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Chang, C.-H.

Publications and source records attributed to Chang, C.-H..

At least 37 records · Page 2Linked to original sources

Germline novelty through recurrent copy-number, protein, and regulatory evolution of the synaptonemal complex

The synaptonemal complex (SC) is a protein-rich structure essential for meiotic recombination and faithful chromosome segregation. Acting like a zipper to paired chromosomes during early prophase, the complex consists of central elements bilaterally tethered by the transverse filaments to the lateral elements anchored on either side to the homologous chromosome axes. Despite being found in most major eukaryotic taxa implying a deeply conserved evolutionary origin, several components of the complex exhibit unusually high rates of sequence turnover. This is puzzlingly exemplified by the SC of Drosophila, where the central elements and transverse filaments display no identifiable homologs outside of the genus. Here, we exhaustively examine the evolutionary history of the SC in Drosophila taking a comparative phylogenomic approach with high species density to circumvent obscured homology due to rapid sequence evolution. Contrasting starkly against other genes involved in meiotic chromosome pairing, SC significantly shows elevated rates of coding evolution due to a combination of relaxed constraint and recurrent, widespread positive selection. In particular, the central element cona and transverse filament c(3)G have diversified through tandem and retro-duplications, repeatedly generating paralogs that likely have novel germline functions. In a striking case of molecular convergence, c(3)G paralogs that independently arose in distant lineages evolved under positive selection to have convergent truncations to the protein termini and elevated testes expression. Surprisingly, the expression of SC genes in the germline is exceedingly prone to change suggesting recurrent regulatory evolution which, in many species, resulted in high testes expression even though Drosophila males are achiasmic. Overall, our study recapitulates the poor conservation of SC components, and further uncovers that the lack of conservation extends to other modalities including copy number, genomic locale, and germline regulation. Considering the elevated testes expression in many Drosophila species and the common ancestor, we suggest that the function of SC genes in the male germline, while still poorly understood, may be a prime target of constant evolutionary pressures driving repeated adaptations and innovations. SummaryThe synaptonemal complex (SC) is essential for meiotic recombination and faithful chromosome segregation across eukaryotes, yet components of the SC are often poorly conserved. Here we show that across the Drosophila phylogeny several SC genes have evolved under recurrent positive selection resulting in orthologs that are barely recognizable. This is partly driven duplications repeatedly generating paralogs that may have adopted novel germline functions, often in the testes. Unexpectedly, while most SC genes are thought to be dispensable in the male germline where recombination is absent in Drosophila, elevated testes expression appears to be the norm across the genus and likely the ancestral state. The evolutionary lability of SC genes in Drosophila is likely a repeated source of adaptive innovations in the germline.

evolutionary biology↗

Rapid turnover of centromeric DNA reveals signatures of genetic conflict in Drosophila

Centromeres reside in rapidly evolving, repeat-rich genomic regions, despite their essential function in chromosome segregation. Across organisms, centromeres are rich in selfish genetic elements such as transposable elements and satellite DNAs that can bias their transmission through meiosis. However, these elements still need to cooperate at some level and contribute to, or avoid interfering with, centromere function. To gain insight into the balance between conflict and cooperation at centromeric DNA, we take advantage of the close evolutionary relationships within the Drosophila simulans clade - D. simulans, D. sechellia, and D. mauritiana - and their relative, D. melanogaster. Using chromatin profiling combined with high resolution fluorescence in situ hybridization on stretched DNA, we characterize all centromeres across these species. We discovered dramatic centromere reorganization involving recurrent shifts between retroelements and satellite DNAs over short evolutionary timescales. We also reveal the recent origin (<240 Kya) of telocentric chromosomes in D. sechellia, where the X and 4th centromeres now sit on telomere-specific retroelements. Finally, the Y chromosome centromeres, which are the only chromosomes that do not experience female meiosis, do not show dynamic cycling between satDNA and TEs. The patterns of rapid centromere turnover in these species are consistent with genetic conflicts in the female germline and have implications for centromeric DNA function and karyotype evolution. Regardless of the evolutionary forces driving this turnover, the rapid reorganization of centromeric sequences over short evolutionary timescales highlights their potential as hotspots for evolutionary innovation.

evolutionary biology↗

Reprogramming human B cells with custom heavy chain antibodies

We describe a genome editing strategy to reprogram the immunoglobulin heavy chain (IgH) locus of human B cells to express custom molecules that respond to immunization. These heavy chain antibodies (HCAbs) comprise a custom antigen-recognition domain linked to an Fc domain derived from the IgH locus and can be differentially spliced to express either B cell receptor (BCR) or secreted antibody isoforms. The HCAb editing platform is highly flexible, supporting antigen-binding domains based on both antibody and non-antibody components, and also allowing alterations in the Fc domain. Using HIV Env protein as a model antigen, we show that B cells edited to express anti-Env HCAbs support the regulated expression of both BCRs and antibodies, and respond to Env antigen in a tonsil organoid model of immunization. In this way, human B cells can be reprogrammed to produce customized therapeutic molecules with the potential for in vivo amplification.

immunology↗

Transcriptome Analysis Reveals Organ-Specific Effects of 2-Deoxyglucose Treatment in Healthy Mice

ObjectiveGlycolytic inhibition via 2-deoxy-D-glucose (2DG) has potential therapeutic benefits for a range of diseases, including cancer, epilepsy, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), and COVID-19, but the systemic effects of 2DG on gene function across different tissues are unclear. MethodsThis study analyzed the transcriptional profiles of nine tissues from C57BL/6J mice treated with 2DG to understand how it modulates pathways systemically. Principal component analysis (PCA), weighted gene co-network analysis (WGCNA), analysis of variance, and pathway analysis were all performed to identify modules altered by 2DG treatment. ResultsPCA revealed that samples clustered predominantly by tissue, suggesting that 2DG affects each tissue uniquely. Unsupervised clustering and WGCNA revealed six distinct tissue-specific modules significantly affected by 2DG, each with unique key pathways and genes. 2DG predominantly affected mitochondrial metabolism in the heart, while in the small intestine, it affected immunological pathways. ConclusionsThese findings suggest that 2DG has a systemic impact that varies across organs, potentially affecting multiple pathways and functions. The study provides insights into the potential therapeutic benefits of 2DG across different diseases and highlights the importance of understanding its systemic effects for future research and clinical applications.

systems biology↗

BRD9-SMAD2/3 orchestrates stemness and tumorigenesis in pancreatic ductal adenocarcinoma

The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGF{beta}/Activin-SMAD2/3 signalling pathway. Inhibition and genetic ablation of BDR9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

cancer biology↗

Inteins in the Loop: A Framework for Engineering Advanced Biomolecular Controllers for Robust Perfect Adaptation

Homeostasis is one of the cornerstones of life shaped by billions of years of evolution. A notion that is similar to homeostasis, but yet more stringent, is Robust Perfect Adaptation (RPA). A system is endowed with RPA if it is capable of driving a variable of interest to a prescribed level despite the presence of disturbances and uncertainties in the environment. Designing and building biomolecular controllers capable of achieving RPA have been identified as an important task which has immediate implications for various disciplines. Here, we develop systematic theoretical and experimental frameworks for custom-built proteins that exploit split inteins -- short amino acid sequences capable of performing protein-splicing reactions -- to design, genetically build and analyze a wide class of RPA-achieving integral feedback controllers. We first lay down a theoretical foundation that facilitates the screening of intein-based controller networks for RPA, and then usher an easy-to-use recipe to simplify their, otherwise complex, underlying mathematical models. Furthermore, we genetically engineer and test various controller circuits based on commonly used transcription factors in mammalian cells. We experimentally and theoretically demonstrate their ability of robustly rejecting external disturbances (that is achieving RPA) over an exquisitely broad dynamic range. Due to their small size, flexibility, modularity, lack of side effects and applicability across various forms of life, inteins serve as promising genetic parts to implement RPA-achieving controllers. To this end, we believe "inteins in the control loop" will leave a significant impact on various disciplines spanning synthetic biology, biofuel production, metabolic engineering and cell therapy among others.

synthetic biology↗

Protein N-Terminomics Reveals Major Proteases in Regulating Beige Adipocyte Differentiation

In this comprehensive study, we present an innovative analytical platform designed to capture the temporal shifts in both the proteome and protein N-terminome during beige adipocyte differentiation. Employing a refined N-terminomics technique, we achieved a high purity of 97% in isolating protein N-terminal peptides. Our data encompassed 7,171 unique N-terminal peptides, with 3,043 from canonical proteins and 4,129 with neo-N-termini. Strikingly, nearly half (44%) of the proteins revealed distinct temporal trajectories between the global proteome and the N-terminome. This underscores the central role of proteolysis in beige adipocyte differentiation. Experimentally, knockdown of either Pmpcb, Plg, or Cstd in preadipocytes attenuated thermogenesis, manifested by reduced levels of beige adipocyte markers like Cidea, Pgc1a, Ucp1, and Tbx1 and an increase in adipogenic proteins, thereby hampering beige adipocyte maturation. A salient discovery was the non-apoptotic role of caspase 8 protease; inhibiting its proteolytic action amplified Ucp1 expression levels. Collectively, our findings spotlight proteases and their proteolytic by-products as vital regulators in beige adipocyte differentiation.

cell biology↗

EZH2/ hSULF1 axis mediates receptor tyrosine kinase signaling to shape cartilage tumor progression

BackgroundChondrosarcomas are primary cancers of cartilaginous tissue and capable of alteration to highly aggressive, metastatic, and treatment-refractory states, leading to a poor prognosis with a five-year survival rate at 11 months for the dedifferentiated subtype. At present, the surgical resection of chondrosarcoma is the only effective treatment, and no other treatment options including targeted therapies, conventional chemotherapies, or immunotherapies are available for these patients. MethodsA non-biased ChIP sequence, cDNA microarray analysis, and validation of chondrosarcoma cell lines identified sulfatase 1(SULF1) as the top EZH2-targeted gene to regulate chondrosarcoma progression. Receptor tyrosine kinase (RTK) array of chondrosarcoma cells with vector control or ectopically expressed SULF1 revealed that cMET was the downstream signal. The regulation of the EZH2/SULF1/cMET axis was further validated in mice and patient samples with mice models and chondrosarcoma tissue array, respectively. ResultsThe EZH2/SULF1/cMET axis is identified, which contributes to the malignancy of chondrosarcoma and provides a potential therapeutic option for the disease. Ectopically expressed SULF1 or pharmaceutical inhibition of the cMET pathway significantly retards the chondrosarcoma growth and extends mice survival. ConclusionsThe results not only established a signal pathway promoting the malignancy of chondrosarcoma but also provided a therapeutic potential for further development of effective target therapy to treat chondrosarcoma.

cancer biology↗

Genetic conflicts between sex chromosomes drive expansion and loss of sperm nuclear basic protein genes in Drosophila

Many animal species employ short, positively charged proteins, called sperm nuclear basic proteins (SNBPs) or protamines, for tighter packaging of genomes in sperm. SNBP repertoires differ dramatically across animal lineages and signatures of rapid evolution have been reported in mammals. Both sperm competition and meiotic drive between sex chromosomes have been proposed as causes of SNBP innovation. We used a phylogenomic approach to investigate SNBP diversification and its underlying causes in Drosophila species. We found unambiguous signatures of positive selection in most SNBP genes except for genes essential for male fertility in D. melanogaster. Unexpectedly, evolutionarily young SNBP genes are more likely to encode essential functions for fertility than ancient, conserved SNBP genes like CG30056, which we found is dispensable for male fertility despite universal retention in Drosophila species. We found 19 independent amplification events involving eight SNBP genes that occurred preferentially on sex chromosomes in 78 Drosophila species. Conversely, we found that otherwise-conserved SNBP genes were lost in the montium group of Drosophila species, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions are prone to degenerate or relocate back to autosomes. We hypothesize that SNBP genes ancestrally encoded by autosomes suppress meiotic drive, whereas sex-chromosomal SNBP expansions directly participate in meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. We conclude that SNBP rapid evolution is driven by genetic conflicts between sex chromosomes during spermatogenesis in Drosophila species.

evolutionary biology↗

Correcting Modification-Mediated Errors in Nanopore Sequencing by Nucleotide Demodification and in silico Correction

The accuracy of Oxford Nanopore Technology (ONT) sequencing has significantly improved thanks to new flowcells, sequencing kits, and basecalling algorithms. However, novel modifications untrained in the basecalling models can seriously reduce the quality. This paper reports a set of ONT-sequenced genomes with unexpected low quality ([~]Q30) due to extensive new modifications. Demodification by whole-genome amplification (WGA) significantly improved the quality of all genomes ([~]Q50-60) while losing the epigenome. We developed a computational method, Modpolish, for correcting modification-mediated errors without WGA. Modpolish produced high-quality genomes and uncovered the underlying modification motifs without loss of epigenome. Our results suggested that novel modifications are prone to ONT errors, which are correctable by WGA or Modpolish without additional short-read sequencing.

bioinformatics↗

Augmented glucose dependency of autoreactive B cells provides a treatment target forlupus

Heightened glycolysis is inherent to immune/inflammatory disorders, but little is known of its role in the pathogenesis of systemic lupus erythematosus (lupus). Here, we profile key autoimmune populations in acute and chronic lupus-prone models and their response to glycolytic inhibition. We demonstrate that glycolysis is specifically required for autoreactive germinal center B cells (GCB), but not for T follicular helper cells (Tfh) to survive. This augmented reliance on glucose oxidation to maintain ATP production in pathogenic GCB renders them highly susceptible to oxidative stress-induced apoptosis triggered by glycolysis blockade via 2-deoxyglucose (2DG). We show that 2DG can preferentially reduce GCB in lupus-prone mice, while sparing other autoreactive populations, including Tfh, but still significantly improving lifespan and kidney function. Furthermore, the subset of GCB expressing B-cell maturation antigen (BCMA) exhibits an exaggerated dependence on glycolysis to sustain their growth. Depletion of these cells with a proliferation-inducing ligand-based CAR T-cells leads to greatly prolonged lifespan of mice with severe autoimmune activation. These results reveal that glycolysis dependent GCB, especially those expressing BCMA, are key lupus mediators and highlight that they can be selectively targeted to improve disease outcomes for lupus patients.

immunology↗

The 24-chain core-shell nanostructure of wood cellulose microfibrils in seed plants

Wood cellulose microfibrils (CMFs) are the most abundant organic substance on earth, but their nanostructures are poorly understood. There are controversies regarding the glucan chain number (N) of CMFs during initial synthesis and whether they become fused afterwards. Here, we combined small-angle X-ray scattering (SAXS), solid-state nuclear magnetic resonance (ssNMR) and X-ray diffraction (XRD) analyses to resolve these controversies. We successfully developed SAXS measurement methods for the cross-section aspect ratio and area of the crystalline-ordered CMF core, which showed higher density than the semi-disordered shell. The 1:1 aspect ratio suggested that CMFs remain mostly segregated, not fused. The area measurement revealed the chain number in the core zone (Ncore). The ratio of ordered cellulose over total cellulose, termed Roc, was determined by ssNMR. Using the formula N = Ncore / Roc, we found that the majority of wood CMFs contain 24 chains, conserved between gymnosperm and angiosperm trees. The average wood CMF has a crystalline-ordered core of [~]2.2 nm diameter and a semi-disordered shell of [~]0.5 nm thickness. In naturally and artificially aged wood, we only observed CMF aggregation (contact without crystalline continuity) but not fusion (forming conjoined crystalline unit). This further argued against the existence of partially fused CMFs in new wood, overturning the recently proposed 18-chain fusion hypothesis. Our findings are important for advancing wood structural knowledge and more efficient utilization of wood resources in sustainable bio-economies.

plant biology↗

Epistatic selection on a selfish Segregation Distorter supergene: drive, recombination, and genetic load

Meiotic drive supergenes are complexes of alleles at linked loci that together subvert Mendelian segregation to gain preferential transmission. In males, the most common mechanism of drive involves the disruption of sperm bearing alternative alleles. While at least two loci are important for male drive-- the driver and the target-- linked modifiers can enhance drive, creating selection pressure to suppress recombination. In this work, we investigate the evolution and genomic consequences of an autosomal multilocus, male meiotic drive system, Segregation Distorter (SD) in the fruit fly, Drosophila melanogaster. In African populations, the predominant SD chromosome variant, SD-Mal, is characterized by two overlapping, paracentric inversion on chromosome arm 2R and nearly perfect (~100%) transmission. We study the SD-Mal system in detail, exploring its components, chromosomal structure, and evolutionary history. Our findings reveal a recent chromosome-scale selective sweep mediated by strong epistatic selection for haplotypes carrying Sd, the main driving allele, and one or more factors within the double inversion. While most SD-Mal chromosomes are homozygous lethal, SD-Mal haplotypes can recombine with other, complementing haplotypes via crossing over and with wildtype chromosomes only via gene conversion. SD-Mal chromosomes have nevertheless accumulated lethal mutations, excess non-synonymous mutations, and excess transposable element insertions. Therefore, SD-Mal haplotypes evolve as a small, semi-isolated subpopulation with a history of strong selection. These results may explain the evolutionary turnover of SD haplotypes in different populations around the world and have implications for supergene evolution broadly.

evolutionary biology↗

Water droplet-in-oil digestion method for single-cell proteomics

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold as well as the number of quantified transmembrane proteins compared to an in-solution digestion (ISD) method. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10000 cells using the ISD method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

systems biology↗

Glutamine metabolism enables NKT cell homeostasis and function through the AMPK-mTORC1 signaling axis

Cellular metabolism is essential in dictating conventional T cell development and function, but its role in natural killer T (NKT) cells has not been well studied. We have previously shown that NKT cells operate distinctly different metabolic programming from CD4 T cells, including a strict requirement for glutamine metabolism to regulate NKT cell homeostasis. However, the mechanisms by which NKT cells regulate glutamine metabolism for their homeostasis and effector functions remain unknown. In this study, we report that steady state NKT cells have higher glutamine levels than CD4 T cells and NKT cells increase glutaminolysis upon activation. Among its many metabolic fates, NKT cells use glutamine to fuel the tricarboxylic acid cycle and glutathione synthesis, and glutamine-derived nitrogen enables protein glycosylation via the hexosamine biosynthesis pathway (HBP). Each of these functions of glutamine metabolism was found to be critical for NKT cell survival and proliferation. Furthermore, we demonstrate that glutaminolysis and the HBP differentially regulate IL-4 and IFN{gamma} production. Finally, glutamine metabolism appears to be controlled by AMP-activated protein kinase (AMPK)-mTORC1 signaling. These findings highlight a unique metabolic requirement of NKT cells which can be potentially serve as an effective immunotherapeutic agent against certain nutrient restricted tumors. SignificanceNKT cells get activated very early during an immune response and produce cytokines and chemokines, which further activate other immune cell types. Although metabolism regulates these functions in other T cell subsets, little is understood about how metabolic pathways are controlled in NKT cells. The present study shows that NKT cells metabolize the amino acid glutamine through two different branches of metabolism, which control NKT cell homeostasis and expansion in a similar manner but control cytokine production differently. This glutamine dependency seems to be regulated by AMP-activated protein kinase (AMPK), which is a central regulator of energy homeostasis. Together, our study demonstrates a unique metabolic profile of glutamine metabolism in NKT cells which could be harnessed for NKT cell-based immunotherapy.

immunology↗

Acoustic frequency-dependent physical mechanism of sub-MHz ultrasound neurostimulation

Ultrasound is an innovative physical modality allowing non-invasive and reversible modulation of neural circuit activity in the brain with high spatial resolution. Despite growing interest in clinical applications, the safe and effective use of ultrasound neuromodulation has been limited by a lack of understanding of the physical mechanisms underlying its effects. Here, we demonstrate acoustic frequency-dependent physical effects that underlie ultrasound neuromodulation, where cavitation and radiation forces are the dominant sources of low- and high-frequency stimulation, respectively. We used 39.5 kHz and 500 kHz acoustic frequencies to stimulate cultured neural and glial cells, excised from rat cortex, to study acoustic frequency-dependent neural responses. We demonstrate increased evoked responses due to increased cavitation activity at the 39.5 kHz acoustic frequency. In contrast, notable cavitation activity was not detected at 500 kHz despite detection of evoked responses. Our work highlights the dependence of ultrasound neuromodulation on acoustic frequencies, with different physical effects underlying cell responses to low and high sub-MHz acoustic frequency ranges.

bioengineering↗

Unique structure and positive selection promote the rapid divergence of Drosophila Y chromosomes

Y chromosomes across diverse species convergently evolve a gene-poor, heterochromatic organization enriched for duplicated genes, LTR retrotransposable elements, and satellite DNA. Sexual antagonism and a loss of recombination play major roles in the degeneration of young Y chromosomes. However, the processes shaping the evolution of mature, already degenerated Y chromosomes are less well-understood. Because Y chromosomes evolve rapidly, comparisons between closely related species are particularly useful. We generated de novo long read assemblies complemented with cytological validation to reveal Y chromosome organization in three closely related species of the Drosophila simulans complex, which diverged only 250,000 years ago and share >98% sequence identity. We find these Y chromosomes are divergent in their organization and repetitive DNA composition and discover new Y-linked gene families whose evolution is driven by both positive selection and gene conversion. These Y chromosomes are also enriched for large deletions, suggesting that the repair of double-strand breaks on Y chromosomes may be biased toward microhomology-mediated end joining over canonical non-homologous end-joining. We propose that this repair mechanism generally contributes to the convergent evolution of Y chromosome organization.

genomics↗

Distinct spermiogenic phenotypes underlie sperm elimination in the Segregation Distorter meiotic drive system

Segregation Distorter (SD) is a male meiotic drive system in Drosophila melanogaster. Males heterozygous for a selfish SD chromosome rarely transmit the homologous SD+ chromosome. It is well established that distortion results from an interaction between Sd, the primary distorting locus on the SD chromosome and its target, a satellite DNA called Rsp, on the SD+ chromosome. However, the molecular and cellular mechanisms leading to post-meiotic SD+ sperm elimination remain unclear. Here we show that SD/SD+ males of different genotypes but with similarly strong degrees of distortion have distinct spermiogenic phenotypes. In some genotypes, SD+ spermatids fail to fully incorporate protamines after the removal of histones, and degenerate during the individualization stage of spermiogenesis. In contrast, in other SD/SD+ genotypes, protamine incorporation appears less disturbed, yet spermatid nuclei are abnormally compacted, and mature sperm nuclei are eventually released in the seminal vesicle. Our analyses of different SD+ chromosomes suggest that the severity of the spermiogenic defects associates with the copy number of the Rsp satellite. We propose that when Rsp copy number is very high (> 2000), spermatid nuclear compaction defects reach a threshold that triggers a checkpoint controlling sperm chromatin quality to eliminate abnormal spermatids during individualization.

genetics↗