Distal Elements Research Articles

To loop or not to loop? CNS-28 is the answer

Regulation of interferon-γ is critical to constrain inflammation and mount effective responses to infection and anti-tumor immunity. In this issue of Immunity, Cui etal. identify a distal silencer element that disrupts promoter-enhancer looping, regulating IFN-γ expression and preventing inappropriate inflammation.

BIND&MODIFY: a long-range method for single-molecule mapping of chromatin modifications in eukaryotes

Epigenetic modifications of histones are associated with development and pathogenesis of disease. Existing approaches cannot provide insights into long-range interactions and represent the average chromatin state. Here we describe BIND&MODIFY, a method using long-read sequencing for profiling histone modifications and transcription factors on individual DNA fibers. We use recombinant fused protein A-M.EcoGII to tether methyltransferase M.EcoGII to protein binding sites to label neighboring regions by methylation. Aggregated BIND&MODIFY signal matches bulk ChIP-seq and CUT&TAG. BIND&MODIFY can simultaneously measure histone modification status, transcription factor binding, and CpG 5mC methylation at single-molecule resolution and also quantifies correlation between local and distal elements.

Programmable protein delivery with a bacterial contractile injection system

Endosymbiotic bacteria have evolved intricate delivery systems that enable these organisms to interface with host biology. One example, the extracellular contractile injection systems (eCISs), are syringe-like macromolecular complexes that inject protein payloads into eukaryotic cells by driving a spike through the cellular membrane. Recently, eCISs have been found to target mouse cells1–3, raising the possibility that these systems could be harnessed for therapeutic protein delivery. However, whether eCISs can function in human cells remains unknown, and the mechanism by which these systems recognize target cells is poorly understood. Here we show that target selection by the Photorhabdus virulence cassette (PVC)—an eCIS from the entomopathogenic bacterium Photorhabdus asymbiotica—is mediated by specific recognition of a target receptor by a distal binding element of the PVC tail fibre. Furthermore, using in silico structure-guided engineering of the tail fibre, we show that PVCs can be reprogrammed to target organisms not natively targeted by these systems—including human cells and mice—with efficiencies approaching 100%. Finally, we show that PVCs can load diverse protein payloads, including Cas9, base editors and toxins, and can functionally deliver them into human cells. Our results demonstrate that PVCs are programmable protein delivery devices with possible applications in gene therapy, cancer therapy and biocontrol.

Current sequence-based models capture gene expression determinants in promoters but mostly ignore distal enhancers

BackgroundThe largest sequence-based models of transcription control to date are obtained by predicting genome-wide gene regulatory assays across the human genome. This setting is fundamentally correlative, as those models are exposed during training solely to the sequence variation between human genes that arose through evolution, questioning the extent to which those models capture genuine causal signals.ResultsHere we confront predictions of state-of-the-art models of transcription regulation against data from two large-scale observational studies and five deep perturbation assays. The most advanced of these sequence-based models, Enformer, by and large, captures causal determinants of human promoters. However, models fail to capture the causal effects of enhancers on expression, notably in medium to long distances and particularly for highly expressed promoters. More generally, the predicted impact of distal elements on gene expression predictions is small and the ability to correctly integrate long-range information is significantly more limited than the receptive fields of the models suggest. This is likely caused by the escalating class imbalance between actual and candidate regulatory elements as distance increases.ConclusionsOur results suggest that sequence-based models have advanced to the point that in silico study of promoter regions and promoter variants can provide meaningful insights and we provide practical guidance on how to use them. Moreover, we foresee that it will require significantly more and particularly new kinds of data to train models accurately accounting for distal elements.

Prediction of Enhancers in DNA Sequence Data using a Hybrid CNN-DLSTM Model.

Enhancer, a distal cis-regulatory element controls gene expression. Experimental prediction of enhancer elements is time-consuming and expensive. Consequently, various inexpensive deep learning-based fast methods have been developed for predicting the enhancers and determining their strength. In this paper, we have proposed a two-stage deep learning-based framework leveraging DNA structural features, natural language processing, convolutional neural network, and long short-term memory to predict the enhancer elements accurately in the genomics data. In the first stage, we extracted the features from DNA sequence data by using three feature representation techniques viz., k-mer based feature extraction along with word2vector based interpretation of underlined patterns, one-hot encoding, and the DNAshape technique. In the second stage, strength of enhancers is predicted from the extracted features using a hybrid deep learning model. The method is capable of adapting itself to varying sizes of datasets. Also, as proposed model can capture long-range sequencing patterns, the robustness of the method remains unaffected against minor variations in the genomics sequence. The method outperforms the other state-of-the-art methods at both stages in terms of performance metrics of prediction accuracy, specificity, Mathew's correlation coefficient, and area under the ROC curve. In summary, the proposed method is a reliable method for enhancer prediction.

Biochemical and Structural Characterization of a Peptidic Inhibitor of the YAP:TEAD Interaction That Binds to the α-Helix Pocket on TEAD.

The TEAD transcription factors are the most distal elements of the Hippo pathway, and their transcriptional activity is regulated by several proteins, including YAP. In some cancers, the Hippo pathway is deregulated and inhibitors of the YAP:TEAD interaction are foreseen as new anticancer drugs. The binding of YAP to TEAD is driven by the interaction of an α-helix and an Ω-loop present in its TEAD-binding domain with two distinct pockets at the TEAD surface. Using the mRNA-based display technique to screen a library of in vitro-translated cyclic peptides, we identified a peptide that binds with a nanomolar affinity to TEAD. The X-ray structure of this peptide in complex with TEAD reveals that it interacts with the α-helix pocket. Under our experimental conditions, this peptide can form a ternary complex with TEAD and YAP. Furthermore, combining it with a peptide binding to the Ω-loop pocket gives an additive inhibitory effect on the YAP:TEAD interaction. Overall, our results show that it is possible to identify nanomolar inhibitors of the YAP:TEAD interaction that bind to the α-helix pocket, suggesting that developing such compounds might be a strategy to treat cancers where the Hippo pathway is deregulated.

Computational predictions of 3D chromatin many-body interactions reveal roles of chromatin hubs in gene expression.

Chromatin 3D organization plays important roles in the regulation of gene expression. Hi-C measurements provide a projection of 3D chromatin organization onto a 2D heatmap, which depicts the population-averaged pairwise contact frequencies between all genomic elements. These 2D heatmaps highlight structural units of chromatin organization such as TADs. As distal cis-regulatory elements such as enhancers interact with promoters to increase the transcriptional output of target genes in a tissue-specific manner, quantifying cell-type specific chromatin spatial organization is therefore critical. However, TADs have been found to be largely invariant across cell types, suggesting that features of 2D Hi-C heatmaps are insufficient to gain understanding of tissue-specific gene expression. Another challenge is when multiple genomic elements are involved: It is not possible to obtain higher-order many-body interactions from Hi-C data. In order to address these problems, we apply CHROMATIX, a computational method which reconstructs ensembles of single-cell chromatin conformations by deconvolving Hi-C data. We reconstructed large ensembles (2×104) of independent 3D single-cell chromatin conformations which reproduce imaging, Dip-C, and 3D FISH single cell measurements. The reconstructed ensembles of single-cell chromatin structures further enable us to uncover statistically significant 3D hubs of higher-order many-body interactions. We uncovered chromatin hubs of many-bodies across hundreds of loci in B-lymphocyte cells. We illustrate the functional roles of these 3D hubs of many-body interactions in regulating gene expression. Using the example of eQTL associated genes, we show that higher-order 3D hubs provide the physical basis of differential gene expression, with detailed examples of hubs of 1-gene-to-many-eQTLs and many-genes-to-1-eQTL. Overall, our results show 3D chromatin many-body hubs are important for regulating gene expression, and they can be discovered through CHROMATIX computational modeling single-cell 3D chromatin conformations using population Hi-C data.

Pneumococcal capsule expression is controlled through a conserved, distal cis-regulatory element during infection.

Streptococcus pneumoniae (the pneumococcus) is the major cause of bacterial pneumonia in the US and worldwide. Studies have shown that the differing chemical make-up between serotypes of its most important virulence factor, the capsule, can dictate disease severity. Here we demonstrate that control of capsule synthesis is also critical for infection and facilitated by two broadly conserved transcription factors, SpxR and CpsR, through a distal cis-regulatory element we name the 37-CE. Strikingly, changing only three nucleotides within this sequence is sufficient to render pneumococcus avirulent. Using in vivo and in vitro approaches, we present a model where SpxR interacts as a unique trimeric quaternary structure with the 37-CE to enable capsule repression in the airways. Considering its dramatic effect on infection, variation of the 37-CE between serotypes suggests this molecular switch could be a critical contributing factor to this pathogen's serotype-specific disease outcomes.

IEnhancer-SKNN: a stacking ensemble learning-based method for enhancer identification and classification using sequence information.

Enhancers, a class of distal cis-regulatory elements located in the non-coding region of DNA, play a key role in gene regulation. It is difficult to identify enhancers from DNA sequence data because enhancers are freely distributed in the non-coding region, with no specific sequence features, and having a long distance with the targeted promoters. Therefore, this study presents a stacking ensemble learning method to accurately identify enhancers and classify enhancers into strong and weak enhancers. Firstly, we obtain the fusion feature matrix by fusing the four features of Kmer, PseDNC, PCPseDNC and Z-Curve9. Secondly, five K-Nearest Neighbor (KNN) models with different parameters are trained as the base model, and the Logistic Regression algorithm is utilized as the meta-model. Thirdly, the stacking ensemble learning strategy is utilized to construct a two-layer model based on the base model and meta-model to train the preprocessed feature sets. The proposed method, named iEnhancer-SKNN, is a two-layer prediction model, in which the function of the first layer is to predict whether the given DNA sequences are enhancers or non-enhancers, and the function of the second layer is to distinguish whether the predicted enhancers are strong enhancers or weak enhancers. The performance of iEnhancer-SKNN is evaluated on the independent testing dataset and the results show that the proposed method has better performance in predicting enhancers and their strength. In enhancer identification, iEnhancer-SKNN achieves an accuracy of 81.75%, an improvement of 1.35% to 8.75% compared with other predictors, and in enhancer classification, iEnhancer-SKNN achieves an accuracy of 80.50%, an improvement of 5.5% to 25.5% compared with other predictors. Moreover, we identify key transcription factor binding site motifs in the enhancer regions and further explore the biological functions of the enhancers and these key motifs. Source code and data can be downloaded from https://github.com/HaoWuLab-Bioinformatics/iEnhancer-SKNN.

Mammalian forelimb evolution is driven by uneven proximal-to-distal morphological diversity.

Vertebrate limb morphology often reflects the environment due to variation in locomotor requirements. However, proximal and distal limb segments may evolve differently from one another, reflecting an anatomical gradient of functional specialization that has been suggested to be impacted by the timing of development. Here, we explore whether the temporal sequence of bone condensation predicts variation in the capacity of evolution to generate morphological diversity in proximal and distal forelimb segments across more than 600 species of mammals. Distal elements not only exhibit greater shape diversity, but also show stronger within-element integration and, on average, faster evolutionary responses than intermediate and upper limb segments. Results are consistent with the hypothesis that late developing distal bones display greater morphological variation than more proximal limb elements. However, the higher integration observed within the autopod deviates from such developmental predictions, suggesting that functional specialization plays an important role in driving within-element covariation. Proximal and distal limb segments also show different macroevolutionary patterns, albeit not showing a perfect proximo-distal gradient. The high disparity of the mammalian autopod, reported here, is consistent with the higher potential of development to generate variation in more distal limb structures, as well as functional specialization of the distal elements.

A genome-wide relay of signalling-responsive enhancers drives hematopoietic specification

Developmental control of gene expression critically depends on distal cis-regulatory elements including enhancers which interact with promoters to activate gene expression. To date no global experiments have been conducted that identify their cell type and cell stage-specific activity within one developmental pathway and in a chromatin context. Here, we describe a high-throughput method that identifies thousands of differentially active cis-elements able to stimulate a minimal promoter at five stages of hematopoietic progenitor development from embryonic stem (ES) cells, which can be adapted to any ES cell derived cell type. We show that blood cell-specific gene expression is controlled by the concerted action of thousands of differentiation stage-specific sets of cis-elements which respond to cytokine signals terminating at signalling responsive transcription factors. Our work provides an important resource for studies of hematopoietic specification and highlights the mechanisms of how and where extrinsic signals program a cell type-specific chromatin landscape driving hematopoietic differentiation.

A distal super-enhancer activates oncogenic ETS2 via recruiting MECOM in inflammatory bowel disease and colorectal cancer

Abnormal activities of distal cis-regulatory elements (CREs) contribute to the initiation and progression of cancer. Gain of super-enhancer (SE), a highly active distal CRE, is essential for the activation of key oncogenes in various cancers. However, the mechanism of action for most tumor-specific SEs still largely remains elusive. Here, we report that a candidate oncogene ETS2 was activated by a distal SE in inflammatory bowel disease (IBD) and colorectal cancer (CRC). The SE physically interacted with the ETS2 promoter and was required for the transcription activation of ETS2. Strikingly, the ETS2-SE activity was dramatically upregulated in both IBD and CRC tissues when compared to normal colon controls and was strongly correlated with the level of ETS2 expression. The tumor-specific activation of ETS2-SE was further validated by increased enhancer RNA transcription from this region in CRC. Intriguingly, a known IBD-risk SNP resides in the ETS2-SE and the genetic variant modulated the level of ETS2 expression through affecting the binding of an oncogenic transcription factor MECOM. Silencing of MECOM induced significant downregulation of ETS2 in CRC cells, and the level of MECOM and ETS2 correlated well with each other in CRC and IBD samples. Functionally, MECOM and ETS2 were both required for maintaining the colony-formation and sphere-formation capacities of CRC cells and MECOM was crucial for promoting migration. Taken together, we uncovered a novel disease-specific SE that distantly drives oncogenic ETS2 expression in IBD and CRC and delineated a mechanistic link between non-coding genetic variation and epigenetic regulation of gene transcription.

In This Issue

In This Issue| January 06 2023 In This Issue Author & Article Information Online Issn: 2643-3249 Print Issn: 2643-3230 ©2023 American Association for Cancer Research2023American Association for Cancer Research Blood Cancer Discov (2023) 4 (1): 1. https://doi.org/10.1158/2643-3230.BCD-4-1-ITI Related Content A commentary has been published: Distinct Genetically Determined Origins of Myd88/BCL2-Driven Aggressive Lymphoma Rationalize Targeted Therapeutic Intervention Strategies A commentary has been published: A Therapeutically Targetable NOTCH1–SIRT1–KAT7 Axis in T-cell Leukemia A commentary has been published: ETV6 Deficiency Unlocks ERG-Dependent Microsatellite Enhancers to Drive Aberrant Gene Activation in B-Lymphoblastic Leukemia View more A commentary has been published: Extracellular Vesicle Secretion by Leukemia Cells In Vivo Promotes CLL Progression by Hampering Antitumor T-cell Responses View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record January 6 2023 Citation In This Issue. Blood Cancer Discov 1 January 2023; 4 (1): 1. https://doi.org/10.1158/2643-3230.BCD-4-1-ITI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Activated NOTCH1 is a common driver of T-cell acute lymphoblastic leukemia (T-ALL). Due to the toxicity of and resistance to NOTCH1 inhibitors, therapies targeting molecules downstream of NOTCH1 might help treat relapsed patients. In this study, Lancho et al. reveal that NOTCH1 directly activates SIRT1 transcription through a distal enhancer element. Genetic loss or inhibition of SIRT1 leads to significant antileukemic effects in mouse models with different SIRT1 allelic dosages. Mechanistically, the oncogenic effects of SIRT1 are mediated, at least in part, by its deacetylation and activation of KAT7. These results uncover the therapeutic potential of SIRT1-KAT7 axis inhibition in T-ALL. ■ See article, p. 12. The regulatory targets of the ETS repressor ETV6, a candidate tumor suppressor in B-lymphoblastic leukemia (B-ALL), have remained poorly understood. Kodgule, Goldman, Monovich et al. show that ETV6 binds to genomic short tandem repeats of the sequence ‘GGAA’ that are repressed in normal... You do not currently have access to this content.

Occlusal load and biomechanics of implant-supported fixed partial dentures: bone stress analysis

Osseointegration emerged in the 1950s, created by Per-Ingvar Branemark, when it was conceptualized as an intimate connection between the implant and the surrounding bone, and with this discovery, it was proposed as a treatment for total or partial edentulism. implants supported. Excessive loads generated by poor adjustment of occlusal forces are recurrent and common, and may generate resorption in the supporting bone; in situations of excess occlusal load, when these loads are distributed at different intensities, on different dental occlusal tables, could this be a reason for the failure of implant-supported rehabilitation? What about the absence of occlusal loading? Could this be a reason for the failure of implant-supported prosthetic rehabilitation? What will be the way to avoid exacerbated stress on occlusal tables, leading to a better generation of treatment prognosis, especially in cases of Fixed Partial Dentures (FPD) with suspended artificial teeth? This study aims, using the finite element methodology, to build a clinical situation of an implant-supported partial denture (FPD) of fixation of 3 elements of regular length, where the location of the occlusal loads (newtons) (total 750N) in the (FPD) ( Fixed Partial Denture) defined in the experimental groups, with higher loads being selected on the occlusal surface of the molar tooth in relation to the premolar, and with different occlusal forces on adjacent teeth, according to an occlusal load proposed in the research. Thus, the occlusal load was distributed, in different intensities, between each tooth of the CAD (Computer Aided Design) model. The biomechanical behavior and stresses of the fragile structures analyzed vary according to the occlusal load exerted and, therefore, controlling such occlusal loads is very relevant. The tensions are always greater and more evident in the posterior regions, whether in the cortical bone or in the medullary bone, especially when occlusal loads are applied unevenly with overload on the posterior elements of the fixed partial denture; therefore, one should always try to preserve these more distal elements.

Role of ZNF143 and Its Association with Gene Expression Patterns, Noncoding Mutations, and the Immune System in Human Breast Cancer.

The function of noncoding sequence variations at ZNF143 binding sites in breast cancer cells is currently not well understood. Distal elements and promoters, also known as cis-regulatory elements, control the expression of genes. They may be identified by functional genomic techniques and sequence conservation, and they frequently show cell- and tissue-type specificity. The creation, destruction, or modulation of TF binding and function may be influenced by genetic modifications at TF binding sites that affect the binding affinity. Therefore, noncoding mutations that affect the ZNF143 binding site may be able to alter the expression of some genes in breast cancer. In order to understand the relationship among ZNF143, gene expression patterns, and noncoding mutations, we adopted an integrative strategy in this study and paid close attention to putative immunological signaling pathways. The immune system-related pathways ErbB, HIF1a, NF-kB, FoxO, JAK-STAT, Wnt, Notch, cell cycle, PI3K-AKT, RAP1, calcium signaling, cell junctions and adhesion, actin cytoskeleton regulation, and cancer pathways are among those that may be significant, according to the overall analysis.

Missing heritability of Wilson disease: a search for the uncharacterized mutations.

Wilson disease (WD), a copper metabolism disorder caused by mutations in ATP7B, manifests heterogeneous clinical features. Interestingly, in a fraction of clinically diagnosed WD patients, mutations in ATP7B appears to be missing. In this review we discuss the plausible explanations of this missing heritability and propose a workflow that can identify the hidden mutations. Mutation analyses of WD generally includes targeted sequencing of ATP7B exons, exon-intron boundaries, and rarely, the proximal promoter region. We propose that variants in the distal cis-regulatory elements and/or deep intronic variants that impact splicing might well represent the hidden mutations. Heterozygous del/ins that remain refractory to conventional PCR-sequencing method may also represent such mutations. In this review, we also hypothesize that mutations in the key copper metabolism genes, like, ATOX1, COMMD1, and SLC31A1, could possibly lead to a WD-like phenotype. In fact, WD does present overlapping symptoms with other rare genetic disorders; hence, the possibility of a misdiagnosis and thus adding to missing heritability cannot be excluded. In this regard, it seems that whole-genome analysis will provide a comprehensive and rapid molecular diagnosis of WD. However, considering the associated cost for such a strategy, we propose an alternative customized screening schema of WD which include targeted sequencing of ATP7B locus as well as other key copper metabolism genes. Success of such a schema has been tested in a pilot study.

Identification of cis‐regulatory elements associated with MAPT expression in human induced pluripotent stem cell derived neural cells.

AbstractBackgroundTau is encoded by MAPT and abnormal aggregates of tau are a hallmark of many neurodegenerative diseases. MAPT is highly expressed in neurons but much more lowly expressed in other cell types, including neural precursor cells (NPCs), suggesting that its expression may be controlled by neuron‐specific transcription factors and cis‐regulatory elements. Tau reduction, even of endogenous tau, is a current therapeutic strategy in both model systems and humans; therefore, understanding MAPT regulation may point to new strategies to achieve tau reduction. Additionally, genome sequencing studies continue to identify disease‐associated variants in non‐coding regions of the genome. These variants are likely located in regulatory regions and affect the expression of target genes whose function contributes to neurodegeneration. Therefore, an additional benefit of this study is that high confidence regulatory regions for MAPT may provide a way to identify important non‐coding variants associated with disease.MethodsThree‐dimensional chromatin conformation capture (Capture‐C), single‐cell multiomics (RNAseq + ATACseq), and chromatin marks including H3K27ac, H3K4me1, and DNase hypersensitivity were used to identify candidate enhancers of MAPT in human NPCs, NPCs differentiated to neurons, and pure inhibitory or excitatory neuron cultures (all lines were H1/H1). Approaches included nomination of elements with signals specific to neurons or neuronal subtypes. The nominated regions were then assessed using luciferase assays to test sufficiency for regulatory activity as well as CRISPR‐dCas9‐KRAB to assess the effect of nominated elements on MAPT expression.ResultsWe identified both proximal and distal regulatory elements for MAPT with function confirmed by up to four orthogonal approaches (chromatin capture, single cell multiomics, a reporter assay, and element inhibition using dCas9‐KRAB). Several of these elements were specific to neurons. One particularly important region was centromeric to MAPT beyond the H2 inversion breakpoint.ConclusionsOverall, these results indicate that MAPT regulation is likely controlled by neuron‐specific cis‐regulatory elements, including both proximal and distal elements. Next steps include study of the effect of trans‐regulatory elements (i.e., transcription factors) that may control recruitment of these cis‐regulatory elements for expression of MAPT and assessment of the effects of the disease‐associated genetic variation on cis‐regulatory element function and expression of MAPT.

MYCN Regulates Cell Survival Via EIF4G1 in Inv(16) Acute Myeloid Leukemia

Transcription factors are frequently deregulated in leukemia and are pivotal to its pathogenesis. The core-binding factor (CBF) transcription factor is a heterodimer of CBFβ and RUNX proteins, is critical to hematopoiesis, and dysregulated in AML. The fusion oncogene CBFβ-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells by sequestering RUNX1 by protein-protein interaction to induce AML. The CBFβ-SMMHC inhibitor, AI-10-49, specifically disrupts its binding to RUNX1 resulting in genome-wide RUNX1 binding, apoptosis induction, and enhanced survival in inv(16) mouse model [Illendula* and Pulikkan* et al, Science 2015]. While previous studies have demonstrated that AI-10-49 induced cell death is partly mediated by repression of MYC expression in inv(16) AML cells [Pulikkan et al, Cell 2018], the function of other MYC family members, in inv(16) cell survival, and overall in AML, remains poorly understood. By conducting Real Time RT-PCR in CD34+ bone marrow cells from human AML samples, we identified MYCN upregulation in inv(16) AML. Analysis of RNA-seq data from inv(16) AML cells treated with AI-10-49 revealed that MYCN expression is downregulated (7-fold). MYCN silencing via CRISPR/Cas9 induced apoptosis in inv(16) AML cells. Furthermore, MYCN genetic targeting by CRISPR/Cas9-RNP approach in human primary inv(16) AML cells transplanted in humanized NSGS xenograft mouse model displayed significant delay in leukemia development, underlining the role of MYCN in inv(16) AML maintenance. ChIP-seq analysis conducted in inv(16) AML cells treated with AI-10-49 demonstrated increased RUNX1 occupancy at MYCN distal genomic element located at +26 Kb (named 'MYCN-e26') downstream from MYCN TSS without any RUNX1 binding at MYCN TSS. ChIP-seq analysis for histone marks such as H3K27ac and H3K4Me1, and chromatin accessibility by ATAC-seq suggested MYCN-e26 acts as a transcriptionally active enhancer element. Analysis of DNase I hypersensitive sites (DHSs) representing accessible chromatin regions in primary AML samples revealed that MYCN-e26 is a bona fide AML enhancer. Finally, deletion of a genomic element in MYCN-e26 surrounding the RUNX1 binding site (200 bp) by CRISPR/Cas9 genome editing downregulated MYCN transcript levels and inhibited inv(16) AML cell survival. Taken together, MYCN-e26 is instrumental in MYCN transcription and cell survival in inv(16) AML cells as a RUNX1 target. To identify the mechanism(s) by which MYCN regulates inv(16) AML cell survival, we performed CUT&Tag sequencing for MYCN and H3K27ac histone mark in inv(16) AML cells. Gene ontology analysis for MYCN binding peaks in transcriptionally active regions revealed mRNA metabolism and stability as the major pathways orchestrated by MYCN in inv(16) AML cells. By comparing the mRNA metabolism/ stability signature genes with transcripts significantly deregulated in human primary inv(16) AML samples, we identified eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) as one of the major genes regulated by MYCN in inv(16) AML cell survival. Our CUT&Tag sequencing analysis demonstrated MYCN binds to EIF4G1 promoter and enhances its transcription. MYCN silencing by CRISPR/Cas9 approach in inv(16) AML cells showed significant downregulation of EIF4G1, suggesting MYCN is required for EIF4G1 expression in inv(16) AML cells. EIF4G1 genetic silencing via CRISPR/Cas9 approach induced apoptosis in inv(16) AML cells in vitro, and significantly delayed leukemia development in NSGS xenograft mouse model, suggesting EIF4G1 is required for the survival of inv(16) leukemia-initiating cells. Our studies demonstrate that CBFβ-SMMHC modulates MYCN levels by inhibition of RUNX1 binding to MYCN distal enhancer in inv(16) AML cells. In addition, our studies bring novel function of MYCN in inv(16) leukemic cell survival by regulating EIF4G1. Future therapeutic approaches targeting MYCN/EIF4G1 axis in combination with AI-10-49 warrant better clinical outcome for the treatment of inv(16) AML.

Learning the histone codes with large genomic windows and three-dimensional chromatin interactions using transformer

The quantitative characterization of the transcriptional control by histone modifications has been challenged by many computational studies, but most of them only focus on narrow and linear genomic regions around promoters, leaving a room for improvement. We present Chromoformer, a transformer-based, three-dimensional chromatin conformation-aware deep learning architecture that achieves the state-of-the-art performance in the quantitative deciphering of the histone codes in gene regulation. The core essence of Chromoformer architecture lies in the three variants of attention operation, each specialized to model individual hierarchy of transcriptional regulation involving from core promoters to distal elements in contact with promoters through three-dimensional chromatin interactions. In-depth interpretation of Chromoformer reveals that it adaptively utilizes the long-range dependencies between histone modifications associated with transcription initiation and elongation. We also show that the quantitative kinetics of transcription factories and Polycomb group bodies can be captured by Chromoformer. Together, our study highlights the great advantage of attention-based deep modeling of complex interactions in epigenomes.

Real-time holographic lensless micro-endoscopy through flexible fibers via fiber bundle distal holography

Fiber-based micro-endoscopes are a critically important tool for minimally-invasive deep-tissue imaging. However, current micro-endoscopes cannot perform three-dimensional imaging through dynamically-bent fibers without the use of bulky optical elements such as lenses and scanners at the distal end, increasing the footprint and tissue-damage. Great efforts have been invested in developing approaches that avoid distal bulky optical elements. However, the fundamental barrier of dynamic optical wavefront-distortions in propagation through flexible fibers limits current approaches to nearly-static or non-flexible fibers. Here, we present an approach that allows holographic, bend-insensitive, coherence-gated, micro-endoscopic imaging using commercially available multi-core fibers (MCFs). We achieve this by adding a partially-reflecting mirror to the distal fiber-tip, allowing to perform low-coherence full-field phase-shifting holography. We demonstrate widefield diffraction-limited reflection imaging of amplitude and phase targets through dynamically bent fibers at video-rate. Our approach holds potential for label-free investigations of dynamic samples.