Intra-chromosomal Interactions Research Articles

Inter3D: Capture of TAD Reorganization Endows Variant Patterns of Gene Transcription

Abstract Topologically associating domain (TAD) reorganization commonly occurs in the cell nucleus and contributes to gene activation and inhibition through the separation or fusion of adjacent TADs. However, identification of functional genes impacted by TAD alteration and the mechanism of TAD reorganization underlying gene transcription remain to be fully elucidated. Here, we first developed a novel approach termed Inter3D to specifically identify genes regulated by TAD reorganization. Our study revealed that the segregation of TADs led to the disruption of intrachromosomal looping at the myosin light chain 12B (MYL12B) locus, via the meticulous reorganization of TADs mediating epigenomic landscapes within tumor cells, thereby exhibiting a significant correlation with the downregulation of its transcriptional activity. Conversely, the fusion of TADs facilitated intrachromosomal interactions, suggesting a potential association with the activation of cytochrome P450 family 27 subfamily B member 1 (CYP27B1). Our study provides comprehensive insight into the capture of TAD rearrangement-mediated gene loci and moves toward understanding the functional role of TAD reorganization in gene transcription. The Inter3D pipeline used in this study is freely available at https://github.com/bm2-lab/inter3D.

Abstract PO3-24-04: Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer

Abstract Title Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer Background Secondary trastuzumab resistance seriously affects the treatment of HER2-positive breast cancer. Although studies have demonstrated several key cellular properties that are closely accompanied with trastuzumab resistance formation, we still have poor knowledge on the genetic and epigenetic variations that promote such transformation. Here we suggested that the epigenetic modification changes on histone H3 together with altered chromatin architecture promote lipid metabolism reprogramming during secondary trastuzumab formation. Materials and methods Induced secondary trastuzumab-resistant SKBR3_HR cell line together with the original trastuzumab-sensitive SKBR3 cell line were applied in this study. Total RNA was collected for transcriptome analysis. Anti-H3K4me3 and K27me3 antibodies were chosen for CUT&Tag sequencing library preparation. Total genome DNA was prepared for Micro-C sequencing library preparation. Activity score of metabolism pathway was calculated as relative gene expression value averaged over all genes in this pathway in certain cell types. Results Upregulation of arachidonic acid metabolism and downregulation of unsaturated fatty acid synthesis, which are mainly characterized by the activation of PTGS1 and PTGES genes and the repression of FASN and SCD genes, respectively, were observed during the formation of secondary trastuzumab resistance from SKBR3 cell to SKBR3_HR cells. Variations of H3k4me3 instead of H3k27me3 regulate the expression of these 4 genes. The accumulation of H3k4me3 was detected at the promoters of PTGS1 and PTGES genes while they were removed from the promoters of FASN and SCD genes in SKBR3_HR cells. More intra-chromosomal interactions were constituted during resistance formation. In detail, 4626 and 4394 topological associated domains, 3125 and 5824 DNA loops were founded in SKBR3 and SKBR3_HR cells, respectively. Furthermore, the lost and gained DNA loops between SCD and PTGS1 genes and distant genome regions may indicate the weaken and strengthen interactions with transcriptional regulators located there. Conclusions During trastuzumab resistance formation, altered histone modifications as well as higher genome structure could regulate the expression of key genes in lipid metabolism pathways, which may further affect cell properties and interactions with cells in tumor microenvironment. Citation Format: Ningjun Duan, Yijia Hua, Yongmei Yin. Altered epigenetic modifications and genome architecture reshape lipid metabolism during secondary trastuzumab resistance formation of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-04.

The zinc-finger protein Z4 cooperates with condensin II to regulate somatic chromosome pairing and 3D chromatin organization.

Chromosome pairing constitutes an important level of genome organization, yet the mechanisms that regulate pairing in somatic cells and the impact on 3D chromatin organization are still poorly understood. Here, we address these questions in Drosophila, an organism with robust somatic pairing. In Drosophila, pairing preferentially occurs at loci consisting of numerous architectural protein binding sites (APBSs), suggesting a role of architectural proteins (APs) in pairing regulation. Amongst these, the anti-pairing function of the condensin II subunit CAP-H2 is well established. However, the factors that regulate CAP-H2 localization and action at APBSs remain largely unknown. Here, we identify two factors that control CAP-H2 occupancy at APBSs and, therefore, regulate pairing. We show that Z4, interacts with CAP-H2 and is required for its localization at APBSs. We also show that hyperosmotic cellular stress induces fast and reversible unpairing in a Z4/CAP-H2 dependent manner. Moreover, by combining the opposite effects of Z4 depletion and osmostress, we show that pairing correlates with the strength of intrachromosomal 3D interactions, such as active (A) compartment interactions, intragenic gene-loops, and polycomb (Pc)-mediated chromatin loops. Altogether, our results reveal new players in CAP-H2-mediated pairing regulation and the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions.

Abstract 1711: Rearranged active enhancers and promoter-enhancer contacts promote lipid metabolic reprogramming during secondary trastuzumab adaptation of HER2 positive breast cancer

Abstract Background: The development of secondary trastuzumab resistance signifies an evolutionary adaptation within HER2-positive breast cancer during anti-HER2 treatment. Our research reveals a comprehensive metabolic reprogramming, with a particular emphasis on the synthesis of unsaturated fatty acids and the degradation of arachidonic acid, accompanying this adaptation. Non-mutational epigenetic variations, particularly alterations in active enhancers, coupled with the rearrangement of enhancer-promoter contacts, demonstrate robust correlations with lipid metabolic reprogramming. These findings suggest that non-mutational epigenetic variations play a crucial role in driving the adaptation to trastuzumab. Materials and Methods: In a prior investigation, we successfully generated secondary trastuzumab-resistant SKBR3_HR cells. In our current study, total RNA was extracted from both trastuzumab-resistant SKBR3_HR and trastuzumab-sensitive SKBR3 cells for transcriptome analysis. Additionally, mass spectrometry was employed to analyze cellular metabolism in both resistant and sensitive cell populations. Simultaneously, we utilized the CUT&Tag kit with an anti-H3K27ac antibody to create a sequencing library for active enhancer measurement. Furthermore, the Micro-C kit was applied to generate a sequencing library for genome contact measurement. Results: In our study, secondary trastuzumab-adaptive SKBR3_HR cells demonstrated a reduction in cellular unsaturated fatty acids, with more than 110 UFAs or UFA-containing lipids showing decreases, while only 36 exhibited increases. This phenomenon may be attributed to the downregulation of SCD, FADS2, and HACD2. Simultaneously, the upregulation of PTGS1 and PTGES may contribute to a higher conversion of arachidonic acid to prostaglandin E2 (PGE2), resulting in immune inhibition. In SKBR3 and SKBR3_HR cells, 10,955 and 10,889 non-promoter H3K27me3 peaks, respectively, were identified as active enhancer regions. Among them, 379 enhancers became silent, and 313 transitioned into an active state during the formation of trastuzumab adaptation. Trastuzumab adaptation led to an increase in intra-chromosomal interactions, particularly short-distance contacts (< 1MB). By employing the activity-by-contact (ABC) algorithm, we observed significant variant contacts between PTGES and SCD promoters and enhancers within 1MB genomic regions in SKBR3_HR cells compared to SKBR3 cells. Conclusion: The formation of secondary trastuzumab adaptation involves the rearrangement of active enhancers and promoter-enhancer contacts, serving as an effective force to drive lipid metabolic reprogramming, especially the synthesis of unsaturated fatty acids and PGE2 production, reshaping the cellular fatty acid composition and anti-tumor immunity. Citation Format: Ningjun Duan, Yijia Hua, Yongmei Yin. Rearranged active enhancers and promoter-enhancer contacts promote lipid metabolic reprogramming during secondary trastuzumab adaptation of HER2 positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1711.

Abstract 405: Translocation detection in cancer using low-pass pore-c sequencing

Abstract Complex chromosomal rearrangements, including translocations, play a critical role in oncogenesis and are often identified as recurrent genetic aberrations in hematologic malignancies and solid tumors[1]. Translocation detection by karyotyping of an individual’s metaphase chromosomes remains challenging for detection of balanced rearrangements, which have no gain or loss of genetic material. Rearrangement detection using whole-genome sequencing is a viable alternative, but it relies on breakpoint-spanning reads and requires high sequencing depth (30-60x) with long reads to achieve high sensitivity, especially in repetitive regions of the genome[2]. Here, we describe a workflow for translocation detection at low sequencing depth with Oxford Nanopore’s long read chromatin conformation capture technique, Pore-C[3]. Importantly, for translocation calling, Pore-C does not rely on breakpoint-spanning reads but rather the high intrachromosomal interaction frequency of genomic regions around the breakpoint, thus largely lowering sequencing depth requirements and reducing mapping issues in low complexity regions. We prepared, barcoded, and sequenced Pore-C libraries of 3 cancer cell lines including lung cancer (A549), Acute Monocytic Leukemia (THP-1), melanoma (COLO 829) and its matching normal (COLO 829 BL) on a single Q20+ MinION flow cell (FLO-MIN114). Genome-wide contact maps of low-pass data (<1.5 Gbps per sample) were compared against higher depth (>30 Gbps per sample) Hi-C maps [4], revealing that low-pass Pore-C successfully captured large-scale genomic rearrangement. For example, chromosomal pairs chr8-chr11 and chr15-chr19 in A549, chr9-chr11 and chr1-chr20 in THP1, and chr7-chr15 in Colo829 were detected at <0.5X depth of sequencing coverage. These translocation events were further validated by breakpoint analysis using adaptive sampling, where targeted regions were previously confirmed by independent studies[4],[5]. Low-pass Pore-C detects translocations in an unbiased manner and does not require prior knowledge of the translocation structure. Combined with its simple sample-preparation workflow and the capability to provide genome-wide copy-number information in a single experiment, it can serve as a cost-effective and comprehensive tool for cancer genomic studies. [1] Chromosomal translocations in human cancer, Nature, 372, 143 (1994) [2] Hi-C as a tool for precise detection and characterisation of chromosomal rearrangements and copy number variation in human tumors, Genome Biology, 18, 125 (2017) [3] Identifying synergistic high-order 3D chromatin conformations from genome-scale nanopore concatemer sequencing, Nature Biotechnology, 40, 1488(2022) [4] Chromosomal translocations detection in cancer cells using chromosomal conformation capture data, Genes (Basel), 13, 7 (2022) [5] A multi-platform reference for somatic structural variation detection, Cell Genomics, 2, 6 (2022) Citation Format: Scott Hickey, Xiaoguang Dai, Sergey Aganezov, John Beaulaurier, Eoghan Harrington, Sissel Juul. Translocation detection in cancer using low-pass pore-c sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 405.

Linker histone H1 drives heterochromatin condensation via phase separation in Arabidopsis.

In the eukaryotic nucleus, heterochromatin forms highly condensed, visible foci known as heterochromatin foci (HF). These HF are enriched with linker histone H1, a key player in heterochromatin condensation and silencing. However, it is unknown how H1 aggregates HF and condenses heterochromatin. In this study, we established that H1 facilitates heterochromatin condensation by enhancing inter- and intrachromosomal interactions between and within heterochromatic regions of the Arabidopsis (Arabidopsis thaliana) genome. We demonstrated that H1 drives HF formation via phase separation, which requires its C-terminal intrinsically disordered region (C-IDR). A truncated H1 lacking the C-IDR fails to form foci or recover HF in the h1 mutant background, whereas C-IDR with a short stretch of the globular domain (18 out of 71 amino acids) is sufficient to rescue both defects. In addition, C-IDR is essential for H1's roles in regulating nucleosome repeat length and DNA methylation in Arabidopsis, indicating that phase separation capability is required for chromatin functions of H1. Our data suggest that bacterial H1-like proteins, which have been shown to condense DNA, are intrinsically disordered and capable of mediating phase separation. Therefore, we propose that phase separation mediated by H1 or H1-like proteins may represent an ancient mechanism for condensing chromatin and DNA.

Three-dimensional chromatin landscapes in hepatocellular carcinoma associated with hepatitis B virus.

Three-dimensional (3D) chromatin architecture frequently altered in cancer. However, its changes during the pathogenesis of hepatocellular carcinoma (HCC) remained elusive. Hi-C and RNA-seq were applied to study the 3D chromatin landscapes and gene expression of HCC and ANHT. Hi-C Pro was used to generate genome-wide raw interaction matrices, which were normalized via iterative correction (ICE). Moreover, the chromosomes were divided into different compartments according to the first principal component (E1). Furthermore, topologically associated domains (TADs) were visualized via WashU Epigenome Browser. Furthermore, differential expression analysis of ANHT and HCC was performed using the DESeq2 R package. Additionally, dysregulated genes associated with 3D genome architecture altered were confirmed using TCGA, qRT-PCR, immunohistochemistry (IHC), etc. RESULTS: First, the intrachromosomal interactions of chr1, chr2, chr5, and chr11 were significantly different, and the interchromosomal interactions of chr4-chr10, chr13-chr21, chr15-chr22, and chr16-chr19 are remarkably different between ANHT and HCC, which resulted in the up-regulation of TP53I3 and ZNF738 and the down-regulation of APOC3 and APOA5 in HCC. Second, 49 compartment regions on 18 chromosomes have significantly switched (A-B or B-A) during HCC tumorigenesis, contributing to up-regulation of RAP2A. Finally, a tumor-specific TAD boundary located on chr5: 6271000-6478000 and enhancer hijacking were identified in HCC tissues, potentially associated with the elevated expression of MED10, whose expression were associated with poor prognosis of HCC patients. This study demonstrates the crucial role of chromosomal structure variation in HCC oncogenesis and potential novel biomarkers of HCC, laying a foundation for cancer precision medicine development.

Chromatin architecture of two different strains of Artemisia annua reveals the alterations in interaction and gene expression.

The hierarchical architecture of chromatins affects the gene expression level of glandular secreting trichomes and the artemisinin biosynthetic pathway-related genes, consequently bringing on huge differences in the content of artemisinin and its derivatives of A. annua. The plant of traditional Chinese medicine "Qinghao" is called Artemisia annua L. in Chinese Pharmacopoeia. High content and the total amount of artemisinin is the main goal of A. annua breeding, nevertheless, the change of chromatin organization during the artemisinin synthesis process has not been discovered yet. This study intended to find the roles of chromatin structure in the production of artemisinin through bioinformatics and experimental validation. Chromosome conformation capture analysis was used to scrutinize the interactions among chromosomes and categorize various scales of chromatin during artemisinin synthesis in A. annua. To confirm the effect of the changes in chromatin structure, Hi-C and RNA-sequencing were performed on two different strains to find the correlation between chromatin structure and gene expression levels on artemisinin synthesis progress and regulation. Our results revealed that the frequency of intra-chromosomal interactions was higher in the inter-chromosomal interactions between the root and leaves on a high artemisinin production strain (HAP) compared to a low artemisinin production strain (LAP). We found that compartmental transition was connected with interactions among different chromatins. Interestingly, glandular secreting trichomes (GSTs) and the artemisinin biosynthetic pathway (ABP) related genes were enriched in the areas which have the compartmental transition, reflecting the regulation of artemisinin synthesis. Topologically associated domain boundaries were associated with various distributions of genes and expression levels. Genes associated with ABP and GST in the adjacent loop were highly expressed, suggesting that epigenetic regulation plays an important role during artemisinin synthesis and glandular secreting trichomes production process. Chromatin structure could show an important status in the mechanisms of artemisinin synthesis process in A. annua.

EccDB: a comprehensive repository for eccDNA-mediated chromatin contacts in multi-species.

We developed the eccDB database to integrate available resources for extrachromosomal circular DNA (eccDNA) data. eccDB is a comprehensive repository for storing, browsing, searching, and analyzing eccDNAs from multispecies. The database provides regulatory and epigenetic information on eccDNAs, with a focus on analyzing intrachromosomal and interchromosomal interactions to predict their transcriptional regulatory functions. Moreover, eccDB identifies eccDNAs from unknown DNA sequences and analyzes the functional and evolutionary relationships of eccDNAs among different species. Overall, eccDB offers web-based analytical tools and a comprehensive resource for biologists and clinicians to decipher the molecular regulatory mechanisms of eccDNAs. eccDB is freely available at http://www.xiejjlab.bio/eccDB.

Long range chromosomal interactions and first passage process in budding yeast.

Chromosomal interaction plays a critical role for nuclear processes including DNA repair, replication, and transcription. One important step during the interaction is the encountering of two chromosome segments, which in statistical physics presents a first passage problem. Here, we use a novel method developed in our lab, chemically induced chromosomal interaction (CICI), to study chromosome motion and the first passage dynamics. We show that both random diffusion and cohesin loop extrusion contribute to intrachromosomal interactions. We also applied polymer physics models to understand the chromosome dynamics.

Umifenovir Epigenetically Targets the IL-10 Pathway in Therapy against Coxsackievirus B4 Infection.

Umifenovir, a broad-spectrum nonnucleoside antiviral drug, has a promising efficacy against coxsackievirus B4 (CVB4) infection, but its mechanism remains unclear. CVB4 is a common human single-stranded RNA virus that belongs to the Picornaviridae family and the Enterovirus genus. Enterovirus can cause severe diseases, such as meningitis, myocarditis, pancreatitis, insulin-dependent diabetes, and several other diseases, in both adults and children. We have previously demonstrated the critical role of interleukin 10 (IL-10) in promoting CVB4 infection and the downregulation of IL-10 by umifenovir. To further explore the underlying mechanisms of umifenovir, we characterized the epigenetic regulation of IL-10 in IL-10 knockout RAW264.7 cells and a BALB/c mouse splenocyte model. Mechanistically, we found that umifenovir inhibited CVB4-activated IL-10 by enhancing the methylation level of the repressive histones H3K9me3 and H3K27me3 while reducing the acetylation level of the activating histone H3K9ac in the promoter region of the IL-10 gene. Furthermore, using a chromosome conformation capture approach, we discovered that CVB4 infection activated the IL-10 gene by forming an intrachromosomal interaction between the IL-10 gene promoter and an intronic enhancer of the downstream MK2 (mitogen-activated protein kinase [MAPK]-activated protein kinase 2 [MAPKAPK2]) gene, a critical component of the p38-MAPK signaling pathway, which is required for IL-10 gene expression. However, umifenovir treatment abolished this spatial conformation and chromatin interaction, thus reducing the continuous expression of IL-10 and subsequent CVB4 replication. In conclusion, this study reveals a novel epigenetic mechanism by which umifenovir controls CVB4 infections, thus laying a theoretical foundation for therapeutic use of umifenovir. IMPORTANCE Viral infections are major threats to human health because of their strong association with a variety of inflammation-related diseases, especially cancer. Many antiviral drugs are performing poorly in treating viral infections. This is probably due to the immunosuppressive effect of highly expressed IL-10, which is caused by viral infection. Umifenovir is a broad-spectrum antiviral drug. Our recent studies showed that umifenovir has a significant inhibitory effect on CVB4 infection and can reduce IL-10 expression caused by CVB4. However, another antiviral drug, rupintrivir, showed good antiviral activity but had no effect on the expression of IL-10. This suggests that the regulation of IL-10 expression is a key part of the antiviral mechanism of umifenovir. Therefore, due to the dual function of the inhibition of CVB4 replication and the regulation of immune antiviral pathway, the mechanism of umifenovir is of great value to study.

NODULIN HOMEOBOX is required for heterochromatin homeostasis in Arabidopsis

Arabidopsis NODULIN HOMEOBOX (NDX) is a nuclear protein described as a regulator of specific euchromatic genes within transcriptionally active chromosome arms. Here we show that NDX is primarily a heterochromatin regulator that functions in pericentromeric regions to control siRNA production and non-CG methylation. Most NDX binding sites coincide with pericentromeric het-siRNA loci that mediate transposon silencing, and are antagonistic with R-loop structures that are prevalent in euchromatic chromosomal arms. Inactivation of NDX leads to differential siRNA accumulation and DNA methylation, of which CHH/CHG hypomethylation colocalizes with NDX binding sites. Hi-C analysis shows significant chromatin structural changes in the ndx mutant, with decreased intrachromosomal interactions at pericentromeres where NDX is enriched in wild-type plants, and increased interchromosomal contacts between KNOT-forming regions, similar to those observed in DNA methylation mutants. We conclude that NDX is a key regulator of heterochromatin that is functionally coupled to het-siRNA loci and non-CG DNA methylation pathways.

HULC targets the IGF1R–PI3K-AKT axis in trans to promote breast cancer metastasis and cisplatin resistance

Insulin-like growth factor I receptor (IGF1R) is frequently upregulated in breast cancer. Due to its intrinsic tyrosine kinase activity, aberrant activation of the IGF1R signaling axis may enhance tumor cell proliferation and cancer stemness, causing tumor relapse, metastasis and resistance to chemotherapy. We utilized a chromatin RNA in situ reverse transcription (CRIST) approach to characterize molecular factors that regulate the IGF1R network. We identified lncRNA HULC (Highly Upregulated in Liver Cancer) as a key trans-regulator of IGF1R in breast cancer cells. Loss of HULC suppressed the expression of IGF1R and the activation of its downstream PI3K/AKT pathway, while HULC overexpression activated the axis in breast cancer cells. Using a transcription-associated trap (RAT) assay, we demonstrated that HULC functioned as a nuclear lncRNA and epigenetically activated IGF1R by directly binding to the intragenic regulatory elements of the gene, orchestrating intrachromosomal interactions, and promoting histone H3K9 acetylation. The activated HULC-IGF1R/PI3K/AKT pathway mediated tumor resistance to cisplatin through the increased expression of cancer stemness markers, including NANOG, SOX2, OCT4, CD44 and ALDH1A1. In immunodeficient mice, stimulation of the HULC-IGF1R pathway promoted tumor metastasis. These data suggest that HULC may be a new epigenetic target for IGF1R axis-targeted therapeutic intervention.

DNA methylation in transposable elements buffers the connection between three-dimensional chromatin organization and gene transcription upon rice genome duplication

IntroductionPolyploidy is a major force in plant evolution and the domestication of cultivated crops. ObjectivesThe study aimed to explore the relationship and underlying mechanism between three-dimensional (3D) chromatin organization and gene transcription upon rice genome duplication. MethodsThe 3D chromatin structures between diploid (2C) and autotetraploid (4C) rice were compared using high-throughput chromosome conformation capture (Hi-C) analysis. The study combined genetics, transcriptomics, whole-genome bisulfite sequencing (WGBS-seq) and 3D genomics approaches to uncover the mechanism for DNA methylation in modulating gene transcription through 3D chromatin architectures upon rice genome duplication. ResultsWe found that 4C rice presents weakened intra-chromosomal interactions compared to its 2C progenitor in some chromosomes. In addition, we found that changes of 3D chromatin organizations including chromatin compartments, topologically associating domains (TADs), and loops, are uncorrelated with gene transcription. Moreover, DNA methylations in the regulatory sequences of genes in compartment A/B switched regions and TAD boundaries are unrelated to their expression. Importantly, although there was no significant difference in the methylation levels in transposable elements (TEs) in differentially expressed gene (DEG) and non-DEG promoters between 2C and 4C rice, we found that the hypermethylated TEs across genes in compartment A/B switched regions and TAD boundaries may suppress the expression of these genes. ConclusionThe study proposed that the rice genome doubling might modulate TE methylation to buffer the effects of chromatin architecture on gene transcription in compartment A/B switched regions and TAD boundaries, resulting in the disconnection between 3D chromatin structure alteration and gene transcription upon rice genome duplication.

P487: SMALL NON-CODING RNAS ASSOCIATE WITH CHROMATIN TO MAINTEIN PROPOGATION OF ACUTE MEYLOID LEUKEMIA

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the clonal expansion of myeloid progenitors without terminal differentiation. The interplay between genome and epigenome alterations drives aberrant gene network linking to leukemia transformation. Altered transcription may occur at three-dimensional chromatin level and be orchestrated by a combination of multiple regulatory factors. Although some RNA molecules, especially non-coding RNAs, were reported to modulate transcription in leukemia cells, the regulatory role of genome-wide RNAs on transcription through chromatin association remains largely unexplored. Aims: To identify novel RNA molecules as chromatin-association factors critical for leukemogenesis through modulating transcription Methods: We performed in situ Mapping of RNA-Genome Interactome (iMARGI) in a human acute leukemia cell line – MV4-11 cells. Chromatin association of candidate RNAs was verified using Chromatin Isolation by RNA Purification (ChIRP) by means of biotinylated oligonucleotides for target capture. A series of ChIP-seq assays were performed in multiple AML cell lines to profile chromatin occupancy by Fibrillarin (FBL), a catalytic component of C/D box ribonucleoproteins (snoRNPs), as well as to observe the remodelling of chromatin states marked by H3K27ac and H3K9me3 upon shRNA-mediated knockdown of FBL. Loss-of-function experiments using antisense oligonucleotides were performed on candidate chromatin-associated RNAs in AML cell lines, AML primary samples, and CD34+ healthy bone marrow cells. Results: In total, about 52.1 million RNA-chromatin interactions were identified out of deep sequencing of four iMARGI libraries. Whereas the vast majority (50.5 million, 97%) of RNA-chromatin interactome were intra-chromosome, a small fraction (1.6 million, 3%) occurred inter chromosome. Though protein-coding mRNAs predominate the chromatin-associated RNA species, other non-coding RNAs were also captured. Intriguingly, different from protein-coding mRNAs and long intergenic non-coding RNAs (lincRNAs) which mainly form intra-chromosome interactions, two small non-coding RNA classes, small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs), were found with prevalent frequency (62% and 92%, respectively) of inter-chromosome associations. SNORD3A (U3) and SNORD118 (U8) were C/D box snoRNAs ranked top by the number of their associated interactions in MV4-11 cells as well as in other cell types. Chromatin association of U3 and U8 was verified using ChIRP, with the observation of unique chromatin binding for both U3 and U8, and of specific binding motifs of transcription factors at their binding sites. ChIP-seq revealed that the snoRNP FBL occupied only a small set of genes (n=118), and shRNA-mediated knockdown of FBL had a minimal impact on H3K27ac and H3K9me3, suggesting that snoRNA-chromatin interactions may function independently on FBL-associated snoRNPs. ASO-mediated loss of U3 or U8 significantly impairs leukemia cells proliferation and colony forming capacity in multiple AML cell lines and primary AML blast samples, whereas only mildly affects CD34+ healthy cells. Summary/Conclusion: We identified chromatin-associated snoRNAs U3 and U8 which were essential for leukemia maintenance and may serve as therapeutic targets for leukemia eradication.

Global profiling of RNA-chromatin interactions reveals co-regulatory gene expression networks in Arabidopsis.

It is increasingly evident that various RNAs can bind chromatin to regulate gene expression and genome organization. Here we adapted a sequencing-based technique to profile RNA-chromatin interactions at a genome-wide scale in Arabidopsis seedlings. We identified more than 10,000 RNA-chromatin interactions mediated by protein-coding RNAs and non-coding RNAs. Cis and intra-chromosomal interactions are mainly mediated by protein-coding RNAs, whereas inter-chromosomal interactions are primarily mediated by non-coding RNAs. Many RNA-chromatin interactions tend to positively correlate with DNA-DNA interactions, suggesting their mutual influence and reinforcement. We further show that some RNA-chromatin interactions undergo alterations in response to biotic and abiotic stresses and that altered RNA-chromatin interactions form co-regulatory networks. Our study provides a global view on RNA-chromatin interactions in Arabidopsis and a rich resource for future investigations of regulatory roles of RNAs in gene expression and genome organization.

Hierarchical chromatin features reveal the toxin production in Bungarus multicinctus

BackgroundBungarus multicinctus, from which a classical Chinese medicine is produced, is known as the most venomous land snake in the world, but the chromatin organization and transcription factor activity during venom replenishment progress have not been explored yet. This study aimed to determine the roles of chromatin structure in toxin activity via bioinformatics and experimental validation.MethodsChromosome conformation capture (Hi-C) analysis was used to examine interactions among chromosomes and identify different scales of chromatin during envenomation in B. multicinctus. Correlations between epigenetic modifications and chromatin structure were verified through ChIP-seq analysis. RNA-seq was used to validate the influence of variation in chromatin structure and gene expression levels on venom production and regulation.ResultsOur results suggested that intra-chromosomal interactions are more intense than inter-chromosomal interactions among the control group, 3-day group of venom glands and muscles. Through this, we found that compartmental transition was correlated with chromatin interactions. Interestingly, the up-regulated genes in more compartmental switch regions reflect the function of toxin activity. Topologically associated domain (TAD) boundaries enriched with histone modifications are associated with different distributions of genes and the expression levels. Toxin-coding genes in the same loop are highly expressed, implying that the importance of epigenetic regulation during envenomination. On a smaller scale, the epigenetic markers affect transcriptional regulation by controlling the recruitment/inhibition of transcription initiation complexes.ConclusionsChromatin structure and epigenetic modifications could play a vital status role in the mechanisms of venom regulation in B. multicinctus.

Histone acetylation dynamics modulates chromatin conformation and allele-specific interactions at oncogenic loci.

In cancer cells, enhancer hijacking mediated by chromosomal alterations and/or increased deposition of acetylated histone H3 lysine 27 (H3K27ac) can support oncogene expression. However, how the chromatin conformation of enhancer-promoter interactions is affected by these events is unclear. In the present study, by comparing chromatin structure and H3K27ac levels in normal and lymphoma B cells, we show that enhancer-promoter-interacting regions assume different conformations according to the local abundance of H3K27ac. Genetic or pharmacological depletion of H3K27ac decreases the frequency and the spreading of these interactions, altering oncogene expression. Moreover, enhancer hijacking mediated by chromosomal translocations influences the epigenetic status of the regions flanking the breakpoint, prompting the formation of distinct intrachromosomal interactions in the two homologous chromosomes. These interactions are accompanied by allele-specific gene expression changes. Overall, our work indicates that H3K27ac dynamics modulates interaction frequency between regulatory regions and can lead to allele-specific chromatin configurations to sustain oncogene expression.

CBCR: A Curriculum Based Strategy For Chromosome Reconstruction.

In this paper, we introduce a novel algorithm that aims to estimate chromosomes’ structure from their Hi-C contact data, called Curriculum Based Chromosome Reconstruction (CBCR). Specifically, our method performs this three dimensional reconstruction using cis-chromosomal interactions from Hi-C data. CBCR takes intra-chromosomal Hi-C interaction frequencies as an input and outputs a set of coordinates that estimate the chromosome’s three dimensional structure in the form of a .pdb file. The algorithm relies on progressively training a distance-restraint-based algorithm with a strategy we refer to as curriculum learning. Curriculum learning divides the Hi-C data into classes based on contact frequency and progressively re-trains the distance-restraint algorithm based on the assumed importance of each curriculum in predicting the underlying chromosome structure. The distance-restraint algorithm relies on a modification of a Gaussian maximum likelihood function that scales probabilities based on the importance of features. We evaluate the performance of CBCR on both simulated and actual Hi-C data and perform validation on FISH, HiChIP, and ChIA-PET data as well. We also compare the performance of CBCR to several current methods. Our analysis shows that the use of curricula affects the rate of convergence of the optimization while decreasing the computational cost of our distance-restraint algorithm. Also, CBCR is more robust to increases in data resolution and therefore yields superior reconstruction accuracy of higher resolution data than all other methods in our comparison.

Sox2-Evf2 lncRNA-mediated mechanisms of chromosome topological control in developing forebrain.

The Evf2 long non-coding RNA directs Dlx5/6 ultraconserved enhancer(UCE)-intrachromosomal interactions, regulating genes across a 27 Mb region on chromosome 6 in mouse developing forebrain. Here, we show that Evf2 long-range gene repression occurs through multi-step mechanisms involving the transcription factor Sox2. Evf2 directly interacts with Sox2, antagonizing Sox2 activation of Dlx5/6UCE, and recruits Sox2 to the Dlx5/6eii shadow enhancer and key Dlx5/6UCE interaction sites. Sox2 directly interacts with Dlx1 and Smarca4, as part of the Evf2 ribonucleoprotein complex, forming spherical subnuclear domains (protein pools, PPs). Evf2 targets Sox2 PPs to one long-range repressed target gene (Rbm28), at the expense of another (Akr1b8). Evf2 and Sox2 shift Dlx5/6UCE interactions towards Rbm28, linking Evf2/Sox2 co-regulated topological control and gene repression. We propose a model that distinguishes Evf2 gene repression mechanisms at Rbm28 (Dlx5/6UCE position) and Akr1b8 (limited Sox2 availability). Genome-wide control of RNPs (Sox2, Dlx and Smarca4) shows that co-recruitment influences Sox2 DNA binding. Together, these data suggest that Evf2 organizes a Sox2 PP subnuclear domain and, through Sox2-RNP sequestration and recruitment, regulates chromosome 6 long-range UCE targeting and activity with genome-wide consequences.