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PubMed2026

AML1-ETO hijacks a distal enhancer of NAT10 to reprogram glutathione metabolism and sustain leukemia stem cell stemness.

Chromosomal translocations produce oncogenic fusion proteins such as AML1-ETO, which predominantly occupy gene promoters to induce transcriptional reprogramming in leukemia stem cells (LSCs), consequently driving the pathogenesis of t(8;21) acute myeloid leukemia (AML). However, whether AML1-ETO is recruited to additional regulatory DNA elements to orchestrate oncogenic gene expression programs has not been fully addressed. Here, we define AML1-ETO and H3K27ac CUT&Tag landscapes in primary t(8;21) AML CD34+ cells and t(8;21) AML cell lines, revealing AML1-ETO binding at a distal enhancer of the RNA N4-acetylcytidine (ac4C) writer N-acetyltransferase 10 (NAT10), thereby driving its transcriptional activation. Genetic ablation or pharmacological inhibition of NAT10 restricted the survival and self-renewal of LSCs in primary t(8;21) AML CD34+ cells, as well as in a retroviral AML1-ETO9a-driven t(8;21) AML mouse model, establishing NAT10 as a potential therapeutic vulnerability. Mechanistically, NAT10 is recruited to glutathione S-transferase omega 2 (GSTO2) mRNA to catalyze ac4C modification, thereby enhancing transcript stability and reprogramming glutathione metabolism, as demonstrated by ac4C profiling, RNA immunoprecipitation (RIP), and dCas13b-NAT10-based analyses. Silencing of GSTO2 in primary t(8;21) AML CD34+ cells decreased intracellular reduced glutathione (GSH) levels and compromised LSC survival and self-renewal, whereas GSTO2 overexpression or GSH supplementation largely rescued LSC maintenance following NAT10 loss. Collectively, these findings enrich and extend the understanding of AML1-ETO regulatory programs by linking distal enhancer activity to a NAT10-GSTO2 ac4C-GSH axis that integrates epigenomic, posttranscriptional, and metabolic reprogramming to sustain LSC stemness, highlighting this circuit as a potential therapeutic vulnerability in t(8;21) AML.

Humans↗
PubMed2026

Gene Cloning, Expression, and Purification of Kunitz Trypsin Inhibitor from Glycine max Using Halo Tag.

Soybean Kunitz Trypsin Inhibitor (SKTI) is one of the most extensively studied protease inhibitors, with applications in pest management, medicine, the food processing industry, and the leather industry. In this study, SKTI was cloned into the pFN29A Flexi vector containing a barnase gene. Genomic DNA was isolated from tender soybean leaves, and SKTI was amplified by PCR to obtain a 671 bp product. After cloning, an internal 380 bp sequence was amplified using specific primers to confirm that the cloned sequence was a functional SKTI, as non-functional SKTI genes also exist in Glycine max. The amplified PCR product, containing an AsiSI site at the 5' end and a PmeI site at the 3' end, was cloned into the pFN29A vector. The resulting colonies were screened by colony PCR, and the insert sequence was confirmed by Sanger sequencing. The recombinant protein, containing a His-tag, Halo-tag, and a TEV protease cleavage site, was expressed in Escherichia coli BL21 cells. Maximum expression was achieved 5 h after induction with 0.5 mM IPTG at 37 °C. The expressed SKTI was purified using affinity chromatography on HaloLink resin, and the bound SKTI was cleaved with HaloTEV protease to obtain pure SKTI. The purified inhibitor effectively inhibited bovine trypsin, with an IC₅₀ of 0.6 ± 0.003 µg/µl, yielding 1.6 mg per gram of bacterial pellet. The 24 kDa inhibitor remained stable up to a temperature of 50 °C. Kinetic analysis revealed that recombinant SKTI competitively inhibits trypsin, with a Kᵢ value of 14 µM.

Cloning, Molecular↗
PubMed2025

Marine-Inspired Antimicrobial Peptides Disrupt Gene Expression at the DNA Level.

Genome mining of Streptomyces sp. H-KF8 combined with sequence engineering yielded two serum-stable, noncytotoxic, nonlytic antimicrobial peptides, L3 and L3-K. Initial studies in uropathogenic Escherichia coli suggested membrane effects and nucleoid relaxation, prompting a comprehensive investigation of their mode of action. In this study tandem mass tag (TMT)-based quantitative proteomics revealed extensive proteome remodeling, with 175 and 120 differentially expressed proteins (DEPs) after treatment with L3 and L3-K, respectively. L3 induced predominantly upregulated responses linked to metabolism, RNA processing, transport, and homeostasis, whereas L3-K mainly caused the downregulation of proteins involved in metabolism, transport, and cell structure. Both peptides disrupted ABC transporter-mediated nutrient uptake and elicited stress responses, while L3 specifically perturbed the mal regulon, indicative of broader transcriptional dysregulation. Complementary fluorescent dye displacement and in vitro transcription/translation assays demonstrated nonspecific DNA binding, stronger for L3 than L3-K, and potent inhibition of transcriptional and translational processes. Strikingly, inhibitory concentrations paralleled their minimum inhibitory concentrations, directly linking DNA binding and interference with central information processing to antimicrobial activity. These findings reveal that L3 and L3-K primarily act by targeting DNA and interfering with the transcription-translation machinery. Beyond offering mechanistic insights, this study underscores peptides' potential to act as scaffolds for next-generation antimicrobial peptides with DNA-binding and nonmembrane-lytic activity.

Antimicrobial Peptides↗
PubMed2024

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells.

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

Animals↗
PubMed1979

The N protein of bacteriophage lambda, defined by its DNA sequence, is highly basic.

Nucleotide sequence has been determined for the restriction fragments and cloned DNA from the pL-N-tL1 region of bacteriophage lambda. A unique reading frame for the N gene is defined by the absence of natural nonsense codons and by the presence of seven nonsense codons generated by mutations in N. This reading frame is initiated at two alternative ATG codons, the second of which is probably the in vivo translation start. Reading is stopped at a single TAG codon. The protein coded is therefore 133 or, more probably, 107 amino acids long, rich in lysine, arginine and proline.

Bacteriophage lambda↗
PubMed1975

[The influence of the rhythm of the damaging agent on the character of hepatic reparative regeneration].

The effect of frequency of administration of tetrachloride of carbon (CCl4) on synthesis of DNA by hepacytes and cells of the stroma, as well as on composition and magnitude of infiltrates in the liver of mice was studied. For this purpose autoradiographic and histological studies of the liver tissue in animals injected with poison once a week, twice a week and every day were carried out dayly. Periodic increases in number of hepacytes tagged with thymidin-H3 and an increase in the number of cells in infiltrates were observed. The rhythm of these changes corresponded to the frequency of administration of CCl4. With the rise in the frequency of injections of the poison the rate of increase in number of cells in infiltrates rose and synthesis of DNA in hepacytes became more intensive. The time interval, however, between the injection of the toxin and the rise of DNA synthesis in hepacytes remained constant irrespective of the frequency of exposure to the damaging factor.

Animals↗
PubMed1992

Insertional mutations in mammals and mammalian cells.

The retroposon sequences, their mechanisms of transposition and the occurrence of insertional mutation in the mammalian genome are reviewed. Insertional mutations fall into two broad categories: those due to the disruption of a gene following the physical integration of a foreign DNA sequence result in loss of gene product and would be expected to be associated with a recessive mutation. A second class of insertional mutation is well documented in which upon integration the promoter/enhancer activities inherent in the retroposon genome exert their influence on neighboring genes. This promoter/enhancer activity of integrated retroposons may have effects over relatively long distances and thus limit the possibilities of establishing an association between retroposon integration and mutation. It is emphasized that a systematic search for insertional mutations in the mammalian genome involves an extensive two-dimensional array of possible retroposon sequences and mutant alleles. Present results represent only a small portion of the total array. Future studies promise to be fruitful in efforts to isolate genes through insertional tagging, to characterize the mechanisms of retroposon transposition, as well as to study the stability of the mammalian genome.

Animals↗
PubMed1992

A survey of expressed genes in Caenorhabditis elegans.

As an adjunct to the genomic sequencing of Caenorhabditis elegans, we have investigated a representative cDNA library of 1,517 clones. A single sequence read has been obtained from the 5' end of each clone, allowing its characterization with respect to the public databases, and the clones are being localized on the genome map. The result is the identification of about 1,200 of the estimated 15,000 genes of C. elegans. More than 30% of the inferred protein sequences have significant similarity to existing sequences in the databases, providing a route towards in vivo analysis of known genes in the nematode. These clones also provide material for assessing the accuracy of predicted exons and splicing patterns and will lead to a more accurate estimate of the total number of genes in the organism than has hitherto been available.

Animals↗
PubMed1978

Correlations of DNA, RNA and protein levels in duodenal mucosa with antiinflammatory potency and disposition to gut damage of non-steroidal agents. Comparative behaviour of glucametacine, indometacin, phenylbutazone and ibuprofen.

As far as molar ratio is concerned, glucametacine was half as potent as indometacin, twice as active as phenylbutazone and four times more effective than ibuprofen in preventing cotton granuloma. Both indometacin and phenylbutazone induced dose related gastrointestinal ulcerations and increase of 51Cr tagged erythrocytes in feces. The former drug displayed gut toxicity at anti-inflammatory doses, the latter at doses approximately four times larger. Glucametacine was still devoid of damaging effects at a dose ten times larger than the minimal one capable of inhibiting granuloma growth. Ibuprofen, too, failed to induce ulcers at all doses examined; however, it displayed a trend toward gut bleeding when doses that increased blood corticosterone were attained. Studies on duodenal mucosa showed that in rats on cotton granuloma, DNA, proteins and DNA:RNA ratio increase as compared to unimplanted rats. Glucametacine and phenylbutazone reversed the increase of DNA and proteins, respectively. Indometacin decreased all forementioned constituents of duodenal mucosa while inducing haemorrhages and ulcers on gut. Furthermore, in naive rats, unlike glucametacine and phenylbutazone, indometacin induced a decrease in protein content of duodenal mucosa. Differences in disposition of gut toxicity among glucametacine and other anti-inflammatory drugs are discussed.

Animals↗
PubMed2025

Multiomics Reveal Associations Between CpG Methylation, Histone Modifications and Transcription in a Species That has Lost DNMT3, the Colorado Potato Beetle.

Insects display exceptional phenotypic plasticity, which can be mediated by epigenetic modifications, including CpG methylation and histone modifications. In vertebrates, both are interlinked and CpG methylation is associated with gene repression. However, little is known about these regulatory systems in invertebrates, where CpG methylation is mainly restricted to gene bodies of transcriptionally active genes. A widely conserved mechanism involves the co-transcriptional deposition of H3K36 trimethylation and the targeted methylation of unmethylated CpGs by the de novo DNA methyltransferase DNMT3. However, DNMT3 has been lost multiple times in invertebrate lineages raising the question of how the links between CpG methylation, histone modifications and gene expression are affected by its loss. Here, we report the epigenetic landscape of Leptinotarsa decemlineata, a beetle species that has lost DNMT3 but retained CpG methylation. We combine RNA-seq, enzymatic methyl-seq and CUT&Tag to study gene expression, CpG methylation and patterns of H3K36me3 and H3K27ac histone modifications on a genome-wide scale. Despite the loss of DNMT3, H3K36me3 mirrors CpG methylation patterns. Together, they give rise to signature profiles for expressed and not expressed genes. H3K27ac patterns show a prominent peak at the transcription start site that is predictive of expressed genes irrespective of their methylation status. Our study provides new insights into the evolutionary flexibility of epigenetic modification systems that urge caution when generalizing across species.

Animals↗
PubMed2026

Development of a cell-based nanoluciferase reporter system for high-throughput screening of HBV cccDNA inhibitors.

Hepatitis B virus (HBV) persistence is sustained by the viral covalently closed circular DNA (cccDNA) minichromosome, which remains a major barrier to curative antiviral therapies. The lack of reliable quantitative cccDNA detection methods and surrogate markers has hindered efforts to target cccDNA in antiviral high-throughput screening (HTS). Here, we established a novel inducible cccDNA-dependent nanoluciferase (NLuc) reporter cell line, designated HepBLE12, by inserting an in-frame 11-amino acid split-NLuc HiBiT tag into the precore (pC) coding region of an HBV transgene. The resulting 1.3-kDa HiBiT tag on pC serves as the detection module of the split NLuc system, generating quantitative luminescence upon high-affinity complementation with the cognate 18-kDa LgBiT subunit in cell lysates. Notably, the HiBiT assay enables direct detection of intracellular HiBiT-pC protein rather than secreted HBeAg, providing a reporter signal more closely linked to cccDNA activity. HepBLE12&#x202f;cells exhibited inducible and robust viral DNA replication, and the cccDNA-dependent HiBiT signal was validated under diverse experimental conditions that modulate cccDNA formation or transcription. We further miniaturized the assay to a 384-well format and optimized key parameters following standard HTS assay development practices. The assay was successfully automated and demonstrated excellent performance in a multi-day variability study and a pilot screen, with signal-to-background (S/B)&#x202f;&#x2248;&#x202f;9, coefficient of variance (CV)&#x202f;<&#x202f;10%, and average Z-factor value of 0.74, exceeding canonical HTS quality benchmarks. Together, the HepBLE12 cell-based HTS platform provides a robust and practical tool for identifying inhibitors targeting HBV cccDNA.

Hepatitis B virus↗
PubMed2004

Conserved function of medaka pink-eyed dilution in melanin synthesis and its divergent transcriptional regulation in gonads among vertebrates.

Medaka is emerging as a model organism for the study of vertebrate development and genetics, and its effectiveness in forward genetics should prove equal to that of zebrafish. Here, we identify by positional cloning a gene responsible for the medaka i-3 albino mutant. i-3 larvae have weakly tyrosinase-positive cells but lack strongly positive and dendritic cells, suggesting loss of fully differentiated melanophores. The region surrounding the i-3 locus is syntenic to human 19p13, but a BAC clone covering the i-3 locus contained orthologs located at 15q11-13, including OCA2 (P). Medaka P consists of 842 amino acids and shares approximately 65% identity with mammalian P proteins. The i-3 mutation is a four-base deletion in exon 13, which causes a frameshift and truncation of the protein. We detected medaka P transcripts in melanin-producing eyeballs and (putative) skin melanophores on embryos and an alternatively spliced form in the non-melanin-producing ovary or oocytes. The mouse p is similarly expressed in gonads, but not alternatively spliced. This is the first isolation of nonmammalian P, the functional mechanism of action of which has not yet been elucidated, even in mammals. Further investigation of the functions of P proteins and the regulation of their expression will provide new insight into body color determination and gene evolution.

Amino Acid Sequence↗
PubMed2026

Cross-target and cell-preferential CRISPR-Cas9 inhibition with carbohydrate-tagged oligonucleotides.

Precise control of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) activity is important for limiting off-target effects and chromosomal rearrangements. Existing inhibitors, including anti-CRISPR proteins and spacer-targeting oligonucleotides, can be constrained by immunogenicity, target-sequence dependence, or delivery challenges. Here, we developed single-stranded DNA oligonucleotides that target conserved regions of the Streptococcus pyogenes Cas9 single-guide RNA (sgRNA) scaffold rather than the variable spacer. Screening identified single-stranded DNA 7 (ssDNA7), which targets stem-loop-1 and the adjacent linker and inhibits Cas9 activity across multiple tested spacer sequences and genomic loci without redesigning the inhibitor. Carbohydrate conjugation improved oligonucleotide stability and preferentially enhanced inhibitory activity in selected liver-derived or cancer cell models. Amplicon sequencing confirmed inhibition of endogenous editing in multiple cell models and normal liver-derived organoids, and supported cell-preferential inhibition in matched cell comparisons. These findings establish sgRNA-scaffold targeting as a strategy for cross-target inhibition of Cas9 and show that carbohydrate conjugation can tune its cellular activity.

CRISPR-Cas9↗
PubMed2026

The Neanderthal-Derived 3p21 Haplotype at LZTFL1 in Modern-Day Moroccans Is Associated With COVID-19 Severity and Further Suggests the Presence of Neanderthals in North Africa.

There is considerable variability in the clinical presentation of COVID-19 among patients infected with SARS-CoV-2. Genome-wide association studies (GWASs) have identified the 12q24.13 and 3p21.31 regions, derived from Neanderthal DNA, as the human genetic loci most strongly associated with COVID-19 severity. We examined in this study the 3p locus in the Moroccan population by analysing allele and haplotype frequencies at the LZTFL1 gene and their associations with COVID-19 outcomes. Three SNPs at LZTFL1, tagging the Neanderthal-derived COVID-19 risk haplotype, were sequenced by Sanger's method in 102 ambulatory participants and 105 hospitalized patients and have been compared to 118 controls negative for SARS-CoV-2 infection using logistic regression analysis. Results showed that the prevalence of the lead variant rs11385942 in this locus was 8.9%, whereas the variants rs35044562 and rs13078854, which tag the Neanderthal haplotype, were present in only 6.3%. Our study showed that only the rs35044562-T and rs13078854-A alleles were associated with a 2.5-fold increased risk of severe COVID-19 (p&#xa0;=&#xa0;0.028). These two alleles, in LD with the rs11385942-AA one, form the haplotype inherited from the Neanderthal, the only haplotype associated with COVID-19 severity in the Moroccan population (p&#xa0;=&#xa0;0.030), whereas sub-Saharan African and the rare local haplotype also containing the rs11385942 variant do not influence the COVID-19 outcomes 19 (p&#xa0;>&#xa0;0.05). Furthermore, our study showed that the Neanderthal haplotype at 3p21 locus exists in the inhabitants of Morocco at a frequency close to that of Europeans and suggests a close connection between North Africa and Eurasia.

Adult↗
PubMed2022

The evolutionary dynamics of extrachromosomal DNA in human cancers.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a common event, driving aggressive tumor growth, drug resistance and shorter survival. Currently, the impact of nonchromosomal oncogene inheritance-random identity by descent-is poorly understood. Also unclear is the impact of ecDNA on somatic variation and selection. Here integrating theoretical models of random segregation, unbiased image analysis, CRISPR-based ecDNA tagging with live-cell imaging and CRISPR-C, we demonstrate that random ecDNA inheritance results in extensive intratumoral ecDNA copy number heterogeneity and rapid adaptation to metabolic stress and targeted treatment. Observed ecDNAs benefit host cell survival or growth and can change within a single cell cycle. ecDNA inheritance can predict, a priori, some of the aggressive features of ecDNA-containing cancers. These properties are facilitated by the ability of ecDNA to rapidly adapt genomes in a way that is not possible through chromosomal oncogene amplification. These results show how the nonchromosomal random inheritance pattern of ecDNA contributes to poor outcomes for patients with cancer.

Biological Evolution↗
PubMed1992

A yeast artificial chromosome contig spanning the Charcot-Marie-Tooth disease type 1A duplication region.

A contiguous set of 43 overlapping yeast artificial chromosome (YAC) clones has been developed for the Charcot-Marie-Tooth disease type 1A (CMT1A) duplication region of chromosome 17p11.2. The contig spans approximately 2.0 Mb and can be represented in a minimum of five overlapping YACs. The YAC clones were isolated from two total human genomic YAC libraries and from YAC libraries made from rodent-human hybrid cell lines. YAC clones were isolated from the libraries by polymerase chain reaction (PCR) technique. Localization to chromosome 17p11.2 was confirmed by fluorescence in situ hybridization. Overlap between the YAC clones was detected by inter-Alu PCR amplification of the YACs and by cross hybridization of the YACs with YAC insert ends obtained by Vectorette PCR. This YAC contig is a useful resource for analyzing and mapping all the genes contained within the CMT1A duplication.

Base Sequence↗
PubMed2026

Endogenous tagging of the TBXT (Brachyury) gene with 2A-EGFP in BJNhem19 human embryonic stem cells using CRISPR-Cas9 genome editing.

Brachyury (TBXT) is a T-box transcription factor essential for directing human embryonic stem cells (hESCs) toward mesodermal lineage specification. Here, we report the generation of a Brachyury/TBXT-2A-EGFP knock-in reporter line in the BJNhem19 hESC line using CRISPR/Cas9-mediated homology-directed repair. Accurate integration of the reporter cassette was confirmed by DNA sequencing and validated by the comparison of EGFP expression to endogenous TBXT gene expression following directed mesodermal differentiation. This reporter line provides a tractable fluorescence-based platform for real-time monitoring of mesodermal commitment, which will help in high-content screening of differentiation protocols.

Journal Article↗