Targeting NHE6 gene expression identifies lysosome and neurodevelopmental mechanisms in a haploid in vitro cell model DOI Creative Commons
Qing Wu, Li Ma, Lena Joesch-Cohen

et al.

Biology Open, Journal Year: 2023, Volume and Issue: 12(11)

Published: Sept. 25, 2023

Christianson syndrome (CS) is an X-linked disorder resulting from loss-of-function (LoF) mutations in SLC9A6 encoding the endosomal Na+/H+ exchanger 6 (NHE6). CS presents with developmental delay, seizures, intellectual disability, nonverbal status, postnatal microcephaly, and ataxia. To define transcriptome signatures of NHE6 LoF, we conducted in-depth RNA-sequencing (RNA-seq) analysis on a haploid null cell model. CRIPSR/Cas9 genome editing introduced multiple LoF into near human line Hap1. Isogenic, paired parental controls were also studied. mutant lines confirmed to have intra-endosomal over-acidification as was seen other cells. RNA-seq performed by two widely used pipelines: HISAT2-StringTie-DEseq2 STAR-HTseq-DEseq2. We identified 1056 differentially expressed genes lines, including associated neurodevelopment, synapse function, voltage-dependent calcium channels, neuronal signaling. Weighted gene co-expression network then applied critical module enriched for governing lysosome function. By identifying significantly changed expression that lysosomal mechanisms NHE6-null cells, our analyses suggest loss function may converge implicated lysosome-related neurologic disease. Further, this model will serve important tool translational science CS.

Language: Английский

A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis DOI Creative Commons
Matthew P. Wilson, Takfarinas Kentache,

Charlotte R. Althoff

et al.

Cell, Journal Year: 2024, Volume and Issue: 187(14), P. 3585 - 3601.e22

Published: May 30, 2024

Dolichol is a lipid critical for N-glycosylation as carrier activated sugars and nascent oligosaccharides. It commonly thought to be directly produced from polyprenol by the enzyme SRD5A3. Instead, we found that dolichol synthesis requires three-step detour involving additional metabolites, where SRD5A3 catalyzes only second reaction. The first third steps are performed DHRSX, whose gene resides on pseudoautosomal regions of X Y chromosomes. Accordingly, report pseudoautosomal-recessive disease presenting congenital disorder glycosylation in patients with missense variants DHRSX (DHRSX-CDG). Of note, has unique dual substrate cofactor specificity, allowing it act NAD+-dependent dehydrogenase NADPH-dependent reductase two non-consecutive steps. Thus, our work reveals unexpected complexity terminal biosynthesis. Furthermore, provide insights into mechanism which metabolism defects contribute disease.

Language: Английский

Citations

9
SETD2 loss-of-function uniquely sensitizes cells to epigenetic targeting of NSD1-directed H3K36 methylation DOI Creative Commons
Ryan T. Wagner, Ryan A. Hlady, Xiaoyu Pan

et al.

Genome biology, Journal Year: 2025, Volume and Issue: 26(1)

Published: Feb. 5, 2025

Language: Английский

Citations

1
Analyzing the functional effects of DNA variants with gene editing DOI Creative Commons

Sarah Cooper,

Sofia Obolenski,

Andrew Waters

et al.

Cell Reports Methods, Journal Year: 2024, Volume and Issue: 4(5), P. 100776 - 100776

Published: May 1, 2024

Continual advancements in genomics have led to an ever-widening disparity between the rate of discovery genetic variants and our current understanding their functions potential roles disease. Systematic methods for phenotyping DNA are required effectively translate data into improved outcomes patients with diseases. To make biggest impact, these approaches must be scalable accurate, faithfully reflect disease biology, define complex mechanisms. We compare analyze function endogenous context using genome editing strategies, such as saturation editing, base prime editing. discuss how technologies can linked high-content readouts gain deep mechanistic insights variant effects. Finally, we highlight key challenges that need addressed bridge genotype phenotype gap, ultimately improve diagnosis treatment

Language: Английский

Citations

4
The PRC2.1 subcomplex opposes G1 progression through regulation of CCND1 and CCND2 DOI Creative Commons
Adam D Longhurst, Kyle Wang, Harsha Garadi Suresh

et al.

eLife, Journal Year: 2025, Volume and Issue: 13

Published: Feb. 4, 2025

Progression through the G1 phase of cell cycle is most highly regulated step in cellular division. We employed a chemogenetic approach to discover novel networks that regulate progression. This uncovered functional clusters genes altered sensitivity cells inhibitors G1/S transition. Mutation components Polycomb Repressor Complex 2 rescued proliferation inhibition caused by CDK4/6 inhibitor palbociclib, but not S or mitosis. In addition its core catalytic subunits, mutation PRC2.1 accessory protein MTF2, PRC2.2 JARID2, rendered resistant palbociclib treatment. found (MTF2), (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and promoters. included promoter cyclins CCND1 CCND2, loss MTF2 lead upregulation both CCND2. Our results demonstrate role PRC2.1, PRC2.2, antagonizing progression diversity linages, including chronic myeloid leukemia (CML), breast cancer, immortalized lines.

Language: Английский

Citations

0
The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition DOI Open Access
Gülnihal Kavaklıoğlu, Alexandra Podhornik, Terezia Vcelkova

et al.

Published: Feb. 17, 2025

Repression of retrotransposition is crucial for the successful fitness a mammalian organism. The domesticated transposon protein L1TD1, derived from LINE-1 (L1) ORF1p, an RNA-binding that expressed only in some cancers and early embryogenesis. In human embryonic stem cells, it found to be essential maintaining pluripotency. cancer, L1TD1 expression highly correlative with malignancy progression as such considered potential prognostic factor tumors. However, its molecular role cancer remains largely unknown. Our findings reveal DNA hypomethylation induces HAP1 tumor cells. depletion significantly modulates both proteome transcriptome thereby reduces cell viability. Notably, associates L1 transcripts interacts ORF1p protein, facilitating retrotransposition. data suggest collaborates ancestral RNA chaperone, ensuring efficient retrotransposons, rather than directly impacting abundance targets. this way, might have important not during development but also tumorigenesis.

Language: Английский

Citations

0
The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition DOI Creative Commons
Gülnihal Kavaklıoğlu, Alexandra Podhornik, Terezia Vcelkova

et al.

eLife, Journal Year: 2025, Volume and Issue: 13

Published: March 20, 2025

Repression of retrotransposition is crucial for the successful fitness a mammalian organism. The domesticated transposon protein L1TD1, derived from LINE-1 (L1) ORF1p, an RNA-binding that expressed only in some cancers and early embryogenesis. In human embryonic stem cells, it found to be essential maintaining pluripotency. cancer, L1TD1 expression highly correlative with malignancy progression as such considered potential prognostic factor tumors. However, its molecular role cancer remains largely unknown. Our findings reveal DNA hypomethylation induces HAP1 tumor cells. depletion significantly modulates both proteome transcriptome thereby reduces cell viability. Notably, associates L1 transcripts interacts ORF1p protein, facilitating retrotransposition. data suggest collaborates ancestral RNA chaperone, ensuring efficient retrotransposons, rather than directly impacting abundance targets. this way, might have important not during development but also tumorigenesis.

Language: Английский

Citations

0
Functional Toxicology: Key Concepts and Applications DOI
Chan Kim, Chris D. Vulpe

Elsevier eBooks, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Language: Английский

Citations

0
A homozygous mutation in the human selenocysteine tRNA gene impairs UGA recoding activity and selenoproteome regulation by selenium DOI Creative Commons
Caroline Vindry, Olivia Guillin, Philippe Wolff

et al.

Nucleic Acids Research, Journal Year: 2023, Volume and Issue: 51(14), P. 7580 - 7601

Published: May 25, 2023

Abstract The selenocysteine (Sec) tRNA (tRNA[Ser]Sec) governs Sec insertion into selenoproteins by the recoding of a UGA codon, typically used as stop codon. A homozygous point mutation (C65G) in human tRNA[Ser]Sec acceptor arm has been reported two independent groups and was associated with symptoms such thyroid dysfunction low blood selenium levels; however, extent altered selenoprotein synthesis resulting from this yet to be comprehensively investigated. In study, we CRISPR/Cas9 technology engineer heterozygous mutant cells, which then compared parental cell lines. This C65G affected many aspects integrity activity. Firstly, expression level significantly reduced due an recruitment RNA polymerase III at promoter. Secondly, strongly altered, but, more surprisingly, it no longer sensitive supplementation. Mass spectrometry analyses revealed isoform unmodified wobble nucleotide U34 cells that correlated activities. Overall, study demonstrates pleiotropic effect single on both phenotype selenoproteome expression.

Language: Английский

Citations

8
Evidence of an intracellular creatine-sensing mechanism that modulates creatine biosynthesis via AGAT expression in human HAP1 cells DOI Creative Commons

Michael B. Tropak,

Ilona Tkachyova,

Ray Gu

et al.

Scientific Reports, Journal Year: 2023, Volume and Issue: 13(1)

Published: Dec. 16, 2023

Cellular homeostasis of creatine (CT), integral part the energy buffering and transducing system connecting intracellular sites ATP production utilization, comprises mechanisms that increase CT, i.e., biosynthesis cellular uptake, CT-lowering processes, such as export non-enzymatic conversion to creatinine. The CT is controlled by negative feedback loop via suppression rate-limiting enzyme arginine:glycine amidinotransferase (AGAT). Although regulatory mechanism involved not well understood, AGAT successfully used in patients with guanidinoacetate methyltransferase (GAMT) deficiency reduce neurotoxic accumulation AGAT-mediated supplementing CT. Utilizing CT-dependent for upregulation expression may represent a therapeutic target an additional syndrome, transporter (CrT) defect, which no effective treatment option available so far. We have CRISPR tag C-terminus nanoluc luciferase (NLuc) reporter HAP1 cells. A biphasic decay AGAT-NLuc response increasing extracellular was observed, whereas decrease directly proportional rise levels approximate IC50 1-2 mM. generated CrT null cells stably expressing CrT-GFP fusion protein further demonstrated mediated high-affinity (Km 9-10 µM) dependent, saturable independent, unsaturable uptake process. direct suggests existence sensing enabling dynamic cell changing concentration relevant homeostasis.

Language: Английский

Citations

7
InsulatedpiggyBacand FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells DOI Creative Commons
Annabel Y. Minard, Stanley C. Winistorfer, Robert C. Piper

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 23, 2024

Abstract Transgene expression in eHAP cells, a haploid cell line popularly used to generate gene knockouts, is difficult owing its low transfection efficiency and propensity for silencing integrated transgenes. To simplify transgene expression, we engineered insulated integrating plasmids that sustain high levels of can be other lines. These vectors are compatible with FLP-FRT piggyBac integration, they flank gene-of interest bilaterally tandem cHS4 core insulators, co-express nuclear-localized blue fluorescent protein identification expressing cells. We further demonstrate transgenic cells fused form heterozygous diploid This method creates carrying the material progenitors could also create defined genotypes. tools expand repertoire experiments performed cultured

Language: Английский

Citations

2