Biology subjects

Hill, C. A.

Publications and source records attributed to Hill, C. A..

10 recordsLinked to original sources

bioRxiv2026

Differential control of both cell cycle-regulated and quantitative histone mRNA expression by Drosophila Mute

Coupling histone gene expression to S phase of the cell cycle is essential for genome duplication and stability. Activation of Cyclin E/Cdk2 at the G1-S transition stimulates high-level expression of histone genes during S phase, but how histone genes are turned off at the end of S phase is not understood. Here we demonstrate that the essential Drosophila gene mute functions to repress inappropriate histone mRNA accumulation outside of S phase by counteracting Cyclin E/Cdk2-dependent phosphorylation of Mxc, which activates histone gene expression. Additionally, Mute plays contrasting roles in histone gene expression during S phase by promoting high levels of H1, H2a and H2b expression but not H3 and H4. Although Mute is present only at replication-dependent histone genes, its loss leads to 801 differentially regulated genes, primarily those involved in muscle related processes in late-stage embryos. Thus, disruptions of histone gene expression control alters the transcriptome resulting in developmental defects.

cell biology↗

bioRxiv2024

Nitrogen enrichment alters selection on rhizobial genes

1Mutualisms evolve over time when individuals belonging to different species derive fitness benefits through the exchange of resources and services. Although prevalent in natural and managed ecosystems, mutualisms can be destabilized by environmental fluctuations that alter the costs and benefits of maintaining the symbiosis. In the rhizobia-legume mutualism, bacteria provide reduced nitrogen to the host plant in exchange for photosynthates that support bacterial metabolism. However, this relationship can be disrupted by the addition of external nitrogen sources to the soil, such as fertilizers. While the molecular mechanisms underpinning the rhizobia-legume symbiosis are well-characterized, the genome-wide fitness effects of nitrogen enrichment on symbiotic rhizobia are less clear. Here, we inoculated a randomly barcoded transposon-site sequencing (RB-TnSeq) library of the bacterium Ensifer (Sinorhizobium) meliloti into soils containing a host plant, alfalfa (Medicago sativa), under conditions of low and high nitrogen availability. Although plant performance remained robust to fertilization, nitrogen enrichment altered gene fitness for specific traits and functions in the rhizobial partner. Genes involved in carbohydrate metabolism showed increased fitness irrespective of soil nutrient content, whereas fitness gains in quorum-sensing genes were only observed in high-nitrogen environments. We also documented reductions in the fitness of nucleotide metabolism and cell-growth genes, while genes from oxidative phosphorylation and various amino-acid biosynthesis pathways were detrimental to fitness under elevated soil nitrogen, underscoring the complex trade-offs in rhizobial responses to nutrient enrichment. Our experimental functional genomics approach identified gene functions and pathways across all E. meliloti replicons that may be associated with the disruption of an agronomically important mutualism. 2 ImportanceUnderstanding the evolutionary dynamics of the rhizobia-legume mutualism is important for elucidating how plant-soil-microbe interactions operate in natural and managed ecosystems. Legumes constitute a significant portion of global food production and generate 25% of all terrestrially fixed nitrogen. The application of chemical fertilizers can disrupt the mutualism by altering the selective pressures experienced by symbiotic rhizobia, potentially affecting gene fitness throughout the microbial genome and leading to the evolution of less productive or cooperative mutualists. To investigate how exogenous nitrogen inputs influence gene fitness during the complex rhizobial lifecycle, we used a barcoded genome-wide mutagenesis screen to quantify gene-level fitness across the rhizobial genome during symbiosis and identify metabolic functions affected by nitrogen enrichment. Our findings provide genomic insight into potential eco-evolutionary mechanisms by which symbioses are maintained or degraded over time in response to changing environmental conditions.

evolutionary biology↗

bioRxiv2024

Drosophila melanogaster Set8 and L(3)mbt function in gene expression independently of histone H4 lysine 20 methylation

Mono-methylation of Lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me-binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of Set8null, H4K20R/A, and l(3)mbt mutant Drosophila melanogaster. We found that the gene expression profiles of H4K20A and H4K20R larvae are markedly different than Set8null larvae despite similar reductions in H4K20me1. Set8null mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo, but these phenotypes are not recapitulated by mutation of H4K20 indicating that the developmental defects of Set8null animals are largely due to H4K20me1-independent effects on gene expression. Further, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in Set8null and H4K20 mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin, and therefore that H4K20me1 does not play a large role in gene expression.

genomics↗

bioRxiv2023

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits

Phenotypic heterogeneity is a common feature of monogenic neurodevelopmental disorders that can arise from differential severity of missense variants underlying disease, but how distinct alleles impact molecular mechanisms to drive variable disease presentation is not well understood. Here, we investigate missense mutations in the DNA methyltransferase DNMT3A associated with variable overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity in neurodevelopmental disease. We generate a DNMT3A P900L/+ mouse model mimicking a disease mutation with mild-to-moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. We show that the P900L mutation leads to disease-relevant overgrowth, obesity, and social deficits shared across DNMT3A disorder models, while the R878H mutation causes more extensive epigenomic disruption leading to differential dysregulation of enhancers elements. We identify distinct gene sets disrupted in each mutant which may contribute to mild or severe disease, and detect shared transcriptomic disruption that likely drives common phenotypes across affected individuals. Finally, we demonstrate that core gene dysregulation detected in DNMT3A mutant mice overlaps effects in other developmental disorder models, highlighting the importance of DNMT3A-deposited methylation in neurodevelopment. Together, these findings define central drivers of DNMT3A disorders and illustrate how variable disruption of transcriptional mechanisms can drive the spectrum of phenotypes in neurodevelopmental disease.

neuroscience↗

bioRxiv2022

Expression of fatty acid synthase genes and their role in development and arboviral infection of Aedes aegypti

BackgroundFatty acids are the building blocks of complex lipids essential for living organisms. In mosquitoes, fatty acids are involved in cell membrane production, energy conservation and expenditure, innate immunity, development, and reproduction. Fatty acids are synthesized by a multifunctional enzyme complex called fatty acid synthase (FAS). Several paralogues of FAS were found in the Aedes aegypti (Ae. aegypti) mosquito. However, the molecular characteristics and the expression of some of these paralogues have not been investigated. MethodsGenome assemblies of Ae. aegypti were analyzed and orthologues of human FAS were identified. Phylogenetic analysis and in silico molecular characterization were performed to identify the functional domains of the Ae. aegypti FAS (AaFAS). Quantitative analysis and loss-of-function experiments were performed to determine the significance of different AaFAS transcripts in various stages of development, expression following different diets and the impact of AaFAS on dengue virus, serotype 2 (DENV2) infection and transmission. ResultsWe identified seven putative FAS genes in the Ae. aegypti genome assembly, based on nucleotide similarity to the FAS proteins (tBLASTn) of humans, other mosquitoes and invertebrates. Bioinformatics and molecular analyses suggested that only five of the FAS genes produce mRNA and therefore represent complete gene models. Expression levels of AaFAS varied among developmental stages and between male and female Ae. aegypti. Quantitative analyses revealed that expression of AaFAS1, the putative orthologue of the human FAS, was highest in adult females. Transient knockdown (KD) of AaFAS1 did not induce a complete compensation by other AaFAS genes but limited DENV2 infection of Aag2 cells in culture and the midgut of the mosquito. ConclusionAaFAS1 is the predominant AaFAS in the adult mosquitoes. It has the highest amino acid similarity to human FAS and contains all enzymatic domains typical of human FAS. AaFAS1 also facilitated DENV2 replication in both cell culture and in mosquito midguts. Our data suggest that AaFAS1 may play a role in transmission of dengue viruses, and could represent a target for intervention strategies.

microbiology↗

bioRxiv2021

SUMM4 complex couples insulator function and DNA replication timingcontrol

AbstractAsynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under- replication of late-replicating intercalary heterochromatin in polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse under-replication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings reveal that DNA replication can be delayed by a chromatin barrier and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for replication timing that does not depend on an asynchronous firing of replication origins.

biochemistry↗

bioRxiv2021

Methanosarcina acetivorans simultaneously produces molybdenum, vanadium, and iron-only nitrogenases in response to fixed nitrogen and molybdenum depletion

All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N2) to ammonia. Some diazotrophs also contain alternative nitrogenases that lack Mo: vanadium (V) and iron-only (Fe) nitrogenases. Among diazotrophs, the regulation and usage of the alternative nitrogenases in methanogens is largely unknown. Methanosarcina acetivorans contains nif, vnf, and anf gene clusters encoding putative Mo-, V-, and Fe-nitrogenases, respectively. This study investigated the effect of fixed nitrogen and Mo/V availability on nitrogenase expression and growth by M. acetivorans. The availability of Mo and V did not affect growth of M. acetivorans with fixed nitrogen but significantly affected growth with N2. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N2 in medium containing Mo. Depletion of Mo (Fe-only condition) resulted in a significant decrease in growth rate and cell yield. The addition of V to Mo-depleted medium stimulated diazotrophic growth but was still less than growth in Mo-replete medium. qPCR analysis revealed transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo. However, vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being the key factor. Immunoblot analysis revealed Mo-nitrogenase is produced when fixed nitrogen is depleted regardless of Mo availability, while V- and Fe-nitrogenases are produced only in the absence of fixed nitrogen and Mo. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and that all three nitrogenases can be simultaneously produced. IMPORTANCEMethanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. As such, methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, even simultaneously. Surprisingly, Mo-nitrogenase was produced in cells grown in the absence of Mo, indicating components of Mo-nitrogenase regulate or are needed to produce V- and Fe-nitrogenases in methanogens. The results provide a foundation to understanding the assembly, regulation, and activity of the alternative nitrogenases in methanogens.

microbiology↗

bioRxiv2020

DNMT3A haploinsufficiency results in behavioral deficits and global epigenomic dysregulation shared across neurodevelopment disorders

Mutations in DNA methyltransferase 3A (DNMT3A) have been detected in autism and related disorders, but how these mutations disrupt nervous system function is unknown. Here we define the effects of neurodevelopmental disease-associated DNMT3A mutations. We show that diverse mutations affect different aspects of protein activity yet lead to shared deficiencies in neuronal DNA methylation. Heterozygous DNMT3A knockout mice mimicking DNMT3A disruption in disease display growth and behavioral alterations consistent with human phenotypes. Strikingly, in these mice we detect global disruption of neuron-enriched non-CG DNA methylation, a binding site for the Rett syndrome protein MeCP2. Loss of this methylation leads to enhancer and gene dysregulation that overlaps with models of Rett syndrome and autism. These findings define effects of DNMT3A haploinsufficiency in the brain and uncover disruption of the non-CG methylation pathway as a convergence point across neurodevelopmental disorders.

neuroscience↗

bioRxiv2020

Tumor-specific changes in Kaposi sarcoma-associated herpesvirus genomes in Ugandan adults with Kaposi sarcoma

Intra-host evolved tumor virus variants have provided insights into the risk, pathogenesis and treatment responses of associated cancers. However, the intra-host variability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma (KS), has not been explored at the whole viral genome level. An accurate and detailed description of KSHV intra-host diversity in whole KSHV genomes from matching tumors and oral swabs from Ugandan adults with HIV-associated KS was obtained by deep, short read sequencing, using duplex unique molecular identifiers (dUMI) - random double-stranded oligonucleotides that barcode individual DNA molecules before library amplification. This allowed suppression of PCR and sequencing errors down to [~]10-9/base. KSHV genomes were assembled de novo, and identified rearrangements were confirmed by PCR. 131-kb KSHV genome sequences, excluding major repeat regions and averaging 2.3 x 104 reads/base, were successfully obtained from 23 specimens from 9 individuals, including 7 tumor-oral pairs. Sampling more than 100 viral genomes in at least one specimen per individual showed that KSHV genomes were virtually homogeneous within samples and within individuals at the point mutational level. Heterogeneity, if present, was due to point mutations and genomic rearrangements in tumors. In 2 individuals, the same mutations were found in distinct KS tumors. The K8.1 gene was inactivated in tumors from 3 individuals, and all KSHV genomic aberrations retained the region surrounding the first major internal repeat (IR1). These findings suggest that lytic gene alterations may contribute to KS tumorigenesis or persistence. Author summaryKaposi sarcoma (KS) is a leading cancer in sub-Saharan Africa and in those with HIV co-infection. Infection by Kaposi sarcoma-associated herpesvirus (KSHV) is necessary for KS, yet why only few KSHV infections develop into KS is largely unknown. While strain differences or mutations in other tumor viruses are known to affect the risk and progression of their associated cancers, whether KSHV genetic variation is important to the natural history of KS is unclear. Most studies of KSHV diversity have characterized only [~]4% of its 165-kb genome and may have been impacted by PCR or cloning artifacts. Here, we performed highly sensitive, single-molecule sequencing of whole KSHV genomes in paired KS tumors and oral swabs from 9 individuals with KS. We found that KSHV genomes were virtually identical within individuals, with no evidence of quasispecies formation nor multistrain infection. However, KSHV genome aberrations and inactivating mutations appeared to be a common, tumor-associated phenomenon, with some mutations shared by distinct tumors within an individual. Certain regions of the KSHV genome featured prominently among tumor-associated mutations, suggesting that they are important contributors to the pathogenesis or persistence of KS.

microbiology↗

bioRxiv2019

Rif1 functions in a tissue-specific manner to control replication timing through its PP1-binding motif

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early and late replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

developmental biology↗