Biology subjects

Shih, Y.-H.

Publications and source records attributed to Shih, Y.-H..

8 recordsLinked to original sources

bioRxiv2025

KRAS withdrawal in Cholangiocarcinoma leads to immune infiltration and tumor regression

Background and AimsCholangiocarcinoma (CCA) is a liver cancer with poor survival rates. Current treatments, including targeted therapies for specific mutations, are limited and benefit only a small subset of patients. KRAS mutations are found in 15-40% of CCA, representing a new potential treatment target. Whether KRAS inhibition leads to CCA tumor regression is unknown partly due to the lack of conditional animal models. Approach and ResultsWe engineered a conditional KrasG12D-driven CCA mouse model by co-delivering plasmids encoding the Sleeping Beauty transposase with a luciferase reporter, a transposon-borne inducible KrasG12D transgene and Cas9 and Trp53 guide RNA into mouse liver by hydrodynamic tail-vein injection. In vivo bioluminescent imaging showed that KrasG12D withdrawal resulted in 99% tumor regression by day 7. KrasG12D withdrawal resulted in infiltration of activated CD8+ T cells by IHC and IF staining. Single cell RNA-Seq result also validated the enrichment of activated CD8+ T cells subpopulation in KrasG12D-withdrawn tumor. RNA-Seq suggested that KrasG12D withdrawal stimulated transforming growth factor beta pathway and induced senescence. We used cytokine array to characterize the secretion of pro-inflammatory factors, including IL-15 and Ccl17, upon KrasG12D withdrawal. Lentiviral overexpression of murine CCL17 delayed CCA tumor progression in a xenograft model, and overexpression of murine IL-15 resulted in tumor regression in a transplant model. Flow cytometry analysis revealed that IL-15 and CCL17 recruited and activated CD8+ T cells in CCA tumor. Expression of IL-15 resulted in blockade of tumor progression in our TKP CCA model. ConclusionsKrasG12D withdrawal results in rapid tumor regression, highlighting the importance of oncogenic Kras in CCA tumor maintenance. KrasG12D withdrawal induces p53-independent senescence, secretion of pro-inflammatory factors, and immune surveillance by activated CD8+ T cells. We identified two secreted factors IL-15 and CCL17 that could recruit and activate T cells and control CCA tumor progression. This study underscores KRAS inhibition as a potential therapeutic approach for CCA.

cancer biology↗

bioRxiv2025

A Drosophila model for Dent disease reveals impaired ER export of Cubilin as pathogenic mechanism

Mutations in the CLCN5 gene encoding the chloride-hydrogen exchanger ClC-5 cause Dents disease, a genetic disorder of the endolysosomal pathway in the proximal tubules of the kidneys. Many patients also develop glomerular lesions, but the underlying mechanism is unclear. We have established an in vivo model for Dents disease using Drosophila nephrocytes that share similarities with podocytes and proximal tubular cells. Upon depletion of ClC-c, the fly homologue of CLCN5, the endocytic receptor Cubilin was lost from the cortex of nephrocytes, which led to a strong decrease in albumin uptake and slit diaphragm (SD) turnover. Moreover, the actin and microtubular cytoskeleton as well as Rab11-marked recycling endosomes showed a strong cortical accumulation, whereas cholesterol-enriched autophagic compartments emerged in the perinuclear area. Cubilin exhibited a mild mislocalization to cortical early and late endosomal compartments and, in addition, strongly accumulated in the endoplasmic reticulum (ER). This was accompanied by a fragmentation of the ER morphology and an increase in ER exit sites and associated Golgi stacks. These secretory pathway phenotypes were also observed upon silencing of a subunit of the vacuolar H+-ATPase (V-ATPase) suggesting that they depend on acidification. Therefore, we speculate that ClC-c and the V-ATPase together acidify the Golgi to allow proper glycosylation and surface trafficking of Cubilin (or its binding partner Amnionless). Interestingly, ER retention of Cubilin was confirmed in ClC-5 knockout mice, underscoring the relevance of this pathomechanism for Dents disease. Translational statementIn this work, we study the function of the fly ortholog of CLCN5 whose mutations cause Dents disease, a devastating hereditary kidney disease. By demonstrating that the protein uptake receptor Cubilin is retained in the ER upon ClC-c/ClC-5 depletion in flies and mice, we provide an unexpected new disease mechanism for this disease. Future therapeutic strategies may be directed at improving ER export through acidification of the Golgi apparatus.

cell biology↗

bioRxiv2025

Arabidopsis REM transcription factors and GDE1 shape the DNA methylation landscape through the recruitment of RNA Polymerase IV transcription complexes.

DNA methylation plays crucial roles in gene regulation and transposon silencing. In plants, the maintenance of DNA methylation is controlled by several self-reinforcing loops involving histone methylation and non-coding RNAs. However, how methylation is initially patterned at specific loci is unknown. Here, we describe four Arabidopsis REM transcription factors, VDD, VAL, REM12 and REM13, that recognize specific sequence regions, and together with the protein GENETICS DETERMINES EPIGENETICS1 (GDE1), recruit RNA polymerase IV transcription complexes to generate 24-nucleotide small interfering RNAs (24nt-siRNAs) that guide DNA methylation to specific loci. In the absence of GDE1, Pol IV transcription complexes redistribute to sites bound by a different factor called REM8. These results suggest that REM proteins act as sequence specific DNA binding proteins that pattern siRNAs and methylation at specific sites in the genome, highlighting the role of genetic information in determining epigenetic patterns.

plant biology↗

bioRxiv2023

Arabidopsis TRB proteins function in H3K4me3 demethylation by recruiting JMJ14

Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.

plant biology↗

bioRxiv2022

Arabidopsis histone H3 lysine 9 methyltransferases KYP/SUVH5/6 are involved in leaf development by interacting with AS1-AS2 to repress KNAT1 and KNAT2

Arabidopsis KRYPTONITE/SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG 4 (KYP/SUVH4), SUVH5 and SUVH6 are H3K9 methyltransferases and they are redundantly involved in silencing of transposable elements (TEs). A recent study indicated that KYP/SUVH5/6 can directly interact with the histone deacetylase HDA6 to synergistically regulate TE expression. However, the function of KYP/SUVH5/6 in plant development is still unclear. The ASYMMETRIC LEAVES1 (AS1) and AS2 form a transcription complex, which is involved in leaf development by repressing the homeobox genes KNOTTED-LIKE FROM ARABIDOPSIS THALIANA 1 (KNAT1) and KNAT2. In this study, we found that KYP and SUVH5/6 directly interact with AS1-AS2 to repress KNAT1 and KNAT2 by altering histone H3 acetylation and H3K9 dimethylation levels. In addition, KYP can directly target on the promoters of KNAT1 and KNAT2, and the binding of KYP is dependent on AS1. Furthermore, the genome-wide occupancy profile of KYP indicated that KYP is enriched in the promoter regions of coding genes, and the binding of KYP is positively correlated with that of AS1 and HDA6. Together, these results indicate that Arabidopsis H3K9 methyltransferases KYP/SUVH5/6 are involved in leaf development by interacting with AS1-AS2 to alter histone H3 acetylation and H3K9 dimethylation from the KNAT1 and KNAT2 loci.

plant biology↗

bioRxiv2021

Discovering Novel Antimicrobial Peptides in Generative Adversarial Network

Due to the growing number of clinical antibiotic resistance cases in recent years, novel antimicrobial peptides (AMPs) can become ideal for next-generation antibiotics. This study trained a deep convolutional generative adversarial network (GAN) with known AMPs to generate novel AMP candidates. The quality of the GAN-designed peptides was evaluated in silico, and eight of them named GAN-pep 1[~]8 were chosen to be synthesized for further experiments. Disk diffusion testing and minimum inhibitory concentration (MIC) determination were used to determine the antibacterial effects of the synthesized GAN-designed peptides. Seven out of the eight synthesized GAN-designed peptides showed antibacterial activities. Additionally, GAN-pep 3 and GAN-pep 8 had a broad spectrum of antibacterial effects. Both of them were also effective against antibiotic-resistant bacteria strains such as methicillin-resistant Staphylococcus aureus (S. aureus) and carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa). GAN-pep 3, the most promising GAN-designed peptide candidate, had low MICs against all the tested bacteria.

microbiology↗

bioRxiv2021

Integrated molecular analysis identifies a conserved pericyte gene signature in zebrafish

Pericytes reside in capillary beds where they share a basement membrane with endothelial cells and regulate their function. However, little is known about embryonic pericyte development, in part, due to lack of specific molecular markers and genetic tools. Here, we applied single cell RNA-sequencing (scRNA-seq) of platelet derived growth factor beta (pdgfrb)-positive cells to molecularly characterize pericytes in zebrafish larvae. scRNA-seq revealed zebrafish cells expressing mouse pericyte gene orthologs while comparison to bulk RNA-seq from wild type and pdgfrb mutant larvae further refined a pericyte geneset. Subsequent integration with mouse pericyte scRNA-seq profiles revealed a core set of conserved pericyte genes. Using transgenic reporter lines, we validated pericyte expression of two genes identified in our analysis: NDUFA4 mitochondrial complex associated like 2a (ndufa4l2a), and potassium voltage-gated channel, Isk-related family, member 4 (kcne4). Both reporter lines exhibited pericyte expression in multiple anatomical locations, while kcne4 was also detected in a subset of vascular smooth muscle cells. Thus, our integrated molecular analysis revealed a molecular profile for zebrafish pericytes and allowed us to develop new tools to observe these cells in vivo. SUMMARY STATEMENTAn integrated molecular analysis in zebrafish identifies new molecular markers for pericytes.

developmental biology↗

bioRxiv2021

Conserved and context-dependent roles for Pdgfrb signaling during zebrafish vascular mural cell development

Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects. Summary statementGenetic analysis in zebrafish demonstrates the conserved role of Pdgfb/Pdgfrb signaling in pericyte and vascular smooth muscle cell formation during vascular development in vertebrates.

developmental biology↗