perturbation-differential perspective, with strengths that are complementary to existing approaches and allow for new insights into gene regulation.

Neuronal MAPT expression is mediated by long-range interactions with cis-regulatory elements.

Tauopathies are a group of neurodegenerative diseases defined by abnormal aggregates of tau, a microtubule-associated protein encoded by MAPT. MAPT expression is near absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression could be controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding disease risk and pathogenesis. Here, we performed chromatin conformation assays (HiC & Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27ac and CTCF in NPCs and differentiated neurons to nominate candidate cis-regulatory elements (cCREs). We assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in neurodegeneration-affected individuals and control subjects. We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the H1/H2 haplotype inversion breakpoint. We also found that rare and predicted damaging genetic variation in nominated CREs was nominally depleted in dementia-affected individuals relative to control subjects, consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduced MAPT expression, may be protective against neurodegenerative disease. Overall, this study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

MAPT expression is mediated by long-range interactions with cis-regulatory elements.

Background: Tauopathies are a group of neurodegenerative diseases driven by abnormal aggregates of tau, a microtubule associated protein encoded by the MAPT gene. MAPT expression is absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression is controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding genetic risk factors. Methods: We performed HiC, chromatin conformation capture (Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27Ac and CTCF in NPCs and neurons differentiated from human iPSC cultures. We nominated candidate cis-regulatory elements (cCREs) for MAPT in human NPCs, differentiated neurons, and pure cultures of inhibitory and excitatory neurons. We then assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in AD cases and controls. Results: Using orthogonal genomics approaches, we nominated 94 cCREs for MAPT, including the identification of cCREs specifically active in differentiated neurons. Eleven regions enhanced reporter gene transcription in luciferase assays. Using CRISPRi, 5 of the 94 regions tested were identified as necessary for MAPT expression as measured by RT-qPCR and RNA-seq. Rare and predicted damaging genetic variation in both nominated and confirmed CREs was depleted in AD cases relative to controls (OR = 0.40, p = 0.004), consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduce MAPT expression, may be protective against neurodegenerative disease. Conclusions: We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the well-described H1/H2 haplotype inversion breakpoint. This study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

Genome-wide strand asymmetry in massively parallel reporter activity favors genic strands.

Massively parallel reporter assays (MPRAs) are useful tools to characterize regulatory elements in human genomes. An aspect of MPRAs that is not typically the focus of analysis is their intrinsic ability to differentiate activity levels for a given sequence element when placed in both of its possible orientations relative to the reporter construct. Here, we describe pervasive strand asymmetry of MPRA signals in data sets from multiple reporter configurations in both published and newly reported data. These effects are reproducible across different cell types and in different treatments within a cell type and are observed both within and outside of annotated regulatory elements. From elements in gene bodies, MPRA strand asymmetry favors the sense strand, suggesting that function related to endogenous transcription is driving the phenomenon. Similarly, we find that within Alu mobile element insertions, strand asymmetry favors the transcribed strand of the ancestral retrotransposon. The effect is consistent across the multiplicity of Alu elements in human genomes and is more pronounced in less diverged Alu elements. We find sequence features driving MPRA strand asymmetry and show its prediction from sequence alone. We see some evidence for RNA stabilization and transcriptional activation mechanisms and hypothesize that the effect is driven by natural selection favoring efficient transcription. Our results indicate that strand asymmetry is a pervasive and reproducible feature in MPRA data. More importantly, the fact that MPRA asymmetry favors naturally transcribed strands suggests that it stems from preserved biological functions that have a substantial, global impact on gene and genome evolution.

TBCRC 002: a phase II, randomized, open-label trial of preoperative letrozole with or without bevacizumab in postmenopausal women with newly diagnosed stage 2/3 hormone receptor-positive and HER2-negative breast cancer.

BACKGROUND: In preclinical studies, the expression of vascular endothelial growth factor (VEGF) in hormone receptor-positive breast cancer is associated with estrogen-independent tumor growth and resistance to endocrine therapies. This study investigated whether the addition of bevacizumab, a monoclonal antibody against VEGF, to letrozole enhanced the antitumor activity of the letrozole in the preoperative setting. METHODS: Postmenopausal women with newly diagnosed stage 2 or 3 estrogen and/or progesterone receptor-positive, HER2-negative breast cancer were randomly assigned (2:1) between letrozole 2.5 mg PO daily plus bevacizumab 15 mg/kg IV every 3 weeks (Let/Bev) and letrozole 2.5 mg PO daily (Let) for 24 weeks prior to definitive surgery. Primary objective was within-arm pathologic complete remission (pCR) rate. Secondary objectives were safety, objective response, and downstaging rate. RESULTS: Seventy-five patients were randomized (Let/Bev n = 50, Let n = 25). Of the 45 patients evaluable for pathological response in the Let/Bev arm, 5 (11%; 95% CI, 3.7-24.1%) achieved pCR and 4 (9%; 95% CI, 2.5-21.2%) had microscopic residual disease; no pCRs or microscopic residual disease was seen in the Let arm (0%; 95% CI, 0-14.2%). The rates of downstaging were 44.4% (95% CI, 29.6-60.0%) and 37.5% (95% CI, 18.8-59.4%) in the Let/Bev and Let arms, respectively. Adverse events typically associated with letrozole (hot flashes, arthralgias, fatigue, myalgias) occurred in similar frequencies in the two arms. Hypertension, headache, and proteinuria were seen exclusively in the Let/Bev arm. The rates of grade 3 and 4 adverse events and discontinuation due to adverse events were 18% vs 8% and 16% vs none in the Let/Bev and Let arms, respectively. A small RNA-based classifier predictive of response to preoperative Let/Bev was developed and confirmed on an independent cohort. CONCLUSION: In the preoperative setting, the addition of bevacizumab to letrozole was associated with a pCR rate of 11%; no pCR was seen with letrozole alone. There was additive toxicity with the incorporation of bevacizumab. Responses to Let/Bev can be predicted from the levels of 5 small RNAs in a pretreatment biopsy. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov (Identifier: NCT00161291), first posted on September 12, 2005, and is completed.

CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries.

In small RNA (smRNA) sequencing studies, highly abundant molecules such as adapter dimer products and tissue-specific microRNAs (miRNAs) inhibit accurate quantification of lowly expressed species. We previously developed a method to selectively deplete highly abundant miRNAs. However, this method does not deplete adapter dimer ligation products that, unless removed by gel-separation, comprise most of the library. Here, we have adapted and modified recently described methods for CRISPR/Cas9-based Depletion of Abundant Species by Hybridization ('DASH') to smRNA-seq, which we have termed miRNA and Adapter Dimer-DASH (MAD-DASH). In MAD-DASH, Cas9 is complexed with single guide RNAs (sgRNAs) targeting adapter dimer ligation products, alongside highly expressed tissue-specific smRNAs, for cleavage in vitro. This process dramatically reduces adapter dimer and targeted smRNA sequences, can be multiplexed, shows minimal off-target effects, improves the quantification of lowly expressed miRNAs from human plasma and tissue derived RNA, and obviates the need for gel-separation, greatly increasing sample throughput. Additionally, the method is fully customizable to other smRNA-seq preparation methods. Like depletion of ribosomal RNA for mRNA-seq and mitochondrial DNA for ATAC-seq, our method allows for greater proportional read-depth of non-targeted sequences.

Discovery and Validation of Circulating Biomarkers of Colorectal Adenoma by High-Depth Small RNA Sequencing.

Purpose: Colorectal cancer is the third most common cancer worldwide, causing approximately 700,000 deaths each year. The majority of colorectal cancers begin as adenomas. Definitive screening for colorectal adenomas is currently accomplished through colonoscopy but, owing largely to costs and invasiveness, is typically limited to patient groups at higher risk by virtue of age or family history. We sought to determine if blood-based small RNA markers could detect colorectal adenoma.Experimental Design: We applied high-depth small RNA sequencing to plasma from a large (n = 189) cohort of patients, balanced for age, sex, and ancestry. Our analytical methodology allowed for the detection of both microRNAs and other small RNA species. We replicated sequencing results by qPCR on plasma samples from an independent cohort (n = 140).Results: We found several small RNA species with significant associations to colorectal adenoma, including both microRNAs and non-microRNA small RNAs. These associations were robust to correction for patient covariates, including age. Among the adenoma-associated small RNAs, two, a miR-335-5p isoform and an un-annotated small RNA, were validated by qPCR in an independent cohort. A classifier trained on measures of these two RNAs in the discovery cohort yields an AUC of 0.755 (0.775 with age) for adenoma detection in the independent cohort. This classifier accurately detects adenomas in patients under 50 and is robust to sex or ancestry.Conclusions: Circulating small RNAs (including but not limited to miRNAs) discovered by sequencing and validated by qPCR identify patients with colorectal adenomas effectively. Clin Cancer Res; 24(9); 2092-9. ©2018 AACR.

Genome-wide DNA methylation measurements in prostate tissues uncovers novel prostate cancer diagnostic biomarkers and transcription factor binding patterns.

BACKGROUND: Current diagnostic tools for prostate cancer lack specificity and sensitivity for detecting very early lesions. DNA methylation is a stable genomic modification that is detectable in peripheral patient fluids such as urine and blood plasma that could serve as a non-invasive diagnostic biomarker for prostate cancer. METHODS: We measured genome-wide DNA methylation patterns in 73 clinically annotated fresh-frozen prostate cancers and 63 benign-adjacent prostate tissues using the Illumina Infinium HumanMethylation450 BeadChip array. We overlaid the most significantly differentially methylated sites in the genome with transcription factor binding sites measured by the Encyclopedia of DNA Elements consortium. We used logistic regression and receiver operating characteristic curves to assess the performance of candidate diagnostic models. RESULTS: We identified methylation patterns that have a high predictive power for distinguishing malignant prostate tissue from benign-adjacent prostate tissue, and these methylation signatures were validated using data from The Cancer Genome Atlas Project. Furthermore, by overlaying ENCODE transcription factor binding data, we observed an enrichment of enhancer of zeste homolog 2 binding in gene regulatory regions with higher DNA methylation in malignant prostate tissues. CONCLUSIONS: DNA methylation patterns are greatly altered in prostate cancer tissue in comparison to benign-adjacent tissue. We have discovered patterns of DNA methylation marks that can distinguish prostate cancers with high specificity and sensitivity in multiple patient tissue cohorts, and we have identified transcription factors binding in these differentially methylated regions that may play important roles in prostate cancer development.

aRNApipe: a balanced, efficient and distributed pipeline for processing RNA-seq data in high-performance computing environments.

SUMMARY: The wide range of RNA-seq applications and their high-computational needs require the development of pipelines orchestrating the entire workflow and optimizing usage of available computational resources. We present aRNApipe, a project-oriented pipeline for processing of RNA-seq data in high-performance cluster environments. aRNApipe is highly modular and can be easily migrated to any high-performance computing (HPC) environment. The current applications included in aRNApipe combine the essential RNA-seq primary analyses, including quality control metrics, transcript alignment, count generation, transcript fusion identification, alternative splicing and sequence variant calling. aRNApipe is project-oriented and dynamic so users can easily update analyses to include or exclude samples or enable additional processing modules. Workflow parameters are easily set using a single configuration file that provides centralized tracking of all analytical processes. Finally, aRNApipe incorporates interactive web reports for sample tracking and a tool for managing the genome assemblies available to perform an analysis. AVAILABILITY AND DOCUMENTATION: https://github.com/HudsonAlpha/aRNAPipe ; DOI: 10.5281/zenodo.202950. CONTACT: rmyers@hudsonalpha.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genomic regulation of invasion by STAT3 in triple negative breast cancer.

Breast cancer is a heterogeneous disease comprised of four molecular subtypes defined by whether the tumor-originating cells are luminal or basal epithelial cells. Breast cancers arising from the luminal mammary duct often express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2). Tumors expressing ER and/or PR are treated with anti-hormonal therapies, while tumors overexpressing HER2 are targeted with monoclonal antibodies. Immunohistochemical detection of ER, PR, and HER2 receptors/proteins is a critical step in breast cancer diagnosis and guided treatment. Breast tumors that do not express these proteins are known as "triple negative breast cancer" (TNBC) and are typically basal-like. TNBCs are the most aggressive subtype, with the highest mortality rates and no targeted therapy, so there is a pressing need to identify important TNBC tumor regulators. The signal transducer and activator of transcription 3 (STAT3) transcription factor has been previously implicated as a constitutively active oncogene in TNBC. However, its direct regulatory gene targets and tumorigenic properties have not been well characterized. By integrating RNA-seq and ChIP-seq data from 2 TNBC tumors and 5 cell lines, we discovered novel gene signatures directly regulated by STAT3 that were enriched for processes involving inflammation, immunity, and invasion in TNBC. Functional analysis revealed that STAT3 has a key role regulating invasion and metastasis, a characteristic often associated with TNBC. Our findings suggest therapies targeting STAT3 may be important for preventing TNBC metastasis.

Distinct gene regulatory programs define the inhibitory effects of liver X receptors and PPARG on cancer cell proliferation.

BACKGROUND: The liver X receptors (LXRs, NR1H2 and NR1H3) and peroxisome proliferator-activated receptor gamma (PPARG, NR1C3) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis. Intriguingly, recent studies suggest that these metabolic regulators also impact tumor cell proliferation. However, a comprehensive temporal molecular characterization of the LXR and PPARG gene regulatory responses in tumor cells is still lacking. METHODS: To better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis, we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics, and nuclear receptor binding patterns. RESULTS: Our results illustrate that the activation of both nuclear receptors inhibited cell proliferation and further decreased glutathione levels, consistent with increased cellular oxidative stress. Despite a common metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points. CONCLUSIONS: Through the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome. These results further provide a detailed molecular map of metabolic reprogramming in cancer cells through LXR and PPARG activation. As ligand-inducible TFs, these nuclear receptors can potentially serve as attractive therapeutic targets for the treatment of various cancers.

USP6 oncogene promotes Wnt signaling by deubiquitylating Frizzleds.

The Wnt signaling pathways play pivotal roles in carcinogenesis. Modulation of the cell-surface abundance of Wnt receptors is emerging as an important mechanism for regulating sensitivity to Wnt ligands. Endocytosis and degradation of the Wnt receptors Frizzled (Fzd) and lipoprotein-related protein 6 (LRP6) are regulated by the E3 ubiquitin ligases zinc and ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43), which are disrupted in cancer. In a genome-wide small interfering RNA screen, we identified the deubiquitylase ubiquitin-specific protease 6 (USP6) as a potent activator of Wnt signaling. USP6 enhances Wnt signaling by deubiquitylating Fzds, thereby increasing their cell-surface abundance. Chromosomal translocations in nodular fasciitis result in USP6 overexpression, leading to transcriptional activation of the Wnt/ß-catenin pathway. Inhibition of Wnt signaling using Dickkopf-1 (DKK1) or a Porcupine (PORCN) inhibitor significantly decreased the growth of USP6-driven xenograft tumors, indicating that Wnt signaling is a key target of USP6 during tumorigenesis. Our study defines an additional route to ectopic Wnt pathway activation in human disease, and identifies a potential approach to modulate Wnt signaling for therapeutic benefit.

Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites.

Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type-specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type-specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites.

CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins.

Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable data sets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIP-seq through epitope tagging of endogenous TFs. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR epitope tagging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several DNA-binding proteins spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIP-quality antibodies are not available.

Blocking of targeted microRNAs from next-generation sequencing libraries.

Highly abundant microRNAs (miRNAs) in small RNA sequencing libraries make it difficult to obtain efficient measurements of more lowly expressed species. We present a new method that allows for the selective blocking of specific, abundant miRNAs during preparation of sequencing libraries. This technique is specific with little off-target effects and has no impact on the reproducibility of the measurement of non-targeted species. In human plasma samples, we demonstrate that blocking of highly abundant hsa-miR-16-5p leads to improved detection of lowly expressed miRNAs and more precise measurement of differential expression overall. Furthermore, we establish the ability to target a second abundant miRNA and to multiplex the blocking of two miRNAs simultaneously. For small RNA sequencing, this technique could fill a similar role as do ribosomal or globin removal technologies in messenger RNA sequencing.

Recurrent read-through fusion transcripts in breast cancer.

Read-through fusion transcripts that result from the splicing of two adjacent genes in the same coding orientation are a recently discovered type of chimeric RNA. We sought to determine if read-through fusion transcripts exist in breast cancer. We performed paired-end RNA-seq of 168 breast samples, including 28 breast cancer cell lines, 42 triple negative breast cancer primary tumors, 42 estrogen receptor positive (ER+) breast cancer primary tumors, and 56 non-malignant breast tissue samples. We analyzed the sequencing data to identify breast cancer associated read-through fusion transcripts. We discovered two recurrent read-through fusion transcripts that were identified in breast cancer cell lines, confirmed across breast cancer primary tumors, and were not detected in normal tissues (SCNN1A-TNFRSF1A and CTSD-IFITM10). Both fusion transcripts use canonical splice sites to join the last splice donor of the 5' gene to the first splice acceptor of the 3' gene, creating an in-frame fusion transcript. Western blots indicated that the fusion transcripts are translated into fusion proteins in breast cancer cells. Custom small interfering RNAs targeting the CTSD-IFITM10 fusion junction reduced expression of the fusion transcript and reduced breast cancer cell proliferation. Read-through fusion transcripts between adjacent genes with different biochemical functions represent a new type of recurrent molecular defect in breast cancer that warrant further investigation as potential biomarkers and therapeutic targets. Both breast cancer associated fusion transcripts identified in this study involve membrane proteins (SCNN1A-TNFRSF1A and CTSD-IFITM10), which raises the possibility that they could be breast cancer-specific cell surface markers.

Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer.

BACKGROUND: Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. RESULTS: We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. CONCLUSION: Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of beta-catenin/GSK3 signaling.

The multi-protein beta-catenin destruction complex tightly regulates beta-catenin protein levels by shuttling beta-catenin to the proteasome. Glycogen synthase kinase 3beta (GSK3beta), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark beta-catenin for ubiquitination and subsequent degradation. Because modulation of both beta-catenin and GSK3beta activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the beta-catenin/GSK3beta interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a beta-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3'oxime (BIO), a specific inhibitor of GSK3beta. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of beta-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear beta-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3beta and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFalpha, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of beta-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.

Bruton's tyrosine kinase revealed as a negative regulator of Wnt-beta-catenin signaling.

Wnts are secreted ligands that activate several receptor-mediated signal transduction cascades. Homeostatic Wnt signaling through beta-catenin is required in adults, because either elevation or attenuation of beta-catenin function has been linked to diverse diseases. To contribute to the identification of both protein and pharmacological regulators of this pathway, we describe a combinatorial screen that merged data from a high-throughput screen of known bioactive compounds with an independent focused small interfering RNA screen. Each screen independently revealed Bruton's tyrosine kinase (BTK) as an inhibitor of Wnt-beta-catenin signaling. Loss of BTK function in human colorectal cancer cells, human B cells, zebrafish embryos, and cells derived from X-linked agammaglobulinemia patients with a mutant BTK gene resulted in elevated Wnt-beta-catenin signaling, confirming that BTK acts as a negative regulator of this pathway. From affinity purification-mass spectrometry and biochemical binding studies, we found that BTK directly interacts with a nuclear component of Wnt-beta-catenin signaling, CDC73. Further, we show that BTK increased the abundance of CDC73 in the absence of stimulation and that CDC73 acted as a repressor of beta-catenin-mediated transcription in human colorectal cancer cells and B cells.

New regulators of Wnt/beta-catenin signaling revealed by integrative molecular screening.

The identification and characterization of previously unidentified signal transduction molecules has expanded our understanding of biological systems and facilitated the development of mechanism-based therapeutics. We present a highly validated small interfering RNA (siRNA) screen that functionally annotates the human genome for modulation of the Wnt/beta-catenin signal transduction pathway. Merging these functional data with an extensive Wnt/beta-catenin protein interaction network produces an integrated physical and functional map of the pathway. The power of this approach is illustrated by the positioning of siRNA screen hits into discrete physical complexes of proteins. Similarly, this approach allows one to filter discoveries made through protein-protein interaction screens for functional contribution to the phenotype of interest. Using this methodology, we characterized AGGF1 as a nuclear chromatin-associated protein that participates in beta-catenin-mediated transcription in human colon cancer cells.