Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins

Biochemical Characterization ◽

Target Dna ◽

Short Palindromic Repeat ◽

Downstream Analysis ◽

Genomic Regions ◽

Efficient Sequence ◽

The clustered regularly interspaced short palindromic repeat (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first demonstrate that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we show that this technology can be employed to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis.

Purification of specific DNA species using the CRISPR system

Biology Methods and Protocols ◽

10.1093/biomethods/bpz008 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 1

Author(s):

Toshitsugu Fujita ◽

Hodaka Fujii

Keyword(s):

Dna Sequences ◽

In Vitro Method ◽

Guide Rna ◽

Crispr System ◽

Target Dna ◽

Genomic Regions ◽

Generation Sequencing ◽

Subsequent Identification

AbstractIn 2013, we developed a new method of engineered DNA-binding molecule-mediated chromatin immunoprecipitation that incorporates the clustered regularly interspaced short palindromic repeats (CRISPR) system to purify specific DNA species. This CRISPR-mediated purification can be performed in-cell or in vitro; CRISPR complexes can be expressed to tag target DNA sequences in the cells to be analyzed, or a CRISPR ribonucleoprotein complex consisting of recombinant nuclease-dead Cas9 (dCas9) and synthetic guide RNA can be used to tag target DNA sequences in vitro. Both methods enable purification of specific DNA sequences in chromatin structures for subsequent identification of molecules (proteins, RNAs, and other genomic regions) associated with the target sequences. The in vitro method also enables enrichment of purified DNA sequences from a pool of heterogeneous sequences for next-generation sequencing or other applications. In this review, we outline the principle of CRISPR-mediated purification of specific DNA species and discuss recent advances in the technology.

Reversible regulation of Cas12a activities by AcrVA5-mediated acetylation and CobB-mediated deacetylation

10.1101/2021.11.16.468554 ◽

2021 ◽

Author(s):

Xiaoman Kang ◽

Lei Yin ◽

Songkuan Zhuang ◽

Tianshuai Hu ◽

Zhile Wu ◽

...

Keyword(s):

Dna Sequences ◽

Wide Spectrum ◽

Defense System ◽

Post Translational Modification ◽

Defense Systems ◽

Target Dna ◽

Short Palindromic Repeat ◽

Type V

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) system protects bacteria and archaea from the invasion of foreign genetic elements. To cope with the host CRISPR systems, phages have evolved many strategies, including the anti-CRISPR (Acr) proteins, to inactivate the Cas nucleases. Recently, it has been reported that the type V-A Cas12a effector can be acetylated and inactivated by AcrVA5, which is a GNAT-family acetyltransferase. However, it is unclear whether the host has any coping strategies to reactivate the defense system. Here we show that the AcrVA5-acetylated Cas12a can be deacetylated by bacterial deacetylase CobB, reactivating Cas12a for both in vitro cleavage of target DNA sequences and in vivo protection of the host from invasion of foreign nucleic acids. Therefore, this study not only shows the reversible regulation of Cas12a activities by post-translational modification but also reveals CobB as a secondary safeguard to bacterial CRISPR defense systems. In addition, we demonstrate that AcrVA5 is a wide-spectrum acetyltransferase, acetylating a large number of target proteins besides Cas12a, and the AcrVA5-acetylated targets can also be deacetylated by CobB.

The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146217 ◽

1993 ◽

Vol 51 ◽

pp. 74-75

Author(s):

Jason R. Swedlow ◽

Neil Osheroff ◽

Tim Karr ◽

John W. Sedat ◽

David A. Agard

Keyword(s):

Topoisomerase Ii ◽

Biochemical Characterization ◽

Developmental Cycle ◽

Dna Topoisomerase Ii ◽

Chromosomal Distribution ◽

Dna Topoisomerase ◽

Ideal System ◽

Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

Ultramicronized Palmitoylethanolamide (um-PEA): A New Possible Adjuvant Treatment in COVID-19 patients

Pharmaceuticals ◽

10.3390/ph14040336 ◽

2021 ◽

Vol 14 (4) ◽

pp. 336

Author(s):

Annalisa Noce ◽

Maria Albanese ◽

Giulia Marrone ◽

Manuela Di Lauro ◽

Anna Pietroboni Zaitseva ◽

...

Keyword(s):

Adjuvant Treatment ◽

Pulmonary Involvement ◽

In Vivo Studies ◽

Pharmacological Treatments ◽

Beneficial Effects ◽

Coronavirus Infection ◽

Ultramicronized Palmitoylethanolamide ◽

The Coronavirus Disease-19 (COVID-19) pandemic has caused more than 100,000,000 cases of coronavirus infection in the world in just a year, of which there were 2 million deaths. Its clinical picture is characterized by pulmonary involvement that culminates, in the most severe cases, in acute respiratory distress syndrome (ARDS). However, COVID-19 affects other organs and systems, including cardiovascular, urinary, gastrointestinal, and nervous systems. Currently, unique-drug therapy is not supported by international guidelines. In this context, it is important to resort to adjuvant therapies in combination with traditional pharmacological treatments. Among natural bioactive compounds, palmitoylethanolamide (PEA) seems to have potentially beneficial effects. In fact, the Food and Drug Administration (FDA) authorized an ongoing clinical trial with ultramicronized (um)-PEA as an add-on therapy in the treatment of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection. In support of this hypothesis, in vitro and in vivo studies have highlighted the immunomodulatory, anti-inflammatory, neuroprotective and pain-relieving effects of PEA, especially in its um form. The purpose of this review is to highlight the potential use of um-PEA as an adjuvant treatment in SARS-CoV-2 infection.

The Novel Arylamidine T-2307 MaintainsIn VitroandIn VivoActivity against Echinocandin-Resistant Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04228-14 ◽

2014 ◽

Vol 59 (2) ◽

pp. 1341-1343 ◽

Cited By ~ 14

Author(s):

Nathan P. Wiederhold ◽

Laura K. Najvar ◽

Annette W. Fothergill ◽

Rosie Bocanegra ◽

Marcos Olivo ◽

...

Keyword(s):

Body Weight ◽

Candida Albicans ◽

Invasive Candidiasis ◽

Placebo Control ◽

The Novel ◽

Content Type ◽

Fungal Burden ◽

ABSTRACTWe evaluated thein vitroandin vivoactivities of the investigational arylamidine T-2307 against echinocandin-resistantCandida albicans. T-2307 demonstrated potentin vitroactivity, and daily subcutaneous doses between 0.75 and 6 mg/kg of body weight significantly improved survival and reduced fungal burden compared to placebo control and caspofungin (10 mg/kg/day) in mice with invasive candidiasis caused by an echinocandin-resistant strain. Thus, T-2307 may have potential use in the treatment of echinocandin-resistantC. albicansinfections.

Is There Potential for Repurposing Statins as Novel Antimicrobials?

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00192-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5111-5121 ◽

Cited By ~ 52

Author(s):

Emma Hennessy ◽

Claire Adams ◽

F. Jerry Reen ◽

Fergal O'Gara

Keyword(s):

Serum Cholesterol ◽

Bacterial Growth ◽

Bacterial Infections ◽

Statin Treatment ◽

Therapeutic Agents ◽

Pleiotropic Effects ◽

Current Information ◽

ABSTRACTStatins are members of a class of pharmaceutical widely used to reduce high levels of serum cholesterol. In addition, statins have so-called “pleiotropic effects,” which include inflammation reduction, immunomodulation, and antimicrobial effects. An increasing number of studies are emerging which detail the attenuation of bacterial growth andin vitroandin vivovirulence by statin treatment. In this review, we describe the current information available concerning the effects of statins on bacterial infections and provide insight regarding the potential use of these compounds as antimicrobial therapeutic agents.

Enzymatic activity in turkey, duck, quail and chicken liver microsomes against four human cytochrome P450 prototype substrates and aflatoxin B1

Journal of Xenobiotics ◽

10.4081/xeno.2011.e4 ◽

2011 ◽

Vol 1 (1) ◽

pp. 4 ◽

Cited By ~ 10

Author(s):

Hansen W. Murcia ◽

Gonzalo J. Díaz ◽

Sandra Milena Cepeda

Keyword(s):

Cytochrome P450 ◽

Aflatoxin B1 ◽

Enzyme Activities ◽

High Performance ◽

Biochemical Characterization ◽

Liver Microsomes ◽

Cytochrome P450 Enzymes ◽

Catalytic Rate

Cytochrome P450 enzymes (CYP) are a group of monooxygenases able to biotransform several kinds of xenobiotics including aflatoxin B1 (AFB1), a highly toxic mycotoxin. These enzymes have been widely studied in humans and others mammals, but there is not enough information in commercial poultry species about their biochemical characteristics or substrate specificity. The aim of the present study was to identify CYPs from avian liver microsomes with the use of prototype substrates specific for human CYP enzymes and AFB1. Biochemical characterization was carried out in vitro and biotransformation products were detected by high-performance liquid chromatography (HPLC). Enzymatic constants were calculated and comparisons between turkey, duck, quail and chicken activities were done. The results demonstrate the presence of four avian ortholog enzyme activities possibly related with a CYP1A1, CYP1A2, CYP2A6 (activity not previously identified) and CYP3A4 poultry orthologs, respectively. Large differences in enzyme kinetics specific for prototype substrates were found among the poultry species studied. Turkey liver microsomes had the highest affinity and catalytic rate for AFB1 whereas chicken enzymes had the lowest affinity and catalytic rate for the same substrate. Quail and duck microsomes showed intermediate values. These results correlate well with the known in vivo sensitivity for AFB1 except for the duck. A high correlation coefficient between 7-ethoxyresorufin-Odeethylase (EROD) and 7-methoxyresorufin- O-deethylase (MROD) activities was found in the four poultry species, suggesting that these two enzymatic activities might be carried out by the same enzyme. The results of the present study indicate that four prototype enzyme activities are present in poultry liver microsomes, possibly related with the presence of three CYP avian orthologs. More studies are needed in order to further characterize these enzymes.

Smad9 is a key player of follicular selection in goose via keeping the balance of LHR transcription

10.1101/213546 ◽

2017 ◽

Author(s):

Daolun Yu ◽

Fanghui Chen ◽

Li Zhang ◽

Hejian Wang ◽

Jie Chen ◽

...

Keyword(s):

Hormone Receptor ◽

Egg Production ◽

Follicular Development ◽

Luteinizing Hormone Receptor ◽

Summary Statement ◽

Follicular Selection ◽

New Strategies

ABSTRACTThe egg production of poultry depends on follicular development and selection. However, the mechanism of selecting the priority of hierarchical follicles is completely unknown. Smad9 is one of the important transcription factors in BMP/Smads pathway and involved in goose follicular initiation. To explore its potential role in goose follicle hierarchy determination, we first blocked Smad9 expression using BMP typeⅠreceptor inhibitor LDN–193189 both in vivo and in vitro. Unexpectedly, LDN–193189 administration could dramatically suppress Smad9 level and elevate egg production (7.08 eggs / bird, P< 0.05) of animals, and the estradiol (E2) and luteinizing hormone receptor (LHR) level were significantly increased (P< 0.05), but the progesterone (P4) and follicle stimulating hormone receptor (FSHR) mRNA remain unchanged. Surprisingly, Smad9 knockdown notably attenuated (P< 0.05) in E2, P4, FSHR and LHR level in goose granulosa cells (gGCs). Further chromatin immunoprecipitation (ChIP) assay of gGCs revealed that Smad9, served as a sensor of balance, bound to the LHR promoter regulating its transcription. These findings demonstrated that Smad9 is differentially expressed in goose follicles, and acts as a key player in controlling goose follicular selection.SUMMARY STATEMENTTo study the hierarchical development mechanism of avian follicle, new strategies can be found to improve the egg production of low-yielding poultry, such as geese.

lncRNA MIR22HG-Derived miR-22-5p Enhances the Radiosensitivity of Hepatocellular Carcinoma by Increasing Histone Acetylation Through the Inhibition of HDAC2 Activity

Frontiers in Oncology ◽

10.3389/fonc.2021.572585 ◽

2021 ◽

Vol 11 ◽

Author(s):

Qiao Jin ◽

Hao Hu ◽

Siqi Yan ◽

Long Jin ◽

Yuliang Pan ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Histone Acetylation ◽

Promoter Region ◽

Animal Experiments ◽

Primary Hepatocellular Carcinoma ◽

Acetylation Level ◽

Expression Levels

BackgroundWith the development of radiotherapy technology, radiotherapy has been increasingly used to treat primary hepatocellular carcinoma (HCC). However, due to radioresistance and the intolerance of the adjacent organs to radiation, the effects of radiotherapy are often unsatisfactory. Therefore, it is necessary to study radiosensitization in HCC.MethodA microarray was used to analyze the genes that were significantly associated with radiosensitivity. HCC cells, HepG2 and MHCC97H, were subjected to radiation in vitro. Real-time PCR was performed to determine MIR22HG (microRNA22 host gene) and miR-22-5p expression levels. Western blotting was performed to determine histone expression levels. A histone deacetylase (HDAC) whole cell assay was used to determine the activity of HDAC2. MTT, colony formation, 5-ethynyl-2′-deoxyuridine, and wound healing assays were performed to examine the function of MIR22HG and miR-22-5p in cellular radiosensitivity. Chromatin immunoprecipitation-PCR was used to confirm that HDAC2 affects the acetylation level of the MIR22HG promoter region. Finally, animal experiments were performed to demonstrate the in vivo effect of MIR22HG on the radiosensitivity of hepatoma.ResultsIrradiation can up-regulate MIR22HG expression and down-regulate HDAC2 expression. Inhibition of HDAC2 expression promotes histone acetylation in the MIR22HG promoter region and up-regulates MIR22HG expression. MIR22HG can increase radiosensitivity via miR-22-5p in HCC.ConclusionInhibition of HDAC2 expression promotes histone acetylation in the MIR22HG promoter region, thereby up-regulating the expression of MIR22HG and promoting the production of miR-22-5p, and ultimately increasing the sensitivity of liver cancer radiotherapy.

Elucidation of the BMI1 interactome identifies novel regulatory roles in glioblastoma

NAR Cancer ◽

10.1093/narcan/zcab009 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Verónica Freire-Benéitez ◽

Nicola Pomella ◽

Thomas O Millner ◽

Anaëlle A Dumas ◽

Maria Victoria Niklison-Chirou ◽

...

Keyword(s):

Protein Composition ◽

Mrna Splicing ◽

Cholesterol Transport ◽

Normal Brain ◽

Regulatory Functions ◽

Polycomb Repressive Complex 1 ◽

Normal Brain Development

Abstract Glioblastoma (GBM) is the most common and aggressive intrinsic brain tumour in adults. Epigenetic mechanisms controlling normal brain development are often dysregulated in GBM. Among these, BMI1, a structural component of the Polycomb Repressive Complex 1 (PRC1), which promotes the H2AK119ub catalytic activity of Ring1B, is upregulated in GBM and its tumorigenic role has been shown in vitro and in vivo. Here, we have used protein and chromatin immunoprecipitation followed by mass spectrometry (MS) analysis to elucidate the protein composition of PRC1 in GBM and transcriptional silencing of defining interactors in primary patient-derived GIC lines to assess their functional impact on GBM biology. We identify novel regulatory functions in mRNA splicing and cholesterol transport which could represent novel targetable mechanisms in GBM.