Annotation of nuclear lncRNAs based on chromatin interactions.

The human genome is pervasively transcribed and produces a wide variety of long non-coding RNAs (lncRNAs), constituting the majority of transcripts across human cell types. Some specific nuclear lncRNAs have been shown to be important regulatory components acting locally. As RNA-chromatin interaction and Hi-C chromatin conformation data showed that chromatin interactions of nuclear lncRNAs are determined by the local chromatin 3D conformation, we used Hi-C data to identify potential target genes of lncRNAs. RNA-protein interaction data suggested that nuclear lncRNAs act as scaffolds to recruit regulatory proteins to target promoters and enhancers. Nuclear lncRNAs may therefore play a role in directing regulatory factors to locations spatially close to the lncRNA gene. We provide the analysis results through an interactive visualization web portal at https://fantom.gsc.riken.jp/zenbu/reports/#F6_3D_lncRNA.

A conserved HOTAIRM1-HOXA1 regulatory axis contributes early to neuronal differentiation.

HOTAIRM1 is unlike most long non-coding RNAs in that its sequence is highly conserved across mammals. Such evolutionary conservation points to it having a role in key cellular processes. We previously reported that HOTAIRM1 is required to curb premature activation of downstream HOXA genes in a cell model recapitulating their sequential induction during development. We found that it regulates 3' HOXA gene expression by a mechanism involving epigenetic and three-dimensional chromatin changes. Here we show that HOTAIRM1 participates in proper progression through the early stages of neuronal differentiation. We found that it can associate with the HOXA1 transcription factor and contributes to its downstream transcriptional program. Particularly, HOTAIRM1 affects the NANOG/POU5F1/SOX2 core pluripotency network maintaining an undifferentiated cell state. HOXA1 depletion similarly perturbed expression of these pluripotent factors, suggesting that HOTAIRM1 is a modulator of this transcription factor pathway. Also, given that binding of HOTAIRM1 to HOXA1 was observed in different cell types and species, our results point to this ribonucleoprotein complex as an integral part of a conserved HOTAIRM1-HOXA1 regulatory axis modulating the transition from a pluripotent to a differentiated neuronal state.

The Talented LncRNAs: Meshing into Transcriptional Regulatory Networks in Cancer.

As a group of diseases characterized by uncontrollable cell growth, cancer is highly multifaceted in how it overrides checkpoints controlling proliferation. Amongst the regulators of these checkpoints, long non-coding RNAs (lncRNAs) can have key roles in why natural biological processes go haywire. LncRNAs represent a large class of regulatory transcripts that can localize anywhere in cells. They were found to affect gene expression on many levels from transcription to mRNA translation and even protein stability. LncRNA participation in such control mechanisms can depend on cell context, with given transcripts sometimes acting as oncogenes or tumor suppressors. Importantly, the tissue-specificity and low expression levels of lncRNAs make them attractive therapeutic targets or biomarkers. Here, we review the various cellular processes affected by lncRNAs and outline molecular strategies they use to control gene expression, particularly in cancer and in relation to transcription factors.

Antisense-oligonucleotide-mediated perturbation of long non-coding RNA reveals functional features in stem cells and across cell types.

Within the scope of the FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem cells (iPSCs) and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition. From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.3%) show molecular phenotypes. Integrating the molecular phenotypes with chromatin-interaction assays further reveals cis- and trans-interacting partners as potential primary targets. Additionally, cell-type enrichment analysis identifies lncRNAs associated with pluripotency, while the knockdown of LINC02595, CATG00000090305.1, and RP11-148B6.2 modulates colony formation of iPSCs. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit a consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights that molecular phenotyping is more comprehensive in revealing affected pathways.

Profiling Chromatin Landscape at High Resolution and Throughput with 2C-ChIP.

Chromatin immunoprecipitation (ChIP) is used to probe the presence of proteins and/or their posttranslational modifications on genomic DNA. This method is often used alongside chromosome conformation capture approaches to obtain a better-rounded view of the functional relationship between chromatin architecture and its landscape. Since the inception of ChIP, its protocol has been modified to improve speed, sensitivity, and specificity. Combining ChIP with deep sequencing has recently improved its throughput and made genome-wide profiling possible. However, genome-wide analysis is not always the best option, particularly when many samples are required to study a given genomic region or when quantitative data is desired. We recently developed carbon copy-ChIP (2C-ChIP), a new form of the high-throughput ChIP analysis method ideally suited for these types of studies. 2C-ChIP applies ligation-mediated amplification (LMA) followed by deep sequencing to quantitatively detect specified genomic regions in ChIP samples. Here, we describe the generation of 2C-ChIP libraries and computational processing of the resulting sequencing data.

Functional annotation of human long noncoding RNAs via molecular phenotyping.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

LAMPS: an analysis pipeline for sequence-specific ligation-mediated amplification reads.

OBJECTIVE: Ligation-Mediated Amplification (LMA) is a versatile biochemical tool for amplifying selected DNA sequences. LMA has increased in popularity due to its integration within chromosome conformation capture (5C) and chromatin immunoprecipitation (2C-ChIP) methodologies. The output of either 5C or 2C-ChIP protocols is a single-read sequencing library of ligated primer pairs that may or may not be multiplexed. While many computational tools currently exist for read mapping and analysis, these tools neither fully support multiplexed libraries nor provide qualitative reporting on the LMA primers involved. Typically, the task of library demultiplexing or primer analysis is offloaded on to the user. Our aim was to develop an easy-to-use pipeline for processing (multiplexed) single-read sequencing data produced by sequence-specific LMA. RESULTS: Here, we describe the Ligation-mediated Amplified, Multiplexed Primer-pair Sequence (LAMPS) analysis pipeline. LAMPS facilitates the analysis of multiplexed LMA sequencing data and provides a thorough assessment of a library's reads for a variety of experimental parameters (e.g., primer-pair efficiency). The standardized output of LAMPS allows for easy integration with downstream analyses, such as data track visualization on a genome browser. LAMPS is made publicly available on GitHub: https://github.com/BlanchetteLab/LAMPS.

HIFI: estimating DNA-DNA interaction frequency from Hi-C data at restriction-fragment resolution.

Hi-C is a popular technique to map three-dimensional chromosome conformation. In principle, Hi-C's resolution is only limited by the size of restriction fragments. However, insufficient sequencing depth forces researchers to artificially reduce the resolution of Hi-C matrices at a loss of biological interpretability. We present the Hi-C Interaction Frequency Inference (HIFI) algorithms that accurately estimate restriction-fragment resolution Hi-C matrices by exploiting dependencies between neighboring fragments. Cross-validation experiments and comparisons to 5C data and known regulatory interactions demonstrate HIFI's superiority to existing approaches. In addition, HIFI's restriction-fragment resolution reveals a new role for active regulatory regions in structuring topologically associating domains.

PRC2-Associated Chromatin Contacts in the Developing Limb Reveal a Possible Mechanism for the Atypical Role of PRC2 in HoxA Gene Expression.

Preventing inappropriate gene expression in time and space is as fundamental as triggering the activation of tissue- or cell-type-specific factors at the correct developmental stage and in the correct cells. Here, we study the impact of Polycomb repressive complex 2 (PRC2) function on HoxA gene regulation. We analyze chromatin conformation of the HoxA cluster and its regulatory regions and show that in addition to the well-known role of PRC2 in silencing Hox genes via direct binding, its function is required for the changes in HoxA long-range interactions distinguishing proximal limbs from distal limbs. This effect stems from the differential PRC2 occupancy over the HoxA cluster and, at least in part, from the ability of PRC2-bound loci to engage in long-range contacts. Unexpectedly, PRC2 also impacts chromatin conformation in a way that promotes enhancer-promoter contacts required for proper HoxA expression, pointing to a dual role of PRC2 in gene regulation.

2C-ChIP: measuring chromatin immunoprecipitation signal from defined genomic regions with deep sequencing.

BACKGROUND: Understanding how transcription occurs requires the integration of genome-wide and locus-specific information gleaned from robust technologies. Chromatin immunoprecipitation (ChIP) is a staple in gene expression studies, and while genome-wide methods are available, high-throughput approaches to analyze defined regions are lacking. RESULTS: Here, we present carbon copy-ChIP (2C-ChIP), a versatile, inexpensive, and high-throughput technique to quantitatively measure the abundance of DNA sequences in ChIP samples. This method combines ChIP with ligation-mediated amplification (LMA) and deep sequencing to probe large genomic regions of interest. 2C-ChIP recapitulates results from benchmark ChIP approaches. We applied 2C-ChIP to the HOXA cluster to find that a region where H3K27me3 and SUZ12 linger encodes HOXA-AS2, a long non-coding RNA that enhances gene expression during cellular differentiation. CONCLUSIONS: 2C-ChIP fills the need for a robust molecular biology tool designed to probe dedicated genomic regions in a high-throughput setting. The flexible nature of the 2C-ChIP approach allows rapid changes in experimental design at relatively low cost, making it a highly efficient method for chromatin analysis.

SMARCA4 loss is synthetic lethal with CDK4/6 inhibition in non-small cell lung cancer.

Tumor suppressor SMARCA4 (BRG1), a key SWI/SNF chromatin remodeling gene, is frequently inactivated in cancers and is not directly druggable. We recently uncovered that SMARCA4 loss in an ovarian cancer subtype causes cyclin D1 deficiency leading to susceptibility to CDK4/6 inhibition. Here, we show that this vulnerability is conserved in non-small cell lung cancer (NSCLC), where SMARCA4 loss also results in reduced cyclin D1 expression and selective sensitivity to CDK4/6 inhibitors. In addition, SMARCA2, another SWI/SNF subunit lost in a subset of NSCLCs, also regulates cyclin D1 and drug response when SMARCA4 is absent. Mechanistically, SMARCA4/2 loss reduces cyclin D1 expression by a combination of restricting CCND1 chromatin accessibility and suppressing c-Jun, a transcription activator of CCND1. Furthermore, SMARCA4 loss is synthetic lethal with CDK4/6 inhibition both in vitro and in vivo, suggesting that FDA-approved CDK4/6 inhibitors could be effective to treat this significant subgroup of NSCLCs.

CDK4/6 inhibitors target SMARCA4-determined cyclin D1 deficiency in hypercalcemic small cell carcinoma of the ovary.

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor suppressor, may also be exploited therapeutically.

Novel long-range regulatory mechanisms controlling PKD2 gene expression.

BACKGROUND: Cis-regulatory elements control gene expression over large distances through the formation of chromatin loops, which allow contact between enhancers and gene promoters. Alterations in cis-acting regulatory systems could be linked to human genetic diseases. Here, we analyse the spatial organization of a large region spanning the polycystic kidney disease 2 (PKD2) gene, one of the genes responsible of autosomal dominant polycystic kidney disease (ADPKD). RESULTS: By using chromosome conformation capture carbon copy (5C) technology in primary human renal cyst epithelial cells, we identify novel contacts of the PKD2 promoter with chromatin regions, which display characteristics of regulatory elements. In parallel, by using functional analysis with a reporter assay, we demonstrate that three DNAse I hypersensitive sites regions are involved in the regulation of PKD2 gene expression. CONCLUSIONS: Finally, through alignment of CCCTC-binding factor (CTCF) sites, we suggest that these novel enhancer elements are brought to the PKD2 promoter by chromatin looping via the recruitment of CTCF.

Double-Stranded Biotinylated Donor Enhances Homology-Directed Repair in Combination with Cas9 Monoavidin in Mammalian Cells.

Homology-directed repair (HDR) induced by site specific DNA double-strand breaks with CRISPR-Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non-homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered a Cas9 protein fused to a monoavidin domain to bind biotinylated donor DNA. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9-donor DNA complexes into cells. Improved HDR percentages of up to 90% in three loci tested (CXCR4, EMX1, and TLR) in standard HEK293T cells were observed. Our results suggest that donor DNA biotinylation and Cas9-donor conjugation in addition to delivery influence HDR efficiency.

Complex multi-enhancer contacts captured by genome architecture mapping.

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

Reciprocal regulation of chromatin state and architecture by HOTAIRM1 contributes to temporal collinear HOXA gene activation.

Thousands of long non-coding RNAs (lncRNAs) have been identified in mammals, many of which represent important regulators of gene expression. However, the mechanisms used by lncRNAs to control transcription remain largely uncharacterized. Here, we report on HOTAIRM1, a promising lncRNA biomarker in leukemia and solid tumors. We find that HOTAIRM1 contributes to three-dimensional chromatin organization changes required for the temporal collinear activation of HOXA genes. We show that distinct HOTAIRM1 variants preferentially associate with either UTX/MLL or PRC2 complexes to modulate the levels of activating and silencing marks at the bivalent domain. HOTAIRM1 contributes to physical dissociation of chromatin loops at the cluster proximal end, which delays recruitment of the histone demethylase UTX and transcription of central HOXA genes. Interestingly, we find overall proximal HOXA gene activation without chromatin conformation changes by HOTAIRM1 in a different cell type. Our results reveal a previously unappreciated relationship between chromatin structure, architecture and lncRNA function.

Chromosome folding and its regulation in health and disease.

There are many ways in which cells may not adequately behave or respond to their environment, and the molecular mechanisms leading to these defects are as diverse as they are many. In this review, we report on how spatial chromatin organization contributes to the proper expression of genes, relating how CTCF-one of its main architects-contributes to gene regulation. We also touch on the emerging role of long noncoding RNAs in shaping chromatin organization and activity. The HOX gene clusters have been used as paradigm in the study of various biological pathways, and the overview we provide gives emphasis to what research on these loci has revealed about chromatin architecture and its regulation in the control of gene expression.

Spatial Organization of Epigenomes.

The role of genome architecture in transcription regulation has become the focus of an increasing number of studies over the past decade. Chromatin organization can have a significant impact on gene expression by promoting or restricting the physical proximity between regulatory DNA elements. Given that any change in chromatin state has the potential to alter DNA folding and the proximity between control elements, the spatial organization of chromatin is inherently linked to its molecular composition. In this review, we explore how modulators of chromatin state and organization might keep gene expression in check. We discuss recent findings and present some of the less well-studied aspects of spatial genome organization such as chromatin dynamics and regulation by non-coding RNAs.

Analysis of long-range interactions in primary human cells identifies cooperative CFTR regulatory elements.

A mechanism by which control DNA elements regulate transcription over large linear genomic distances is by achieving close physical proximity with genes, and looping of the intervening chromatin paths. Alterations of such regulatory 'chromatin looping' systems are likely to play a critical role in human genetic disease at large. Here, we studied the spatial organization of a ≈790 kb locus encompassing the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Dysregulation of CFTR is responsible for cystic fibrosis, which is the most common lethal genetic disorder in Caucasian populations. CFTR is a relatively large gene of 189 kb with a rather complex tissue-specific and temporal expression profile. We used chromatin conformation at the CFTR locus to identify new DNA sequences that regulate its transcription. By comparing 5C chromatin interaction maps of the CFTR locus in expressing and non-expressing human primary cells, we identified several new contact points between the CFTR promoter and its surroundings, in addition to regions featuring previously described regulatory elements. We demonstrate that two of these novel interacting regions cooperatively increase CFTR expression, and suggest that the new enhancer elements located on either side of the gene are brought together through chromatin looping via CTCF.