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bioRxiv2024

Simultaneous protein and RNA analysis in single extracellular vesicles, including viruses: SPIRFISH

Interest in using nanoparticles for delivery of therapeutic RNA has been steadily growing, provoking a need to precisely understand their structure and contents. Single-particle and single-molecule analysis techniques provide snapshots of single biological nanoparticles, including viruses, liposomes, and extracellular vesicles (EVs). While existing methods primarily focus on protein detection, RNA delivery is becoming increasingly prevalent. A method to simultaneously detect protein and internal RNA in the same particle would reveal variability in size, structure, and RNA packaging efficiency, enabling optimization of nanoparticle delivery. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor (SP-IRIS) with single-molecule fluorescence in-situ hybridization (smFISH). Using SPIRFISH, we detect HIV-1 envelope protein and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNA within EVs. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles. Teaser: A new single particle analysis technique simultaneously detects specific RNA and protein in biological nanoparticles.

molecular biology↗

bioRxiv2024

Development of antisense tools to study Bodo saltans and its intracellular symbiont

Obligate symbioses are common in nature and present a particular challenge for functional genetic analysis. In many cases, the host is a non-model species with poor tools for genetic manipulation and the symbiont cannot be cultured or its gene expression manipulated to investigate function. Here we investigated the potential for using antisense inhibition to analyse host and symbiont gene function within an obligate aquatic symbiosis. We focused on the kinetoplastid host Bodo saltans and its bacterial symbiont, Candidatus Bodocaedibacter vickermanii, a member of Rickettsiales. We conclude that antisense inhibition is not feasible in the B. saltans and its symbiont, as the holobiont feeds on the antisense molecules - and increases in numbers - upon treatment with the antisense construct. Although our approach has proven unsuccessful, we have developed an array of protocols which can be used to study the biology of this microeukaryote and its microbial associates.

molecular biology↗

bioRxiv2025

Glycosomal Aquaglyceroporin 1 Dual Role in Iron Homeostasis and Antimony Susceptibility in Leishmania amazonensis

Leishmania parasites cause a spectrum of diseases known as leishmaniases and must acquire nutrients like iron while surviving host defenses. Aquaglyceroporin 1 (AQP1) is a membrane channel that, in L. major, localizes to the flagellum and mediates antimony uptake and cell-volume regulation. Here, we show that in L. amazonensis AQP1 is instead targeted to glycosomes and that its expression is modulated by iron availability. A CRISPR-Cas9-mediated knockout of AQP1 in L. amazonensis revealed its multifunctional importance. AQP1-null promastigotes displayed a significant growth defect, particularly under iron-depleted conditions, and were impaired in regulating cell volume under osmotic stress. The mutant parasites contained approximately 50% less intracellular iron than wild-type cells and showed an increase in total superoxide dismutase activity, underscoring a role for AQP1 in iron homeostasis and oxidative stress management. AQP1 deletion also markedly reduced virulence in murine macrophages and in infected mice. Strikingly, loss of AQP1 increased resistance to trivalent antimony (SbIII), a first-line antileishmanial drug. AQP1-knockout promastigotes showed a 70% increase in SbIII EC50 and accumulated more Sb intracellularly than wild-type, suggesting an altered antimony handling. Altogether, L. amazonensis AQP1 is a glycosomal protein that links iron metabolism, osmoregulation, and antimony susceptibility. Its glycosomal targeting and multifaceted roles differ from those of AQP1 orthologs in other Leishmania species. These findings suggest the existence of additional antimony uptake mechanisms beyond AQP1, with implications for understanding drug resistance. Author SummaryLeishmaniases are neglected tropical diseases caused by parasites that survive and multiply inside vertebrates cells. These parasites rely on hosts nutrients like iron and must resist both host defenses and treatment with toxic drugs such as antimony. We studied a protein called Aquaglyceroporin 1 (AQP1) in Leishmania amazonensis, a species that causes skin lesions in South America. Unlike related species, where AQP1 is found on the parasites surface, we discovered that in L. amazonensis AQP1 is located in an internal organelle called glycosome. By deleting this protein from the parasite, we found that it plays a crucial role in iron balance, sensitivity to antimony drugs, and the parasites ability to cause disease. Unexpectedly, parasites without AQP1 were more resistant to antimony but still accumulated high levels of the drug, suggesting that Leishmania has other ways of taking up antimony. Our findings challenge the assumption that all Leishmania species use the same strategies to survive, and highlight the need to understand species-specific differences when designing treatments or analyzing parasite biology.

molecular biology↗

bioRxiv2022

Comprehensive analysis of Arabidopsis thaliana DNA polymerase epsilon catalytic subunit A and B mutants - an insight into differentially expressed genes and protein-protein interactions

One of the main replicative enzymes in most eukaryotes, DNA polymerase {varepsilon} (POLE), is composed of four subunits, namely a single catalytic and three regulatory subunits. In Arabidopsis, the catalytic subunit of POLE is encoded by two genes: Arabidopsis thaliana DNA POLYMERASE EPSILON CATALYTIC SUBUNIT A (AtPOL2A) and B (AtPOL2B). Although studies have shown AtPOL2A to be involved in various biological processes, the role of AtPOL2B is unclear. Here, we investigated the transcriptomes of both atpol2a and atpol2b mutants, and the promoter sequences to provide a better insight into the targets of AtPOL2s at the molecular level. In the present study, leaf cDNA libraries of four AtPOL2 mutants (atpol2a-1 and atpol2b-1, -2 and - 3) were sequenced using the Illumina platform. Analysis of gene expression profiles identified a total of 198, 76, 141 and 67 differentially expressed genes in atpol2a-1, atpol2b-1, atpol2b-2 and atpol2b-3, respectively; the majority of pericentromeric transposable elements were transcriptionally active in atpol2a-1 as compared to atpol2b mutants and wild type. Protein-protein interaction network analysis and molecular docking identified three (CER1, RPA1E and AT5G60250) and two (PR1 and AT5G48490) proteins as potential interactors (cluster size > 60 and balanced score < -900) of AtPOL2A and AtPOL2B, respectively; Interestingly, these five proteins also showed a significant interaction between POLE catalytic subunit of Saccharomyces cerevisiae. Our in silico promoter analysis showed that the AtPOL2A promoter sequence is overrepresented with cis-acting regulatory elements (CREs) associate with cell cycle regulation, meristematic/reproductive tissue-specific pattern of expression and MYB protein recognition, whereas the AtPOL2B promoter sequence was mainly enriched with stress-responsive elements. The information provided here has led to the identification of targets of AtPOL2s at the molecular level and CREs putatively associated with the regulation of AtPOL2s. To our knowledge, this study provides the first comparative transcriptome profiling of single-gene mutants of AtPOL2s.

molecular biology↗

bioRxiv2020

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells

The emergence of a novel, highly pathogenic coronavirus, 2019-nCoV, in China, and its rapid national and international spread pose a global health emergency. Coronaviruses use their spike proteins to select and enter target cells and insights into nCoV-2019 spike (S)-driven entry might facilitate assessment of pandemic potential and reveal therapeutic targets. Here, we demonstrate that 2019-nCoV-S uses the SARS-coronavirus receptor, ACE2, for entry and the cellular protease TMPRSS2 for 2019-nCoV-S priming. A TMPRSS2 inhibitor blocked entry and might constitute a treatment option. Finally, we show that the serum form a convalescent SARS patient neutralized 2019-nCoV-S-driven entry. Our results reveal important commonalities between 2019-nCoV and SARS-coronavirus infection, which might translate into similar transmissibility and disease pathogenesis. Moreover, they identify a target for antiviral intervention. One sentence summaryThe novel 2019 coronavirus and the SARS-coronavirus share central biological properties which can guide risk assessment and intervention.

molecular biology↗

bioRxiv2020

HSATII RNA is induced via E2F3a and a non-canonical ATM-regulated DNA damage response pathway

Pericentromeric human satellite II (HSATII) repeats are normally silent, but can be actively transcribed in tumor cells, where increased HSATII copy number is associated with a poor prognosis in colon cancer, and in human cytomegalovirus (HCMV)-infected cells, where the RNA facilitates viral replication. Here, we report that HCMV infection or treatment of ARPE-19 diploid epithelial cells with the DNA-damaging agents, etoposide and zeocin, induced HSATII RNA expression, and a kinase-independent function of ATM was required for the induction. Additionally, various breast cancer cell lines growing in adherent, 2-dimensional cell culture expressed HSATII RNA at different levels, and levels were markedly increased when cells were either infected with HCMV or treated with zeocin. High levels of HSATII RNA expression correlated with enhanced migration of breast cancer cells, and knockdown of HSATII RNA reduced cell migration and the rate of cell proliferation. Our investigation links high expression of HSATII RNA to the DNA damage response, centered on a non-canonical function of ATM, and demonstrates a role for the satellite RNA in tumor cell proliferation and movement. SIGNIFICANCEHSATII RNA is associated with cancer progression, immunostimulation and, as we recently reported, it plays an important role in herpesvirus infections. However, the understanding of cellular processes responsible for the expression of HSATII RNA has been limited. Our current investigation identified a non-canonical, ATM kinase-independent DNA-damage response pathway as a common cellular mechanism regulating HSATII RNA induction in virus-infected cells or cells treated with DNA-damaging agents. Additionally, our study provides a link between expression of HSATII RNA and the cellular growth and migration phenotypes of cancer cells, establishing a new paradigm to study the biological consequences of HSATII RNA expression, i.e., treatment of normal diploid and tumor cells with DNA-damaging agents.

molecular biology↗

bioRxiv2021

Human Tear Metabolomics Using Liquid Chromatography-Q Exactive-HF Mass Spectrometry

AimTo putatively identify and characterise human tear metabolites in a normal subject on an untargeted platform of liquid chromatography-Q exactive-HF mass spectrometry. MethodsFour samples of unstimulated tears were collected from both eyes on four consecutive days between 1 - 2 pm using a microcapillary tube and pooled from both eyes each day. Untargeted analysis of the tears was performed by chromatographic separation of constituent metabolites in both CSH-C18RP (Charged Surface Hybrid-C18 Reversed Phase) and SeQuant ZIC-pHILIC (Zwitterionic-polymeric Hydrophilic Interaction Liquid Chromatography) columns, followed by heated electrospray ionization (HESI) and the acquisition of mass spectra using QExactive-HF mass spectrometer. Compound Discoverer software (v2.0) was used for data analysis. ResultEighty-two metabolites were tentatively identified. Seventy compounds (85.4 %) were observed in all four samples with a coefficient of variation (CV) less than 25 %. Fifty-nine metabolites (71.9 %) were novel in the healthy tears. Amino acids were the most frequently detected metabolites in the tears (28 %), followed by carbohydrates (12.2 %), carboxylic acids (8.5 %), carnitines (6.1 %) and glycerophospholipids (4.9 %), respectively. ConclusionThe current untargeted platform is capable of detecting a range of tear metabolites across several biological categories. This study provides a baseline for further ocular surface studies.

molecular biology↗

bioRxiv2021

Programmable Cleavage of Double-stranded DNA by Combined Action of Argonaute CbAgo from Clostridium butyricum and Nuclease Deficient RecBC Helicase from E.coli

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Discovery of mesophilic Argonautes active at physiological temperature places pAgos closer to their possible application for genome editing as a simpler alternative to CRISPR/Cas nucleases. Currently, the use of mesophilic pAgos as programmable DNA endonucleases is hampered by their poor action on double-stranded DNA (dsDNA), mainly due to their inability to invade the DNA duplex. The present study demonstrates that efficient in vitro cleavage of double-stranded DNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecBexo-C). Properties of CbAgo and characteristics of simultaneous cleavage of complementary DNA strands in concurrence with DNA strand unwinding by RecBexo-C were thoroughly explored using 0.3-25 kb DNA substrates. When combined with RecBexo-C helicase, CbAgo was capable of cleaving target sequences located 11-12.5 kb from the ends of linear dsDNA at 37{o}C. Our study demonstrates that CbAgo with RecBexo-C can be programmed to generate dsDNA fragments flanked with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. At present, the combination of CbAgo and RecBexo-C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of dsDNA at any desired location.

molecular biology↗

bioRxiv2022

Claspin-dependent and -independent Chk1 activation by a panel of biological stresses

Replication stress has been suggested to be an ultimate trigger of carcinogenesis. Oncogenic signal, such as overexpression of CyclinE, has been shown to induce replication stress. Here, we show that various biological stresses, including heat, oxidative stress, osmotic stress, LPS, hypoxia, and arsenate induce activation of Chk1, a key effector kinase for replication checkpoint. Some of these stresses indeed reduce the fork rate, inhibiting DNA replication. Analyses of Chk1 activation in the cell population with western analyses showed that Chk1 activation by these stresses is largely dependent on Claspin. On the other hand, single cell analyses with Fucci cells indicated that while Chk1 activation during S phase is dependent on Claspin, that in G1 is mostly independent of Claspin. We propose that various biological stresses activate Chk1 either directly by stalling DNA replication fork or by some other mechanism that does not involve replication inhibition. The former pathway predominantly occurs in S phase and depends on Claspin, while the latter pathway, which may occur throughout the cell cycle, is largely independent of Claspin. Our findings provide evidence for novel links between replication stress checkpoint and other biological stresses and points to the presence of unknown mechanisms of Chk1 activation in mammalian cells.

molecular biology↗

bioRxiv2023

Development of QSAR Models to Identify Mycobacterium tuberculosis enoyl-ACP-reductase Enzyme Inhibitors

Tuberculosis is a global concern due to its high prevalence in developing countries and the ability of mycobacteria to develop resistance to current treatment regimens. In this project, we propose the use of QSAR (Quantitative Structure-Activity Relationships) modeling as a means to identify and evaluate the inhibitory activity of candidate molecules for molecular improvement stages and/or in vitro assays. This approach allows for in silico estimation, reducing research time and costs. To achieve this, we utilized the SAR (Structure-Activity Relationships) study conducted by He, Alian, and Montellano (2007), which focused on a series of arylamides tested as inhibitors of the enzyme enoyl-ACP-reductase (InhA) in Mycobacterium tuberculosis. We developed both the Hansh-Fujita (classical) and CoMFA (Comparative Molecular Field Analysis) QSAR models. The classical QSAR model produced the most favorable statistical results using Multiple Linear Regression (MLR). It achieved an internal validation correlation factor R2 of 0.9012 and demonstrated predictive quality with a Stone-Geisser indicator Q2 of 0.8612. External validation resulted in a correlation factor R2 of 0.9298 and Q2 of 0.720, indicating a highly predictive mathematical model. The CoMFA Model obtained a Q2 of 0.6520 in internal validation, enabling the estimation of energy fields around the molecules. This information is crucial for molecular improvement efforts. We constructed a library of small molecules, analogous to those used in the SAR study, and subjected them to the classic QSAR function. As a result, we identified ten molecules with high estimated biological activity. Molecular docking analysis suggests that these ten analogs, identified by the classical QSAR model, exhibit favorable estimated free energy of binding. In conclusion, the QSAR methodology proves to be an efficient and effective tool for searching and identifying promising drug-like molecules.

bioinformatics↗

bioRxiv2024

Transglutaminase 2 is an RNA-binding protein: Experimental verification and characterisation of a novel transglutaminase feature

Transglutaminase 2 (TG2) is a uniquely versatile protein with diverse catalytic activities, such as transglutaminase, protein disulfide isomerase, GTPase, protein kinase, and participates in several biological processes. According to information available in the RBP2GO database, TG2 can be an RNA-binding protein (RBP). RBPs participate in posttranscriptional gene expression regulation, influencing RNAs function, while RNA molecules can also modulate RBPs biological activity. Our goal was to confirm this novel character of TG2 in human umbilical cord vein endothelial cells (HUVEC), which physiologically express TG2. First, UV cross-linked RNA-protein complexes were isolated from immortalised HUVEC using orthogonal organic phase separation. Compared with the RBP2GO database, mass spectrometry identified 392 potential RBPs, including TG2 and 20 novel, endothelium-related RBPs. Total RNA from HUVEC pulled down recombinant human TG2. Complex formation between TG2 and a 43-mer RNA molecule with a secondary structure as well as a homo-oligomeric single-stranded poly(dG), but not poly(dA), could be observed in magnetic RNA-protein pull-down experiments. Experiments with TG2 inhibitors NC9 and GTP{gamma}S, which stabilise its open and closed conformation, respectively, revealed that the open conformation of the enzyme favoured RNA-binding. Biolayer interferometry revealed a high binding affinity between TG2 and RNA with a KD value of 88 nM. We propose that superficial residues on the catalytic core and C-terminal {beta}-barrel domains, being in a hidden position in the closed TG2, are involved in RNA binding. Our study demonstrates TG2s previously uncharacterised RNA-binding ability, opening new avenues for understanding its multi-functionality. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/591323v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@3c7acaorg.highwire.dtl.DTLVardef@a165f2org.highwire.dtl.DTLVardef@2c0293org.highwire.dtl.DTLVardef@15d3d81_HPS_FORMAT_FIGEXP M_FIG C_FIG Transglutaminase 2 (TG2) is a unique multifunctional protein demonstrating large conformational changes. It has various transglutaminase and other catalytic and non-catalytic activities which show conformation dependency. Our study has characterised a novel, open conformation-related biological activity of TG2 and its RNA-binding ability.

molecular biology↗

bioRxiv2024

Foxg1 and companions: not only transcription factors

Here, moving from our most recent results on Foxg1 biology, we firstly summarize available information about a few, special pleiotropic effectors of neurodevelopmental interest, involved in control of both transcription and post-transcriptional steps of gene expression. Next, upon further scanning of literature, we report evidence that, not strictly limited to neurodevelopmental processes, such functional pleiotropy also applies to other transcription factors, involved in physiology and homeostasis. Besides, by systematic mining of a major public protein-protein interaction database, we collect robust evidence that an involvement of "canonical" transcription factors in post-transcriptional control of gene expression may be a pervasive phenomenon, characterizing hundreds of effectors. Finally, we discuss the biological meaning of these findings and propose three evolutionary mechanisms that may have conspired to such unexpected scenario.

molecular biology↗

bioRxiv2025

Advanced eTAM-seq enables high-fidelity, low-input N6-methyladenosine profiling in human cells and embryonic mouse tissues

Functional dissection of N6-methyladenosine (m6A), the most abundant internal messenger RNA modification in mammals, demands quantitative, scalable detection technology. We previously reported eTAM-seq, which supports transcriptome-wide quantification of m6A by enzyme-assisted adenosine deamination. While effective, TadA8.20--the enzyme used in our first-generation technology--is sensitive not only to m6A but also to RNA structure, making accurate detection dependent on the inclusion of control transcriptomes. Here, we introduce eTAM-seq-v2, in which we replace TadA8.20 with TadA8r, a further evolved adenosine deaminase with superior catalytic efficiency. eTAM-seq-v2 supports control-free m6A calling with high fidelity. Because enzyme treatment preserves RNA integrity, eTAM-seq surveys >51% of A sites in all expressed genes with moderate sequencing depth (60 million uniquely mapped reads) and delivers robust performance with as little as 10 ng of total RNA ([~]500 cells). With eTAM-seq-v2, we delineate the m6A landscape across six human cell lines and seven embryonic mouse tissues. While uncovering broadly conserved m6A patterns, we reveal that most neighboring m6A sites are independently deposited at the single-molecule level. Moving forward, we envision that eTAM-seq-v2 will enable researchers to survey m6A in diverse biological contexts and uncover new insights into its regulatory roles.

molecular biology↗

bioRxiv2025

Fish from the sky: Airborne eDNA tracks aquatic life

Water and air are generally treated as separate reservoirs of environmental DNA (eDNA) derived from the species resident in those respective environments. However, it is likely that eDNA routinely crosses the air-water boundary in both directions as a result of deposition, evaporation, or other processes. Here, we systematically tested methods of sampling eDNA at the air-water interface, showing for the first time that aquatic life can be reliably detected from passive air samples collected nearby. We deployed four simple air samplers -- three different kinds of filters and one open tray of deionized water -- alongside paired water samples and visual counts over a six-week peak run of Coho salmon (Oncorhynchus kisutch) at a local spawning stream. We then quantified eDNA concentrations in both air and water (air: copies/day/cm2; water: copies/L) using quantitative PCR, to estimate (1) the concentration of target eDNA in air vs. water, and (2) the capture performance of each filter type. Despite an approximate 25,000-times dilution versus water, passive air collectors captured quantitative airborne eDNA signals that closely paralleled salmon counts, although recovery varied with sampler design and orientation. We show the air-water interface is a quantifiable source of aquatic genetic information using simple, passive samplers that do not require electricity, making them appealing for biomonitoring in remote or resource-limited settings. This work points the way to using airborne eDNA as a robust pathway for biological information critical to conservation, resource management, and public-health protection.

molecular biology↗

bioRxiv2025

OpenFISH enables integrated high-resolution spatial transcriptomics and metabolomics on a single tissue section

Spatial transcriptomics enables in situ mapping of gene expression, yet no current platform provides single-cell, same-section integration with metabolomics, limiting direct links between transcriptional programsand metabolic phenotypesin native tissue. We present OpenFISH, a rapid, imaging-based spatial transcriptomics method operable on standard microscopes, requiring no proprietary hardware, and fully compatible with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). OpenFISH resolves hundreds of transcripts at subcellular resolution within 24 h and can be performed after MALDI-MSI, preserving metabolite distributions for cell-accurate co-registration on the same section. In mouse brain, integration with MALDI-MSI resolved metabolic heterogeneity at the level of individual cells. OpenFISH also quantified cell type-specific transcriptional activation of transposable elements after systemic lipopolysaccharide (LPS) challenge and detected disrupted spatial organization of D1 striatal neurons in Reeler mutants. Benchmarking showed performance comparable to or exceeding commercial platforms at [~]0.5% of per-sample cost. By enabling same-section, near-single-cell co-mapping of transcripts and metabolites in an accessible workflow, OpenFISH provides a scalable framework for high-content spatial multi-omics across neuroscience, immunology, cancer biology, and beyond.

molecular biology↗

bioRxiv2025

Rapid degradation of 6 class I HDAC complexes reveals minimal functional overlap between complexes

The class 1 HDACs 1, 2 and 3 form seven families of distinct large multiprotein complexes that regulate gene expression via deacetylation of lysines in histone tails. The degree of redundancy and functional overlap between complexes and their primary gene targets, remains unknown. We used CRISPR/Cas9 to independently tag HDAC complexes with FKBP12F36V in HCT116 cells enabling rapid (<1 hr), PROTAC-mediated, degradation. RNA sequencing at 6 h reveals that together, the 4 major complexes (CoREST, NuRD, NCoR/SMRT and SIN3A) perturbed >50% of expressed genes. More than 60% of these are specific to an individual complex. Of genes regulated by more than one complex, approaching 50% are reciprocally regulated such that HDAC complexes act as antagonistic regulators. Homer analysis strongly suggests that the complexes are reliant on different transcription factors. This is the first study to identify the primary targets of individual HDAC complexes and directly compare the effects of rapid degradation on gene regulation in the same biological system.

molecular biology↗

bioRxiv2019

Interrogation of Enhancer Function by Enhanced CRISPR Epigenetic Editing

Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe enhanced CRISPR/dCas9-based epigenetic editing systems, enCRISPRa and enCRISPRi, for multiplexed analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the new systems display more robust perturbation of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Multiplexed perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoietic lineage specification. Hence, enhanced CRSIPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.

molecular biology↗

bioRxiv2019

Ctcf Haploinsufficiency Mediates Intron Retention in A Tissue-specific Manner

CTCF is a master regulator of gene transcription and chromatin organization with occupancy at thousands of DNA target sites. CTCF is essential for embryonic development and somatic cell viability and has been characterized as a haploinsufficient tumor suppressor. Increasing evidence demonstrates CTCF as a key player in several alternative splicing (AS) regulatory mechanisms, including transcription elongation, regulation of splicing factors, and epigenetic regulation. However, the genome-wide impact of Ctcf dosage on AS has not been investigated. We examined the effect of Ctcf haploinsufficiency on gene expression and AS in multiple tissues from Ctcf hemizygous (Ctcf+/-) mice. Distinct tissue-specific differences in gene expression and AS were observed in Ctcf+/- mice compared to wildtype mice. We observed a surprisingly large number of increased intron retention (IR) events in Ctcf+/- liver and kidney, specifically in genes associated with cytoskeletal organization, splicing and metabolism. This study provides further evidence for Ctcf dose-dependent and tissue-specific regulation of gene expression and AS. Our data provide a strong foundation for elucidating the mechanistic role of CTCF in AS regulation and its biological consequences.

molecular biology↗