Biology subjects

Whitehead, J.

Publications and source records attributed to Whitehead, J..

8 recordsLinked to original sources

bioRxiv2026

Biosensor-guided evolution of chalcone synthase enhances biosynthesis of natural and non-natural flavanones

Flavonoids constitute a large class of natural products widely investigated for their bioactive properties, with microbial production offering a potentially scalable alternative to plant extraction. However, achieving structural diversification of these compounds in microbial systems remains challenging, as modification of the flavonoid B-ring typically relies on downstream tailoring enzymes. An alternative strategy is to exploit the intrinsic promiscuity of the canonical flavanone biosynthesis pathway to introduce structural variation at an early stage. Here, we sought to improve microbial production of diverse flavanones by systematically leveraging pathway promiscuity. By constructing a combinatorial library of pathways comprising 4-coumarate-CoA ligase, chalcone synthase, and chalcone isomerase, we enabled the conversion of a panel of ring-substituted cinnamic acid precursors into ten natural and non-natural flavanones. In parallel, we established a genetically encoded biosensor based on the transcriptional regulator FdeR and demonstrated its responsiveness across all ten flavanones. Leveraging this biosensor for high-throughput screening, we performed directed evolution of chalcone synthases from Hordeum vulgare and Arabidopsis thaliana, identifying enzyme variants that led to improved production of O-methylated flavanones, including isosakuranetin, hesperetin, and homoeriodictyol, as well as fluoro-substituted flavanones. In addition, we demonstrated that specific variants of H. vulgare chalcone synthase promoted the formation of isoferuloyl-derived derailment products. Collectively, this work establishes the FdeR-based biosensor as a versatile platform for pathway and enzyme engineering, enabling efficient early-stage diversification of flavanones in microbial systems and providing insight into the mutational landscape of chalcone synthases.

Synthetic Biology↗

bioRxiv2026

Conserved dimerization architecture in C-type lectins from virus-vector mosquitoes

C-type lectins (CTLs) play key roles in innate immunity and microbial carbohydrate recognition. In the disease vector mosquito Aedes aegypti, the CTLD-S family comprises 34 soluble CTLs whose members are implicated in flavivirus dissemination and microbial homeostasis, yet their structure and organization remain uncharacterized. Here, we combine X-ray crystallography, small-angle X-ray scattering (SAXS), molecular dynamics, and machine learning-based structure prediction to characterize CTLs in Aedes aegypti. We determined the crystal structures of four representative CTLD-S proteins: mosGCTL-1, -3, -6, and -20. All crystals featured mosGCTL proteins in an identical homodimer arrangement, positioning both carbohydrate-binding sites on the same molecular face. Dimerization was confirmed in solution and AlphaFold predictions across the entire CTLD-S family indicated that dimer formation may be a unifying feature of mosquito CTLD-S proteins. For one mosGCTL structure, paucimannose glycans bound at a Ca2+-dependent site, demonstrating bi-dentate glycan-binding through one dimer. Finally, machine learning based predictions indicated hundreds of possible CTLD-S heterodimers may be viable, with wide-ranging implications for preferred glycan binding through one dimer. Our findings reveal a conserved dimeric arrangement among mosquito lectins that may underpin carbohydrate recognition relevant to vector-pathogen interactions.

biochemistry↗

bioRxiv2024

Genetic Basis, Quantitative Nature, and Functional Relevance of Evolutionarily Conserved DNA Methylation

DNA methylation (DNAm) is a key epigenetic mark that modulates regulatory elements and gene expression, playing a crucial role in mammalian development and physiological function. Despite extensive characterization of DNAm profiles across species, little is known about its evolutionary conservation. Here, we conducted a comparative epigenome-wide analysis of great apes to identify and characterize sequence- and methylation-conserved CpGs (MCCs). Using 202 DNAm arrays, alongside 6 matched genotype and 13 matched transcriptomic datasets, we identified 11,500 MCCs for which methylation was evolutionarily related to sequences of CpGs and methylation quantitative trait loci. MCCs were the most stable across healthy human tissues and exhibited weaker genetic associations than other CpGs. Moreover, MCCs showed minimal associations with demographic, environmental factors, and noncancer diseases, yet demonstrated stronger associations with certain cancers than other CpGs, particularly gastrointestinal cancers. Functional enrichment analysis revealed that genes associated with MCC methylation in cancer were enriched for cancer driver genes and canonical cancer pathways, highlighting a significant regulatory role for MCCs in tumorigenesis. Collectively, our findings reveal the extent of DNAm conservation in great ape evolution, its association with genetic conservation, and its relevance to human diseases. These integrative analyses offer evolutionary insights into epigenetic variation and its functional implications in human populations.

genetics↗

bioRxiv2024

Complete Reference Genome and Pangenome Expand Biologically Relevant Information for Genome-Wide DNA Methylation Analysis Using Short-Read Sequencing and Array Data

BackgroundThe new complete telomere-to-telomere human genome assembly, T2T-CHM13, and the first draft of the human pangenome reference provide unique opportunities to update the reference genome for epigenetics investigations and clinical research. However, it is largely unclear how these reference genome updates may impact DNA methylation (DNAm) analysis. ResultsCompared to the previous GRCh38 assembly, we found an average increase of 7.4% (range 5.4%-9.9% across samples and sequencing methods) in the number of CpGs genome-wide using T2T-CHM13 with data from four commonly used short-read sequencing DNAm profiling methods. The increase in number of CpGs facilitated discovery of 88 new differentially methylated CpGs within cancer driver genes in an epigenome-wide association study (EWAS) of colon cancer. Further, by aligning probe sequences from the commonly used and recently released Illumina DNAm arrays to T2T-CHM13 and GRCh38, we showed the enhanced utility of T2T-CHM13 for evaluation of potential probe cross-reactivity (i.e., where probes match multiple regions) and mismatch (i.e., where probes do not perfectly match the target region), resulting in the identification of new and more reproducible sets of unambiguous probes (i.e., probes uniquely mapping to the target region) (HM450K, n = 430,719; EPIC, n = 777,491; EPICv2, n = 859,216). In EWASs of 24 cancer types, an average of 945 additional differentially methylated CpG sites were identified in the new unambiguous probe set rather than in the GRCh38-based unambiguous probe set, with enrichments in cancer driver genes and cancer signaling pathways. Moreover, the pangenome called 4.5% more CpGs on average in short-read sequencing data than T2T-CHM13 and identified cross-population and population-specific unambiguous probes in DNAm arrays, owing to its improved representation of genetic diversity. These additional CpGs were overlapped with the promoters and gene bodies of various biologically and medically relevant genes and pangenome-based unambiguous probes can potentially facilitate the discovery of DNAm alterations in more than 200 cancer driver genes in each cancer type. ConclusionsUse of T2T-CHM13 and pangenome references can benefit epigenome-wide association studies by including CpGs previously unobserved in short-read sequencing data and by improving the identification of unambiguous probes for DNAm arrays, thus expanding biologically relevant information. This study highlights the practical applications of T2T-CHM13 and pangenome for genome biology and provides a basis for expansion of epigenetics investigations.

genomics↗

bioRxiv2024

Comparison of Infinium MethylationEPIC v2.0 to v1.0 for human population epigenetics: considerations for addressing EPIC version differences in DNA methylation-based tools

The recently launched Illumina Infinium MethylationEPIC v2.0 (EPICv2), successor of MethylationEPIC v1.0 (EPICv1), retains a majority of probes in EPICv1, while expanding coverage of regulatory elements. The concordance between the two EPIC versions in DNA methylation-based tools has not yet been investigated. To address this, DNA methylation was profiled on both versions using matched blood samples across four cohorts spanning early to late adulthood. High concordance between versions at the array level but variable agreement at the individual probe level was noted. A significant contribution of EPIC version to DNA methylation variation was observed, though it was to a smaller extent compared to sample relatedness and cell type composition. Modest but significant differences in DNA methylation-based estimates between versions were observed, irrespective of the data preprocessing method used. Adjustments for EPIC version or calculation of estimates separately for each version largely mitigated these version-specific discordances. This work emphasizes the importance of accounting for EPIC version differences in research scenarios, especially in meta-analyses and longitudinal studies that require data harmonization across versions. Summary blurbOur work illustrates the importance of accounting for EPIC version in DNA methylation-based tools, specifically in research scenarios when samples profiled across different versions are harmonized.

genomics↗

bioRxiv2024

An engineered human cardiac tissue model reveals contributions of systemic lupus erythematosus autoantibodies to myocardial injury

Systemic lupus erythematosus (SLE) is a highly heterogenous autoimmune disease that affects multiple organs, including the heart. The mechanisms by which myocardial injury develops in SLE, however, remain poorly understood. Here we engineered human cardiac tissues and cultured them with IgG fractions containing autoantibodies from SLE patients with and without myocardial involvement. We observed unique binding patterns of IgG from two patient subgroups: (i) patients with severe myocardial inflammation exhibited enhanced binding to apoptotic cells within cardiac tissues subjected to stress, and (ii) patients with systolic dysfunction exhibited enhanced binding to the surfaces of viable cardiomyocytes. Functional assays and RNA sequencing (RNA-seq) revealed that IgGs from patients with systolic dysfunction exerted direct effects on engineered tissues in the absence of immune cells, altering tissue cellular composition, respiration and calcium handling. Autoantibody target characterization by phage immunoprecipitation sequencing (PhIP-seq) confirmed distinctive IgG profiles between patient subgroups. By coupling IgG profiling with cell surface protein analyses, we identified four pathogenic autoantibody candidates that may directly alter the function of cells within the myocardium. Taken together, these observations provide insights into the cellular processes of myocardial injury in SLE that have the potential to improve patient risk stratification and inform the development of novel therapeutic strategies.

bioengineering↗

bioRxiv2020

Population genomics of North American northern pike: variation and sex-specific signals from a chromosome-level, long read genome assembly

The northern pike Esox lucius is a freshwater fish renowned for having low genetic diversity but ecological success throughout the Northern Hemisphere. Here we generate an annotated chromosome-level genome assembly of 941 Mbp in length with 25 chromosome-length scaffolds using long-reads and chromatin capture technology. We then align whole-genome resequencing data against this reference to genotype northern pike from Alaska through New Jersey (n = 47). A striking decrease in genetic diversity occurs along the sampling range, whereby samples to the west of the North American Continental Divide have substantially higher diversity than populations to the east. As an example, individuals from Interior Alaska in the west and St. Lawrence River in the east have on average 181K and 64K heterozygous SNPs per individual, respectively (i.e., a SNP variant every 3.2 kbp and 11.2 kbp, respectively). Even with such low diversity, individuals clustered with strong support within each population, and this may be related to numerous private alleles in each population. Evidence for recent population expansion was observed for a Manitoba hatchery and the St. Lawrence population (Tajimas D = -1.07 and -1.30, respectively). Non-uniform patterns of diversity were observed across the genome, with large regions showing elevated diversity in several chromosomes, including LG24. In populations with the master sex determining gene amhby still present in the genome, amhby is in LG24. As expected, amhby was largely male-specific in Alaska and the Yukon and absent southeast to these populations, but we also document some amhby(-) males in Alaska and amhby(+) males in the Columbia River. This indicates that rather than a discrete boundary after which amhby was lost in North America, there is a patchwork of presence of this system in the western region. These results support the theory that northern pike recolonized North America from refugia in Alaska and expanded following deglaciation from west to east, with probable founder effects resulting in loss of both neutral and functional diversity including the loss of the sex determination system.

genomics↗

bioRxiv2020

Hydrogel mechanical properties are more important than osteoinductive growth factors for bone formation with MSC spheroids

Mesenchymal stromal cells (MSCs) can promote tissue repair in regenerative medicine, and their therapeutic potential is further enhanced via spheroid formation. We demonstrated that intraspheroidal presentation of Bone Morphogenetic Protein-2 (BMP-2) on hydroxyapatite (HA) nanoparticles resulted in more spatially uniform MSC osteodifferentiation, providing a method to internally influence spheroid phenotype. Stress relaxation of hydrogels has emerged as a potent stimulus to enhance monodispersed MSC spreading and osteogenic differentiation, but the effect of hydrogel viscoelasticity on MSC spheroids has not been reported. Herein, we describe a materials-based approach to augment the osteogenic potential of entrapped MSC spheroids by leveraging the mechanical properties of alginate hydrogels. Compared to spheroids entrapped in covalently crosslinked, elastic alginate, calcium deposition of MSC spheroids was consistently increased in ionically crosslinked, viscoelastic alginate. We observed significant increases in calcium deposition by MSC spheroids loaded with BMP-2-HA in viscoelastic gels compared to soluble BMP-2, which was higher than all elastic alginate gels. Upon implantation in critically sized calvarial bone defects, we observed enhanced bone formation in all animals treated with viscoelastic hydrogels. Increases in bone formation were evident in viscoelastic gels, regardless of the mode of presentation of BMP-2 (i.e., soluble delivery or HA nanoparticles). These studies demonstrate that the dynamic mechanical properties of viscoelastic alginate are an effective strategy to enhance the therapeutic potential of MSC spheroids for bone formation and repair.

bioengineering↗