Specific Genomic Loci Research Articles

IPSC-Derived Human Hepatocytes as a Novel Model to Investigate Thyroid Hormone Action and Signaling

Thyroid hormone (TH) actions are essential to normal metabolism and neurologic function. Abnormal TH levels can lead to significant morbidity including metabolic abnormalities, cardiac disease, and obesity. TH action is mediated by the thyroid hormone receptor (TR) isoforms and their coregulators. In the disease models of Resistance to Thyroid Hormone (RTH) it is clear that the TR isoforms play tissue-specific roles in humans, however a platform for drug discovery to potentially treat the diseases does not exist. We hypothesize that human hepatocytes (iHeps) derived from induced pluripotent stem cells (iPSCs) will serve as an ideal model to study TH signaling and to delineate mechanisms of TH diseases in humans. To investigate the TH action in humans, we have developed a serum free human iPSC hepatic differentiation protocol that utilizes sequential exposure to growth factors to mimic actual human development. iHeps are robustly responsive to TH with over 1000 regulated genes. Importantly, they allow us to use CRISPR/Cas9 to determine molecular physiology and mechanism of disease. To demonstrate the role of TRs in TH signaling in human liver cells we have focused on TRβ1 the main TR isoform expressed in liver. we knocked out TRβ1 (THRBKO) using guide RNAs (gRNAs) we engineered to specific genomic loci to truncate the expression of this isoform at the DNA binding domain. Also, to define mechanisms of TR action in the context of RTH, we used this same approach to introduce the Δ337T mutation found in human TRβ1 that causes RTH. Both THRBKO and Δ337T lines are karyotypically normal and flow cytometry analysis in both lines demonstrated that these lines differentiated normally into hepatocytes after gene editing with CRISPR/Cas9. Once clones were identified the edited iHeps were treated with PBS (vehicle) or T3 (10nM) for 24 hours. Real-time quantitative PCR (RT qPCR) was done to assess mRNA expression of T3 target genes. RT qPCR analysis confirmed the success of TRβ1 deletion as the response to T3 was lost on both positive and negative THRB targets. In the Δ337T iHeps, the response to TH was diminshed indicating the successful generation of this line. These preliminary results confirm our ability to edit IPSCs and then to differentiate into hepatocytes, allowing us to further study the action of TH and the mutations involved in RTH.

CRISPR/Cas9 technology in disease research and therapy: a review

Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level, including insertion, knockout, replacement and point mutation of specific DNA fragments. The ultimate principle of genome editing technology relying on engineered nucleases is to generate double-stranded DNA breaks at specific locations in genome and then repair them through non-homologous end joining or homologous recombination. With the intensive study of these nucleases, genome editing technology develops rapidly. The most used nucleases include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats associated Cas proteins. Based on introducing the development and principles of above mentioned genome editing technologies, we review the research progress of CRISPR/Cas9 system in the application fields of identification of gene function, establishment of disease model, gene therapy, immunotherapy and its prospect.

From the Literature

From the Literature

Gain of LINC00624 Enhances Liver Cancer Progression by Disrupting the Histone Deacetylase 6/Tripartite Motif Containing 28/Zinc Finger Protein 354C Corepressor Complex.

Long noncoding RNAs (lncRNAs) are involved in almost every stage of tumor initiation and progression. Here, we have identified an antisense lncRNA, LINC00624, that arises from the antisense strand of chromo-domain-helicase-DNA-binding protein 1-like (CHD1L), located on chr1q21.1, with significant copy number gain and transcriptional activation of CHD1L and B-cell CLL/lymphoma 9 protein (BCL9), in hepatocellular carcinoma (HCC). Overexpression of LINC00624 enhances tumor growth and metastasis in vitro and in vivo. Mechanistically, higher levels of LINC00624 strengthen the interaction between histone deacetylase 6 (HDAC6) and tripartite motif containing 28 (TRIM28), which accelerates HDAC6 ubiquitination and degradation. Moreover, LINC00624 binds to the RBCC domain of TRIM28, inhibits trimer formation, and weakens the interaction between TRIM28 and zinc finger protein 354C (ZNF354C). Thus, LINC00624 overexpression disrupts the formation of the HDAC6-TRIM28-ZNF354C transcriptional corepressor complex, resulting in the dissociation of the complex from the promoter of CHD1L and BCL9, thereby removing transcription inhibition. Our findings suggest that LINC00624 acts as a molecular decoy that sequesters the HDAC6-TRIM28-ZNF354C transcriptional corepressor complex away from the specific genomic loci, and that it can potentially be a therapeutic target in HCC.

Chromatin remodeling factors regulate environmental stress responses in plants.

Environmental stress from climate change and agricultural activity threatens global plant biodiversity as well as crop yield and quality. As sessile organisms, plants must maintain the integrity of their genomes and adjust gene expression to adapt to various environmental changes. In eukaryotes, nucleosomes are the basic unit of chromatin around which genomic DNA is packaged by condensation. To enable dynamic access to packaged DNA, eukaryotes have evolved Snf2 (sucrose nonfermenting 2) family proteins as chromatin remodeling factors (CHRs) that modulate the position of nucleosomes on chromatin. During plant stress responses, CHRs are recruited to specific genomic loci, where they regulate the distribution or composition of nucleosomes, which in turn alters the accessibility of these loci to general transcription or DNA damage repair machinery. Moreover, CHRs interplay with other epigenetic mechanisms, including DNA methylation, histone modifications, and deposition of histone variants. CHRs are also involved in RNA processing at the post-transcriptional level. In this review, we discuss major advances in our understanding of the mechanisms by which CHRs function during plants' response to environmental stress.

Development of CRISPR technology for precise single-base genome editing: a brief review

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPR-mediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications.

Super-resolution microscopy of genome organization.

Recent advancements in sequencing and imaging technologies are providing new perspectives in solving the mystery of three-dimensional folding of genome in a nucleus. Chromosome conformation capture sequencing has discovered new chromatin structures such as topologically associated domains and loops in hundreds of kilobases. Super-resolution fluorescence microscopy with nanometer resolutions, in particular multiplexed approaches with sequence-specificity, has visualized chromatin structures from the rough folds of whole chromosomes to the fine loops of cis-regulatory elements in intact individual nuclei. Here, recent advancements in genome visualization tools with highly multiplexed labeling and reading are introduced. These imaging technologies have found ensemble behavior consistent to sequencing results, while unveiling single-cell variations. But, they also generated contradictory results on the roles of architectural proteins (like cohesion and CTCF) and enhancer-promoter interactions. Live-cell labeling methods for imaging specific genomic loci, especially the CRISPR/dCas9 system, are reviewed in order to give perspectives in the emergence of tools for visualizing genome structural dynamics.

CRISPR/Cas9 uses

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated system (Cas) is a system that provides immunity to most prokaryoticorganisms against viral attacks and other foreign bodies. CRISPR systems consist of a scissor-like protein called Cas9 and a genetic GPS guide “The guide RNA”. However, researchers have reoriented and repurposed the primordial immune system to precisely manipulate genomes in most organisms by introducing DNA double-strand breaks at specific genome locations to introduce specific DNA modifications. More applications of CRISPR have arisen since its discovery, from disabling parasites to correcting mutations and improving crop yields. This review was conceived as a guide to CRISPR technology, from its discovery to the latest breakthroughs. It is hoped that this study will provide a general-based view for this life changing technology, inspiring scientists to go further with CRISPR in the sake of a better life.

Use of SNP chips to detect rare pathogenic variants: retrospective, population based diagnostic evaluation

ObjectiveTo determine whether the sensitivity and specificity of SNP chips are adequate for detecting rare pathogenic variants in a clinically unselected population.DesignRetrospective, population based diagnostic evaluation.Participants49 908 people recruited to...

Nuclear Long Non-Coding RNAs as Epigenetic Regulators in Cancer.

Transcriptome analyses have revealed the presence of numerous long non-coding RNAs (lncRNAs) in mammalian cells. Many lncRNAs are expressed in development-, differentiation-, and disease-specific manners, suggesting their importance as cell regulators. Some nuclear lncRNAs are bound to specific genomic loci, either near or distant from their own transcription sites, and regulate gene expression in cis or trans. These lncRNAs recruit epigenetic factors, including the DNA methyl transferase and histone modification complex, and mediate both the 3D genome structure and nuclear domains. LncRNAs are now considered as an emerging member of epigenetic regulators. LncRNAs are dysregulated in various types of cancer and act as either oncogenic or tumor- suppressing factors. They are involved in virtually all of the cancer hallmarks and are potential diagnostic markers and therapeutic targets. In this review, we describe several representative lncRNAs and provide a current overview of the mechanisms by which lncRNAs participate in epigenetic regulation and contribute to cancer development.

A Systems Biology Approach to Identify Essential Epigenetic Regulators for Specific Biological Processes in Plants.

Upon sensing developmental or environmental cues, epigenetic regulators transform the chromatin landscape of a network of genes to modulate their expression and dictate adequate cellular and organismal responses. Knowledge of the specific biological processes and genomic loci controlled by each epigenetic regulator will greatly advance our understanding of epigenetic regulation in plants. To facilitate hypothesis generation and testing in this domain, we present EpiNet, an extensive gene regulatory network (GRN) featuring epigenetic regulators. EpiNet was enabled by (i) curated knowledge of epigenetic regulators involved in DNA methylation, histone modification, chromatin remodeling, and siRNA pathways; and (ii) a machine-learning network inference approach powered by a wealth of public transcriptome datasets. We applied GENIE3, a machine-learning network inference approach, to mine public Arabidopsis transcriptomes and construct tissue-specific GRNs with both epigenetic regulators and transcription factors as predictors. The resultant GRNs, named EpiNet, can now be intersected with individual transcriptomic studies on biological processes of interest to identify the most influential epigenetic regulators, as well as predicted gene targets of the epigenetic regulators. We demonstrate the validity of this approach using case studies of shoot and root apical meristem development.

Engineering 3D genome organization.

Cancers and developmental disorders are associated with alterations in the 3D genome architecture in space and time (the fourth dimension). Mammalian 3D genome organization is complex and dynamic and plays an essential role in regulating gene expression and cellular function. To study the causal relationship between genome function and its spatio-temporal organization in the nucleus, new technologies for engineering and manipulating the 3D organization of the genome have been developed. In particular, CRISPR-Cas technologies allow programmable manipulation at specific genomic loci, enabling unparalleled opportunities in this emerging field of 3D genome engineering. We review advances in mammalian 3D genome engineering with a focus on recent manipulative technologies using CRISPR-Cas and related technologies.

Histone modification dynamics at H3K27 are associated with altered transcription of in planta induced genes in Magnaporthe oryzae.

Transcriptional dynamic in response to environmental and developmental cues are fundamental to biology, yet many mechanistic aspects are poorly understood. One such example is fungal plant pathogens, which use secreted proteins and small molecules, termed effectors, to suppress host immunity and promote colonization. Effectors are highly expressed in planta but remain transcriptionally repressed ex planta, but our mechanistic understanding of these transcriptional dynamics remains limited. We tested the hypothesis that repressive histone modification at H3-Lys27 underlies transcriptional silencing ex planta, and that exchange for an active chemical modification contributes to transcription of in planta induced genes. Using genetics, chromatin immunoprecipitation and sequencing and RNA-sequencing, we determined that H3K27me3 provides significant local transcriptional repression. We detail how regions that lose H3K27me3 gain H3K27ac, and these changes are associated with increased transcription. Importantly, we observed that many in planta induced genes were marked by H3K27me3 during axenic growth, and detail how altered H3K27 modification influences transcription. ChIP-qPCR during in planta growth suggests that H3K27 modifications are generally stable, but can undergo dynamics at specific genomic locations. Our results support the hypothesis that dynamic histone modifications at H3K27 contributes to fungal genome regulation and specifically contributes to regulation of genes important during host infection.

Characterization of Centromeres and Telomeres in Human Cells

The nucleus of eukaryotic cells stores genetic information in chromatin, the functional form of DNA in cells. Chromatin loci have been observed to exhibit anomalous motion that is often ascribed to the viscoelastic nature of the chromatin, transient interactions of DNA-associated proteins and/or physical obstructions [1,2]. However, a direct link between a chromatin locus motion and the rheology of its local environment is missing. In this work, we investigate dynamics of specific genomic loci, centromeres and telomeres, the centers and ends of the linear interphase chromosomes, respectively, in the context of their local rheological environment.

Nanobody-mediated control of gene expression and epigenetic memory

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells.

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.

Genome Editing in Y. lipolytica Using TALENs.

TALENs (Transcription Activator-Like EndoNuclease) are molecular scissors designed to recognize and introduce a double-strand break at a specific genome locus. They represent tools of interest in the frame of genome edition. Upon cleavage, two different pathways lead to DNA repair: Non-homologous End Joining (NHEJ) repair, leading to efficient introduction of short insertion/deletion mutations which can disrupt translational reading frame and Homology Recombination (HR)-directed repair that occurs when exogenous DNA is supplied. Here we introduce how to use TALENs in the oleaginous yeast Yarrowia lipolytica by presenting a step-by-step method allowing to knock out or to introduce in vivo a point mutation in a gene of Yarrowia lipolytica. This chapter describes the material required, the transformation procedure, and the screening process.

EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error.

Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with significantly higher error rates and hypothesized to have implications in cancer, aging, and diseases such as Down syndrome and Alzheimer's. Therefore, research into transcription errors is of growing importance within the field of genetics. Nevertheless, methodological barriers limit the progress in accurately identifying transcription errors. Pro-Seq and NET-Seq can purify nascent RNA and map RNA polymerases along the genome but cannot be used to identify transcriptional mutations. Here we present background Error Model-coupled Precision nuclear run-on Circular-sequencing (EmPC-seq), a method combining a nuclear run-on assay and circular sequencing with a background error model to precisely detect nascent transcription errors and effectively discern TEELs within the genome.

Using CRISPR-Cas9-based genome engineering tools in Drosophila melanogaster.

Drosophila melanogaster has been used as a model organism for over a century. Mutant-based analyses have been used extensively to understand the genetic basis of different cellular processes, including development, neuronal function and diseases. Most of the earlier genetic mutants and specific tools were generated by random insertions and deletion strategies and then mapped to specific genomic loci. Since all genomic regions are not equally accessible to random mutations and insertions, many genes still remain uncharacterized. Low efficiency of targeted genomic manipulation approaches that rely on homologous recombination, and difficulty in generating resources for sequence-specific endonucleases, such as ZFNs (Zinc Finger Nucleases) and TALENs (Transcription Activator-Like Effector Nucleases), could not make these gene targeting techniques very popular. However, recently RNA directed DNA endonucleases, such as CRISPR-Cas, have transformed genome engineering owing to their comparative ease, versatility, and low expense. With the added advantage of preexisting genetic tools, CRISPR-Cas-based manipulations are being extensively used in Drosophila melanogaster and simultaneously being fine-tuned for specific experimental requirements. In this chapter, I will discuss various uses of CRISPR-Cas-based genetic engineering and specific design methods in Drosophila melanogaster. I will summarize various already available tools that are being utilized in conjunction with CRISPR-Cas technology to generate specific genetic manipulation and are being optimized to address specific questions. Finally, I will discuss the future directions of Drosophila genetics research and how CRISPR-Cas can be utilized to target specific questions, addressing which has not been possible thus far.

ProbeDealer is a convenient tool for designing probes for highly multiplexed fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) is a powerful method to visualize the spatial positions of specific genomic loci and RNA species. Recent technological advances have leveraged FISH to visualize these features in a highly multiplexed manner. Notable examples include chromatin tracing, RNA multiplexed error-robust FISH (MERFISH), multiplexed imaging of nucleome architectures (MINA), and sequential single-molecule RNA FISH. However, one obstacle to the broad adoption of these methods is the complexity of the multiplexed FISH probe design. In this paper, we introduce an easy-to-use, versatile, and all-in-one application called ProbeDealer to design probes for a variety of multiplexed FISH techniques and their combinations. ProbeDealer offers a one-stop shop for multiplexed FISH design needs of the research community.