技术资料

Erythroid Krüppel-like factor (EKLF) is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf(-/-)) mice die at day 14.5 of gestation from severe anemia. In this study,we demonstrate that early progenitor cells fail to undergo terminal erythroid differentiation in Eklf(-/-) embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis,transcriptional profiling was performed with RNA from wild-type and Eklf(-/-) early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation,with the critical regulator of the cell cycle,E2f2,at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf(-/-) early erythroid progenitor cells,which showed a delay in the G(1)-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier,EKLF binding sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation. View Publication

Focal adhesion kinase (FAK) is up-regulated in thyroid cancer and small molecule FAK scaffolding inhibitor,Y15,was shown to decrease cancer growth in vitro and in vivo. We sought to test the effectiveness of Y15 in thyroid cancer cell lines,profile gene expression with Y15 compared with clinical trial FAK inhibitor PF-04554878,and use Y15 in novel drug combinations. Cell viability was decreased in a dose dependent manner in four thyroid cancer cell lines with Y15 and with higher doses in PF-04554878. Y397 FAK and total FAK were decreased with Y15 and decreased less with PF-04554878. Detachment and necrosis were increased in a dose-dependent manner in all cell lines with Y15. Clonogenicity was decreased in a dose-dependent manner for both Y15 and PF-04554878. We compared gene profiles between papillary thyroid cell lines,TPC1,BCPAP and K1,and 380,109,and 74 genes were significantly textgreater2-fold changed with Y15 treatment,respectively. Common up-regulated genes were involved in apoptosis,cell cycle,transcription and heat shock; down-regulated genes were involved in cell cycle,cell-to-cell interactions,and cancer stem cell markers. We also compared gene profiles of TT cells treated with Y15 versus PF-04554878. Y15 caused 144 genes to change over 4 fold and PF-04554878 caused 208 gene changes textgreater4-fold (ptextless0.05). Among genes changed 4 fold,11 were shared between the treatments,including those involved in metabolism,cell cycle,migration and transcription. Y15 demonstrated synergy with PF-04554878 in TT cells and also synergy with Cabozantinib,Sorafenib,Pazopanib,and strong synergy with Sunitinib in resistant K1 cells. This report revealed the biological effect of Y15 inhibitor,detected the unique and common gene signature profiles in response to Y15 in 4 different thyroid cancer cell lines,demonstrated differential response changes with Y15 and PF-04554878 treatment,and showed the synergy of Y15 with PF-04554878,Cabozantinib,Sorafenib,Pazopanib,and Sunitinib. View Publication

We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse,we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding,at the molecular level,how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here,we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally,we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization,as well as T cell migration in vitro. Altogether,our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility. View Publication

A diverse antibody repertoire is essential for humoral immunity. Antibody diversification requires the introduction of deoxyuridine (dU) mutations within immunoglobulin genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR). dUs are normally recognized and excised by the base excision repair (BER) protein uracil-DNA glycosylase 2 (UNG2). However,FAM72A downregulates UNG2 permitting dUs to persist and trigger SHM and CSR. How FAM72A promotes UNG2 degradation is unknown. Here,we show that FAM72A recruits a C-terminal to LisH (CTLH) E3 ligase complex to target UNG2 for proteasomal degradation. Deficiency in CTLH complex components result in elevated UNG2 and reduced SHM and CSR. Cryo-EM structural analysis reveals FAM72A directly binds to MKLN1 within the CTLH complex to recruit and ubiquitinate UNG2. Our study further suggests that FAM72A hijacks the CTLH complex to promote mutagenesis in cancer. These findings show that FAM72A is an E3 ligase substrate adaptor critical for humoral immunity and cancer development. Antibody diversification relies on the intentional mutagenesis of immunoglobulin genes for adaptive immune responses. Here,the authors identified a CTLH E3 ubiquitin ligase complex that co-opts FAM72A to recruit and degrade the UNG2 base excision repair factor to permit mutagenesis. View Publication

Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood,aside from numerous congenital abnormalities. FA mouse models have been generated; however,they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero,a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology,we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential,disruption of the second allele causes a severe growth disadvantage. As a result,heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele,quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning,this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage. ?? 2014. View Publication

BackgroundFANCA mutations have been detected in a variety of cancers and found to be pro-carcinogenic. However,no functional studies have been identified regarding the involvement of FANCA in the occurrence and the immune response of LUAD.MethodsThe mRNA expression and overall survival rates of FANCA were evaluated by the TIMER,PrognoScan and TCGA database in LUAD tissues,and FANCA expression was further validated by clinical serum samples using ELISA. The correlation between FANCA and immune infiltration level was investigated via TISIDB database and CIBERSORT algorithm. The Kaplan–Meier plotter was used to further evaluate the prognostic value based on the expression levels of FANCA in related immune cells. Then,the influence of FANCA knockout on the proliferation,migration,and invasion of A549 and H1299 cells was validated using CCK8,cloning formation,and Transwell assays. Subsequently,HLA-A2-restricted FANCA antigenic peptides were predicted and synthesized by NetMHC4.0 and SYFPEITHI,and DCs were induced and cultured in vitro. Finally,DCs loaded with HLA-A2-restricted FANCA antigenic peptides were co-cultured with autologous peripheral blood lymphocyte to generate specific CTLs. The killing effects of different CTLs on LUAD cells were studied.ResultsThe results showed that high levels of FANCA in patients with LUAD were significantly correlated with worse OS survival,which was correlated with age,clinical stage,pathological T stage,M stage,and N stage in LUAD. Knockdown of FANCA in A549 and H1299 cells significantly inhibited proliferation,metastasis,and invasion in vitro. In addition,FANCA was significantly related to immune infiltrate,genomic alterations and TMB. FANCA expression infuenced the prognosis of LUAD patients by directly affecting immune cell infltration. Finally,HLA-A2-restricted FANCA antigenic peptides were synthesized. And FANCA 146–154 (SLLEFAQYL) antigenic peptide exhibit a stronger affinity for DCs,and induce CTLs to produce stronger targeted killing ability for LUAD cells at an effector-to-target ratio of 40:1.ConclusionThese results demonstrated that the elevation of FANCA promotes malignant phenotype of LUAD,and the potential peptide P2 (SLLEFAQYL) derived from FANCA may be used as an epitope vaccine for the treatment of LUAD. View Publication

Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure,we found that Fancd2(-/-) mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit(+)Sca-1(+)Lineage(-) (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2(-/-) KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition,the supportive function of the marrow microenvironment was compromised in Fancd2(-/-) mice. Treatment with Sirt1-mimetic and the antioxidant drug,resveratrol,maintained Fancd2(-/-) KSL cells in quiescence,improved the marrow microenvironment,partially corrected the abnormal cell cycle status,and significantly improved the spleen colony-forming capacity of Fancd2(-/-) bone marrow cells. We conclude that Fancd2(-/-) mice have readily quantifiable hematopoietic defects,and that this model is well suited for pharmacologic screening studies. View Publication

Although brain development abnormalities and brain cancer predisposition have been reported in some Fanconi patients,the possible role of Fanconi DNA repair pathway during neurogenesis is unclear. We thus addressed the role of fanca and fancg,which are involved in the activation of Fanconi pathway,in neural stem and progenitor cells during brain development and adult neurogenesis. Fanca(-/-) and fancg(-/-) mice presented with microcephalies and a decreased neuronal production in developing cortex and adult brain. Apoptosis of embryonic neural progenitors,but not that of postmitotic neurons,was increased in the neocortex of fanca(-/-) and fancg(-/-) mice and was correlated with chromosomal instability. In adult Fanconi mice,we showed a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cells exhaustion with ageing. In addition,embryonic and adult Fanconi neural stem cells showed a reduced capacity to self-renew in vitro. Our study demonstrates a critical role for Fanconi pathway in neural stem and progenitor cells during developmental and adult neurogenesis. View Publication

The airways are lined by secretory and multiciliated cells which function together to remove particles and debris from the respiratory tract. The transcriptome of multiciliated cells has been extensively studied,but the function of many of the genes identified is unknown. We have established an assay to test the ability of over-expressed transcripts to promote multiciliated cell differentiation in mouse embryonic tracheal explants. Overexpression data indicated that Fibronectin type 3 and ankyrin repeat domains 1 (Fank1) and JAZF zinc finger 1 (Jazf1) promoted multiciliated cell differentiation alone,and cooperatively with the canonical multiciliated cell transcription factor Foxj1. Moreover,knock-down of Fank1 or Jazf1 in adult mouse airway epithelial cultures demonstrated that these factors are both required for ciliated cell differentiation in vitro This analysis identifies Fank1 and Jazf1 as novel regulators of multiciliated cell differentiation. Moreover,we show that they are likely to function downstream of IL6 signalling and upstream of Foxj1 activity in the process of ciliated cell differentiation. In addition,our in vitro explant assay provides a convenient method for preliminary investigation of over-expression phenotypes in the developing mouse airways.This article has an associated First Person interview with the first author of the paper. View Publication

Genetically encoded calcium ion (Ca 2+ ) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca 2+ dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs,FR-GECO1a and FR-GECO1c,based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm,respectively,display large responses to Ca 2+ in vitro (Δ F / F 0 = 6 for FR-GECO1a,18 for FR-GECO1c),are bright under both one-photon and two-photon illumination,and have high affinities (apparent K d = 29 nM for FR-GECO1a,83 nM for FR-GECO1c) for Ca 2+ . FR-GECOs offer sensitive and fast detection of single action potentials in neurons,and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators. Subject terms: Fluorescent proteins,Optogenetics,Zebrafish,Molecular neuroscience,Calcium signalling View Publication

We demonstrate a highly efficient method for gene delivery into clinically relevant human cell types,such as induced pluripotent stem cells (iPSCs) and fibroblasts,reducing the protocol time by one full day. To preserve cell physiology during gene transfer,we designed a microfluidic strategy,which facilitates significant gene delivery in a short transfection time (textless1 min) for several human cell types. This fast,optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications. View Publication

BACKGROUND/AIMS The Rho-ROCK signaling pathways play an important role in the activation of hepatic stellate cells (HSCs). We investigated the effects of fasudil hydrochloride hydrate (fasudil),a Rho-kinase (ROCK) inhibitor,on cell growth,collagen production,and collagenase activity in HSCs. METHODS Rat HSCs and human HSC-derived TWNT-4 cells were cultured for studies on stress fiber formation and alpha-smooth muscle actin (alpha-SMA) expression. Proliferation was measured by BrdU incorporation,and apoptosis by TUNEL assay. The phosphorylation states of the MAP kinases (MAPKs),extra cellular signal -regulated kinase 1/2 (ERK1/2),c-jun kinase (JNK),and p38 were evaluated by western blot analysis. Type I collagen,matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production and gene expression were evaluated by ELISA and real-time PCR,respectively. Collagenase activity (active MMP-1) was also evaluated. RESULTS Fasudil (100 microM) inhibited cell spreading,the formation of stress fibers,and expression of alpha-SMA with concomitant suppression of cell growth,although it did not induce apoptosis. Fasudil inhibited phosphorylation of ERK1/2,JNK,and p38. Treatment with fasudil suppressed the production and transcription of collagen and TIMP,stimulated the production and transcription of MMP-1,and enhanced collagenase activity. CONCLUSION These findings demonstrated that fasudil not only suppresses proliferation and collagen production but also increases collagenase activity. View Publication