Biology subjects

Bui, M.

Publications and source records attributed to Bui, M..

5 recordsLinked to original sources

bioRxiv2018

Elastic and Rigidified CENP-A Nucleosomes govern Centromeric Chromatin Plasticity

Histone variants fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether and how nucleosome variants encode unique mechanical properties to their cognate chromatin structures remains elusive. Here, using novel in silico and in vitro nanoindentation methods, extending to in vivo dissections, we report that histone variant nucleosomes are intrinsically more elastic than their canonical counterparts. Furthermore, binding proteins which discriminate between histone variant nucleosomes suppress this innate elasticity and also compact chromatin. Interestingly, when we overexpress the binding proteins in vivo, we also observe increased compaction of chromatin enriched for histone variant nucleosomes, correlating with diminished access. Together, these data suggest a plausible link between innate mechanical properties possessed by histone variant nucleosomes, the adaptability of chromatin states in vivo, and the epigenetic plasticity of the underlying locus.\n\nSignificanceNucleosomes are the base unit which organize eukaryotic genomes. Besides the canonical histone, histone variants create unique local chromatin domains that fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. We developed computational and single-molecule nanoindentation tools to determine mechanical properties of histone variant nucleosomes. We found that the CENP-A nucleosome variant is more elastic than the canonical H3 nucleosome but becomes stiffer when bound to its partner CENP-C. In addition, CENP-C induces cross-array clustering, creating a chromatin state that less accessible. These data suggest that innate material properties of nucleosomes can influence the ultimate chromatin state, thereby influence biological outcomes.

biochemistry↗

bioRxiv2018

Live calcium imaging of Aedes aegypti neuronal tissues reveals differential importance of chemosensory systems for life-history-specific foraging strategies

Aedes aegypti have a wide variety of sensory pathways that have supported success as a species as well as a highly competent vector of numerous debilitating infectious pathogens. Investigations into mosquito sensory systems and their effects on behavior are valuable resources for the advancement of mosquito control strategies. Numerous studies have elucidated key aspects of mosquito sensory systems, however there remains critical gaps within the field. In particular, compared to that of the adult form, there has been a lack of studies directed towards the immature life stages. Additionally, although numerous studies have pinpointed specific sensory receptors as well as relevant response behaviors, there has been a lack of studies able to monitor both concurrently. To begin filling aforementioned gaps, here we engineered Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s. Using this strain, combined with advanced confocal microscopy, we were able to simultaneously measure live stimulus-evoked calcium responses in both neuronal and muscle cells with a wide spatial range and resolution. Moreover, by coupling in vivo calcium imaging with behavioral assays we were able to gain functional insights into how stimulus-evoked neural and muscle activities are represented, modulated, and transformed in mosquito larvae enabling us to elucidate mosquito sensorimotor properties important for life-history-specific foraging strategies.\n\nSignificance StatementUnderstanding mosquito sensory systems and resulting behavior has been a major factor in the advancement of mosquito control innovations. Aedes aegypti larvae offer an effective life stage for further elucidating information on mosquito sensory systems. Due to their relatively simplified nervous system, mosquito larvae are ideal for studying neural signal transduction, coding, and behavior. Moreover, a better understanding of the larval sensory system may enable the development of novel control methodologies able to target mosquitoes before they reach a vector-competent stage. Here we engineer Ae. aegypti to ubiquitously express a genetically encoded calcium indicator, GCaMP6s and use this tool to observe links between sensorimotor responses and behavior by exploiting live calcium imaging as well as live tracking based behavioral assays.

neuroscience↗

bioRxiv2017

Inference about causation between body mass index and DNA methylation in blood from a twin family study

BackgroundSeveral studies have reported DNA methylation in blood to be associated with body mass index (BMI), but only a few have investigated causal aspects of the association. We used a twin family design to assess this association at two life points and applied a novel analytical approach to investigate the evidence for causality.\n\nMethodsThe methylation profile of DNA from peripheral blood was measured for 479 Australian women (mean age 56 years) from 130 twin families. Linear regression was used to estimate the associations of methylation at ~410 000 cytosine-guanine dinucleotides (CpG), and of the average methylation at ~20 000 genes, with current BMI, BMI at age 18-21 years, and the change between the two (BMI change). A novel regression-based methodology for twins, Inference about Causation through Examination of Familial Confounding (ICE FALCON), was used to assess causation.\n\nResultsAt 5% false discovery rate, nine, six and 12 CpGs at 24 loci were associated with current BMI, BMI at age 18-21 years and BMI change, respectively. The average methylation of BHLHE40 and SOCS3 loci was associated with current BMI, and of PHGDH locus was associated with BMI change. From the ICE FALCON analyses with BMI as the predictor and methylation as the outcome, a womans methylation level was associated with her co-twins BMI, and the association disappeared conditioning on her own BMI, consistent with BMI causing methylation. To the contrary, using methylation as the predictor and BMI as the outcome, a womans BMI was not associated with her co-twins methylation level, consistent with methylation not causing BMI.\n\nConclusionFor middle-aged women, peripheral blood DNA methylation at several genomic locations is associated with current BMI, BMI at age 18-21 years and BMI change. Our study suggests that BMI has a causal effect on peripheral blood DNA methylation.

epidemiology↗

bioRxiv2017

Germline Cas9 Expression Yields Highly Efficient Genome Engineering in a Major Worldwide Disease Vector, Aedes aegypti

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti. Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we built, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a first step towards the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.\n\nSignificance StatementAedes aegypti is the principal vector of multiple arboviruses that significantly affect human health including dengue, chikungunya, and zika. Development of tools for efficient genome engineering in this mosquito will not only lay the foundation for the application of novel genetic control strategies that do not rely on insecticides, but will also accelerate basic research on key biological processes involved in disease transmission. Here, we report the development of a transgenic CRISPR approach for rapid gene disruption in this organism. Given their high editing efficiencies, the Cas9 strains we developed can be used to quickly generate novel genome modifications allowing for high-throughput gene targeting, and can possibly facilitate the development of gene drives, thereby accelerating comprehensive functional annotation and development of innovative population control strategies for Ae. aegypti.

bioengineering↗

bioRxiv2017

Internal modifications in the CENP-A nucleosome modulate centromeric dynamics

Post-translational modifications (PTMs) of core histones have studied for over 2 decades, and are correlated with changes in transcriptional status, chromatin fiber folding, and nucleosome dynamics. However, within the centromere-specific histone H3 variant CENP-A, few modifications have been reported, and their functions remain largely unexplored. In this multidisciplinary report, we utilize in silico computational and in vivo approaches to dissect lysine 124 of human CENP-A, which was previously reported to be acetylated in advance of replication. Computational modeling demonstrates that acetylation of K124 causes tightening of the histone core, and hinders accessibility to its C-terminus, which in turn diminishes CENP-C binding. Additionally, CENP-A K124ac/H4 K79ac containing nucleosomes are prone to DNA sliding. In vivo experiments using an acetyl or unacetylatable mimic (CENP-A K124Q and K124A respectively) reveal alterations in CENP-C levels, and a modest increase in mitotic errors. Furthermore, mutation of K124 results in alterations in centromeric replication timing, with the permanently acetylated form replicating centromeres early, and the unacetylable form replicating centromeres late. Purification of native CENP-A proteins followed by mass spectrometry analysis reveal that while CENP-A K124 is acetylated at G1/S, it switches to monomethylation during early and mid-S phase. Finally, we provide evidence that the HAT p300 is involved in this cycle. Taken together, our data suggest that cyclical modifications within the CENP-A nucleosome can influence the binding of key kinetochore proteins, the integrity of mitosis and centromeric replication. These data support the emerging paradigm that core modifications in histone variant nucleosomes transduce defined changes to key biological processes.

biochemistry↗