MethNet: a robust approach to identify regulatory hubs and their distal targets from cancer data.

Aberrations in the capacity of DNA/chromatin modifiers and transcription factors to bind non-coding regions can lead to changes in gene regulation and impact disease phenotypes. However, identifying distal regulatory elements and connecting them with their target genes remains challenging. Here, we present MethNet, a pipeline that integrates large-scale DNA methylation and gene expression data across multiple cancers, to uncover cis regulatory elements (CREs) in a 1 Mb region around every promoter in the genome. MethNet identifies clusters of highly ranked CREs, referred to as 'hubs', which contribute to the regulation of multiple genes and significantly affect patient survival. Promoter-capture Hi-C confirmed that highly ranked associations involve physical interactions between CREs and their gene targets, and CRISPR interference based single-cell RNA Perturb-seq validated the functional impact of CREs. Thus, MethNet-identified CREs represent a valuable resource for unraveling complex mechanisms underlying gene expression, and for prioritizing the verification of predicted non-coding disease hotspots.

Brain and cancer associated binding domain mutations provide insight into CTCF's relationship with chromatin and its ability to act as a chromatin organizer.

Although only a fraction of CTCF motifs are bound in any cell type, and approximately half of the occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique properties of each protein, but also on the genomic context of bound sites. Our studies also highlight the reciprocal relationship between CTCF and chromatin, demonstrating that the unique binding properties of WT and mutant proteins have a distinct impact on accessibility, TF binding, cohesin overlap, chromatin interactivity and gene expression programs, providing insight into their cancer and brain related effects.

MethNet: a robust approach to identify regulatory hubs and their distal targets in cancer.

Aberrations in the capacity of DNA/chromatin modifiers and transcription factors to bind non-coding regions can lead to changes in gene regulation and impact disease phenotypes. However, identifying distal regulatory elements and connecting them with their target genes remains challenging. Here, we present MethNet, a pipeline that integrates large-scale DNA methylation and gene expression data across multiple cancers, to uncover novel cis regulatory elements (CREs) in a 1Mb region around every promoter in the genome. MethNet identifies clusters of highly ranked CREs, referred to as 'hubs', which contribute to the regulation of multiple genes and significantly affect patient survival. Promoter-capture Hi-C confirmed that highly ranked associations involve physical interactions between CREs and their gene targets, and CRISPRi based scRNA Perturb-seq validated the functional impact of CREs. Thus, MethNet-identified CREs represent a valuable resource for unraveling complex mechanisms underlying gene expression, and for prioritizing the verification of predicted non-coding disease hotspots.

Cell-type-specific prediction of 3D chromatin organization enables high-throughput in silico genetic screening.

Investigating how chromatin organization determines cell-type-specific gene expression remains challenging. Experimental methods for measuring three-dimensional chromatin organization, such as Hi-C, are costly and have technical limitations, restricting their broad application particularly in high-throughput genetic perturbations. We present C.Origami, a multimodal deep neural network that performs de novo prediction of cell-type-specific chromatin organization using DNA sequence and two cell-type-specific genomic features-CTCF binding and chromatin accessibility. C.Origami enables in silico experiments to examine the impact of genetic changes on chromatin interactions. We further developed an in silico genetic screening approach to assess how individual DNA elements may contribute to chromatin organization and to identify putative cell-type-specific trans-acting regulators that collectively determine chromatin architecture. Applying this approach to leukemia cells and normal T cells, we demonstrate that cell-type-specific in silico genetic screening, enabled by C.Origami, can be used to systematically discover novel chromatin regulation circuits in both normal and disease-related biological systems.

CRISPR and biochemical screens identify MAZ as a cofactor in CTCF-mediated insulation at Hox clusters.

CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxA cluster, complemented by biochemical approaches. Among the candidates, we identified Myc-associated zinc-finger protein (MAZ) as a cofactor in CTCF insulation. MAZ colocalizes with CTCF at chromatin borders and, similar to CTCF, interacts with the cohesin subunit RAD21. MAZ KO disrupts gene expression and local contacts within topologically associating domains. Similar to CTCF motif deletions, MAZ motif deletions lead to derepression of posterior Hox genes immediately after CTCF boundaries upon differentiation, giving rise to homeotic transformations in mouse. Thus, MAZ is a factor contributing to appropriate insulation, gene expression and genomic architecture during development.

The art of chromosome dynamics: an interview with Jane Skok.

In this interview, Professor Jane Skok speaks with Storm Johnson, commissioning editor for Epigenomics, on her work to date in the field of chromosome architecture and regulatory elements. Jane Skok's lab uses sophisticated microscopic techniques to visualize recombination in individual cells, tracing the dynamic changes in chromosome architecture and nuclear location at different stages of this complex process. This line of research unites two lifelong passions: science and art. After completing her PhD in immunology and genetics at the Imperial Cancer Research Fund in Lincoln's Inn Fields, Dr Skok took 12 years off and pursued training in art while caring for her young children. She then returned to science, joining David Gray's lab at Imperial College London as a postdoctoral fellow to study B cell biology and acquired expertise in Mandy Fisher's lab to understand how nuclear organization of the antigen receptor genes regulate V(D)J recombination and allelic exclusion. Dr Skok continued to pursue these questions in her own lab at University College London and elucidated the roles of Pax5, locus contraction and nuclear subcompartmentalization in maintaining allelic exclusion. In 2006, Dr Skok was recruited to New York University School of Medicine, where her lab has revealed the activities of several signaling factors in guiding B cell development and they made the surprising discovery that the RAG proteins and the DNA damage response factor ATM help ensure allelic exclusion at the immunoglobulin gene loci. More recently, those at the Skok lab have turned their attention to understanding how localized and long-range chromatin contacts impact gene regulation in health and disease settings.

Ontogeny and Vulnerabilities of Drug-Tolerant Persisters in HER2+ Breast Cancer.

Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. "Drug-tolerant persisters" (DTP), a subpopulation of cancer cells that survive via reversible, nongenetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKI) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single-cell RNA sequencing reveals that HER2+ breast cancer cells cycle stochastically through a "pre-DTP" state, characterized by a G0-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting shows that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation. SIGNIFICANCE: DTPs are implicated in resistance to anticancer therapies, but their ontogeny and vulnerabilities remain unclear. We find that HER2 TKI-DTPs emerge from stochastically arising primed cells ("pre-DTPs") that engage either of two distinct transcriptional programs upon TKI exposure. Our results provide new insights into DTP ontogeny and potential therapeutic vulnerabilities. This article is highlighted in the In This Issue feature, p. 873.

Simultaneous Tagmentation-Based Detection of ChIP/ATAC Signal with Bisulfite Sequencing.

DNA methylation is thought to regulate accessibility of chromatin and binding of regulatory elements; however, it is difficult to determine if chromatin accessibility or transcription factor (TF) binding overlap with methylated or unmethylated DNA if the assays are performed separately. In order to examine accessibility or TF binding simultaneously with methylation on the same DNA molecule, we developed EpiMethylTag which combines ATAC-Seq or ChIP-Seq (M-ATAC or M-ChIP) with bisulfite conversion. Our approach provides a fast, low-input, low sequencing depth method to determine whether DNAme and accessibility/TF binding are mutually exclusive or can coexist in certain locations.

Somatic Focal Copy Number Gains of Noncoding Regions of Receptor Tyrosine Kinase Genes in Treatment-Resistant Epilepsy.

Epilepsy is a heterogenous group of disorders defined by recurrent seizure activity due to abnormal synchronized activity of neurons. A growing number of epilepsy cases are believed to be caused by genetic factors and copy number variants (CNV) contribute to up to 5% of epilepsy cases. However, CNVs in epilepsy are usually large deletions or duplications involving multiple neurodevelopmental genes. In patients who underwent seizure focus resection for treatment-resistant epilepsy, whole genome DNA methylation profiling identified 3 main clusters of which one showed strong association with receptor tyrosine kinase (RTK) genes. We identified focal copy number gains involving epidermal growth factor receptor (EGFR) and PDGFRA loci. The dysplastic neurons of cases with amplifications showed marked overexpression of EGFR and PDGFRA, while glial and endothelial cells were negative. Targeted sequencing of regulatory regions and DNA methylation analysis revealed that only enhancer regions of EGFR and gene promoter of PDGFRA were amplified, while coding regions did not show copy number abnormalities or somatic mutations. Somatic focal copy number gains of noncoding regulatory represent a previously unrecognized genetic driver in epilepsy and a mechanism of abnormal activation of RTK genes. Upregulated RTKs provide a potential avenue for therapy in seizure disorders.

Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.

The Ig heavy chain protein but not its message controls early B cell development.

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.

Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency.

Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.

Author Correction: Defining the relative and combined contribution of CTCF and CTCFL to genomic regulation.

An amendment to this paper has been published and can be accessed via the original article.

Defining the relative and combined contribution of CTCF and CTCFL to genomic regulation.

BACKGROUND: Ubiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL, is normally only present in the testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription. RESULTS: We demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem, we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C, and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites to regulate transcription, chromatin looping, and insulation. CONCLUSION: This study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

CTCF and CTCFL in cancer.

CTCF plays a key role in organizing chromatin into TAD structures but it can also function as a transcription factor. CTCFL (CTCF-like), the paralog of CTCF, is normally transiently expressed in pre-meiotic male germ cells together with ubiquitously expressed CTCF. It plays a unique role in spermatogenesis by regulating expression of testis-specific genes. Genetic alterations in CTCF and its paralog CTCFL have both been found in numerous cancers, but it remains unknown to what extent CTCFL deregulates transcription on its own or by opposing CTCF. Here, we discuss some of the potential mechanisms by which these two proteins could alter gene regulation and contribute to oncogenic transcriptional programs.

DNA methylation disruption reshapes the hematopoietic differentiation landscape.

Mutations in genes involved in DNA methylation (DNAme; for example, TET2 and DNMT3A) are frequently observed in hematological malignancies1-3 and clonal hematopoiesis4,5. Applying single-cell sequencing to murine hematopoietic stem and progenitor cells, we observed that these mutations disrupt hematopoietic differentiation, causing opposite shifts in the frequencies of erythroid versus myelomonocytic progenitors following Tet2 or Dnmt3a loss. Notably, these shifts trace back to transcriptional priming skews in uncommitted hematopoietic stem cells. To reconcile genome-wide DNAme changes with specific erythroid versus myelomonocytic skews, we provide evidence in support of differential sensitivity of transcription factors due to biases in CpG enrichment in their binding motif. Single-cell transcriptomes with targeted genotyping showed similar skews in transcriptional priming of DNMT3A-mutated human clonal hematopoiesis bone marrow progenitors. These data show that DNAme shapes the topography of hematopoietic differentiation, and support a model in which genome-wide methylation changes are transduced to differentiation skews through biases in CpG enrichment of the transcription factor binding motif.

The novel lncRNA BlackMamba controls the neoplastic phenotype of ALK- anaplastic large cell lymphoma by regulating the DNA helicase HELLS.

The molecular mechanisms leading to the transformation of anaplastic lymphoma kinase negative (ALK-) anaplastic large cell lymphoma (ALCL) have been only in part elucidated. To identify new culprits which promote and drive ALCL, we performed a total transcriptome sequencing and discovered 1208 previously unknown intergenic long noncoding RNAs (lncRNAs), including 18 lncRNAs preferentially expressed in ALCL. We selected an unknown lncRNA, BlackMamba, with an ALK- ALCL preferential expression, for molecular and functional studies. BlackMamba is a chromatin-associated lncRNA regulated by STAT3 via a canonical transcriptional signaling pathway. Knockdown experiments demonstrated that BlackMamba contributes to the pathogenesis of ALCL regulating cell growth and cell morphology. Mechanistically, BlackMamba interacts with the DNA helicase HELLS controlling its recruitment to the promoter regions of cell-architecture-related genes, fostering their expression. Collectively, these findings provide evidence of a previously unknown tumorigenic role of STAT3 via a lncRNA-DNA helicase axis and reveal an undiscovered role for lncRNA in the maintenance of the neoplastic phenotype of ALK-ALCL.