Nuclear Protein Extracts Research Articles

Abstract 5638: The BAF complex mediates intertwined activity of YAP1 and β-catenin in synovial sarcoma

Abstract Introduction: Synovial sarcoma (SySa) is a rare malignant soft tissue tumor characterized by a specific reciprocal translocation t(X;18). The resulting chimeric SS18::SSX fusion protein drives SySa tumorigenesis by integrating into BAF chromatin remodeling complexes, changing their composition and dysregulating gene transcription. The role of BAF complexes in cancer steadily increases with ∼20% of all malignancies having alterations in BAF complex subunits. BAF complexes are multiprotein complexes with several interaction partners determining their localization and functionality. Since previous analyses revealed an induced activation of the transcriptional coregulators YAP1 and β-catenin in SySa, the aim of this study was to analyze if nuclear YAP1 and β-catenin are interacting with BAF complexes and if this has an impact on their transcriptional activity. Experimental procedures: Co-immunoprecipitation experiments on nuclear protein extracts and proximity ligation assays were performed to investigate interactions between YAP1, β-catenin, and BAF complex subunits in SySa cells. Knockdown of common and specific BAF complex subunits was conducted employing RNA interference. To assess changes in YAP1 and β-catenin transcriptional activities, TEAD and TOPFlash luciferase reporter assays were performed, respectively, and protein expression levels were detected by immunoblotting. Results: In SySa, protein-protein interaction experiments revealed nuclear interactions between β-catenin, YAP1, the SS18::SSX fusion protein and various BAF subunits. YAP1 and β-catenin’s transcriptional activities were found to be interdependent as they mutually enhanced each other’s activation. Knockdown of SS18::SSX as well as specific BAF complex subunits was associated with diminished transcriptional activities of these coregulators and, interestingly, substantially affected their mutual activation. This indicates that SS18::SSX-containing BAF complex assembly is crucial for interdependent YAP1 and β-catenin activation in SySa cells. Conclusions: This study reveals a significant role of the SySa-specific fusion protein and BAF chromatin remodeling complexes in mediating intertwined activation of YAP1 and β-catenin. Citation Format: Ilka Isfort, Ruth Berthold, Lorena Heinst, Anna Kuntze, Eva Wardelmann, Marcel Trautmann, Wolfgang Hartmann. The BAF complex mediates intertwined activity of YAP1 and β-catenin in synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5638.

The role of fibrinogen in neurodegeneration during a neuroinflammatory disease

AbstractBackgroundFibrinogen is not only a marker of inflammation, but also a cause of inflammatory responses. We previously showed that when elevated during traumatic brain injury (TBI), fibrinogen increased cerebrovascular permeability mainly via caveolar transcytosis and, after extravasation and deposition in vasculo‐astrocyte interface, it activated astrocytes causing neurodegeneration that was associated with memory reduction.MethodTo define the possible mechanisms of fibrinogen‐induced activation of neurons, primary mouse brain cortical neurons from C57BL/6 mice were plated in poly‐d‐lysine‐ and laminin‐coated cell culture plates. Cells were treated for 1 hour with fibrinogen (0.5 or 1 mg/mL) in the presence or absence of a nuclear factor‐κB (NF‐kB) blocker, caffeic acid phenethyl ester (CAPE), or media alone (control). All groups contained hirudin to prevent the conversion of fibrinogen to fibrin. Levels of interleukin‐6 (IL‐6) and chemokine (C‐C motif) ligand 2 (CCL2) were assessed by qRT‐PCR. NF‐kB activity was measured in neuronal nuclear protein and cell extracts by the TransAMTM NF‐kB p65 protein assay and Western blot (WB). In parallel, using a cortical contusion injury (CCI), a mouse model of mild‐to‐moderate TBI was created in C576J mice. NF‐kB was detected with immunocytochemistry in cultured cells and brain samples collected 14 days after CCI. The specific association of fibrinogen with its neuronal receptors intercellular adhesion molecule‐1 (ICAM‐1) and cellular prion protein (PrPC) was assessed with a proximity ligation assay in cells.ResultFibrinogen specifically bound to neurons via its receptors ICAM‐1 and PrPC and increased NF‐kB levels. Fibrinogen caused upregulation of pro‐inflammatory cytokines IL‐6 and CCL2 mRNA. ELISA and WB NF‐kB p65 analysis demonstrated increased DNA binding by p65 and protein expression in fibrinogen‐treated neurons compared to that in control and CAPE groups. Fibrinogen caused translocation of NF‐kB p65 to the nucleus.ConclusionResults suggest that a specific interaction of fibrinogen with neurons results in neuroinflammation through activation of NF‐kB.

Self-assembly of promoter DNA and RNA Pol II machinery into transcriptionally active biomolecular condensates.

Transcription in the nucleus occurs in a concentrated, dense environment, and no reasonable biochemical facsimile of this milieu exists. Such a biochemical environment would be important for further understanding transcriptional regulation. We describe here the formation of dense, transcriptionally active bodies in vitro with only nuclear extracts and promoter DNA. These biomolecular condensates (BMCs) are 0.5 to 1 μm in diameter, have a macromolecular density of approximately 100 mg/ml, and are a consequence of a phase transition between promoter DNA and nuclear extract proteins. BMCs are physically associated with transcription as any disruption of one compromised the other. The BMCs contain RNA polymerase II and elongation factors, as well as factors necessary for BMC formation in vivo. We suggest that BMCs are representative of the in vivo nuclear environment and a more physiologically relevant manifestation of the preinitiation complex/elongation machinery.

Mechanisms of Pol II Recruitment to the Human Beta-Globin Locus Control Region Super-Enhancer

The human ꞵ-globin gene locus consists of five genes that encode subunits of hemoglobin. The locus control region (LCR) regulates these genes, acting as a super-enhancer to promote high-level of β-globin expression in erythroid cells. The LCR, like other super-enhancers, is known to recruit transcription complexes and generate non-coding enhancer RNA (eRNA). Past research has suggested that super-enhancers form phase-separated transcription initiation domains that concentrate RNA polymerase II (Pol II) and transfer Pol II to target gene promoters during transient looping interactions. The aim of this project is to examine the role of TFIIH, involved transcription initiation, in enhancer RNA production and Pol II transfer in the globin gene locus. The LCR was immobilized on magnetic beads and incubated with erythroid nuclear protein extracts. Proteins not bound were washed away and bound proteins were subjected to western blotting experiments. The data show that TFIIH is bound at the LCR and thus may be involved in eRNA production. Cut & Run experiments were performed to examine the localization of TFIIH in the globin gene locus in intact cells. In the future, the laboratory plans to deplete TFIIH and examine production of eRNA and Pol II recruitment at the LCR.

Systemic sclerosis and primary biliary cholangitis share an antibody population with identical specificity.

Anti-centromere (ACA) and antimitochondrial antibodies (AMA) are specific for limited-cutaneous systemic sclerosis (lcSSc) and primary biliary cholangitis (PBC), respectively, and can coexist in up to 25 and 30% of SSc and PBC patients. Here, we evaluated whether anti-centromeric protein A (CENP-A) antibodies cross-react with mitochondrial antigens. To this end, sera from two lcSSc patients (pt1 and pt4), one of them (pt4) also affected by PBC, were used as the source of ACA, previously shown to recognize different groups of amino acids (motifs) in the CENP-A region spanning amino acids 1-17 (Ap1-17). Pt1 and pt4 Ap1-17-specific IgG were purified by affinity-chromatography on insolubilized Ap1-17-peptide column and tested by western blotting with nuclear and cytoplasmic protein extract from HeLa cells. Immunoreactive proteins were identified by mass spectrometry and validated by immunodot. The results showed that affinity-purified SSc/PBC pt4 anti-Ap1-17 and not SSc pt1 anti-Ap1-17 Ab, specifically cross-reacted with the E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC-E2), the major mitochondrial autoantigen in PBC. Sequence homology analysis indicated that the motif A-x-x-P-x-A-P recognized by pt4 anti-Ap1-17 IgG and shared by CENP-A and PDC-E2, is also expressed by some members of the Human Herpesvirus family, suggesting that they may trigger the production of these cross-reacting antibodies.

GLP-2 Improves Hepatic Inflammation and Fibrosis in Mdr2-/- Mice Via Activation of NR4a1/Nur77 in Hepatic Stellate Cells and Intestinal FXR Signaling

Glucagon-like peptide (GLP)-2 may exert antifibrotic effects on hepatic stellate cells (HSCs). Thus, we aimed to test whether application of the GLP-2 analogue teduglutide has hepatoprotective and antifibrotic effects in the Mdr2/Abcb4-/- mouse model of sclerosing cholangitis displaying hepatic inflammation and fibrosis. Mdr2-/- mice were injected daily for 4 weeks with teduglutide followed by gene expression profiling (bulk liver; isolated HSCs) and immunohistochemistry. Activated HSCs (LX2 cells) and immortalized human hepatocytes and human intestinal organoids were treated with GLP-2. mRNA profiling by reverse transcription polymerase chain reaction and electrophoretic mobility shift assay using cytosolic and nuclear protein extracts was performed. Hepatic inflammation, fibrosis, and reactive cholangiocyte phenotype were improved in GLP-2-treated Mdr2-/- mice. Primary HSCs isolated from Mdr2-/- mice and LX2 cells exposed to GLP-2 invitro displayed significantly increased mRNA expression levels of NR4a1/Nur77 (P < .05). Electrophoretic mobility shift assay revealed an increased nuclear NR4a1 binding after GLP-2 treatment in LX2 cells. Moreover, GLP-2 alleviated the Tgfβ-mediated reduction of NR4a1 nuclear binding activity. Invivo, GLP-2 treatment of Mdr2-/- mice resulted in increased intrahepatic levels of muricholic acids (accordingly Cyp2c70 mRNA expression was significantly increased), and in reduced mRNA levels of Cyp7a1 and FXR. Serum Fgf15 levels were increased in Mdr2-/- mice treated with GLP-2. Accordingly, GLP-2 treatment of human intestinal organoids activated their FXR-FGF19 signaling axis. GLP-2 treatment increased NR4a1/Nur77 activation in HSCs, subsequently attenuating their activation. GLP-2 promoted intestinal Fxr-Fgf15/19 signaling resulting in reduced Cyp7a1 and increased Cyp2c70 expression in the liver, contributing to hepatoprotective and antifibrotic effects of GLP-2 in the Mdr2-/- mouse model.

The XPO7/Npat Axis Is a Potential Therapeutic Target for TP53-Mutated AML

Acute myeloid leukemia (AML) is a devastating disease with a long-term survival rate below 30%. While AML is a genetically heterogeneous disease, TP53 mutations are among the most powerful risk factors in AML, which underscores the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes/pathways whose loss is vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. To generate AML lines with a relatively "clean" genetic background, we established mouse AML lines harboring WT Trp53 with normal karyotype (Yamauchi et al. Cancer Cell, 2018). We then generated Trp53-KO AML lines using a single guide RNA (sgRNA) targeting Trp53. Genome-wide CRISPR-Cas9 dropout screens were performed using these lines to identify genes/pathways whose loss is vulnerable to TP53 deficiency. We identified Xpo7, a putative nuclear/cytoplasmic transporter, as a factor necessary for the survival of Trp53-KO AML cells. sgRNAs targeting Xpo7 were enriched in Trp53-WT AML cells after the 16-day culture, while they were significantly depleted in Trp53-KO cells. Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. As expected, CRISPR/Cas9-mediated Xpo7 depletion and shRNA-mediated Xpo7 knockdown significantly delayed cell cycle progression and suppressed cell growth only in Trp53-KO AML cells, but not in WT cells. We next performed CRISPR-Cas9 saturation mutagenesis scan targeting all Xpo7 exons using Trp53-WT and -KO mouse AML cells. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT cells, namely in the presence of an MDM2 inhibitor. In stark contrast, the same sgRNAs which target coding exons including the C-terminal importin-β domain were mostly depleted in Trp53-KO AMLs. Since Xpo7 presumably mediates the nuclear import and export of proteins, we hypothesized that Xpo7 promotes retention of Trp53 protein in the nucleus in Trp53-WT AML cells. As expected, Trp53 protein levels in the nucleus and mRNA levels of Trp53-targeting genes were significantly decreased upon Xpo7 depletion. Furthermore, Xpo7 depletion resulted in resistance to Selinexor, a XPO1 inhibitor that reportedly blocks Trp53 protein export from the nucleus. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AML. Next, to elucidate the mechanisms underlying the toxic effects observed in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose cytoplasmic/nuclear localization was potentially regulated by Xpo7 in Trp53-KO AML cells. We first performed IP-mass spectrometry using an anti-Xpo7 antibody to identify Xpo7 interacting proteins. Then, we obtained both cytoplasmic and nuclear protein extracts from both Xpo7-WT and -KO AML cells and identified proteins whose localization was significantly altered upon Xpo7 deletion by mass spectrometry. We found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and localized more in the cytoplasm upon Xpo7 depletion in Trp53-KO AMLs to a significant extent. Strikingly, Npat protein levels were robustly upregulated following Trp53 depletion, and Npat depletion significantly delayed the progression of the G1/S transition and proliferation in Trp53-KO AML cells. Finally, to explore the functional significance of the XPO7/NPAT axis in human TP53-mutated AML cells, we assessed the correlation between XPO7 expression levels and AML subtypes and/or genetic background using publicly-available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets (Ley et al. NEJM, 2013). Moreover, XPO7 and NPAT mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature, 2018 and Iacobucci et al. Nat Genet. 2019). In fact, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed proliferation of HEL, a human AEL cell line harboring TP53 mutation, in vitro and in vivo. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML, such as AEL.

PRMT5 Interacting Partners and Substrates in Oligodendrocyte Lineage Cells.

The protein arginine methyl transferase PRMT5 is an enzyme expressed in oligodendrocyte lineage cells and responsible for the symmetric methylation of arginine residues on histone tails. Previous work from our laboratory identified PRMT5 as critical for myelination, due to its transcriptional regulation of genes involved in survival and early stages of differentiation. However, besides its nuclear localization, PRMT5 is found at high levels in the cytoplasm of several cell types, including oligodendrocyte progenitor cells (OPCs) and yet, its interacting partners in this lineage, remain elusive. By using mass spectrometry on protein eluates from extracts generated from primary oligodendrocyte lineage cells and immunoprecipitated with PRMT5 antibodies, we identified 1196 proteins as PRMT5 interacting partners. These proteins were related to molecular functions such as RNA binding, ribosomal structure, cadherin and actin binding, nucleotide and protein binding, and GTP and GTPase activity. We then investigated PRMT5 substrates using iTRAQ-based proteomics on cytosolic and nuclear protein extracts from CRISPR-PRMT5 knockdown immortalized oligodendrocyte progenitors compared to CRISPR-EGFP controls. This analysis identified a similar number of peptides in the two subcellular fractions and a total number of 57 proteins with statistically decreased symmetric methylation of arginine residues in the CRISPR-PRMT5 knockdown compared to control. Several PRMT5 substrates were in common with cancer cell lines and related to RNA processing, splicing and transcription. In addition, we detected ten oligodendrocyte lineage specific substrates, corresponding to proteins with high expression levels in neural tissue. They included: PRC2C, a proline-rich protein involved in methyl-RNA binding, HNRPD an RNA binding protein involved in regulation of RNA stability, nuclear proteins involved in transcription and other proteins related to migration and actin cytoskeleton. Together, these results highlight a cell-specific role of PRMT5 in OPC in regulating several other cellular processes, besides RNA splicing and metabolism.

High Vitamin D Concentrations Restore the Ability to Express LL37 by M. tuberculosis-Infected Human Macrophages.

Vitamin D has an immunomodulatory function and is involved in eliminating pathogens. Vitamin D deficiencies reported in Type 2 diabetes mellitus (T2DM) patients make them more susceptible to developing tuberculosis (TB). The macrophages are the immune cells that control intracellular pathogens by producing the antimicrobial peptide cathelicidin-LL37. This pathway involves TLR activation by pathogens, vitamin D receptor (VDR) ligation, and the enzyme 1α-hydroxylase Cytochrome P450 Family 27 Subfamily B Member 1 (CYP27B1). However, it is not clear whether the biological actions of vitamin D are affected by high glucose concentrations. This study aimed to evaluate the vitamin D contribution in the expression of VDR and CYP27B1, involved in the conversion of an inactive to an active form of vitamin D in the infected macrophages using M. tuberculosis as an infection model. The expression of LL37 and the nucleus translocation of VDR were evaluated as the readout of the response of vitamin D and determined if those processes are affected by glucose concentrations. Macrophages from healthy donors were cultured under glucose concentrations of 5.5, 15, or 30 mM, stimulated with vitamin D in inactive (25(OH)D3) or active (1,25(OH)2D3) forms, and infected with M. tuberculosis. The vitamin D-dependent induction of LL37 and the expression of VDR and CYP27B1 genes were analyzed by qPCR, and VDR translocation was analyzed in nuclear protein extracts by ELISA. M. tuberculosis downregulated the expression of LL37 regardless of the glucose concentration, whereas VDR and CYP27B1 upregulated it regardless of the glucose concentration. After evaluating two concentrations of vitamin D, 1 nM or 1 μM, the high concentration (1 μM) was necessary to restore the induction of LL37 expression in M. tuberculosis-infected macrophages. High concentrations of the inactive form of vitamin D restore the infected macrophages’ ability to express LL37 regardless of the glucose concentration. This finding supports the idea that vitamin D administration in patients with T2DM could benefit TB control and prevention.

Polymorphisms of the FCN2 Gene 3'UTR Region and Their Clinical Associations in Preterm Newborns.

Ficolin-2 is regarded as an important innate immunity factor endowed with both lectin (carbohydrate recognition) qualities and ability to induce complement activation. The aim of this study was to investigate the association of the FCN2 3’-untranslated region (3’UTR) polymorphisms with ficolin-2 expression and perinatal complications in preterm neonates. The sequencing analysis allowed us to identify six 3’UTR polymorphisms with minor allele frequency (MAF) >1%: rs4521835, rs73664188, rs11103564, rs11103565, rs6537958 and rs6537959. Except for rs4521835, all adhered to Hardy-Weinberg expectations. Moreover, rs6537958 and rs6537959 were shown to be in perfect linkage disequilibrium (LD) with nine other genetic polymorphisms: rs7040372, rs7046516, rs747422, rs7847431, rs6537957, rs6537960, rs6537962, rs11462298 and rs7860507 together stretched on a distance of 1242 bp and very high LD with rs11103565. The 3’UTR region was shown to bind nuclear extract proteins. The polymorphisms at rs4521835 and rs73664188 were found to influence serum ficolin-2 concentration significantly. All polymorphisms identified create (together with exon 8 polymorphism, rs7851696) two haplotype blocks. Among 49 diplotypes (D1-D49) created from rs7851696 (G>T), rs4521835 (T>G), rs73664188 (T>C), rs11103564 (T>C), rs11103565 (G>A) and rs6537959 (T>A), twenty two occurred with frequency >1%. Two diplotypes: D13 (GTTTGT/GGTCGT) and D10 (GTTTGT/GGTCGA), were significantly more frequent among preterm neonates with early onset of infection and pneumonia, compared with newborns with no infectious complications (OR 2.69 and 2.81, respectively; both p<0.05). The minor (C) allele at rs73664188 was associated with an increased risk of very low (≤1500 g) birthweight (OR=1.95, p=0.042) but was associated with the opposite effect at rs11103564 (OR=0.11, p=0.005).

Abstract 08: Functional Characterisation Of Uromodulin Promoter Risk Variants Associated With Blood Pressure

Using a genome wide association study (GWAS), we identified a locus on the promoter of the human uromodulin (UMOD) gene associating with primary hypertension. In this study we aimed to identify genotype-differential protein binding partners which influence UMOD expression and to examine pathways linking UMOD expression to blood pressure variability in humans.We examined a variant on this locus, by incubating 50mer oligonucleotides featuring alternate alleles (C/G) with HEK-293 nuclear protein extract. Subsequently, we performed an electrophoretic mobility shift assay (EMSA) and purified protein complexes to analyse by Tandem-mass-spectroscopy (MS2). Filtering for peptide matches &gt;20, we interpreted these data by Ingenuity Pathway Analysis (IPA). From a bank of 98 human renal biopsy samples, we assessed UMOD expression by qRT-PCR. RNA-seq was performed using the NextSeq-500 system on a subset of high and low UMOD expressors (n=3/ group). Differentially expressed (false-discovery-rate (FDR) &lt;0.05) genes were identified, with strong correlations in these data validated by qRT-PCR. Implicated pathways were interpreted using both IPA and gene set enrichment analysis (GSEA).The C allele at our risk variant displayed greater binding affinity versus the G allele; 0.77 (0.13, 1.40, 95%CI). In contrast no difference in binding affinity was observed between the T and C alleles of our control variant, 0.08 ( -1.22, 1.39, 95%CI). Mass spectroscopy of EMSA complexes identified PARP1 binding at this target. By IPA we identified a complex between PARP1 and HNF1β as driving expression of UMOD. RNA-sequencing identified positive correlation in expression of UMOD and the Na-K-Cl cotransporter (NKCC2) (FDR=2.58e-9, log2-fold-change [L2FC]= 3.3), the renal outer medullary potassium channel (ROMK) (FDR=3.69e-2, L2FC= 2.79) and the epithelial sodium channel (ENaC) (FDR=1.83e-2, L2FC= 1.25). We validated this coexpression between UMOD and NKCC2 by qRT-PCR and show a significant correlation (p=2.2e-16, R2= 0.844, n=84).Together these data suggest our risk variant contributes to differences in UMOD expression via a PARP1:HNF1β transcriptional complex and these differences associate with blood pressure through sodium homeostasis in the human kidney.

Tanshinone IIA protects human coronary artery endothelial cells from ferroptosis by activating the NRF2 pathway

The pathogenesis of atherosclerosis is closely related to endothelial cell injury caused by lipid peroxidation-induced ferroptosis. Tanshinone IIA (TSA) protects endothelial tissues from damage. In this study, we investigated whether TSA exerts its protective effect on endothelial cells by inhibiting ferroptosis. Ferroptosis was induced in human coronary artery endothelial cells (HCAECs), and cells were treated with TSA. Morphological examination indicated that TSA exerted a significant protective effect on the HCAECs. This was further confirmed by LDH release and cell death detection assays. Flow cytometry revealed that TSA significantly reduced the excessive accumulation of total cellular ROS and lipid ROS caused by ferroptosis inducers. TSA also restored the reduction of glutathione (GSH), a potent and abundant reductant in cells. In addition, we found that TSA promoted the expression of NRF2, an essential player in response to oxidative stress, and its downstream genes. Immunofluorescent staining revealed that TSA promoted the nuclear translocation of NRF2. Increased nuclear translocation of NRF2 was validated by Western blot evaluation of cytoplasmic and nuclear protein extracts. Furthermore, NRF2 inhibition abolished the protective effects of TSA on HCAECs. These data demonstrate that TSA represses ferroptosis via activation of NRF2 in HCAECs.

Transcriptional downregulation of FAM3C/ILEI in the Alzheimer's brain.

Amyloid-β (Aβ) accumulation in the brain triggers the pathogenic cascade for Alzheimer's disease (AD) development. The secretory protein FAM3C (also named ILEI) is a candidate for an endogenous suppressor of Aβ production. In this study, we found that FAM3C expression was transcriptionally downregulated in the AD brain. To determine the transcriptional mechanism of the human FAM3C gene, we delineated the minimal 5'-flanking sequence required for basal promoter activity. From a database search for DNA-binding motifs, expression analysis using cultured cells, and promoter DNA-binding assays, we identified SP1 and EBF1 as candidate basal transcription factors for FAM3C, and found that SMAD1 was a putative inducible transcription factor and KLF6 was a transcription repressor for FAM3C. Genomic deletion of the basal promoter sequence from HEK293 and Neuro-2a cells markedly reduced endogenous expression of FAM3C and abrogated SP1- or EBF1-mediated induction of FAM3C. Nuclear protein extracts from AD brains contained lower levels of SP1 and EBF1 than did those from control brains, although the relative mRNA levels of these factors did not differ significantly between the groups. Additionally, the ability of nuclear SP1 and EBF1 in AD brains to bind with the basal promoter sequence-containing DNA probe was reduced compared with the binding ability of these factors in control brains. Thus, the transcriptional downregulation of FAM3C in the AD brain is attributable to the reduced nuclear levels and genomic DNA binding of SP1 and EBF1. An expressional decline in FAM3C may be a risk factor for Aβ accumulation and eventually AD development.

Histone Deacetylase Inhibitors Abolish HBZ Protein Expression and Induce Cell Death in HTLV-1 Related Adult T-Cell Leukemia-Lymphoma

Background:Adult T-cell leukemia-lymphoma (ATL) is a malignancy with dismal prognosis caused by HTLV-1. ATL is incurable by conventional drugs. Histone deacetylase (HDAC) inhibitors (HDIs) are broadly active anti-neoplastic agents that can be cautiously exploited in the treatment of viral related lymphomas. HTLV-1 usually establishes lifelong latency in human CD4+ T-cells, which in turn propagate and persist as result of the multifaceted functions of HTLV-1 basic zipper factor (HBZ) through various signaling pathways that promote growth and proliferation, and block apoptosis and cell senescence. All ATL cells harbor at least one copy of latent intact HTLV-1 provirus in most cases, and unlike Tax, all tumors express HBZ from the negative 3‘ proviral strand via SP-1 activity. The HTLV-1 promoter is negatively regulated by HDACs at the 5‘ long terminal repeat (LTR), and by HBZ, which disrupts Tax positive interaction with the transcriptional activator p300/CBP, thus blocking viral transcription. Therefore, treatment of HTLV-1 infected cells with HDIs can induce expression of Tax protein, which is highly immunogenic, and which could promote immune-mediated clearance of ATL cells in vivo.Methods:Low-passage IL-2 dependent ATL cell lines established from patients with acute type ATL and freshly isolated ATL cells were treated with several HDIs (VPA, sodium butyrate, vorinostat, belinostat, panabinostat, and mocetinostat) at various doses and time points. Apoptosis was measured by annexin V and FACS analysis. Expression of HBZ protein, Tax, acetyl and total histone 3 subunits (H3), BIM, caspase 3, caspase 9, Jun B, c-Jun, Jun D, and Sp-1 were measured by Western blots before and after HDI treatment. HBZ and Tax mRNA expression was measured by quantitative RT-PCR. SP-1 binding to HTLV-1 specific oligonucleotide consensus sequence was measured by gel shift assay using nuclear protein extracts before and after HDI treatment.Results:HDIs sequentially increased acetylated H3 levels, blocked HBZ protein, upregulated Tax expression, stabilized BIM, and induced cleavage of pro-caspases 9 and 3 (intrinsic apoptotic pathway), resulting in apoptosis of ATL cells. HDIs tested (VPA and belinostat) blocked expression of Jun-B, which is known to be upregulated by HBZ, while no significant effects were observed on c-Jun and Jun D protein levels. HDIs tested (VPA, belinostat, and panabinostat) did not significantly alter spliced-HBZ RNA levels or affect SP-1 binding to HBZ promoter-specific oligonucleotide sequence, suggesting a post-translational mechanism may contribute to abrogation of HBZ in ATL cells.Conclusions:Our data demonstrate that HDIs target ATL cells in part by abrogating HBZ protein, which could negatively impact the longevity of ATL and HTLV-1 cells in vivo. Long-term clinical use of HDIs might be beneficial for eliminating residual ATL cells after definitive treatment. A pilot clinical trial testing belinostat-based consolidation therapy for aggressive leukemic ATL types is currently ongoing at our institution (available at Clinicaltrials.gov , NCT02737046). DisclosuresNo relevant conflicts of interest to declare.

Identifying Cytoplasmic and Nuclear C‐Terminal to LisH (CTLH) Complex Interactors Using High‐Throughput Proteomics

The C-terminal to LisH (CTLH) complex is a tumour-suppressive E3 Really Interesting New Gene (RING) ubiquitin ligase that regulates intracellular signaling pathways and maintains cellular homeostasis. It is comprised of 8 individual proteins that have been shown to mediate a variety of intracellular protein-protein interactions; of these proteins, Required for Meiotic Division 5 Homolog A (RMND5A) and Macrophage Erythroblast Attacher (MAEA) contain the RING domains required for the CTLH complex to mediate ubiquitin transfer to specific protein substrates. Although its E3 ligase activity has been recently characterized, the endogenous subcellular localization of the complex and compartment-specific protein interaction network – which encompasses many unidentified E3 ligase substrates – has not been identified. In this study, we hypothesized that the CTLH complex is present in the cytoplasm and nucleus, and that the CTLH complex mediates compartment-specific protein-protein interactions with cytoplasmic and nuclear proteins. To test this hypothesis, we used a chemical-based subcellular fractionation method combined with low- and high-throughput proteomics. We first optimized the chemical fractionation method and used Western blot to detect each endogenous CTLH complex member in HeLa cell cytoplasmic and nuclear protein extracts. We then identified compartment-specific CTLH complex interactors using Affinity Purification Coupled to Mass Spectrometry (AP-MS) using the main CTLH complex scaffold protein – Ran Binding Protein M (RanBPM) – as bait in cytoplasmic and nuclear HeLa protein extracts. Following Liquid Chromatography Electrospray Ionizing Tandem Mass Spectrometry (LC-ESI-MS/MS) and subsequent in silico filtering using the MiST algorithm, we identified 27 cytoplasmic, 155 nuclear, and 31 nucleocytoplasmic high-confidence (MiST Score ≥ 0.75) RanBPM interactors. STRING enrichment analysis of the high-confidence interactors revealed that RanBPM and its associated CTLH complex may be involved in a variety of conserved Gene Ontology (GO) processes including Epigenetic Regulation of Gene Expression and Chromatin Assembly. As such, we validated a number of newly-identified compartment-specific RanBPM interactors associated with the enriched GO terms by co-immunoprecipitation – using RanBPM as bait – and resulting Western blot. Furthermore, we focussed on an interesting novel putative interactor that is present in the two aforementioned GO processes – histone variant MacroH2A1 – and further validated its interaction with the CTLH complex in vivo using Proximity Ligation Assay (PLA), suggesting that this interaction may be physiologically relevant. Overall, by using this workflow, we identified and validated a number of novel compartment-specific potential CTLH complex interactors in vitro, further validated a promising novel interaction between the CTLH complex and MacroH2A1 in vivo, and successfully implicated the CTLH complex in a number of previously unexplored cellular mechanisms.

Human METTL18 is a histidine-specific methyltransferase that targets RPL3 and affects ribosome biogenesis and function.

Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance.

C1QBP inhibits proliferation of porcine circovirus type 2 by restricting nuclear import of the capsid protein.

Complement component 1 Q subcomponent-binding protein (C1QBP) has been shown to interact with the porcine circovirus type 2 (PCV2) Cap protein. Here, using yeast two-hybrid (Y2H) and co-immunoprecipitation assays, as well as laser confocal microscopy, the interaction between C1QBP and Cap was confirmed. Furthermore, overexpression of C1QBP in cells altered the intracellular location of Cap, which was observed using confocal microscopy and verified by detection of Cap in nuclear protein extracts in a Western blot assay. By inhibiting nuclear transport of Cap, overexpression of C1QBP downregulated PCV2 proliferation in PK-15 cells, as determined by quantitative polymerase chain reaction (qPCR). As C1QBP plays a similar role in a fusion of green fluorescent protein (GFP) with the Cap nuclear localisation signal (NLS) sequence, (CapNLS-GFP), we propose that the target site for C1QBP in Cap is possibly located in the NLS region. Considering all the results together, this study demonstrated that C1QBP interacts with the Cap NLS region, resulting in changes in the intracellular localisation of the Cap protein. We confirmed that overexpression of C1QBP inhibits the proliferation of PCV2, and this is possibly related to the function of C1QBP in controlling nuclear transport of Cap.

The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana.

A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium-mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes β-ketoacyl-CoA synthase (ShKCS20) and caffeic acid-O-methyltransferase (ShCOMT). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.

Abstract 286: Regulation of APOBEC3B gene expression through the Rb/E2F pathway

Abstract The propensity of tumors to increase genomic diversity allows for a bottleneck-type adaptation to anti-cancer treatments. The genomic alterations fueling these adaptations in many different tumor types are, in part, brought about by ongoing endogenous activity of the DNA cytosine deaminase APOBEC3B (A3B). The ability of A3B to convert DNA cytosine bases into uracils has been shown to facilitate C-to-T transitions, C-to-G transversions, and DNA breakage. While low in healthy tissues, A3B expression and activity is elevated in tumors and may increase further throughout metastatic disease. However, molecular mechanisms responsible for transcriptional upregulation of A3B in tumor cells are poorly understood. Here, we investigate the possibility that the Rb/E2F pathway, which is often dysregulated in breast cancer, contributes to A3B gene expression. First, we cloned the A3B promoter upstream of a luciferase reporter and used a combination of deletion and site-directed mutagenesis to demonstrate that only one out of five in silico predicted E2F binding sites is functionally relevant, as disrupting this site caused constitutive reporter activation. Moreover, expression of the BK polyomavirus T-antigen, which inactivates Rb, induced the reporter and additional activation was not observed by mutating the functional E2F binding site. Second, SILAC-labelled nuclear protein extracts were subjected to affinity purification coupled to quantitative mass spectrometry to compare transcriptional complexes that bound wild-type promoter sequences, but not sequences with mutant E2F binding site promoter elements. These proteomics experiments showed that proteins belonging to the DREAM and PRC1.6-complexes strongly bind the wild-type but not the mutant E2F binding sites. Third, RNAi-mediated knock-down of candidate E2F family members implicated E2F6, and to a lesser extend E2F4, which are components of the PRC1.6 and DREAM complexes, respectively, in A3B gene repression. Taken together, these studies indicate that A3B expression may be suppressed by the concerted effort of two different E2F binding complexes and that Rb inactivation, by viral T-antigen here and genetic mutation as often seen in breast cancer, is able to upregulate A3B gene expression, promote genomic DNA mutagenesis, and fuel tumor evolution. Citation Format: Pieter A. Roelofs, William L. Brown, Paul N. Span, John W. Martens, Chai Y. Goh, Boon H. Chua, Dennis Kappei, Reuben S. Harris. Regulation of APOBEC3B gene expression through the Rb/E2F pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 286.

FURIN Expression in Vascular Endothelial Cells Is Modulated by a Coronary Artery Disease-Associated Genetic Variant and Influences Monocyte Transendothelial Migration.

BackgroundGenome‐wide association studies have shown an association between the single‐nucleotide polymorphism rs17514846 on chromosome 15q26.1 and coronary artery disease susceptibility. The underlying biological mechanism is, however, not fully understood. rs17514846 is located in the FES Upstream Region (FURIN) gene, which is expressed in vascular endothelial cells (ECs). We investigated whether rs17514846 has an influence on FURIN expression in ECs and whether FURIN affects EC behavior.Methods and ResultsQuantitative reverse transcription–polymerase chain reaction analysis showed that cultured vascular ECs from individuals carrying the coronary artery disease risk allele of rs17514846 had higher FURIN expression than cells from noncarriers. In support, luciferase reporter analyses in ECs indicated that the risk allele had higher transcriptional activity than the nonrisk allele. Electrophoretic mobility shift assays using EC nuclear protein extracts detected a DNA‐protein complex with allele‐specific differential binding of a nuclear protein. Knockdown of FURIN in ECs reduced endothelin‐1 secretion, nuclear factor‐κB activity, vascular cell adhesion molecule‐1, and MCP1 (monocyte chemotactic protein‐1) expression and monocyte‐endothelial adhesion and transmigration. A population‐based study showed an association of the rs17514846 risk allele with higher circulating MCP1 levels and greater carotid intima‐media thickness.ConclusionsThe coronary artery disease risk variant at the 15q26.1 locus modulates FURIN expression in vascular ECs. FURIN levels in ECs affect monocyte‐endothelial adhesion and migration.