IFDlong: an isoform and fusion detector for accurate annotation and quantification of long-read RNA-seq data.

Advancements in long-read transcriptome sequencing (long-RNA-seq) technology have revolutionized the study of isoform diversity. These full-length transcripts enhance the detection of various transcriptome structural variations, including novel isoforms, alternative splicing events, and fusion transcripts. By shifting the open reading frame or altering gene expressions, studies have proved that these transcript alterations can serve as crucial biomarkers for disease diagnosis and therapeutic targets. In this project, we proposed IFDlong, a bioinformatics and biostatistics tool to detect isoform and fusion transcripts using bulk or single-cell long-RNA-seq data. Specifically, the software performed gene and isoform annotation for each long-read, defined novel isoforms, quantified isoform expression by a novel expectation-maximization algorithm, and profiled the fusion transcripts. For evaluation, IFDlong pipeline achieved overall the best performance when compared with several existing tools in large-scale simulation studies. In both isoform and fusion transcript quantification, IFDlong is able to reach more than 0.8 Spearman's correlation with the truth, and more than 0.9 cosine similarity when distinguishing multiple alternative splicing events. In novel isoform simulation, IFDlong can successfully balance the sensitivity (higher than 90%) and specificity (higher than 90%). Furthermore, IFDlong has proved its accuracy and robustness in diverse in-house and public datasets on healthy tissues, cell lines and multiple types of diseases. Besides bulk long-RNA-seq, IFDlong pipeline has proved its compatibility to single-cell long-RNA-seq data. This new software may hold promise for significant impact on long-read transcriptome analysis. The IFDlong software is available at https://github.com/wenjiaking/IFDlong.

Nuclear GAPDH in cortical microglia mediates cellular stress-induced cognitive inflexibility.

We report a mechanism that underlies stress-induced cognitive inflexibility at the molecular level. In a mouse model under subacute cellular stress in which deficits in rule shifting tasks were elicited, the nuclear glyceraldehyde dehydrogenase (N-GAPDH) cascade was activated specifically in microglia in the prelimbic cortex. The cognitive deficits were normalized with a pharmacological intervention with a compound (the RR compound) that selectively blocked the initiation of N-GAPDH cascade without affecting glycolytic activity. The normalization was also observed with a microglia-specific genetic intervention targeting the N-GAPDH cascade. At the mechanistic levels, the microglial secretion of High-Mobility Group Box (HMGB), which is known to bind with and regulate the NMDA-type glutamate receptors, was elevated. Consequently, the hyperactivation of the prelimbic layer 5 excitatory neurons, a neural substrate for cognitive inflexibility, was also observed. The upregulation of the microglial HMGB signaling and neuronal hyperactivation were normalized by the pharmacological and microglia-specific genetic interventions. Taken together, we show a pivotal role of cortical microglia and microglia-neuron interaction in stress-induced cognitive inflexibility. We underscore the N-GAPDH cascade in microglia, which causally mediates stress-induced cognitive alteration.

Application and perspective of CRISPR/Cas9 genome editing technology in human diseases modeling and gene therapy.

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) mediated Cas9 nuclease system has been extensively used for genome editing and gene modification in eukaryotic cells. CRISPR/Cas9 technology holds great potential for various applications, including the correction of genetic defects or mutations within the human genome. The application of CRISPR/Cas9 genome editing system in human disease research is anticipated to solve a multitude of intricate molecular biology challenges encountered in life science research. Here, we review the fundamental principles underlying CRISPR/Cas9 technology and its recent application in neurodegenerative diseases, cardiovascular diseases, autoimmune related diseases, and cancer, focusing on the disease modeling and gene therapy potential of CRISPR/Cas9 in these diseases. Finally, we provide an overview of the limitations and future prospects associated with employing CRISPR/Cas9 technology for diseases study and treatment.

Population-based screening for colorectal cancer in Wuhan, China.

Fecal DNA test has emerged as a non-invasive alternative for colorectal cancer (CRC) screening in average-risk population. However, there is currently insufficient evidence in China to demonstrate the effectiveness of population-based CRC screening using fecal DNA based test. Here, a large-scale real-world study for CRC screening was implemented in Wuhan, Hubei province, China. A total of 98,683 subjects aged between 45 and 60 years were screened by a fecal DNA test (ColoTect®) which detected methylation status of SDC2, ADHFE1, and PPP2R5C. Participants who tested positive were advised to receive diagnostic colonoscopy. 4449 (4.5%) subjects tested positive for fecal DNA test, and 3200 (71.9%) underwent colonoscopy. Among these, 2347 (73.3%) had abnormal colonoscopy findings, of which 1330 (56.7%) subjects received pathological diagnosis. Detection rates for CRC and advanced precancerous lesions were 1.3% and 2.3%, respectively. Detection rates for nonadvanced adenomas and polyps were 14.0% and 21.6%, respectively. 28.0% of all colonoscopies showed colorectal neoplasm but lack pathological diagnosis. 6.1% showed other abnormalities such as enteritis. In conclusion, preliminary real-world evidence suggested that fecal DNA tests had promising diagnostic yield in population-based CRC screening. Clinical trial registration: https://www.chictr.org.cn/showproj.html?proj=192838, identifier ChiCTR2300070520.

Myoglobin modulates the Hippo pathway to promote cardiomyocyte differentiation.

The endogenous mechanisms that propagate cardiomyocyte differentiation and prevent de-differentiation remain unclear. While the expression of the heme protein myoglobin increases by over 50% during cardiomyocyte differentiation, a role for myoglobin in regulating cardiomyocyte differentiation has not been tested. Here, we show that deletion of myoglobin in cardiomyocyte models decreases the gene expression of differentiation markers and stimulates cellular proliferation, consistent with cardiomyocyte de-differentiation. Mechanistically, the heme prosthetic group of myoglobin catalyzes the oxidation of the Hippo pathway kinase LATS1, resulting in phosphorylation and inactivation of yes-associated protein (YAP). In vivo, myoglobin-deficient zebrafish hearts show YAP dephosphorylation and accelerated cardiac regeneration after apical injury. Similarly, myoglobin knockdown in neonatal murine hearts shows increased YAP dephosphorylation and cardiomyocyte cycling. These data demonstrate a novel role for myoglobin as an endogenous driver of cardiomyocyte differentiation and highlight myoglobin as a potential target to enhance cardiac development and improve cardiac repair and regeneration.

Porcine Kidney Organoids Derived from Naïve-like Embryonic Stem Cells.

The scarcity of donor kidneys greatly impacts the survival of patients with end-stage renal failure. Pigs are increasingly becoming potential organ donors but are limited by immunological rejection. Based on the human kidney organoid already established with the CHIR99021 and FGF9 induction strategy, we generated porcine kidney organoids from porcine naïve-like ESCs (nESCs). The derived porcine organoids had a tubule-like constructure and matrix components. The porcine organoids expressed renal markers including AQP1 (proximal tubule), WT1 and PODO (podocyte), and CD31 (vascular endothelial cells). These results imply that the organoids had developed the majority of the renal cell types and structures, including glomeruli and proximal tubules. The porcine organoids were also identified to have a dextran absorptive function. Importantly, porcine organoids have a certain abundance of vascular endothelial cells, which are the basis for investigating immune rejection. The derived porcine organoids might serve as materials for immunosuppressor screening for xenotransplantation.

Reduced acetylation of TFAM promotes bioenergetic dysfunction in the failing heart.

General control of amino acid synthesis 5-like 1 (GCN5L1) was previously identified as a key regulator of protein lysine acetylation in mitochondria. Subsequent studies demonstrated that GCN5L1 regulates the acetylation status and activity of mitochondrial fuel substrate metabolism enzymes. However, the role of GCN5L1 in response to chronic hemodynamic stress is largely unknown. Here, we show that cardiomyocyte-specific GCN5L1 knockout mice (cGCN5L1 KO) display exacerbated heart failure progression following transaortic constriction (TAC). Mitochondrial DNA and protein levels were decreased in cGCN5L1 KO hearts after TAC, and isolated neonatal cardiomyocytes with reduced GCN5L1 expression had lower bioenergetic output in response to hypertrophic stress. Loss of GCN5L1 expression led to a decrease in the acetylation status of mitochondrial transcription factor A (TFAM) after TAC in vivo, which was linked to a reduction in mtDNA levels in vitro. Together, these data suggest that GCN5L1 may protect from hemodynamic stress by maintaining mitochondrial bioenergetic output.

Species origin of exogenous transcription factors affects the activation of endogenous pluripotency markers and signaling pathways of porcine induced pluripotent stem cells.

The incomplete silencing of exogenous transcription factors (TFs) and the lack of endogenous counterpart activation hampers the application of porcine induced pluripotent stem cells (piPSCs). We used porcine, bovine and murine TFs to reprogram porcine fetal fibroblasts. Porcine TFs-derived piPSCs (ppiPSCs) showed the highest levels of endogenous pluripotency markers activation, were able to differentiate into three germ layers and primordial germ cell-like cells (PGCLCs) and integrated into neural ectoderm of E7.5 mouse embryos in vitro. The bovine TFs derived piPSCs (bpiPSCs) expressed endogenous pluripotency markers higher than murine TFs derived piPSCs (mpiPSCs), but both had limited differentiation ability in vitro and depended on continuous expression of exogenous TFs for the maintenance. RNA sequencing confirmed ppiPSCs had distinct global transcriptional profiling, upregulated Hippo, PI3K-Akt, MAPK and relevant pluripotency signaling pathways as porcine blastocyst inner cell mass and expressed PGC early related genes. In addition, a positive and a negative correlation between exogenous and endogenous TFs' expression level were observed in ppiPSCs and bpiPSCs lines, respectively. The TFs' protein structures in pig were more similar to cattle than to mouse. In conclusion, the species affinity of the exogenous TFs is a key element, and the own species origin of TFs is optimal for iPSCs generation and application.

Mechanistic insights into DNA binding and cleavage by a compact type I-F CRISPR-Cas system in bacteriophage.

CRISPR-Cas systems are widespread adaptive antiviral systems used in prokaryotes. Some phages, in turn, although have small genomes can economize the use of genetic space to encode compact or incomplete CRISPR-Cas systems to inhibit the host and establish infection. Phage ICP1, infecting Vibrio cholerae, encodes a compact type I-F CRISPR-Cas system to suppress the antiphage mobile genetic element in the host genome. However, the mechanism by which this compact system recognizes the target DNA and executes interference remains elusive. Here, we present the electron cryo-microscopy (cryo-EM) structures of both apo- and DNA-bound ICP1 surveillance complexes (Aka Csy complex). Unlike most other type I surveillance complexes, the ICP1 Csy complex lacks the Cas11 subunit or a structurally homologous domain, which is crucial for dsDNA binding and Cas3 activation in other type I CRISPR-Cas systems. Structural and functional analyses revealed that the compact ICP1 Csy complex alone is inefficient in binding to dsDNA targets, presumably stalled at a partial R-loop conformation. The presence of Cas2/3 facilitates dsDNA binding and allows effective dsDNA target cleavage. Additionally, we found that Pseudomonas aeruginosa Cas2/3 efficiently cleaved the dsDNA target presented by the ICP1 Csy complex, but not vice versa. These findings suggest a unique mechanism for target dsDNA binding and cleavage by the compact phage-derived CRISPR-Cas system.

Myocardial brain-derived neurotrophic factor regulates cardiac bioenergetics through the transcription factor Yin Yang 1.

AIMS: Brain-derived neurotrophic factor (BDNF) is markedly decreased in heart failure patients. Both BDNF and its receptor, tropomyosin-related kinase receptor (TrkB), are expressed in cardiomyocytes; however, the role of myocardial BDNF signalling in cardiac pathophysiology is poorly understood. Here, we investigated the role of BDNF/TrkB signalling in cardiac stress response to exercise and pathological stress. METHODS AND RESULTS: We found that myocardial BDNF expression was increased in mice with swimming exercise but decreased in a mouse heart failure model and human failing hearts. Cardiac-specific TrkB knockout (cTrkB KO) mice displayed a blunted adaptive cardiac response to exercise, with attenuated upregulation of transcription factor networks controlling mitochondrial biogenesis/metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). In response to pathological stress (transaortic constriction, TAC), cTrkB KO mice showed an exacerbated heart failure progression. The downregulation of PGC-1α in cTrkB KO mice exposed to exercise or TAC resulted in decreased cardiac energetics. We further unravelled that BDNF induces PGC-1α upregulation and bioenergetics through a novel signalling pathway, the pleiotropic transcription factor Yin Yang 1. CONCLUSION: Taken together, our findings suggest that myocardial BDNF plays a critical role in regulating cellular energetics in the cardiac stress response.

Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2.

A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.

GPER-dependent estrogen signaling increases cardiac GCN5L1 expression.

Reversible lysine acetylation regulates the activity of cardiac metabolic enzymes, including those controlling fuel substrate metabolism. Mitochondrial-targeted GCN5L1 and SIRT3 have been shown to regulate the acetylation status of mitochondrial enzymes, but the role that lysine acetylation plays in driving metabolic differences between male and female hearts is not currently known. In this study, we describe a significant difference in GCN5L1 levels between male and female mouse hearts, and in the hearts of women between post- and premenopausal age. We further find that estrogen drives GCN5L1 expression in a cardiac cell line and uses pharmacological approaches to determine the mechanism to be G protein-coupled estrogen receptor (GPER) activation, via translational regulation.NEW & NOTEWORTHY We demonstrate here for the first time that mitochondrial protein acetylation is increased in female hearts, associated with an increase in GCN5L1 levels through a GPER-dependent mechanism. These findings reveal a new potential mediator of divergent cardiac mitochondrial function between men and women.

Diet-induced obese mice are resistant to improvements in cardiac function resulting from short-term adropin treatment.

Previous studies have shown that treatment with recombinant adropin, a circulating peptide secreted by the liver and brain, restores glucose utilization in the hearts of diet-induced obese mice. This restoration of fuel substrate flexibility, which is lost in obese and diabetic animals, has the potential to improve contractile function in the diabetic heart. Using an ex vivo approach, we examined whether short-term adropin treatment could enhance cardiac function in a mouse model of diet-induced obesity. Our study showed that acute adropin treatment reduces inhibitory phosphorylation of pyruvate dehydrogenase in primary neonatal cardiomyocytes, and leads to moderate improvements in ex vivo cardiac function in mice fed a low fat diet. Conversely, short-term exposure to adropin led to a small decrease in cardiac function in mice fed a long-term high fat diet. Insulin treatment did not significantly alter cardiac function in adropin treated hearts from either low or high fat diet mice, however acute adropin treatment did moderately restore some aspects of downstream insulin signaling in high fat diet fed mice. Overall, these data suggest that in an ex vivo setting, acute adropin treatment alone is not sufficient to promote improved cardiac function in obese animals.

The mitochondrial regulator PGC1α is induced by cGMP-PKG signaling and mediates the protective effects of phosphodiesterase 5 inhibition in heart failure.

Phosphodiesterase 5 inhibition (PDE5i) activates cGMP-dependent protein kinase (PKG) and ameliorates heart failure; however, its impact on cardiac mitochondrial regulation has not been fully determined. Here, we investigated the role of the mitochondrial regulator peroxisome proliferator-activated receptor γ co-activator-1α (PGC1α) in the PDE5i-conferred cardioprotection, utilizing PGC1α null mice. In PGC1α+/+ hearts exposed to 7 weeks of pressure overload by transverse aortic constriction, chronic treatment with the PDE5 inhibitor sildenafil improved cardiac function and remodeling, with improved mitochondrial respiration and upregulation of PGC1α mRNA in the myocardium. By contrast, PDE5i-elicited benefits were abrogated in PGC1α-/- hearts. In cultured cardiomyocytes, PKG overexpression induced PGC1α, while inhibition of the transcription factor CREB abrogated the PGC1α induction. Together, these results suggest that the PKG-PGC1α axis plays a pivotal role in the therapeutic efficacy of PDE5i in heart failure.

A Novel TLR4-SYK Interaction Axis Plays an Essential Role in the Innate Immunity Response in Bovine Mammary Epithelial Cells.

Mammary gland epithelium, as the first line of defense for bovine mammary gland immunity, is crucial in the process of mammary glands' innate immunity, especially that of bovine mammary epithelial cells (bMECs). Our previous studies successfully marked SYK as an important candidate gene for mastitis traits via GWAS and preliminarily confirmed that SYK expression is down-regulated in bMECs with LPS (E. coli) stimulation, but its work mechanism is still unclear. In this study, for the first time, in vivo, TLR4 and SYK were colocalized and had a high correlation in mastitis mammary epithelium; protein−protein interaction results also confirmed that there was a direct interaction between them in mastitis tissue, suggesting that SYK participates in the immune regulation of the TLR4 cascade for bovine mastitis. In vitro, TLR4 also interacts with SYK in LPS (E. coli)-stimulated or GBS (S. agalactiae)-infected bMECs, respectively. Moreover, TLR4 mRNA expression and protein levels were little affected in bMECsSYK- with LPS stimulation or GBS infection, indicating that SYK is an important downstream element of the TLR4 cascade in bMECs. Interestingly, IL-1ß, IL-8, NF-κB and NLRP3 expression in LPS-stimulated or GBS-infected bMECsSYK- were significantly higher than in the control group, while AKT1 expression was down-regulated, implying that SYK could inhibit the IL-1ß, IL-8, NF-κB and NLRP3 expression and alleviate inflammation in bMECs with LPS and GBS. Taken together, our solid evidence supports that TLR4/SYK/NF-κB signal axis in bMECs regulates the innate immunity response to LPS or GBS.

Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States.

The inner cell mass of the pre-implantation blastocyst consists of the epiblast and hypoblast from which embryonic stem cells (ESCs) and extra-embryonic endoderm (XEN) stem cells, respectively, can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of its in vivo tissue origin. We have developed a novel approach for deriving porcine XEN (pXEN) cells via culturing the blastocysts with a chemical cocktail culture system. The pXEN cells were positive for XEN markers, including Gata4, Gata6, Sox17, and Sall4, but not for pluripotent markers Oct4, Sox2, and Nanog. The pXEN cells also retained the ability to undergo visceral endoderm (VE) and parietal endoderm (PE) differentiation in vitro. The maintenance of pXEN required FGF/MEK+TGFß signaling pathways. The pXEN cells showed a stable phenotype through more than 50 passages in culture and could be established repeatedly from blastocysts or converted from the naïve-like ESCs established in our lab. These cells provide a new tool for exploring the pathways of porcine embryo development and differentiation and providing further reference to the establishment of porcine ESCs with potency of germline chimerism and gamete development.

Generation of Urine-Derived Induced Pluripotent Stem Cell Line from Patients with Acute Kidney Injury.

Acute kidney injury (AKI) is mainly characterized by rapid decline of renal function. Currently, the strategy of stem cells might be a therapy to treat AKI. The objective of this study was to obtain human urine-derived cells (HUCs) from patients with AKI, followed by establishing induced pluripotent stem (iPS) cell line. We isolated urine cells from patients with AKI and found that the cells could survive long term with epithelioid morphology and maintain a normal karyotype. The cell line had expression of renal-specific markers and renal development-related genes. After induction, the urine cells cotransfecting with TET-ON vectors were converted into iPS cells. The HUC-derived iPS (HUC-iPS) was positive for alkaline phosphatase staining, and had expression of pluripotency markers, consistent with human embryonic fibroblast-derived iPS cell. Notably, HUC-iPS could be induced to undergo directional kidney precursor cells (KPCs) differentiation under defined conditions, and transplantation of KPCs resulted in reducing kidney damage from ischemia-reperfusion injury in mice. Therefore, we successfully established HUC-iPS cell from patients with AKI and provided a novel stem cell resource for cell therapy in AKI.

SIX1 Activation Is Involved in Cell Proliferation, Migration, and Anti-inflammation of Acute Ischemia/Reperfusion Injury in Mice.

Nephrogenic proteins are re-expressed after ischemia/reperfusion (I/R) injury; however, the role of these proteins is still unknown. We found that sine oculis homeobox 1 (SIX1), a developmentally regulated homeoprotein, is reactivated in tubular epithelial cells after I/R injury associated with cell proliferation/migration and anti-inflammation. We demonstrated that SIX1 promoted cell proliferation by upregulating cyclin and glycolytic genes, and might increase cell migration by upregulating the expression of matrix metalloproteinase 9 (MMP9) directly or indirectly in the cell model. Notably, SIX1 targeted the promoters of the amino-terminal enhancer of split (AES) and fused in sarcoma (FUS), which are cofactors of nuclear factor-κB (NF-κB) subunit RELA, and then inhibited the transactivation function of RELA. The expression of monocyte chemotactic protein-1 (MCP-1) was decreased by the SIX1-mediated NF-κB pathway. Our results showed that the expression of cyclin, glycolytic genes, and MMP9 were significantly increased, and the infiltration of monocytes/macrophages (Mophs) was suppressed in SIX1 overexpression kidney at 1, 2, and 3 days after reperfusion. The overexpression of SIX1 resulted in reducing kidney damage from I/R injury in mice by promoting cell proliferation and migration and by inhibiting inflammation. Our study provides evidence that SIX1 involved in cell proliferation, migration, and anti-inflammation in the I/R model, which might be a potential therapeutic target that could be used to ameliorate kidney damage.

Outpatient Intravenous Diuretic Clinic: An Effective Strategy for Management of Volume Overload and Reducing Immediate Hospital Admissions.

BACKGROUND: Heart failure (HF) readmissions pose a major burden to patients and the healthcare system. We evaluated whether outpatient intravenous (IV) diuretic clinic is a safe and effective strategy to reduce HF hospitalizations. METHODS: We reviewed 34 clinic encounters with 27 unique patients (median age 72) who had volume overload refractory to oral diuretics that were treated with IV furosemide in the outpatient clinic. One patient (2.9%) was admitted to the hospital directly, and the rest were discharged home. RESULTS: More than 80% of the patients had continued weight loss for 7 days (median weight loss 5.4 lb). During the median follow-up period of 15 months, 15 patients (56%) had subsequent HF hospitalizations. HF admission was delayed by a median of 22 days from the clinic visit and 138 days from the previous HF admission prior to clinic visit. Estimated cost saving per admission avoided was $10,395. One patient developed severe hypokalemia (< 3.0 mmol/L), and the remaining had no adverse events. CONCLUSION: Outpatient IV diuresis is effective and well tolerated. It leads to significant weight loss, persisting in the majority of patients for 7 days. In select patients, it should be considered as a strategy to rapidly improve symptoms, reduce hospitalizations and decrease costs.

USF3 modulates osteoporosis risk by targeting WNT16, RANKL, RUNX2, and two GWAS lead SNPs rs2908007 and rs4531631.

Osteoporotic fractures cause major morbidity and mortality in the aging population. Genome-wide association studies (GWAS) have identified USF3 as the novel susceptibility gene of osteoporosis. However, the functional role in bone metabolism and the target gene of the basic helix-loop-helix transcription factor USF3 are unclear. Here, we show that USF3 enhances osteoblast differentiation and suppresses osteoclastogenesis in cultured human osteoblast-like U-2OS cells. Mechanistic studies revealed that transcription factor USF3 antagonistically interacts with anti-osteogenic TWIST1/TCF12 heterodimer in the WNT16 and RUNX2 promoter, and counteracts CREB1 and JUN/FOS in the RANKL promoter. Importantly, the osteoporosis GWAS variant g.1744A>G (rs2908007A>G) located in the WNT16 promoter confers G-allele-specific transcriptional modulation by USF3, TWIST1/TCF12 and TBX5/TBX15, and USF3 transactivates the osteoclastogenesis suppressor WNT16 promoter activity and antagonizes the repression of WNT16 by TWIST1 and TCF12. The risk G allele of osteoporosis GWAS variant g.3260A>G (rs4531631A>G) in the RANKL promoter facilitates the binding of CREB1 and JUN/FOS and enhances transactivation of the osteoclastogenesis contributor RANKL that is inhibited by USF3. Our findings uncovered the functional mechanisms of osteoporosis novel GWAS-associated gene USF3 and lead single nucleotide polymorphisms rs2908007 and rs4531631 in the regulation of bone formation and resorption.