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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Phosphorothioate-Based Site-Specific Labeling of Large RNAs for Structural and Dynamic Studies

Pulsed electron-electron double resonance (PELDOR) spectroscopy, X-ray scattering interferometry (XSI), and single-molecule Forster resonance energy transfer (smFRET) are molecular rulers that provide inter- or intramolecular pair-wise distance distributions in the nanometer range, thus being ideally suitable for structural and dynamic studies of biomolecules including RNAs. The prerequisite for such applications requires site-specific labeling of biomolecules with spin labels, gold nanoparticles, and fluorescent tags, respectively. Recently, site-specific labeling of large RNAs has been achieved by a combination of transcription of an expanded genetic alphabet containing A-T/G-C base pairs and NaM-TPT3 unnatural base pair (UBP) with posttranscriptional modifications at UBP bases by click chemistry or amine-NHS ester reactions. However, due to the bulky sizes of functional groups or labeling probes used, such strategies might cause structural perturbation and decrease the accuracy of distance measurements. Here, we synthesize an a-thiophosphorylated variant of rTPT3TP (rTPT3aS), which allows for post-transcriptional site-specific labeling of large RNAs at the internal a-phosphate backbone via maleimide-modified probes. Subsequent PELDOR, XSI, and smFRET measurements result in narrower distance distributions than labeling at the TPT3 base. The presented strategy provides a new route to empower the molecular rulers for structural and dynamic studies of large RNA and its complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Biochemical and structural basis for YTH domain of human YTHDC1 binding to methylated adenine in DNA

The recently characterized mammalian writer (methyltransferase) and eraser (demethylase) of the DNA N6-methyladenine (N6mA) methyl mark act on single-stranded (ss) and transiently-unpaired DNA. As YTH domain-containing proteins bind N6mA-containing RNA in mammalian cells, we investigated whether mammalian YTH domains are also methyl mark readers of N6mA DNA. Here, we show that the YTH domain of YTHDC1 (known to localize in the nucleus) binds ssDNA containing N6mA, with a 10 nM dissociation constant. This binding is stronger by a factor of 5 than in an RNA context, tested under the same conditions. However, the YTH domains of YTHDF2 and YTHDF1 (predominantly cytoplasmic) exhibited the opposite effect with ~1.5–2× stronger binding to ssRNA containing N6mA than to the corresponding DNA. We determined two structures of the YTH domain of YTHDC1 in complex with N6mA-containing ssDNA, which illustrated that YTHDC1 binds the methylated adenine in a single-stranded region flanked by duplexed DNA. We discuss the hypothesis that the writer-reader-eraser of N6mA-containining ssDNA is associated with maintaining genome stability. Structural comparison of YTH and SRA domains (the latter a DNA 5-methylcytosine reader) revealed them to be diverse members of a larger family of DNA/RNA modification readers, apparently having originated from bacterial modification-dependent restriction enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Data for "Viral Delivery of Recombinases Activates Heritable Genetic Switches in Plants"

Viral vectors provide an increasingly versatile platform for transformation-free reagent delivery to plants. RNA viral vectors can be used to induce gene silencing, overexpress proteins, or introduce gene editing reagents; however, they are often constrained by carrying capacity or restricted tropism in germline cells. Site-specific recombinases that catalyze precise genetic rearrangements are powerful tools for genome engineering that vary in size and, potentially, efficacy in plants. In this work, we show that viral vectors based on tobacco rattle virus (TRV) deliver and stably express four recombinases ranging in size from ∼0.6 to ∼1.5 kb and achieve simultaneous marker removal and reporter activation through targeted excision in transgenic Nicotiana benthamiana lines. TRV vectors with Cre, FLP, CinH, and Integrase13 efficiently mediated recombination in infected somatic tissue and led to heritable modifications at high frequency. An excision-activated Ruby reporter enabled simple and high-resolution tracing of infected cell lineages without the need for molecular genotyping. Together, our experiments broaden the scope of viral recombinase delivery and offer insights into infection dynamics that may be useful in developing future viral vectors.

gene editing↗
DOE OSTI2025

Viral delivery of recombinases activates heritable genetic switches in plants

Viral vectors provide an increasingly versatile platform for transformation-free reagent delivery to plants. RNA viral vectors can be used to induce gene silencing, overexpress proteins, or introduce gene editing reagents; however, they are often constrained by carrying capacity or restricted tropism in germline cells. Site-specific recombinases that catalyze precise genetic rearrangements are powerful tools for genome engineering that vary in size and, potentially, efficacy in plants. In this work, we show that viral vectors based on tobacco rattle virus (TRV) deliver and stably express four recombinases ranging in size from ~0.6kb to ~1.5kb and achieve simultaneous marker removal and reporter activation through targeted excision in transgenic Nicotiana benthamiana lines. TRV vectors with Cre, FLP, CinH, and Integrase13 efficiently mediated recombination in infected somatic tissue and led to heritable modifications at high frequency. Here, an excision-activated Ruby reporter enabled simple and high-resolution tracing of infected cell lineages without the need for molecular genotyping. Together, our experiments broaden the scope of viral recombinase delivery and offer insights into infection dynamics that may be useful in developing future viral vectors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Spaceflight-Associated Changes of snoRNAs in Peripheral Blood Mononuclear Cells and Plasma Exosomes—A Pilot Study

During spaceflight, astronauts are exposed to various physiological and psychological stressors that have been associated with adverse health effects. Therefore, there is an unmet need to develop novel diagnostic tools to predict early alterations in astronauts’ health. Small nucleolar RNA (snoRNA) is a type of short non-coding RNA (60–300 nucleotides) known to guide 2'-O-methylation (Nm) or pseudouridine (ψ) of ribosomal RNA (rRNA), small nuclear RNA (snRNA), or messenger RNA (mRNA). Emerging evidence suggests that dysregulated snoRNAs may be key players in regulating fundamental cellular mechanisms and in the pathogenesis of cancer, heart, and neurological disease. Therefore, we sought to determine whether the spaceflight-induced snoRNA changes in astronaut’s peripheral blood (PB) plasma extracellular vesicles (PB-EV) and peripheral blood mononuclear cells (PBMCs). Using unbiased small RNA sequencing (sRNAseq), we evaluated changes in PB-EV snoRNA content isolated from astronauts (n = 5/group) who underwent median 12-day long Shuttle missions between 1998 and 2001. Using stringent cutoff (fold change > 2 or log 2 -fold change >1, FDR < 0.05), we detected 21 down-and 9—up-regulated snoRNAs in PB-EVs 3 days after return (R + 3) compared to 10 days before launch (L-10). qPCR validation revealed that SNORA74A was significantly down-regulated at R + 3 compared to L-10. We next determined snoRNA expression levels in astronauts’ PBMCs at R + 3 and L-10 (n = 6/group). qPCR analysis further confirmed a significant increase in SNORA19 and SNORA47 in astronauts’ PBMCs at R + 3 compared to L-10. Notably, many downregulated snoRNA-guided rRNA modifications, including four Nms and five ψs. Our findings revealed that spaceflight induced changes in PB-EV and PBMCs snoRNA expression, thus suggesting snoRNAs may serve as potential novel biomarkers for monitoring astronauts’ health.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Disruption of evolutionarily correlated tRNA elements impairs accurate decoding

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. In this work, we solved structures of the bacterial ribosome containing either wild-type tRNA$^{\mathrm{Ala}}_{\mathrm{GGC}}$ or tRNA$^{\mathrm{Ala}}_{\mathrm{GGC}}$ containing a reversed 32–38 pair on cognate and near-cognate codons. Structures of wild-type tRNA$^{\mathrm{Ala}}_{\mathrm{GGC}}$ bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32–38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in tRNA$^{\mathrm{Ala}}_{\mathrm{GGC}}$ ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32–38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits — TASOR, MPP8 and Periphilin — contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The essential Rhodobacter sphaeroides CenKR two-component system regulates cell division and envelope biosynthesis

Bacterial two-component systems (TCSs) often function through the detection of an extracytoplasmic stimulus and the transduction of a signal by a transmembrane sensory histidine kinase. This kinase then initiates a series of reversible phosphorylation modifications to regulate the activity of a cognate, cytoplasmic response regulator as a transcription factor. Several TCSs have been implicated in the regulation of cell cycle dynamics, cell envelope integrity, or cell wall development in Escherichia coli and other well-studied Gram-negative model organisms. However, many α-proteobacteria lack homologs to these regulators, so an understanding of how α-proteobacteria orchestrate extracytoplasmic events is lacking. In this work we identify an essential TCS, CenKR ( C ell en velope K inase and R egulator), in the α-proteobacterium Rhodobacter sphaeroides and show that modulation of its activity results in major morphological changes. Using genetic and biochemical approaches, we dissect the requirements for the phosphotransfer event between CenK and CenR, use this information to manipulate the activity of this TCS in vivo , and identify genes that are directly and indirectly controlled by CenKR in Rb . sphaeroides . Combining ChIP-seq and RNA-seq, we show that the CenKR TCS plays a direct role in maintenance of the cell envelope, regulates the expression of subunits of the Tol-Pal outer membrane division complex, and indirectly modulates the expression of peptidoglycan biosynthetic genes. CenKR represents the first TCS reported to directly control the expression of Tol-Pal machinery genes in Gram-negative bacteria, and we predict that homologs of this TCS serve a similar function in other closely related organisms. We propose that Rb . sphaeroides genes of unknown function that are directly regulated by CenKR play unknown roles in cell envelope biosynthesis, assembly, and/or remodeling in this and other α-proteobacteria.

Lakey, Bryan D. (ORCID:0000000332795242)↗
DOE OSTI2023

Oxamniquine derivatives overcome Praziquantel treatment limitations for Schistosomiasis

Human schistosomiasis is a neglected tropical disease caused by Schistosoma mansoni, S. haematobium, and S. japonicum. Praziquantel (PZQ) is the method of choice for treatment. Due to constant selection pressure, there is an urgent need for new therapies for schistosomiasis. Previous treatment of S. mansoni included the use of oxamniquine (OXA), a drug that is activated by a schistosome sulfotransferase (SULT). Guided by data from X-ray crystallography and Schistosoma killing assays more than 350 OXA derivatives were designed, synthesized, and tested. We were able to identify CIDD-0150610 and CIDD-0150303 as potent derivatives in vitro that kill (100%) of all three Schistosoma species at a final concentration of 71.5 μM. We evaluated the efficacy of the best OXA derivates in an in vivo model after treatment with a single dose of 100 mg/kg by oral gavage. The highest rate of worm burden reduction was achieved by CIDD -150303 (81.8%) against S. mansoni, CIDD-0149830 (80.2%) against S. haematobium and CIDD-066790 (86.7%) against S. japonicum. We have also evaluated the ability of the derivatives to kill immature stages since PZQ does not kill immature schistosomes. CIDD-0150303 demonstrated (100%) killing for all life stages at a final concentration of 143 μM in vitro and effective reduction in worm burden in vivo against S. mansoni. To understand how OXA derivatives fit in the SULT binding pocket, X-ray crystal structures of CIDD-0150303 and CIDD-0150610 demonstrate that the SULT active site will accommodate further modifications to our most active compounds as we fine tune them to increase favorable pharmacokinetic properties. Treatment with a single dose of 100 mg/kg by oral gavage with co-dose of PZQ + CIDD-0150303 reduced the worm burden of PZQ resistant parasites in an animal model by 90.8%. Therefore, we conclude that CIDD-0150303, CIDD-0149830 and CIDD-066790 are novel drugs that overcome some of PZQ limitations, and CIDD-0150303 can be used with PZQ in combination therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis. We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-β) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation. We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-β1 induced collagen expression/deposition. OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evidence for Lignocellulose-Decomposing Enzymes in the Genome and Transcriptome of the Aquatic Hyphomycete Clavariopsis aquatica

Fungi are ecologically outstanding decomposers of lignocellulose. Fungal lignocellulose degradation is prominent in saprotrophic Ascomycota and Basidiomycota of the subkingdom Dikarya. Despite ascomycetes dominating the Dikarya inventory of aquatic environments, genome and transcriptome data relating to enzymes involved in lignocellulose decay remain limited to terrestrial representatives of these phyla. We sequenced the genome of an exclusively aquatic ascomycete (the aquatic hyphomycete Clavariopsis aquatica), documented the presence of genes for the modification of lignocellulose and its constituents, and compared differential gene expression between C. aquatica cultivated on lignocellulosic and sugar-rich substrates. We identified potential peroxidases, laccases, and cytochrome P450 monooxygenases, several of which were differentially expressed when experimentally grown on different substrates. Additionally, we found indications for the regulation of pathways for cellulose and hemicellulose degradation. Our results suggest that C. aquatica is able to modify lignin to some extent, detoxify aromatic lignin constituents, or both. Such characteristics would be expected to facilitate the use of carbohydrate components of lignocellulose as carbon and energy sources.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii , an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3′−5′ exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5′−3′ exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identifiedidentifiedidentifiedin the archaeon Haloferax volcanii , which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3′−5′ exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5′−3′ exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Spectroscopic characterization of Mn 2+ and Cd 2+ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme

Phosphorothioate modifications have widespread use in the field of nucleic acids. As substitution of sulfur for oxygen can alter metal coordination preferences, the phosphorothioate metal-rescue experiment is a powerful method for identifying metal coordination sites that influence specific properties in a large RNAs. The A9/G10.1 metal binding site of the hammerhead ribozyme (HHRz) has previously been shown to be functionally important through phosphorothioate rescue experiments. While an A9-S Rp substitution is inhibitory in Mg 2+ , thiophilic Cd 2+ rescues HHRz activity. Mn 2+ is also often used in phosphorothioate metal-rescue studies but does not support activity for the A9-S Rp HHRz. Here, we use EPR, electron spin-echo envelope modulation (ESEEM), and X-ray absorption spectroscopic methods to directly probe the structural consequences of Mn 2+ and Cd 2+ coordination to R p and S p phosphorothioate modifications at the A9/G10.1 site in the truncated hammerhead ribozyme (tHHRz). The results demonstrate that while Cd 2+ does indeed bind to S in the thio-substituted ligand, Mn 2+ coordinates to the non–sulfur oxo group of this phosphorothioate, regardless of isomer. Computational models demonstrate the energetic preference of Mn—O over Mn—S coordination in metal-dimethylthiophosphate models. In the case of the tHHRz, the resulting Mn 2+ coordination preference of oxygen in either R p or S p A9 phosphorothioates differentially tunes catalytic activity, with Mn—O coordination in the A9-S Rp phosphorothioate enzyme being inhibitory.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

A crucial role for dynamic expression of components encoding the negative arm of the circadian clock

In the Neurospora circadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm component frequency ( frq ). FRQ interacts with FRH (FRQ-interacting RNA helicase) and CKI, forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated as brd-8 , that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex. Loss of brd-8 reduces H4 acetylation and RNA polymerase (Pol) II occupancy at frq and other known circadian genes, and leads to a long circadian period, delayed phase, and defective overt circadian output at some temperatures. In addition to strongly associating with the NuA4 histone acetyltransferase complex, BRD-8 is also found complexed with the transcription elongation regulator BYE-1. Expression of brd-8, bye-1, histone h2a.z , and several NuA4 subunits is controlled by the circadian clock, indicating that the molecular clock both regulates the basic chromatin status and is regulated by changes in chromatin. Taken together, our data identify auxiliary elements of the fungal NuA4 complex having homology to mammalian components, which along with conventional NuA4 subunits, are required for timely and dynamic frq expression and thereby a normal and persistent circadian rhythm.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The cholesterol transporter NPC1 is essential for epigenetic regulation and maturation of oligodendrocyte lineage cells

The intracellular cholesterol transporter NPC1 functions in late endosomes and lysosomes to efflux unesterified cholesterol, and its deficiency causes Niemann–Pick disease Type C, an autosomal recessive lysosomal disorder characterized by progressive neurodegeneration and early death. Here, we use single-nucleus RNA-seq on the forebrain of Npc1 –/– mice at P16 to identify cell types and pathways affected early in pathogenesis. Our analysis uncovers significant transcriptional changes in the oligodendrocyte lineage during developmental myelination, accompanied by diminished maturation of myelinating oligodendrocytes. We identify upregulation of genes associated with neurogenesis and synapse formation in Npc1 –/– oligodendrocyte lineage cells, reflecting diminished gene silencing by H3K27me3. Npc1 –/ – oligodendrocyte progenitor cells reproduce impaired maturation in vitro, and this phenotype is rescued by treatment with GSK-J4, a small molecule inhibitor of H3K27 demethylases. Moreover, mobilizing stored cholesterol in Npc1 –/– mice by a single administration of 2-hydroxypropyl-β-cyclodextrin at P7 rescues myelination, epigenetic marks, and oligodendrocyte gene expression. Our findings highlight an important role for NPC1 in oligodendrocyte lineage maturation and epigenetic regulation, and identify potential targets for therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The landscape of Chlamydomonas histone H3 lysine 4 methylation reveals both constant features and dynamic changes during the diurnal cycle

Chromatin modifications are epigenetic regulatory features with major roles in various cellular events, yet they remain understudied in algae. Here, we interrogated the genome-wide distribution pattern of mono- and trimethylated histone H3 lysine 4 (H3K4) using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) during key phases of the Chlamydomonas cell cycle: early G 1 phase, Zeitgeber Time 1 (ZT1), when cells initiate biomass accumulation, S/M phase (ZT13) when cells are replicating DNA and undergoing mitosis, and late G 0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at transcription start sites of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at some point during the life cycle but not necessarily by continuous active transcription, as exemplified by early zygotic genes, which may remain transcriptionally dormant for thousands of generations between sexual cycles. The exceptions to this rule were around 120 loci, some of which encode non-poly-adenylated transcripts, such as small nuclear RNAs and replication-dependent histones that had H3K4me3 peaks only when they were being transcribed. Mono-methylated H3K4 was the default state for the vast majority of histones that were bound outside of transcription start sites and terminator regions of genes. A small fraction of the genome that was depleted of any H3 lysine 4 methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Besides marking protein coding genes, H3K4me3 ChIP-seq data served also as a annotation tool for validation of hundreds of long non-coding RNA genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Multi-scale signaling and tumor evolution in high-grade gliomas

Although genomic anomalies in glioblastoma (GBM) have been well studied for over a decade, its 5-year survival rate remains lower than 5%. We seek to expand the molecular landscape of high-grade glioma, composed of IDH-wildtype GBM and IDH-mutant grade 4 astrocytoma, by integrating proteomic, metabolomic, lipidomic, and post-translational modifications (PTMs) with genomic and transcriptomic measurements to uncover multi-scale regulatory interactions governing tumor development and evolution. Applying 14 proteogenomic and metabolomic platforms to 228 tumors (212 GBM and 16 grade 4 IDH-mutant astrocytoma), including 28 at recurrence, plus 18 normal brain samples and 14 brain metastases as comparators, reveals heterogeneous upstream alterations converging on common downstream events at the proteomic and metabolomic levels and changes in protein-protein interactions and glycosylation site occupancy at recurrence. Recurrent genetic alterations and phosphorylation events on PTPN11 map to important regulatory domains in three dimensions, suggesting a central role for PTPN11 signaling across high-grade gliomas.

60 APPLIED LIFE SCIENCES↗