Genome-wide Fashion Research Articles

Differential Analysis of Protein-DNA Binding Using ChIP-Seq Data.

Chromatin immunoprecipitation in combination with next-generation sequencing (ChIP-Seq) allows probing of protein-DNA binding in a rapid and genome-wide fashion. Herein we describe the required steps to preprocess ChIP-Seq data and to analyze the differential binding of proteins to DNA for perturbation experiments. In these experiments, different conditions are compared to find the underlying biological mechanisms caused by the stimulus or treatment. In addition, we provide a sample analysis using the steps outlined in the chapter.

P07.11.B A NOVEL CRISPR/CAS9 KNOCKOUT LIBRARY UNCOVERS PAN-ESSENTIAL MIRNAS IN HUMAN CANCER CELL LINES

Abstract BACKGROUND MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play an important role during cancer initiation and progression. Genome-wide functional screening using the CRISPR/Cas9 system is a powerful tool to uncover genetic dependencies for distinct tumor entities, and has revealed novel insights into pan-cancer fitness genes. Current libraries are focused on single guide RNAs (sgRNAs) targeting protein-coding genes, thereby limiting functional genomics-based investigations of miRNA function. MATERIAL AND METHODS We here designed a novel CRISPR/Cas9 knockout library of 8,107 distinct sgRNAs targeting a total of 1,769 human miRNAs. Using a total of 45 human cancer cell lines, representing 16 different tumor entities, we performed negative selection screens to identify miRNA fitness genes. Screen hits per cell line were scored on the basis of a combination of a supervised Bayesian gene essentiality analysis (BAGEL2) and an unsupervised gene ranking algorithm (MAGeCK). miRNA genes that scored across all cell lines, i.e. potentially pan-essential miRNAs, were identified using a combination of the Adaptive Daisy Model (ADaM) algorithm and the Fitness Percentile (FiPer) method. RESULTS In silico analyses revealed that our library has significantly lower off-target activity for protein-coding genes as compared to previously described miRNA-targeting sgRNA libraries, while providing a higher coverage of miRNAs present in the human genome. In total, only 13% (n=230) of all targeted miRNAs induced a fitness effect on one or more cell lines, and the majority (83%) of these genes induced a dependency in less than 50% of the tested cell lines. Pan-cancer fitness gene detection by ADaM or FiPer differed solely on the level of stringency. As such, ADaM and FiPer identified 34 core fitness or 54 common essential miRNAs, respectively, and almost all core fitness genes (97%) were identified as common essential. Preliminary network analyses revealed that protein-coding genes that are regulated by core fitness miRNAs are significantly enriched for cell cycle and p53 signaling networks, providing a first rationale for their dependency profiles in human cancer cell lines. CONCLUSION We here established a novel method for functional investigation of miRNAs by means of genome-wide CRISPR/Cas9 knockout screens. Our sgRNA library outperforms previously established libraries in terms of specificity and overall miRNA coverage, making it a novel tool to investigate miRNA function in a genome-wide fashion. We here use this library to define a first-of-its-kind genome-wide functional annotation of miRNAs, revealing a subset of pan-cancer fitness miRNAs that might regulate essential cellular processes such as cell cycle progression. Our data highlight the wide potential of this library in investigating miRNA function in diverse biological settings.

CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells.

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines-K562, ST486, HepG2, and MCF7-which revealed several essential E-boxes and genes. Among them, we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression, and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.

Prediction accuracy of regulatory elements from sequence varies by functional sequencing technique.

Various sequencing based approaches are used to identify and characterize the activities of cis-regulatory elements in a genome-wide fashion. Some of these techniques rely on indirect markers such as histone modifications (ChIP-seq with histone antibodies) or chromatin accessibility (ATAC-seq, DNase-seq, FAIRE-seq), while other techniques use direct measures such as episomal assays measuring the enhancer properties of DNA sequences (STARR-seq) and direct measurement of the binding of transcription factors (ChIP-seq with transcription factor-specific antibodies). The activities of cis-regulatory elements such as enhancers, promoters, and repressors are determined by their sequence and secondary processes such as chromatin accessibility, DNA methylation, and bound histone markers. Here, machine learning models are employed to evaluate the accuracy with which cis-regulatory elements identified by various commonly used sequencing techniques can be predicted by their underlying sequence alone to distinguish between cis-regulatory activity that is reflective of sequence content versus secondary processes. Models trained and evaluated on D. melanogaster sequences identified through DNase-seq and STARR-seq are significantly more accurate than models trained on sequences identified by H3K4me1, H3K4me3, and H3K27ac ChIP-seq, FAIRE-seq, and ATAC-seq. These results suggest that the activity detected by DNase-seq and STARR-seq can be largely explained by underlying DNA sequence, independent of secondary processes. Experimentally, a subset of DNase-seq and H3K4me1 ChIP-seq sequences were tested for enhancer activity using luciferase assays and compared with previous tests performed on STARR-seq sequences. The experimental data indicated that STARR-seq sequences are substantially enriched for enhancer-specific activity, while the DNase-seq and H3K4me1 ChIP-seq sequences are not. Taken together, these results indicate that the DNase-seq approach identifies a broad class of regulatory elements of which enhancers are a subset and the associated data are appropriate for training models for detecting regulatory activity from sequence alone, STARR-seq data are best for training enhancer-specific sequence models, and H3K4me1 ChIP-seq data are not well suited for training and evaluating sequence-based models for cis-regulatory element prediction.

A new pipeline denoted SPICE identifies novel JUN-IKZF1 composite elements

Abstract DNA composite elements allow combinatorial transcription factor binding, which is important for fine tuning gene expression in response to physiological stimuli and in pathological conditions. Moreover, composite elements may contribute to epigenetic regulation. Accordingly, developing screening algorithms to identify novel composite elements and their compositions is crucial. Here we report a pipeline termed Spacing Preference Identification of Composite Elements (SPICE), that systematically predicts novel protein binding partners along with their DNA motifs and spacing preferences in a genome wide fashion. Besides predicting known composite elements like AP1-IRF composite elements (AICE) and STAT5 tetramers, SPICE de novo predicted a range of novel binding partners, including JUN-IKZF1 composite elements. We validated JUN-IKZF1 association by co-immunoprecipitation and showed cooperative binding of these factors to an upstream conserved noncoding region, CNS9, in the human IL10 gene based on electrophoretic mobility shift assays. Furthermore, the activity of an IL10 luciferase reporter depended on both AP1 and IKZF1 binding sites within this composite element in mouse B and T cells, confirming its transcriptional relevance. These data establish SPICE as a valuable pipeline for predicting novel transcription factor complexes that regulate gene transcription and provide insights into the biological cooperation of IKZF1 and AP1. Division of Intramural Research, NHLBI.

Abstract 4311: Using multiomics analysis to identify dysregulated transcription factors in non-small cell lung cancer (NSCLC) to drive the expression of a cancer-activated synthetic biomarker

Abstract Early detection of cancer is an important driver of increased survival, quality of life and reduced healthcare costs. Earli is developing a highly sensitive, orthogonal approach that uses a genetic construct that usurps dysregulated pathways and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. Identification of abnormally elevated transcription factors (TF) in cancer is crucial when developing a cancer-activated expression platform. Evaluating TF function in a genome wide fashion is a challenge, especially since TF activity is not solely reflected by just its RNA expression or even by protein abundance, but also its post-translational modification including phosphorylation. In this study, we use the publicly available multiomics dataset from Clinical Proteomic Tumor Analysis Consortium (CPTAC) which includes RNA-seq based transcriptional profiling, MS based protein abundance and phosphorylation from 211 paired tumor and normal adjacent samples derived from NSCLC patients. Performing Multiomics Factor Analysis (MOFA) analysis, we identified a short list of 34 dysregulated TFs in NSCLC that displayed high expression levels in at least two of the three omics platforms relative to normal tissues, six of which scored highly across all three. A subset of candidate TF binding sequences were subcloned as multimers into expression constructs with a basal promoter and empirically evaluated in multiple cancer cell lines, including patient-derived xenograft (PDX) cell lines and normal cell lines.Transfection experiments demonstrated that these novel chimeric synthetic promoters could produce robust levels of expression in PDX-derived cell lines that was 10-20x higher than expression mediated by known cancer-activated promoters such as the endogenous survivin (BIRC5) promoter. Furthermore, levels achieved were only 3-4 fold lower than a control EF-1a promoter, one of the strongest promoters, typically used to drive constitutive expression in mammalian cells. Currently, a small subset of these chimeric constructs is being tested in murine tumor models and in large animal cancer models such as oncopigs. These experiments will further drive the development of this approach for use in early cancer detection. Citation Format: Yue Wendy Zhang, Shireen Rudina, Dariusz Wodziak, Chloe Xia, Maggie Louie, Albert Park, David Suhy. Using multiomics analysis to identify dysregulated transcription factors in non-small cell lung cancer (NSCLC) to drive the expression of a cancer-activated synthetic biomarker. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4311.

Capture Methylation-Sensitive Restriction Enzyme Sequencing (Capture MRE-Seq) for Methylation Analysis of Highly Degraded DNA Samples.

Understanding the impact of DNA methylation within different disease contexts often requires accurate assessment of these modifications in a genome-wide fashion. Frequently, patient-derived tissues stored in long-term hospital tissue banks have been preserved using formalin-fixation paraffin-embedding (FFPE). While these samples can comprise valuable resources for studying disease, the fixation process ultimately compromises the DNA's integrity and leads to degradation. Degraded DNA can complicate CpG methylome profiling using traditional techniques, particularly when performing methylation-sensitive restriction enzyme sequencing (MRE-seq), yielding high backgrounds and resulting in lowered library complexity. Here, we describe Capture MRE-seq, a new MRE-seq protocol tailored to preserving unmethylated CpG information when using samples with highly degraded DNA. The results using Capture MRE-seq correlate well (0.92) with traditional MRE-seq calls when profiling non-degraded samples, and can recover unmethylated regions in highly degraded samples when traditional MRE-seq fails, which we validate using bisulfite sequencing-based data (WGBS) as well as methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq).

Temporal analysis suggests a reciprocal relationship between 3D chromatin structure and transcription.

To infer potential causal relationships between 3D chromatin structure, enhancers, and gene transcription, we mapped each feature in a genome-wide fashion across eight narrowly spaced time points of macrophage activation. Enhancers and genes connected by loops exhibit stronger correlations between histone H3K27 acetylation and expression than can be explained by genomic distance or physical proximity alone. At these looped enhancer-promoter pairs, changes in acetylation at distal enhancers precede changes in gene expression. Changes in gene expression exhibit a directional bias at differential loop anchors; gained loops are associated with increased expression of genes oriented away from the center of the loop, and lost loops are often accompanied by high levels of transcription within the loop boundaries themselves. These results are consistent with a reciprocal relationship where loops can facilitate increased transcription by connecting promoters to distal enhancers, whereas high levels of transcription can impede loop formation.

Stress Conditions Modulate the Chromatin Interactions Network in Arabidopsis.

Stresses have been known to cause various responses like cellular physiology, gene regulation, and genome remodeling in the organism to cope and survive. Here, we assessed the impact of stress conditions on the chromatin-interactome network of Arabidopsis thaliana. We identified thousands of chromatin interactions in native as well as in salicylic acid treatment and high temperature conditions in a genome-wide fashion. Our analysis revealed the definite pattern of chromatin interactions and stress conditions could modulate the dynamics of chromatin interactions. We found the heterochromatic region of the genome actively involved in the chromatin interactions. We further observed that the establishment or loss of interactions in response to stress does not result in the global change in the expression profile of interacting genes; however, interacting regions (genes) containing motifs for known TFs showed either lower expression or no difference than non-interacting genes. The present study also revealed that interactions preferred among the same epigenetic state (ES) suggest interactions clustered the same ES together in the 3D space of the nucleus. Our analysis showed that stress conditions affect the dynamics of chromatin interactions among the chromatin loci and these interaction networks govern the folding principle of chromatin by bringing together similar epigenetic marks.

The role of reciprocal fusions in MLL-r acute leukemia: studying the chromosomal translocation t(6;11)

Leukemia patients bearing t(6;11)(q27;q23) translocations can be divided in two subgroups: those with breakpoints in the major breakpoint cluster region of MLL (introns 9–10; associated mainly with AML M1/4/5), and others with breakpoints in the minor breakpoint cluster region (introns 21–23), associated with T-ALL. We cloned all four of the resulting fusion genes (MLL-AF6, AF6-MLL, exMLL-AF6, AF6-shMLL) and subsequently transfected them to generate stable cell culture models. Their molecular function was tested by inducing gene expression for 48 h in a Doxycycline-dependent fashion. Here, we present our results upon differential gene expression (DGE) that were obtained by the “Massive Analyses of cDNA Ends” (MACE-Seq) technology, an established 3′-end based RNA-Seq method. Our results indicate that the PHD/BD domain, present in the AF6-MLL and the exMLL-AF6 fusion protein, is responsible for chromatin activation in a genome-wide fashion. This led to strong deregulation of transcriptional processes involving protein-coding genes, pseudogenes, non-annotated genes, and RNA genes, e.g., LincRNAs and microRNAs, respectively. While cooperation between the MLL-AF6 and AF6-MLL fusion proteins appears to be required for the above-mentioned effects, exMLL-AF6 is able to cause similar effects on its own. The exMLL-AF6/AF6-shMLL co-expressing cell line displayed the induction of a myeloid-specific and a T-cell specific gene signature, which may explain the T-ALL disease phenotype observed in patients with such breakpoints. This again demonstrated that MLL fusion proteins are instructive and allow to study their pathomolecular mechanisms.

INCISOR: An Algorithm to Identify Synthetic Rescue Mediators of Resistance to Targeted and Immunotherapy.

Despite the success of targeted therapies including immunotherapies in cancer treatments, tumor resistance to targeted therapies remains a fundamental challenge. Tumors can evolve resistance to a therapy that targets one gene by acquiring compensatory alterations in another gene, such compensatory interaction between two genes is referred to as synthetic rescue (SR) interactions. To identify SRs, here we describe an algorithm, INCISOR, that leverages tumor transcriptomics and clinical information from10,000 patients as well as data from experimental screens. INCISOR can identify SRs that are common across several cancer-types in genome-wide fashion by sifting through half a billion possible gene-gene combinations and provide a framework to design therapies to tackle resistance.

Measuring Chromatin Accessibility: ATAC-Seq.

Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) is a method to investigate the accessibility of chromatin in a genome-wide fashion. In this chapter, we provide a brief history of the chromatin accessibility field followed by a detailed protocol to perform ATAC-Seq assay.

Breast Cancer Detection and Treatment Monitoring Using a Noninvasive Prenatal Testing Platform: Utility in Pregnant and Nonpregnant Populations.

Numerous publications have reported the incidental detection of occult malignancies upon routine noninvasive prenatal testing (NIPT). However, these studies were not designed to evaluate the NIPT performance for cancer detection. We investigated the sensitivity of a genome-wide NIPT pipeline, called GIPSeq, for detecting cancer-specific copy number alterations (CNAs) in plasma tumor DNA (ctDNA) of patients with breast cancer. To assess whether a pregnancy itself, with fetal cell-free DNA (cfDNA) in the maternal circulation, might influence the detection of ctDNA, results were compared in pregnant (n = 25) and nonpregnant (n = 25) cancer patients. Furthermore, the ability of GIPSeq to monitor treatment response was assessed. Overall GIPSeq sensitivity for detecting cancer-specific CNAs in plasma cfDNA was 26%. Fifteen percent of detected cases were asymptomatic at the time of blood sampling. GIPSeq sensitivity mainly depended on the tumor stage. Also, triple negative breast cancers (TNBC) were more frequently identified compared to hormone-positive or HER2-enriched tumors. This might be due to the presence of high-level gains and losses of cfDNA or high ctDNA loads in plasma of TNBC. Although higher GIPSeq sensitivity was noted in pregnant (36%) than in nonpregnant women (16%), the limited sample size prohibits a definite conclusion. Finally, GIPSeq profiling of cfDNA during therapy allowed monitoring of early treatment response. The results underscore the potential of NIPT-based tests, analyzing CNAs in plasma cfDNA in a genome-wide and unbiased fashion for breast cancer detection, cancer subtyping and treatment monitoring in a pregnant and nonpregnant target population.

The human malaria parasite genome is configured into thousands of coexpressed linear regulatory units

The human malaria parasite Plasmodium falciparum thrives in radically different host environments in mosquitoes and humans, with only a limited set of transcription factors. The nature of regulatory elements or their target genes in the P. falciparum genome remains elusive. Here, we found that this eukaryotic parasite uses an efficient way to maximally use genetic and epigenetic regulation to form regulatory units (RUs) during blood infections. Genes located in the same RU tend to have the same pattern of expression over time and are associated with open chromatin along regulatory elements. To precisely define and quantify these RUs, a novel hidden Markov model was developed to capture the regulatory structure in a genome-wide fashion by integrating expression and epigenetic evidence. We successfully identified thousands of RUs and cross-validated with previous findings. We found more genes involved in red blood cell (RBC) invasion located in the same RU as the PfAP2-I (AP2-I) transcription factor, demonstrating that AP2-I is responsible for regulating RBC invasion. Our study has provided a regulatory mechanism for a compact eukaryotic genome and offers new insights into the in vivo transcriptional regulation of the P. falciparum intraerythrocytic stage.

3076 – FINE-TUNING GENE EXPRESSION BY CRISPRA IN MOUSE EMBRYONIC STEM CELLS FOR SPECIFIC TISSUE INDUCTION

Hematopoietic stem cells (HSCs) constitute the base of the hematopoietic system, giving rise to every blood cell type. Because of their clinical potential, numerous approaches have been tackled trying to generate HSCs in vitro for regenerative medicine purposes. However, due to the complexity HSCs development has, still there is not a robust way to generate bona-fide HSCs, bringing out the necessity to explore alternatives to identify genetic programmes that could lead in a robust generation of this kind of cells. In this way, gene activation by alternative CRISPR (clustered regularly interspaced short palindromic repeats) tools is a novel approach to activate endogenous genes by the simple use of gRNAs targeting promoter regions. This platform can be used in CRISPRa-based screening using library of gRNAs targeting gene promoters in a genome-wide and unbiased fashion, or alternatively can be used to activate the transcription of specific groups of genes. To test the best CRISPRa system to be used in mESCs, we generated mESCs containing the two most widely used CRISPRa systems (SunTag and SAM) that can induce a gene activation constitutively. In mESCs, SunTag induces a more robust gene expression than SAM in all the genes tested. In parallel, we have generated two inducible CRISPRa (iCRISPRa) mESC lines, VPR and iSAM. We will present the comparison of all the different CRISPRa systems in mESC and in hemogenic differentiated cells to determine the most robust iCRISPRa system in mouse embryonic stem cells differentiation system.

Systematic identification of silencers in human cells.

The majority of the human genome does not encode proteins. Many of these noncoding regions contain important regulatory sequences that control gene expression. To date, most studies have focused on activators such as enhancers, but regions that repress gene expression—silencers—have not been systematically studied. We have developed a system that identifies silencer regions in a genome-wide fashion on the basis of silencer-mediated transcriptional repression of caspase 9. We found that silencers are widely distributed and may function in a tissue-specific fashion. These silencers harbor unique epigenetic signatures and are associated with specific transcription factors. Silencers also act at multiple genes, and at the level of chromosomal domains and long-range interactions. Deletion of silencer regions linked to the drug transporter genes ABCC2 and ABCG2 caused chemo-resistance. Overall, our study demonstrates that tissue-specific silencing is widespread throughout the human genome and likely contributes substantially to the regulation of gene expression and human biology.

Transcriptional effects of copy number alterations in a large set of human cancers

Copy number alterations (CNAs) can promote tumor progression by altering gene expression levels. Due to transcriptional adaptive mechanisms, however, CNAs do not always translate proportionally into altered expression levels. By reanalyzing >34,000 gene expression profiles, we reveal the degree of transcriptional adaptation to CNAs in a genome-wide fashion, which strongly associate with distinct biological processes. We then develop a platform-independent method—transcriptional adaptation to CNA profiling (TACNA profiling)—that extracts the transcriptional effects of CNAs from gene expression profiles without requiring paired CNA profiles. By applying TACNA profiling to >28,000 patient-derived tumor samples we define the landscape of transcriptional effects of CNAs. The utility of this landscape is demonstrated by the identification of four genes that are predicted to be involved in tumor immune evasion when transcriptionally affected by CNAs. In conclusion, we provide a novel tool to gain insight into how CNAs drive tumor behavior via altered expression levels.

A comparative epigenome analysis of gammaherpesviruses suggests cis-acting sequence features as critical mediators of rapid polycomb recruitment.

Latent Kaposi sarcoma-associated herpesvirus (KSHV) genomes rapidly acquire distinct patterns of the activating histone modification H3K4-me3 as well as repressive H3K27-me3 marks, a modification linked to transcriptional silencing by polycomb repressive complexes (PRC). Interestingly, PRCs have recently been reported to restrict viral gene expression in a number of other viral systems, suggesting they may play a broader role in controlling viral chromatin. If so, it is an intriguing possibility that latency establishment may result from viral subversion of polycomb-mediated host responses to exogenous DNA.To investigate such scenarios we sought to establish whether rapid repression by PRC constitutes a general hallmark of herpesvirus latency. For this purpose, we performed a comparative epigenome analysis of KSHV and the related murine gammaherpesvirus 68 (MHV-68). We demonstrate that, while latently replicating MHV-68 genomes readily acquire distinct patterns of activation-associated histone modifications upon de novo infection, they fundamentally differ in their ability to efficiently attract H3K27-me3 marks. Statistical analyses of ChIP-seq data from in vitro infected cells as well as in vivo latency reservoirs furthermore suggest that, whereas KSHV rapidly attracts PRCs in a genome-wide manner, H3K27-me3 acquisition by MHV-68 genomes may require spreading from initial seed sites to which PRC are recruited as the result of an inefficient or stochastic recruitment, and that immune pressure may be needed to select for latency pools harboring PRC-silenced episomes in vivo.Using co-infection experiments and recombinant viruses, we also show that KSHV’s ability to rapidly and efficiently acquire H3K27-me3 marks does not depend on the host cell environment or unique properties of the KSHV-encoded LANA protein, but rather requires specific cis-acting sequence features. We show that the non-canonical PRC1.1 component KDM2B, a factor which binds to unmethylated CpG motifs, is efficiently recruited to KSHV genomes, indicating that CpG island characteristics may constitute these features. In accord with the fact that, compared to MHV-68, KSHV genomes exhibit a fundamentally higher density of CpG motifs, we furthermore demonstrate efficient acquisition of H2AK119-ub by KSHV and H3K36-me2 by MHV-68 (but not vice versa), furthermore supporting the notion that KSHV genomes rapidly attract PRC1.1 complexes in a genome-wide fashion. Collectively, our results suggest that rapid PRC silencing is not a universal feature of viral latency, but that some viruses may rather have adopted distinct genomic features to specifically exploit default host pathways that repress epigenetically naive, CpG-rich DNA.

BAT Hi-C maps global chromatin interactions in an efficient and economical way.

Chromosome Conformation Capture (3C)-based technologies, such as Hi-C, have represented a significant breakthrough in investigating the structure and function of higher-order genome architecture. However, the mapping of global chromatin interactions remains challenging across many biological conditions due to high background noise and financial constraints, especially for small laboratories. Here, we describe the Bridge linker-Alul-Tn5 Hi-C (BAT Hi-C) method, which is a simple and efficient method for delineating chromatin conformational features of mouse embryonic stem (mES) cells and uncover DNA loops. This protocol combines Alul fragmentation and biotinylated linker-mediated proximity ligation to obtain kilobase (kb) resolution with a marked increase in the amount of unique read pairs. The protocol also includes chromatin isolation to reduce background noise and Tn5 tagmentation to cut down on preparation time. Importantly, with only one-third sequencing depth, our method revealed the same spectrum of chromatin contacts as in situ Hi-C. BAT Hi-C is an economical (i.e., approximately $40 for library preparation) and straightforward (total hands-on time of 3 days) tool that is ideal for the in-depth analysis of long-range chromatin looping events in a genome-wide fashion.

MYC deregulates TET1 and TET2 expression to control global DNA (hydroxy)methylation and gene expression to maintain a neoplastic phenotype in T-ALL

BackgroundWhile aberrant DNA methylation is a characteristic feature of tumor cells, our knowledge of how these DNA methylation patterns are established and maintained is limited. DNA methyltransferases and ten-eleven translocation methylcytosine dioxygenases (TETs) function has been found altered in a variety of cancer types.ResultsHere, we report that in T cell acute lymphoblastic leukemia (T-ALL) the MYC oncogene controls the expression of TET1 and TET2 to maintain 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) patterns, which is associated with tumor cell-specific gene expression. We found that cellular senescence and tumor regression upon MYC inactivation in T-ALL was associated with genome-wide changes in 5mC and 5hmC patterns. Correlating with the changes in DNA (hydroxy)methylation, we found that T-ALL overexpress TET1, while suppressing TET2 in a MYC-dependent fashion. Consequently, MYC inactivation led to an inverse expression pattern, decreasing TET1, while increasing TET2 levels. Knockdown of TET1 or ectopic expression of TET2 in T-ALL was associated with genome-wide changes in 5mC and 5hmC enrichment and decreased cell proliferation, suggesting a tumor promoting function of TET1, and a tumor suppressing role for TET2. Among the genes and pathways controlled by TET1, we found ribosomal biogenesis and translational control of protein synthesis highly enriched.ConclusionsOur finding that MYC directly deregulates the expression of TET1 and TET2 in T-ALL provides novel evidence that MYC controls DNA (hydroxy)methylation in a genome-wide fashion. It reveals a coordinated interplay between the components of the DNA (de)methylating machinery that contribute to MYC-driven tumor maintenance, highlighting the potential of specific TET enzymes for therapeutic strategies.