Biology subjects

Hwang, S.

Publications and source records attributed to Hwang, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Mechanism-guided engineering of a minimal biological particle for genome editing

The widespread application of genome editing to treat or even cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped Delivery Vehicles (EDVs) are engineered virally-derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication components in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of [~]80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs, and demonstrated increased editing in cell lines and therapeutically-relevant primary human T cells. These results show that virally-derived particles can be streamlined to create efficacious genome editing delivery vehicles that could simplify production and manufacturing. SIGNIFICANCE STATEMENTOur results highlight the importance of understanding how virally-derived particles function to eliminate unnecessary viral proteins and create more efficacious and easier-to-produce delivery vehicles for therapeutic genome editing.

bioengineering↗

bioRxiv2024

Context-dependent roles for autophagy in myeloid cells in tumor progression

Autophagy is known to suppress tumor initiation by removing genotoxic stresses in normal cells. Conversely, autophagy is also known to support tumor progression by alleviating metabolic stresses in neoplastic cells. Centered on this pro-tumor role of autophagy, there have been many clinical trials to treat cancers through systemic blocking of autophagy. Such systemic inhibition affects both tumor cells and non-tumor cells, and the consequence of blocked autophagy in non-tumor cells in the context of tumor microenvironment is relatively understudied. Here, we examined the effect of autophagy-deficient myeloid cells on the progression of autophagy-competent tumors. We found that blocking autophagy only in myeloid cells modulated tumor progression markedly but such effects were context dependent. In a tumor implantation model, the growth of implanted tumor cells was substantially reduced in mice with autophagy-deficient myeloid cells; T cells infiltrated deeper into the tumors and were responsible for the reduced growth of the implanted tumor cells. In an oncogene-driven tumor induction model, however, tumors grew faster and metastasized more in mice with autophagy- deficient myeloid cells. These data demonstrate that the autophagy status of myeloid cells plays a critical role in tumor progression, promoting or suppressing tumor growth depending on the context of tumor-myeloid cell interactions. This study indicates that systemic use of autophagy inhibitors in cancer therapy may have differential effects on rates of tumor progression in patients due to effects on myeloid cells and that this warrants more targeted use of selective autophagy inhibitors in a cancer therapy in a clinical setting.

cancer biology↗

bioRxiv2024

Lineage-specific genomic imprinting in the ZNF791 locus

BackgroundGenomic imprinting is the primary epigenetic phenomenon that results in parent-of-origin effects on mammalian development and growth. Research on genomic imprinting in domesticated animals has lagged due to a primary focus on orthologous imprinted genes. This emphasis has limited the discovery of imprinted genes specific to livestock. To identify genomic imprinting in pigs, we generated parthenogenetic porcine embryos alongside biparental normal embryos, and then performed whole-genome bisulfite sequencing and RNA sequencing on these samples. ResultsIn our analyses, we discovered a maternally methylated differentially methylated region within the orthologous ZNF791 locus in pigs. Additionally, we identified both a major imprinted isoform of the ZNF791-like gene and an unannotated antisense transcript that has not been previously annotated. Importantly, our comparative analyses of the orthologous ZNF791 gene in various eutherian mammals, including humans, non-human primates, rodents, artiodactyls, and dogs, revealed that this gene is subjected to genomic imprinting exclusively in domesticated animals, thereby highlighting lineage-specific imprinting. Furthermore, we explored the potential mechanisms behind the establishment of maternal DNA methylation imprints in porcine and bovine oocytes, supporting the notion that integration of transposable elements, active transcription, and histone modification may collectively contribute to the methylation of embedded intragenic CpG island promoters. ConclusionsOur findings convey fundamental insights into molecular and evolutionary aspects of livestock species-specific genomic imprinting and provide critical agricultural implications.

genetics↗

bioRxiv2024

MEM-based pangenome indexing for k-mer queries

Pangenomes are growing in number and size, thanks to the prevalence of high-quality long-read assemblies. However, current methods for studying sequence composition and conservation within pangenomes have limitations. Methods based on graph pangenomes require a computationally expensive multiple-alignment step, which can leave out some variation. Indexes based on k-mers and de Bruijn graphs are limited to answering questions at a specific substring length k. We present Maximal Exact Match Ordered (MEMO), a pangenome indexing method based on maximal exact matches (MEMs) between sequences. A single MEMO index can handle arbitrary-length queries over pangenomic windows. MEMO enables both queries that test k-mer presence/absence (membership queries) and that count the number of genomes containing k-mers in a window (conservation queries). MEMOs index for a pangenome of 89 human autosomal haplotypes fits in 2.04 GB, 8.8x smaller than a comparable KMC3 index and 11.4x smaller than a PanKmer index. MEMO indexes can be made smaller by sacrificing some counting resolution, with our decile-resolution HPRC index reaching 0.67 GB. MEMO can conduct a conservation query for 31-mers over the human leukocyte antigen locus in 13.89 seconds, 2.5x faster than other approaches. MEMOs small index size, lack of k-mer length dependence, and efficient queries make it a flexible tool for studying and visualizing substring conservation in pangenomes.

bioinformatics↗

bioRxiv2024

The impact of ancestral, environmental and genetic influences on germline de novo mutation rates and spectra

De novo germline mutation is an important factor in the evolution of allelic diversity and disease predisposition in a population. Here, we study the influence of genetically-inferred ancestry and environmental factors on de novo mutation rates and spectra. Using a genetically diverse sample of [~]10K whole-genome sequenced trios, one of the largest de novo mutation catalogues to date, we found that genetically-inferred ancestry is associated with modest but significant changes in both germline mutation rate and spectra across continental populations. These effects may be due to genetic or environmental factors correlated with ancestry. We find epidemiological evidence that exposure to tobacco smoke is significantly associated with increased de novo mutation rate, but it does not mediate the observed ancestry effects. Investigation of several other potential mutagenic factors using Mendelian randomisation showed no consistent effects, except for age of menopause, where increased age corresponded to a reduction in de novo mutation rate. Overall, our study presents evidence on new factors influencing de novo mutational rate and spectra.

genetics↗

bioRxiv2024

FGF2 binds to the allosteric site (site 2) and activates αvβ3 integrin and FGF1 binds to site 2 but suppresses integrin activation by FGF2: a potential mechanism of anti-inflammatory action of FGF1

FGF1 is known as an anti-inflammatory and has suppresses insulin resistance. Its homologue FGF2 is pro-inflammatory. Mechanism of FGF1s anti-inflammatory action and FGF2s pro-inflammatory action are unknown. Several inflammatory cytokines (e.g., CX3CL1, CCL5, and CXCL12, and CD40L) bind to the classical ligand (RGD)-binding site (site 1) of integrin v{beta}3. In addition, they bind to the allosteric site (site 2) of v{beta}3, which is distinct from site 1, and allosterically activate v{beta}3. Site 2 is involved in inflammatory signals since inflammatory lipid mediator 5-hydroxycholesterol binds to site 2 and induces integrin activation and inflammatory signals (e.g., TNF and IL-6 secretion). We thus hypothesized that FGF1 and FGF2 bind to site 2 and affect activation status of integrins. Here we describe that FGF2 bound to site 2 and allosterically activated v{beta}3 integrin. Point mutations in the site 2-binding interface of FGF2 suppressed this activation, indicating that FGF2 binding to site 2 is required for inducing integrin activation. In contrast, FGF1 bound to site 2 but did not activate v{beta}3, and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2. These findings suggest that FGF1s anti-inflammatory action is mediated by blocking site 2. FGF1 has potential as an anti-inflammatory agent, but is not appropriate for long-term use since it is potent mitogen. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of v{beta}3, suppressed v{beta}3 activation by FGF2 as effectively as WT FGF1. We propose that FGF1 R50E has therapeutic potential for inflammatory diseases.

biochemistry↗

bioRxiv2024

FGF2 binds to the allosteric site (site 2) and activates integrin αIIbβ3 and FGF1 binds to site 2 but suppresses integrin activation by FGF2: A potential mechanism of anti-thrombotic action of FGF1.

It has been believed that platelet integrin IIb{beta}3 recognizes fibrinogen and several ECM proteins, and we recently showed that IIb{beta}3 binds to several inflammatory cytokines (e.g., CCL5, and CXCL12), which are stored in platelet granules. These ligands bind to the classical ligand (RGD)-binding site (site 1) of integrin IIb{beta}3. Also, they bind to the allosteric site (site 2) of IIb{beta}3, which is distinct from site 1, and allosterically activate IIb{beta}3. Site 2 is known to be involved in allosteric integrin activation and inflammatory signaling. FGF2 is also stored in platelet granules and known to be pro-thrombotic, but it is unclear if FGF2 binds to IIb{beta}3. We studied if FGF2 and its homologue FGF1 bind to IIb{beta}3 and induce allosteric activation. FGF1 (not stored in platelet granules) is known to be anti-thrombotic. Mechanism of FGF1s anti-thrombotic action is unknown. Here we describe that FGF1 and FGF2 bound to site 1 of IIb{beta}3, indicating that IIb{beta}3 is a new receptor for FGF1/2. Notably, FGF2 bound to site 2 and allosterically activated IIb{beta}3. Point mutations in the site 2-binding interface of FGF2 suppressed this activation, indicating that FGF2 binding to site 2 is required for activation (FGF2 is an agonist to site 2). In contrast, FGF1 bound to site 2 but did not activate IIb{beta}3, and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of v{beta}3, suppressed IIb{beta}3 activation by FGF2 as effectively as WT FGF1. We propose that FGF1 R50E has therapeutic potential for anti-thrombosis.

biochemistry↗

bioRxiv2024

Transcript-specific enrichment enables profiling rare cell states via scRNA-seq

Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing (scRNA-seq) identifies many rare populations of interest that express specific marker transcript combinations, traditional flow sorting limits our ability to enrich these populations for further profiling, including requiring cell surface markers with high-fidelity antibodies. Additionally, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. To address these limitations, we describe Programmable Enrichment via RNA Flow-FISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations from complex cellular mixtures defined by the presence or absence of specific RNA transcripts. Across immune populations (n = 141,227 cells) and fresh-frozen and formalin-fixed paraffin-embedded brain tissue (n = 29,522 nuclei), we demonstrate the sorting logic that can be used to enrich for cell populations via RNA-based cytometry followed by high-throughput scRNA-seq. Our approach provides a rational, programmable method for studying rare populations identified by one or more marker transcripts.

genomics↗

bioRxiv2023

Autophagy regulates tumor growth and metastasis

The role of autophagy in tumorigenesis and tumor metastasis remains poorly understood. Here we show that inhibition of autophagy stabilizes the transcription factor Twist1 through Sequestosome-1 (SQSTM1, also known as p62) and thus increases cell proliferation, migration, and epithelial-mesenchymal transition (EMT) in tumor development and metastasis. Inhibition of autophagy or p62 overexpression blocks Twist1 protein degradation in the proteasomes, while p62 inhibition enhances it. SQSTM1/p62 interacts with Twist1 via the UBA domain of p62, in a Twist1-ubiquitination-dependent manner. Lysine 175 in Twist1 is critical for Twist1 ubiquitination, degradation, and SQSTM1/p62 interaction. For squamous skin cancer and melanoma cells that express Twist1, SQSTM1/p62 increases tumor growth and metastasis in mice. Together, our results identified Twist1 as a key downstream protein for autophagy and suggest a critical role of the autophagy/p62/Twist1 axis in cancer development and metastasis.

cancer biology↗

bioRxiv2023

Growth Dynamics of Ductal Carcinoma in Situ Recapitulate Normal Breast Development

Ductal carcinoma in situ (DCIS) and invasive breast cancer share many morphologic, proteomic, and genomic alterations. Yet in contrast to invasive cancer, many DCIS tumors do not progress and may remain indolent over decades. To better understand the heterogenous nature of this disease, we reconstructed the growth dynamics of 18 DCIS tumors based on the geo-spatial distribution of their somatic mutations. The somatic mutation topographies revealed that DCIS is multiclonal and consists of spatially discontinuous subclonal lesions. Here we show that this pattern of spread is consistent with a new Comet model of DCIS tumorigenesis, whereby multiple subclones arise early and nucleate the buds of the growing tumor. The discontinuous, multiclonal growth of the Comet model is analogous to the branching morphogenesis of normal breast development that governs the rapid expansion of the mammary epithelium during puberty. The branching morphogenesis-like dynamics of the proposed Comet model diverges from the canonical model of clonal evolution, and better explains observed genomic spatial data. Importantly, the Comet model allows for the clinically relevant scenario of extensive DCIS spread, without being subjected to the selective pressures of subclone competition that promote the emergence of increasingly invasive phenotypes. As such, the normal cell movement inferred during DCIS growth provides a new explanation for the limited risk of progression in DCIS and adds biologic rationale for ongoing clinical efforts to reduce DCIS overtreatment.

cancer biology↗

bioRxiv2023

Unveiling the enigma of Brain-resident immune cells

The immune system has been extensively studied in traditional immune hubs like the spleen and lymph nodes. However, recent advances in immunology highlight unique immune cell characteristics across anatomical compartments. In this study, we challenged conventional thinking by uncovering distinct immune cell populations within the brain parenchyma, separate from those in the blood, meninges, and choroid plexus, with unique transcriptional profiles. Brain-resident immune cells are not derived from maternal immune cells, and age-related changes, with an increase in CD8+ T cells in aged mice, are noted. Alzheimers disease (AD) alters microglias interaction with brain-resident immune cells, emphasizing immune-brain dynamics. Furthermore, we reveal dynamic immune cell interactions and essential cytokine roles in brain homeostasis, with stable cytokine expression but emerging signaling pathways in AD. In summary, this study advances our understanding of brain-resident immune cells in both normal and pathological conditions.

immunology↗

bioRxiv2023

PKC modulator bryostatin-1 therapeutically targets CNS innate immunity to attenuate neuroinflammation and promote remyelination

In multiple sclerosis (MS), microglia and macrophages within the central nervous system (CNS) play an important role in determining the balance between myelin repair and demyelination/neurodegeneration. Phagocytic and regenerative functions of these CNS innate immune cells support remyelination, whereas chronic and maladaptive inflammatory activation promotes lesion expansion and disability, particularly in the progressive forms of MS. No currently approved drugs convincingly target microglia and macrophages within the CNS, contributing to the critical lack of therapies promoting remyelination and slowing progression in MS. Here, we found that the protein kinase C (PKC)-modulating drug bryostatin-1 (bryo-1), a CNS-penetrant compound with an established human safety profile, produces a shift in microglia and CNS macrophage transcriptional programs from pro-inflammatory to regenerative phenotypes, both in vitro and in vivo. Treatment of microglia with bryo-1 prevented the activation of neurotoxic astrocytes while stimulating scavenger pathways, phagocytosis, and secretion of factors that promote oligodendrocyte differentiation. In line with these findings, systemic treatment with bryo-1 augmented remyelination following a focal demyelinating injury in vivo. Our results demonstrate the potential of bryo-1 and functionally related PKC modulators as myelin regenerative and neuroprotective agents in MS and other neurologic diseases through therapeutic targeting of microglia and CNS-associated macrophages. One Sentence SummaryPKC modulation in CNS innate immune cells favors the activation of a beneficial phenotype that promotes myelin regeneration and neuroprotection.

neuroscience↗

bioRxiv2023

TroR is the primary regulator of the iron homeostasis transcription network in the halophilic archaeon Haloferax volcanii

Maintaining intracellular iron concentration within the homeostatic range is vital to meet cellular metabolic needs and reduce oxidative stress. Previous research revealed that the haloarchaeon Halobacterium salinarum encodes four diphtheria toxin repressor (DtxR) family transcription factors (TFs) that together regulate the iron response through an interconnected transcriptional regulatory network (TRN). However, the metal specificity of DtxR TFs and the conservation of the TRN remained poorly understood. Here we identified and characterized the TRN of Haloferax volcanii for comparison. Genetic analysis demonstrated that Hfx. volcanii relies on three DtxR transcriptional regulators (Idr, SirR, and TroR), with TroR as the primary regulator of iron homeostasis. Bioinformatics and molecular approaches revealed that TroR binds a conserved cis-regulatory motif located [~]100 nt upstream of the start codon of iron-related target genes. Transcriptomics analysis demonstrated that, under conditions of iron sufficiency, TroR repressed iron uptake and induced iron storage mechanisms. TroR repressed the expression of one other DtxR TF, Idr. This reduced DtxR TRN complexity relative to that of Hbt. salinarum appeared correlated with natural variations in iron availability. Based on these data, we hypothesize that increasing TRN complexity appears selected for under variable environmental conditions such as iron availability.

microbiology↗

bioRxiv2023

KoNA: Korean Nucleotide Archive as a New Data Repository for Nucleotide Sequence Data

During the last decade, generation and accumulation of petabase-scale high-throughput sequencing data have resulted in ethical and technical challenges, including access to human data, and transfer, storage, and sharing of enormous amount of data. To promote data-driven research in biology, the Korean government announced that all the biological data generated from government-funded research projects should be deposited in the Korea BioData Station (K-BDS), which consists of multiple databases for individual data types. We introduce the Korean Nucleotide Archive (KoNA), a repository for nucleotide sequence data. As of July 2022, the Korean Read Archive in KoNA has collected over 477 TB of raw next generation sequencing data from several national genome projects. To ensure data quality and prepare for international alignment, a standard operating procedure (SOP) was adopted, which is similar to the International Nucleotide Sequence Database Collaboration. The SOP includes quality control processes for submitted data and metadata using an automated pipeline followed by manual examination. To ensure fast and stable data transfer, a high-speed transmission system called GBox is used in KoNA. Furthermore, the data uploaded to or downloaded from KoNA through GBox can be readily processed in a cloud-computing service for genomic data analysis called Bio-Express. This seamless coupling of KoNA, GBox, and Bio-Express enhances data experience including submission, access, and analysis of raw nucleotide sequences. KoNA not only satisfies the unmet needs for a national sequence repository in Korea, but also provides datasets to researchers globally and contribute to advances in genomics. KoNA is available at https://www.kobic.re.kr/kona/.

genomics↗

bioRxiv2022

Heritability of de novo germline mutation reveals a contribution from paternal but not maternal genetic factors

De novo mutations (DNMs) in the germline have long been identified as a key element in the causes of developmental and other genetic disorders. Previous attempts to investigate genetic factors affecting DNMs have suffered from a lack of statistical power, due to the difficulty of obtaining a sufficient number of parent-offspring trios. Thus, the rare disease cohort of the UKs 100k Genomes Project (100kGP), comprising more than 10,000 trios, represents an unprecedented opportunity to investigate the genetics of germline mutation. Here we estimate SNP heritability of DNM count in offspring, as a measure of the relative contribution of genetic factors to the variance of the trait, in a PCA-selected subset of the 100kGP cohort. We estimate separate SNP heritabilities for paternally and maternally transmitted mutations (based on parentally phased DNMs in offspring), computed using parental genetic variants at a range of minimum frequencies and a variety of methodologies. We estimate a heritability of 10-20% for paternal DNMs; by contrast, for maternal DNMs we find no significant evidence for non-zero heritability. We investigated the partitioning of heritability among genes with different expression profiles in different tissue or cell states, and found a relative heritability enrichment for genes expressed in gonadal tissues, particularly testis. Among germ cells in adult testes we observed relative enrichment of heritability in genes associated with the (undifferentiated) spermatogonial stem cell state.

genetics↗

bioRxiv2022

The complete sequence of a human Y chromosome

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure including long palindromes, tandem repeats, and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4, 5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029 base pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, revealing the complete ampliconic structures of TSPY, DAZ, and RBMY gene families; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a prior assembly of the CHM13 genome4 and mapped available population variation, clinical variants, and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

genomics↗

bioRxiv2022

Structure of the Wnt-Frizzled-LRP6 initiation complex reveals the basis for co-receptor discrimination

Wnt morphogens are critical for embryonic development and tissue regeneration. Canonical Wnts form ternary receptor complexes composed of tissue-specific Frizzled receptors together with the shared LRP5/6 co-receptors to initiate {beta}-catenin signaling. The structure of a ternary complex of an affinity-matured XWnt8-Frizzled8-LRP6 complex elucidates the basis of co-receptor discrimination by canonical Wnts by means of their N-termini and linker domains that engage the LRP6 E1E2 domain funnels. Chimeric Wnts bearing modular linker grafts were able to transfer LRP6 domain specificity between different Wnts and enable non-canonical Wnt5a to signal through the canonical pathway. Synthetic peptides comprising the linker domain serve as Wnt-specific antagonists. The structure of the ternary complex provides a topological blueprint for the orientation and proximity of Frizzled and LRP6 within the Wnt cell surface signalosome.

biophysics↗

bioRxiv2022

Therapeutic Potential of Blocking GAPDH Nitrosylation with CGP3466b in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS). Although classically considered a demyelinating disease, neuroaxonal injury occurs in both the acute and chronic phases and represents a pathologic substrate of disability not targeted by current therapies. Nitric oxide (NO) generated by CNS macrophages and microglia contributes to neuroaxonal injury in all phases of MS, but candidate therapies that prevent NO-mediated injury have not been identified. Here, we demonstrate that the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is robustly nitrosylated in the CNS in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. GAPDH nitrosylation is blocked in vivo with daily administration of CGP3466b, a CNS-penetrant compound with an established safety profile in humans. Consistent with the known role of nitrosylated GAPDH (SNO-GAPDH) in neuronal cell death, blockade of SNO-GAPDH with CGP3466b attenuates neurologic disability and reduces axonal injury in EAE independent of effects on the immune system. Our findings suggest that SNO-GAPDH contributes to neuroaxonal injury during neuroinflammation and identify CGP3466b as a candidate neuroprotective therapy in MS.

neuroscience↗