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DOE OSTI2025

The Gene Ontology knowledgebase in 2026

Abstract The Gene Ontology (GO) knowledgebase (https://geneontology.org) is a comprehensive resource describing the functions of genes. The GO knowledgebase is regularly updated and improved. We describe here the major updates that have been made in the past 3 years. The ontology and annotations have been expanded and revised, particularly in several areas of biology: cellular metabolism, multi-organism interactions (e.g. host-pathogen), extracellular matrix proteins, chromatin remodeling (e.g. the “histone code”), and noncoding RNA functions. We have released version 2 of a comprehensive set of integrated, reviewed annotations for human genes, which we call the “functionome.” We have also dramatically increased the number of GO-CAM models, with over 1500 models of metabolic and signaling pathways, primarily in human, mouse, budding and fission yeast, and fruit fly. Finally, we discuss our current recommendations and future prospects of AI in the use and development of GO.

Aleksander, Suzi A (ORCID:0000000167872901)↗

DOE OSTI2020

The cis-regulatory codes of response to combined heat and drought stress in Arabidopsis thaliana

Abstract Plants respond to their environment by dynamically modulating gene expression. A powerful approach for understanding how these responses are regulated is to integrate information about cis-regulatory elements (CREs) into models called cis-regulatory codes. Transcriptional response to combined stress is typically not the sum of the responses to the individual stresses. However, cis-regulatory codes underlying combined stress response have not been established. Here we modeled transcriptional response to single and combined heat and drought stress in Arabidopsis thaliana. We grouped genes by their pattern of response (independent, antagonistic and synergistic) and trained machine learning models to predict their response using putative CREs (pCREs) as features (median F-measure = 0.64). We then developed a deep learning approach to integrate additional omics information (sequence conservation, chromatin accessibility and histone modification) into our models, improving performance by 6.2%. While pCREs important for predicting independent and antagonistic responses tended to resemble binding motifs of transcription factors associated with heat and/or drought stress, important synergistic pCREs resembled binding motifs of transcription factors not known to be associated with stress. These findings demonstrate how in silico approaches can improve our understanding of the complex codes regulating response to combined stress and help us identify prime targets for future characterization.

Azodi, Christina B.↗

DOE OSTI2021

Modeling temporal and hormonal regulation of plant transcriptional response to wounding

Plants respond to wounding stress by changing gene expression patterns and inducing the production of hormones including jasmonic acid. This wounding transcriptional response activates specialized metabolism pathways such as the glucosinolate pathways in Arabidopsis thaliana. While the regulatory factors and sequences controlling a subset of wound-response genes are known, it remains unclear how wound response is regulated globally. Here, we how these responses are regulated by incorporating putative cis-regulatory elements, known transcription factor binding sites, in vitro DNA affinity purification sequencing, and DNase I hypersensitive sites to predict genes with different wound-response patterns using machine learning. We observed that regulatory sites and regions of open chromatin differed between genes upregulated at early and late wounding time-points as well as between genes induced by jasmonic acid and those not induced. Expanding on what we currently know, we identified cis-elements that improved model predictions of expression clusters over known binding sites. Using a combination of genome editing, in vitro DNA-binding assays, and transient expression assays using native and mutated cis-regulatory elements, we experimentally validated four of the predicted elements, three of which were not previously known to function in wound-response regulation. Our study provides a global model predictive of wound response and identifies new regulatory sequences important for wounding without requiring prior knowledge of the transcriptional regulators.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

A DE1 BINDING FACTOR 1–GLABRA2 module regulates rhamnogalacturonan I biosynthesis in Arabidopsis seed coat mucilage

Abstract The mucilage surrounding hydrated Arabidopsis thaliana seeds is a specialized extracellular matrix composed mainly of the pectic polysaccharide rhamnogalacturonan I (RG-I). Although, several genes responsible for RG-I biosynthesis have been identified, the transcriptional regulatory mechanisms controlling RG-I production remain largely unknown. Here we report that the trihelix transcription factor DE1 BINDING FACTOR 1 (DF1) is a key regulator of mucilage RG-I biosynthesis. RG-I biosynthesis is significantly reduced in loss-of-function mutants of DF1. DF1 physically interacts with GLABRA2 (GL2) and both proteins transcriptionally regulate the expression of the RG-I biosynthesis genes MUCILAGE MODIFIED 4 (MUM4) and GALACTURONOSYLTRANSFERASE-LIKE5 (GATL5). Through chromatin immunoprecipitation-quantitative PCR and transcriptional activation assays, we uncover a cooperative mechanism of the DF1–GL2 module in activating MUM4 and GATL5 expression, in which DF1 binds to the promoters of MUM4 and GATL5 through interacting with GL2 and facilitates the transcriptional activity of GL2. The expression of DF1 and GL2 is directly regulated by TRANSPARENT TESTA GLABRA2 (TTG2) and, in turn, DF1 directly represses the expression of TTG2. Taken together, our data reveal that the transcriptional regulation of mucilage RG-I biosynthesis involves a regulatory module, comprising DF1, GL2, and TTG2.

Biochemistry & Molecular Biology↗

DOE OSTI2022

Shade triggers posttranscriptional PHYTOCHROME-INTERACTING FACTOR-dependent increases in H3K4 trimethylation

The phytochrome (phy)-PHYTOCHROME-INTERACTING FACTOR (PIF) sensory module perceives and transduces light signals to direct target genes (DTGs), which then drive the adaptational responses in plant growth and development appropriate to the prevailing environment. These signals include the first exposure of etiolated seedlings to sunlight upon emergence from subterranean darkness and the change in color of the light that is filtered through, or reflected from, neighboring vegetation (“shade”). Previously, we identified three broad categories of rapidly signal-responsive genes: those repressed by light and conversely induced by shade; those repressed by light, but subsequently unresponsive to shade; and those responsive to shade only. Here, we investigate the potential role of epigenetic chromatin modifications in regulating these contrasting patterns of phy-PIF module-induced expression of DTGs in Arabidopsis (Arabidopsis thaliana). Using RNA-seq and ChIP-seq to determine time-resolved profiling of transcript and histone 3 lysine 4 trimethylation (H3K4me3) levels, respectively, we show that, whereas the initial dark-to-light transition triggers a rapid, apparently temporally coincident decline of both parameters, the light-to-shade transition induces similarly rapid increases in transcript levels that precede increases in H3K4me3 levels. Together with other recent findings, these data raise the possibility that, rather than being causal in the shade-induced expression changes, H3K4me3 may function to buffer the rapidly fluctuating shade/light switching that is intrinsic to vegetational canopies under natural sunlight conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Distant-acting Enhancers in Development and Disease

Multiple converging lines of evidence from experimental and human genetic studies indicate that both common and rare sequence variants involving enhancers play major roles in Mendelian and complex human disease phenotypes. We use a combination of sequence-based molecular approaches including chromatin mapping and synthesis-enabled modification of enhancer sequences, large-scale transgenic mouse studies (http://enhancer.lbl.gov), and CRISPR genome editing in the mouse model to study the in vivofunction of enhancers in developmental, evolutionary, and disease-related processes. Using examples from our ongoing work, I will illustrate how these methods provide insight into the function and evolution of distant-acting regulatory sequences and offer a starting point for understanding their role in human disease. I will discuss how improved in vivo mouse assays can be used to study candidate pathogenic human mutations in enhancers at scale, revealing the extent to which human genetic studies alone can be insufficient to identify truly causal mutations in enhancers. I will also show how such assays can be used to explore the sensitivity of enhancers that are extremely conserved ("ultraconserved") in evolution to mutations, revealing a remarkable functional robustness to sequence changes that contrasts with apparent evolutionary constraints.

Visel, A↗

DOE OSTI2020

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

ABSTRACT The Shox2 homeodomain transcriptional regulator is known for its critical functions during mouse embryogenesis, enabling accurate development of limbs, craniofacial structures, neural populations and the cardiac conduction system. At the genomic level, the Shox2 gene is flanked by an extensive gene desert, a continuous non-coding genomic region spanning over 500 kilobases that contains a multitude of evolutionarily conserved elements with predicted cis -regulatory activities. However, the transcriptional enhancer potential of the vast majority of these elements in combination with the biological necessity of the gene desert have not yet been explored. Using transgenic reporter assays in mouse embryos to validate an extensive set of stringent epigenomic enhancer predictions, we identify several novel gene desert enhancers with distinct tissue-specific activities in Shox2 expressing tissues. 4C-seq chromatin conformation capture further uncovers a repertoire of gene desert enhancers with overlapping activities in the proximal limb, in a compartment essential for Shox2 -mediated stylopod formation. Leveraging CRISPR/Cas9 to delete the gene desert region contained in the Shox2 topologically associated domain (TAD), we demonstrate that this complex cis -regulatory platform is essential for embryonic survival and required for control of region-specific Shox2 expression in multiple developing tissues. While transcription of Shox2 in the embryonic limb is only moderately affected by gene desert loss, Shox2 expression in craniofacial and cardiac domains is nearly abolished. In particular, Shox2 transcripts in the sinus venosus (SV) encompassing the sinoatrial node (SAN) were depleted in embryos lacking the gene desert, likely accounting for the embryonic lethality due to Shox2 -dependency of the SAN pacemaker. Finally, we discover a 1.5kb SV enhancer within the deleted gene desert region, which may act as a genomic module controlling the development of the cardiac conduction system. In summary, our results identify a gene desert indispensable for pleiotropic patterning and highlight the importance of these extensive regulatory landscapes for embryonic development and viability.

Abassah-Oppong, Samuel↗

DOE OSTI2021

Topologically Associating Domain Boundaries are Commonly Required for Normal Genome Function

Topologically associating domain (TAD) boundaries are thought to partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotype, but the overall extent to which this occurs remains unknown. Here we show that TAD boundary deletions commonly disrupt normal genome function in vivo . We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80kb in size) from the genome in mice. All deletions examined resulted in at least one detectable molecular or organismal phenotype, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. For 5 of 8 (62%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and suggest that noncoding deletions affecting TAD boundaries should be carefully considered for potential pathogenicity in clinical genetics screening.

Rajderkar, Sudha↗

DOE OSTI2025

Ultrastructure of the Endoplasmic Reticulum in Eukaryotic Microalgae

ABSTRACT The endoplasmic reticulum (ER) is a large and highly dynamic component of the eukaryotic endomembrane system. In eukaryotic microalgae, it plays six distinct roles: (1) It envelopes the chromatin to form thenucleus. (2) It forms cisternae in the cytoplasm, some of which scaffold the synthesis of proteins destined for incorporation into membranes or for secretion. (3) It associates withGolgicisternae to scaffold the synthesis of glycosylated proteins. (4) It associates with theplasma membraneto mediate the synthesis and secretion of hydrophobic molecules. (5) It mediates the synthesis of cytoplasmiclipid bodies. (6) In lineages harboring complex plastids of red algal ancestry, it forms thechloroplast ER, which envelops the primary chloroplast envelope. In this review, these systems are illustrated using the quick‐freeze deep‐etch electron microscopy (QFDEEM) technique, which lifts up the topological configurations adopted by this gossamer system. A key finding is that in all the complex microalgae examined except dinoflagellates, the inner nuclear envelope membrane associates directly with the plastid‐contiguous membrane of the chloroplast ER at foci designated as chloroplast‐nuclear junctions. These junctions may play a role in regulating the maintenance and physiology of the complex organelles.

Microbiology↗

DOE OSTI2024

PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar

The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. Here, this network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription – quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector–reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen–fixing clade

Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root–nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

DNA hypomethylation of the host tree impairs interaction with mutualistic ectomycorrhizal fungus

Summary Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown. By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 ( DDM1 ) and two demethylases DEMETER‐LIKE ( DML ) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor . We compared the ectomycorrhizas formed between transgenic and wild‐type (WT) trees and analyzed their methylomes and transcriptomes. The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi‐ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX‐ dml and RNAi‐ ddm1 ) and 120 genes/1441 TEs in the fungal genome suggesting a host‐induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes ( cis and trans effects) in common with 17 being differentially methylated ( cis ). Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate‐mediated pathways, and immune response but also terpenoid metabolism.

Vigneaud, Julien↗

DOE OSTI2025

The architecture of resilience: a genome assembly of Myrothamnus flabellifolia sheds light on desiccation tolerance and sex determination

Myrothamnus flabellifolia is a dioecious resurrection plant endemic to southern Africa that has become an important model for understanding desiccation tolerance. Despite its ecological and medicinal significance, genomic and transcriptomic resources for the species are limited. We generated a chromosome-level, haplotype-resolved reference genome assembly and annotation for M. flabellifolia and conducted transcriptomic profiling across a natural dehydration–rehydration time course in the field. Genome architecture and sex determination were characterized, and co-expression network and cis-regulatory element (CRE) enrichment analyses were used to investigate dynamic responses to desiccation. The 1.28-Gb genome exhibits unusually consistent chromatin architecture with unique chromosome organization across highly divergent haplotypes. We identified an XY sexual system with a small sex-determining region on Chromosome 8. Transcriptomic responses varied with dehydration severity, pointing to early suppression of growth, progressive activation of protective mechanisms, and subsequent return to homeostasis upon rehydration. Late embryogenesis abundant and early light-induced protein transcripts were dynamically regulated and showed enrichment of abscisic acid and stress-responsive CREs pointing toward conserved responses. Together, this study provides foundational resources for understanding the genomic architecture and reproductive biology of M. flabellifolia and offers new insights into the mechanisms of desiccation tolerance.

chromosome structure↗

DOE OSTI2020

The CCR4‐NOT complex component NOT1 regulates RNA‐directed DNA methylation and transcriptional silencing by facilitating Pol IV‐dependent siRNA production

Summary Small interfering RNAs (siRNAs) are responsible for establishing and maintaining DNA methylation through the RNA‐directed DNA methylation (RdDM) pathway in plants. Although siRNA biogenesis is well known, it is relatively unclear about how the process is regulated. By a forward genetic screen in Arabidopsis thaliana , we identified a mutant defective in NOT1 and demonstrated that NOT1 is required for transcriptional silencing at RdDM target genomic loci. We demonstrated that NOT1 is required for Pol IV‐dependent siRNA accumulation and DNA methylation at a subset of RdDM target genomic loci. Furthermore, we revealed that NOT1 is a constituent of a multi‐subunit CCR4‐NOT deadenylase complex by immunoprecipitation combined with mass spectrometry and demonstrated that the CCR4‐NOT components can function as a whole to mediate chromatin silencing. Therefore, our work establishes that the CCR4‐NOT complex regulates the biogenesis of Pol IV‐dependent siRNAs, and hence facilitates DNA methylation and transcriptional silencing in Arabidopsis.

Zhou, Hao‐Ran↗

DOE OSTI2020

Programming PAM antennae for efficient CRISPR-Cas9 DNA editing

Bacterial CRISPR-Cas9 nucleases have been repurposed as powerful genome editing tools. Whereas engineering guide RNAs or Cas nucleases have proven to improve the efficiency of CRISPR editing, modulation of protospacer-adjacent motif (PAM), indispensable for CRISPR, has been less explored. Here, we develop a DNA origami–based platform to program a PAM antenna microenvironment and address its performance at the single-molecule level with submolecular resolution. To mimic spatially controlled in vivo PAM distribution as may occur in chromatin, we investigate the effect of PAM antennae surrounding target DNA. We find that PAM antennae effectively sensitize the DNA cleavage by recruiting Cas9 molecules. Super-resolution tracking of single single-guide RNA/Cas9s reveals localized translocation of Cas9 among spatially proximal PAMs. We find that the introduction of the PAM antennae effectively modulates the microenvironment for enhanced target cleavage (up to ~50%). These results provide insight into factors that promote more efficient genome editing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich’s ataxia

Disruptions to iron-sulfur (Fe-S) clusters, essential cofactors for a broad range of proteins, cause widespread cellular defects resulting in human disease. A source of damage to Fe-S clusters is cuprous (Cu 1+ ) ions. Since histone H3 enzymatically produces Cu 1+ for copper-dependent functions, we asked whether this activity could become detrimental to Fe-S clusters. Here, we report that histone H3–mediated Cu 1+ toxicity is a major determinant of cellular functional pool of Fe-S clusters. Inadequate Fe-S cluster supply, due to diminished assembly as occurs in Friedreich’s ataxia or defective distribution, causes severe metabolic and growth defects in Saccharomyces cerevisiae. Decreasing Cu 1+ abundance, through attenuation of histone cupric reductase activity or depletion of total cellular copper, restored Fe-S cluster–dependent metabolism and growth. Our findings reveal an interplay between chromatin and mitochondria in Fe-S cluster homeostasis and a potential pathogenic role for histone enzyme activity and Cu 1+ in diseases with Fe-S cluster dysfunction.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Cryo-EM structure of the human Sirtuin 6–nucleosome complex

Sirtuin 6 (SIRT6) is a multifaceted protein deacetylase/deacylase and a major target for small-molecule modulators of longevity and cancer. In the context of chromatin, SIRT6 removes acetyl groups from histone H3 in nucleosomes, but the molecular basis for its nucleosomal substrate preference is unknown. Our cryo–electron microscopy structure of human SIRT6 in complex with the nucleosome shows that the catalytic domain of SIRT6 pries DNA from the nucleosomal entry-exit site and exposes the histone H3 N-terminal helix, while the SIRT6 zinc-binding domain binds to the histone acidic patch using an arginine anchor. In addition, SIRT6 forms an inhibitory interaction with the C-terminal tail of histone H2A. The structure provides insights into how SIRT6 can deacetylate both H3 K9 and H3 K56.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Mechanisms of HIV-1 integrase resistance to dolutegravir and potent inhibition of drug-resistant variants

HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

59 BASIC BIOLOGICAL SCIENCES↗