Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) strategy to ...comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CCCTC-binding factor (CTCF) and RNA polymerase II (RNAPII) with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription. Furthermore, we show that haplotype variants and allelic interactions have differential effects on chromosome configuration, influencing gene expression, and may provide mechanistic insights into functions associated with disease susceptibility. 3D genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.
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•ChIA-PET is inclusive in mapping 3D genome at multi-scale and nucleotide resolution•CTCF foci spatially arrange RNAPII transcription concordant in CTCF-motif direction•SNPs alter haplotype chromatin topology and function that link to disease risks•3D genome models elucidate topological framework for transcriptional regulation
Advanced ChIA-PET shows that CTCF/cohesin and RNA polymerase II arrange spatial organization for coordinated transcription. Haplotype variants exhibit allelic effects on chromatin topology and transcription that link disease susceptibility.
Abstract
Alternative splicing allows expression of mRNA isoforms from a single gene, expanding the diversity of the proteome. Its prevalence in normal biological and disease processes warrant precise ...tools for modulation. Here we report the engineering of CRISPR Artificial Splicing Factors (CASFx) based on RNA-targeting CRISPR-Cas systems. We show that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also create inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing. Finally, we demonstrate the activation of
SMN2
exon 7 splicing in spinal muscular atrophy (SMA) patient fibroblasts, suggesting a potential application of the CASFx system.
Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable ...markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.
The three-dimensional (3D) genome structure plays a fundamental role in gene regulation and cellular functions. Recent studies in 3D genomics inferred the very basic functional chromatin folding ...structures known as chromatin loops, the long-range chromatin interactions that are mediated by protein factors and dynamically extruded by cohesin. We combined the use of FISH staining of a very short (33 kb) chromatin fragment, interferometric photoactivated localization microscopy (iPALM), and traveling salesman problem-based heuristic loop reconstruction algorithm from an image of the one of the strongest CTCF-mediated chromatin loops in human lymphoblastoid cells. In total, we have generated thirteen good quality images of the target chromatin region with 2-22 nm oligo probe localization precision. We visualized the shape of the single chromatin loops with unprecedented genomic resolution which allowed us to study the structural heterogeneity of chromatin looping. We were able to compare the physical distance maps from all reconstructed image-driven computational models with contact frequencies observed by ChIA-PET and Hi-C genomic-driven methods to examine the concordance between single cell imaging and population based genomic data.
Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human ...estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.
Supratentorial ependymoma (ST-EPN) is a type of malignant brain tumor mainly seen in children. Since 2014, it has been known that an intrachromosomal fusion
C11orf95-RELA
is an oncogenic driver in ...ST-EPN Parker et al. Nature 506:451–455 (2014); Pietsch et al. Acta Neuropathol 127:609–611 (2014) but the molecular mechanisms of oncogenesis are unclear. Here we show that the C11orf95 component of the fusion protein dictates DNA binding activity while the RELA component is required for driving the expression of ependymoma-associated genes. Epigenomic characterizations using ChIP-seq and HiChIP approaches reveal that C11orf95-RELA modulates chromatin states and mediates chromatin interactions, leading to transcriptional reprogramming in ependymoma cells. Our findings provide important characterization of the molecular underpinning of
C11orf95-RELA
fusion and shed light on potential therapeutic targets for
C11orf95-RELA
subtype ependymoma.
Acute promyeloid leukemia (APL) is characterized by the oncogenic fusion protein PML-RARα, a major etiological agent in APL. However, the molecular mechanisms underlying the role of PML-RARα in ...leukemogenesis remain largely unknown.
Using an inducible system, we comprehensively analyze the 3D genome organization in myeloid cells and its reorganization after PML-RARα induction and perform additional analyses in patient-derived APL cells with native PML-RARα. We discover that PML-RARα mediates extensive chromatin interactions genome-wide. Globally, it redefines the chromatin topology of the myeloid genome toward a more condensed configuration in APL cells; locally, it intrudes RNAPII-associated interaction domains, interrupts myeloid-specific transcription factors binding at enhancers and super-enhancers, and leads to transcriptional repression of genes critical for myeloid differentiation and maturation.
Our results not only provide novel topological insights for the roles of PML-RARα in transforming myeloid cells into leukemia cells, but further uncover a topological framework of a molecular mechanism for oncogenic fusion proteins in cancers.
The unique virus-cell interaction in Epstein-Barr virus (EBV)-associated malignancies implies targeting the viral latent-lytic switch is a promising therapeutic strategy. However, the lack of ...specific and efficient therapeutic agents to induce lytic cycle in these cancers is a major challenge facing clinical implementation. We develop a synthetic transcriptional activator that specifically activates endogenous BZLF1 and efficiently induces lytic reactivation in EBV-positive cancer cells. A lipid nanoparticle encapsulating nucleoside-modified mRNA which encodes a BZLF1-specific transcriptional activator (mTZ3-LNP) is synthesized for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more efficiently activates EBV lytic gene expression in EBV-associated epithelial cancers. Here we show the potency and safety of treatment with mTZ3-LNP to suppress tumor growth in EBV-positive cancer models. The combination of mTZ3-LNP and ganciclovir yields highly selective cytotoxic effects of mRNA-based lytic induction therapy against EBV-positive tumor cells, indicating the potential of mRNA nanomedicine in the treatment of EBV-associated epithelial cancers.
The genomes of multicellular organisms are extensively folded into 3D chromosome territories within the nucleus
. Advanced 3D genome-mapping methods that combine proximity ligation and ...high-throughput sequencing (such as chromosome conformation capture, Hi-C)
, and chromatin immunoprecipitation techniques (such as chromatin interaction analysis by paired-end tag sequencing, ChIA-PET)
, have revealed topologically associating domains
with frequent chromatin contacts, and have identified chromatin loops mediated by specific protein factors for insulation and regulation of transcription
. However, these methods rely on pairwise proximity ligation and reflect population-level views, and thus cannot reveal the detailed nature of chromatin interactions. Although single-cell Hi-C
potentially overcomes this issue, this method may be limited by the sparsity of data that is inherent to current single-cell assays. Recent advances in microfluidics have opened opportunities for droplet-based genomic analysis
but this approach has not yet been adapted for chromatin interaction analysis. Here we describe a strategy for multiplex chromatin-interaction analysis via droplet-based and barcode-linked sequencing, which we name ChIA-Drop. We demonstrate the robustness of ChIA-Drop in capturing complex chromatin interactions with single-molecule precision, which has not been possible using methods based on population-level pairwise contacts. By applying ChIA-Drop to Drosophila cells, we show that chromatin topological structures predominantly consist of multiplex chromatin interactions with high heterogeneity; ChIA-Drop also reveals promoter-centred multivalent interactions, which provide topological insights into transcription.
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