Decreased Promoter Activity Research Articles

Interruption of mycothiol synthesis and intracellular redox status impact iron-regulated reporter activation in Mycobacterium smegmatis.

Iron scavenging is required for full virulence of mycobacterial pathogens. During infection, the host immune response restricts mycobacterial access to iron, which is essential for bacterial respiration and DNA synthesis. The Mycobacterium tuberculosis iron-dependent regulator (IdeR) responds to changes in iron accessibility by repressing iron-uptake genes when iron is available. In contrast, iron-uptake gene transcription is induced when iron is depleted. The ideR gene is essential in M. tuberculosis and is required for bacterial growth. To further study how iron regulates transcription, wee developed an iron responsive reporter system that relies on an IdeR-regulated promoter to drive Cre and loxP mediated recombination in Mycobacterium smegmatis. Recombination leads to the expression of an antibiotic resistance gene so that mutations that activate the IdeR-regulated promoter can be selected. A transposon library in the background of this reporter system was exposed to media containing iron and hemin, and this resulted in the selection of mutants in the antioxidant mycothiol synthesis pathway. We validated that inactivation of the mycothiol synthesis gene mshA results in increased recombination and increased IdeR-regulated promoter activity in the reporter system. Further, we show that vitamin C, which has been shown to oxidize iron through the Fenton reaction, can decrease promoter activity in the mshA mutant. We conclude that the intracellular redox state balanced by mycothiol can alter IdeR activity in the presence of iron.IMPORTANCEMycobacterium smegmatis is a tractable organism to study mycobacterial gene regulation. We used M. smegmatis to construct a novel recombination-based reporter system that allows for the selection of mutations that deregulate a promoter of interest. Transposon mutagenesis and insertion sequencing (TnSeq) in the recombination reporter strain identified genes that impact iron regulated promoter activity in mycobacteria. We found that the mycothiol synthesis gene mshA is required for IdeR mediated transcriptional regulation by maintaining intracellular redox balance. By affecting the oxidative state of the intracellular environment, mycothiol can modulate iron-dependent transcriptional activity. Taken more broadly, this novel reporter system can be used in combination with transposon mutagenesis to identify genes that are required by Mycobacterium tuberculosis to overcome temporary or local changes in iron availability during infection.

RUNX1 Isoforms Regulate RUNX1 and Target-Genes Differentially in Platelets/Megakaryocytes: Association with Clinical Cardiovascular Events

RUNX1 is an essential transcription factor for hematopoiesis. Germline RUNX1 haplodeficiency is associated with thrombocytopenia, platelet dysfunction and predisposition to myeloid malignancies. Three major RUNX1 isoforms (A, B and C) are recognized and share a DNA binding RUNT domain; RUNX1A lacks the transactivation domain. Two distinct promoters regulate RUNX1 expression: distal P1 regulates RUNX1C; proximal P2 regulates RUNX1B and RUNX1A. Little is known regarding the differential effects of isoforms in RUNX1 autoregulation, target-gene regulation and association with clinical events. We performed studies in megakaryocytic HEL cells, HeLa cells (which do not express RUNX1) and in platelets of healthy subjects (n=74) and provide evidence for isoform-specific differential autoregulation and target-gene regulation by RUNX1. We show an association between expression of specific RUNX1-regulated genes in whole blood with cardiovascular events in patients. There are 5 RUNX1 consensus binding sites in P1 promoter and 1 in P2 promoter within ~1000 bp from ATG. Wild type RUNX1 P1 and P2 promoter regions were individually cloned into PGL4 luciferase promoter vector. ChIP studies using PMA-treated megakaryocytic HEL cells showed RUNX1 binding to chromatin regions encompassing the RUNX1 binding sites in both promoters. Mutations of 2 RUNX1 binding sites in intron region of P1 promoter reduced promoter activity; mutations of first 2 RUNX1 binding sites at the exon region of P1 promoter increased activity. Mutation of single RUNX1 binding site in P2 promoter increased promoter activity. Thus, RUNX1 binds to P1 and P2 promoters to regulate activities. To examine the RUNX1 autoregulation by individual isoforms, we co-transfected each isoform with P1 or P2 promoter vector in HeLa cells, which do not express endogenous RUNX1. In response to RUNX1B over-expression, both P1 and P2 promoters showed a dose-dependent decrease in promoter activity. RUNX1C over-expression increased P1 and P2 promoter activities. Thus, RUNX1B and RUNX1C regulate P1 and P2 promoters differentially. In HEL cells, which have endogenous RUNX1, RUNX1B overexpression decreased RUNX1C and RUNX1A mRNA/protein expression by ~50%. RUNX1C overexpression increased RUNX1B and RUNX1A mRNA/protein expression. Thus, RUNX1B decreases RUNX1C and RUNX1A expression; RUNX1C increases RUNX1B and RUNX1A. We studied the regulation by RUNX1 isoforms of target genes PCTP (phosphatidylcholine transfer protein), MYL9 (myosin light chain), PDE5A (phosphodiesterase 5A) and F13A (factor XIIIA) . In HeLa cells. RUNX1B overexpression increased PCTP, MYL9 and PDE5A (protein and mRNA); RUNX1C overexpression reduced PCTP and MYL9. In HEL cells RUNX1B increased PCTP, MYL9, PDE5A and F13A expression; RUNX1C reduced PCTP and MYL9 expression. To obtain evidence in vivo, we studied the relationships between RUNX1 isoforms and their relationships to RUNX1-target genes by RNAseq in leukocyte-poor platelets (74 healthy donors; ages 30-75 yrs). RUNX1B correlated negatively with RUNX1C (R= -0.413 and p=1.5e-06) and RUNX1A (R= -0.493 and p=0.00e+oo); RUNX1C correlated positively with RUNX1A (R=0.45, p<0.0001). RUNX1B correlated positively with PCTP, F131A, PDE5A and RAB1B, and negatively with MYL9 (Figure 1); no significant relationships were observed with RUNX1C or RUNX1A. To understand the relationships to cardiovascular events (MI or death), we explored whole blood gene expression using microarrays in 587 patients (2 cohorts, 190 and 397 patients) presenting to cardiac catherization for chest pain and followed for 3.8 years. In our previous studies RUNX1B transcripts were higher and RUNX1C lower in those with MI or death. We now found that higher expressions of F13A1(Figure 2) and RAB31, which have been shown by us to be RUNX1-targets, were associated with cardiovascular events. Conclusions: RUNX1 isoforms autoregulate RUNX1 expression differentially and regulate target genes in a differential manner, a pattern consistent with in vivo platelet gene expression. The relative expression of RUNX1B and RUNX1C isoforms and of their target genes associate with clinical cardiovascular events. RUNX1-isoform specific effects need to be considered in clinical scenarios where RUNX1 is involved and in its pharmacologic modulation.

Transcriptional Regulation Associated with Subcutaneous Adipogenesis in Porcine ACSL1 Gene.

Long-chain acyl-CoA synthetase 1 (ACSL1) plays an important role in fatty acid metabolism and fat deposition. The transcription of the ACSL1 gene is regulated specifically among cells and physiological processes, and transcriptional regulation of ACSL1 in adipogenesis remains elusive. Here, we characterize transcription factors (TFs) associated with adipogenesis in the porcine ACSL1 gene. CCAAT-enhancer binding protein (C/EBP)α, a well-known adipogenic marker, was found to enhance the expression of the ACSL1 gene via binding two tandem motifs in the promoter. Further, we demonstrate that ACSL1 mediates C/EBPα effects on adipogenesis in preadipocytes cultured from subcutaneous fat tissue of pigs via gain- and loss-of-function analyses. The cAMP-response element binding protein, another TF involved in adipogenesis, was also identified in the regulation of ACSL1 gene expression. Additionally, single nucleotide polymorphisms (SNPs) were screened in the promoter of ACSL1 among four breeds including the Chinese indigenous Min, and Duroc, Berkshire, and Yorkshire pigs through sequencing of PCR products. Two tightly linked SNPs, -517G>T and -311T>G, were found exclusively in Min pigs. The haplotype mutation decreases promoter activity in PK-15 and ST cells, and in vivo the expression of ACSL1, illustrating a possible role in adipogenesis regulated by C/EBPα/ACSL1 axis. Additionally, a total of 24 alternative splicing transcripts were identified, indicating the complexity of alternative splicing in the ACSL1 gene. The results will contribute to further revealing the regulatory mechanisms of ACSL1 during adipogenesis and to the characterization of molecular markers for selection of fat deposition in pigs.

ZEB1 potentiates chemoresistance in breast cancer stem cells by evading apoptosis

Chemoresistance renders a challenge to the clinics to treat breast cancer patients. Current treatment strategies are effective in mitigating tumor growth but remain largely ineffective against cancer-initiating cells or breast Cancer Stem Cells (CSCs). Epithelial-to-mesenchymal-transition (EMT) regulates breast CSC physiology. Zinc finger E-box binding homeobox 1 (ZEB1) is a key EMT-transcription factor that regulates breast CSC - differentiation and metastasis. However, its potential role in modulating tumor chemoresistance has not yet been fully understood. In-silico analysis revealed a higher ZEB1 expression in breast cancer patients that leads to decreased overall and relapse-free survival. We generated sorted breast CSC with stable ZEB1 overexpression (CD24−/CD44+GFP-ZEB1) and/or silencing (CD24−/CD44+ZEB1 shRNA) as well as breast cancer cells with stable ZEB1 overexpression (CD24+GFP-ZEB1) and/or silencing (CD24+ZEB1 shRNA). An increased colony-forming efficiency and doxorubicin accumulation correlated with decreased promoter activity and expression profile of ABCC1 drug-efflux ABC transporter in CD24−/CD44+GFP-ZEB1. Additionally, CD24−/CD44+GFP-ZEB1 demonstrated doxorubicin-induced higher anti-apoptotic and lower pro-apoptotic protein expressions in the mitochondrial and cytosolic fractions. Chemoresistant CD24−/CD44+GFP-ZEB1 cells depicted 1000-fold higher IC-50 values of doxorubicin and decreased activation of JNK-p38 stress kinase molecular signaling-dependent mammosphere forming efficiency to evade apoptosis. Thus, ZEB1 and its downstream effectors are plausible therapeutic targets for the mitigation of breast cancer chemoresistance in patients.

Transcriptional Analysis of the Gene Encoding the Putative Myristoylated Membrane Protein (ORF458R) of Invertebrate Iridescent Virus 6 (IIV6)

Invertebrate iridescent virus 6 (IIV6) is a member of the genus Iridovirus and belongs to the Iridoviridae family. The entirely sequenced dsDNA genome, composed of 212 482 bp, encodes 215 putative open reading frames (ORFs). ORF458R encodes a putative myristoylated membrane protein. RT-PCR analysis of ORF458R expression in the presence of DNA replication and protein synthesis inhibitors showed that this gene is transcribed in the late phase of the virus infection. Time course analysis showed that transcription of ORF458R initiates between 12 and 24 h p.i. and starts to decrease after this point. Transcription of ORF458R initiated 53 nucleotides upstream of the translation start site and ended 40 nucleotides after the stop codon. Dual luciferase reporter gene assay showed that sequences between ‒61st and +18th nucleotides are essential for promoter activity. Interestingly, a remarkable decrease in promoter activity, in the presence of sequences between ‒299th and ‒143rd nucleotides, suggested a repressor activity between these regions. Our results showed that ORF458R is transcriptionally active, and separately located sequences at its upstream region with promoter and repressor activities regulating its expression. This information on the transcriptional analysis of ORF458R will contribute to our understanding of the molecular mechanisms of IIV6 replication.

Nucleotide Context Can Modulate Promoter Strength in Genes Transcribed by RNA Polymerase III.

The small nuclear RNAs 4.5SH and 4.5SI were characterized only in mouse-like rodents; their genes originate from 7SL RNA and tRNA, respectively. Similar to many genes transcribed by RNA polymerase III (pol III), the genes of 4.5SH and 4.5SI RNAs include boxes A and B, forming an intergenic pol III-directed promoter. In addition, their 5'-flanking sequences have TATA-like boxes at position -31/-24, also required for efficient transcription. The patterns of the three boxes notably differ in the 4.5SH and 4.5SI RNA genes. The A, B, and TATA-like boxes were replaced in the 4.5SH RNA gene with the corresponding boxes in the 4.5SI RNA gene to evaluate their effect on the transcription of transfected constructs in HeLa cells. Simultaneous replacement of all three boxes decreased the transcription level by 40%, which indicates decreased promoter activity in a foreign gene. We developed a new approach to compare the promoter strength based on the competition of two co-transfected gene constructs when the proportion between the constructs modulates their relative activity. This method demonstrated that the promoter activity of 4.5SI is 12 times that of 4.5SH. Unexpectedly, the replacement of all three boxes of the weak 4.5SH promoter with those of the strong 4.5SI gene significantly reduced, rather than enhanced, the promoter activity. Thus, the strength of a pol III-directed promoter can depend on the nucleotide environment of the gene.

Integrated transcriptomic and metabolomic profiles reveal anthocyanin accumulation in Scutellaria baicalensis petal coloration

Scutellaria baicalensis is a flowering plant of the Lamiaceae genus whose petals are mostly purple, with a few being pink and white. To elucidate the kinetics behind S. baicalensis petal coloration, an integrative analysis of target flavonoid metabolic and transcriptome profiles was performed to identify the anthocyanin biosynthesis pathway, which was differently activated in S. baicalensis germplasms with three different petal colors (white, pink and purple). Metabolomic profile data revealed that 18 anthocyanins were significantly different in S. baicalensis petals. Transcriptomic data showed that S. baiANS in anthocyanin biosynthesis was significantly downregulated in white flower petals. Interestingly, the sequence variations were found in the S. baiANS promoter region, where it was likely that a mutation reduced S. baiANS expression in white S. baicalensis flowers. Yeast one-hybrid (Y1H) analysis of S. baiMYB indicated that it was not bound to the S. baiANS promoters of white S. baicalensis flowers. Dual-luciferase assays further confirmed that the S. baiANS promoter decreased promoter activity, which resulted in reduced anthocyanin biosynthesis in white S. baicalensis flowers. The findings of this study elucidate the mechanisms behind the loss-of-color phenotype in white S. baicalensis flowers while providing potential leads for further studies on biological and flower evolution.

Transcriptional Analysis of the Gene Encoding the Putative Myristoylated Membrane Protein (ORF458R) of Invertebrate Iridescent Virus 6 (IIV6)

Invertebrate iridescent virus 6 (IIV6) is a member of the genus Iridovirus and belongs to the Iridoviridae family. The entirely sequenced dsDNA genome, composed of 212.482 bp, encodes 215 putative open reading frames (ORFs). ORF458R encodes a putative myristoylated membrane protein. RT-PCR analysis of ORF458R expression in the presence of DNA replication and protein synthesis inhibitors showed that this gene is transcribed in the late phase of the virus infection. Time course analysis showed that transcription of ORF458R initiates between 12 and 24 h p.i. and starts to decrease after this point. Transcription of ORF458R initiated 53 nucleotides upstream of the translation start site and ended 40 nucleotides after the stop codon. Dual luciferase reporter gene assay showed that sequences between -61st and +18th nucleotides are essential for promoter activity. Interestingly, a remarkable decrease in promoter activity, in the presence of sequences between -299th and -143rd nucleotides, suggested a repressor activity between these regions. Our results showed that ORF458R is transcriptionally active, and separately located sequences at its upstream region with promoter and repressor activities regulating its expression. This information on the transcriptional analysis of ORF458R will contribute to our understanding of the molecular mechanisms of IIV6 replication.

Differential RUNX1-Isoform Specific Autoregulation of RUNX1 and Regulation of Target Genes (PCTP, MYL9) in Megakaryocytic Cells

RUNX1 is an essential transcription factor for hematopoiesis. Human germline RUNX1 haplodeficiency is associated with impaired megakaryocyte differentiation, thrombocytopenia, platelet dysfunction and predisposition to myeloid malignancies. The two major RUNX1 isoforms RUNX1C and RUNX1B differ at N-terminus by 14AA, and are regulated by two distinct promoters P1 and P2, respectively. RUNX1B and RUNX1C have distinct roles and tissue expression. Little is known regarding the differential effects of RUNX1 isoforms in RUNX1 autoregulation, and on their regulation of target genes. There are 5 RUNX1 consensus binding sites located in P1 promoter and one RUNX1 site in P2 promoter. To study RUNX1 promoters, both WT RUNX1 P1 and P2 promoter regions were individually cloned into PGL4 luciferase promoter vector. ChIP studies using PMA-treated megakaryocytic HEL cells showed RUNX1 binding to chromatin regions encompassing the RUNX1 binding sites in both promoters. Mutations of 2 RUNX1 binding sites in intron region of P1 promoter reduced promoter activity; mutations of first 2 RUNX1 binding sites in at the exon region of P1 promoter increased activity. Mutation of single RUNX1 binding site in P2 promoter increased promoter activity. Thus, RUNX1 binds to P1 and P2 promoters to regulate their activities. To examine the RUNX1 autoregulation by individual isoforms, we co-transfected each isoform with P1 or P2 promoter vector in HeLa cells, which express negligible endogenous RUNX1. In response to RUNX1B, P1 promoter showed a dose-dependent decrease in promoter activity. RUNX1C expression, increased P1 promoter activity in a dose-dependent manner. With constant RUNX1C expression, increase in RUNX1B co-expression led to a dose-dependent decrease in P1 promoter activity. In HeLa cells RUNX1B decreased P2 promoter activity in a dose-dependent manner. With constant RUNX1B expression, an increase in RUNX1C increased RUNX1 P2 promoter activity. Thus, RUNX1B and RUNX1C regulate both P1 and P2 promoters in a distinct manner. In studies in megakaryocytic HEL cells, which express endogenous RUNX1, RUNX1B overexpression decreased RUNX1C expression by 50%. RUNX1C overexpression increased by 2-fold RUNX1B protein. In studies using RUNX1C-specific antibody, and expression plasmids for hemagglutinin-tagged-RUNX1B and c-Myc-tagged RUNX1C, RUNX1B overexpression decreased RUNX1C; RUNX1C overexpression increased RUNX1B. These studies indicate that RUNX1 isoforms autoregulate in a differential manner at the protein level. We studied the effects of RUNX1 isoforms on two established downstream RUNX1 target genes PCTP (phosphatidylcholine transfer protein) and MYL9(myosin light chain) in HeLa cells. RUNX1B overexpression increased both PCTP and MYL9 protein on immunoblotting, while RUNX1C overexpression reduced both proteins. This suggests RUNX1B and RUNX1C have differential effects in regulating downstream genes. These findings are in line with our studies in whole blood from a large cohort of patients with CAD showing a positive correlation between PCTP (Circulation 2017136(10):927-939) with RUNX1B transcript levels and a negative relationship with those for RUNX1C. Conclusions: RUNX1 isoforms autoregulate RUNX1 expression differentially in an isoform-specific manner. Moreover, they regulate target genes (MYL9 and PCTP) in a differential manner. These RUNX1 isoform-specific effects may be important in understanding the consequences of germline RUNX1 haplodeficiency and in modulating RUNX1 levels for therapeutic purposes.

IKKε Inhibitor Amlexanox Promotes Olaparib Sensitivity through the C/EBP-β-Mediated Transcription of Rad51 in Castrate-Resistant Prostate Cancer.

Simple SummaryMost men with advanced hormone-sensitive prostate cancer (HSPC) treated with androgen deprivation therapy will develop castrate resistant prostate cancer (CRPC), a lethal form of prostate cancer (PC). Our group has previously shown that IKKε expression is stronger in CRPC tumors and correlates with aggressive PC. Moreover, we have shown that IKKε depletion or inhibition (BX795, Amlexanox) decrease CRPC cell proliferation and tumor volume in an in vivo mouse model. We also demonstrate that IKKε inhibitors specifically target CRPC to induce a senescent phenotype as well as DNA damage and genomic instability. In this study, we demonstrated that IKKε depletion or inhibition block C/EBP-β recruitment on Rad51 promoter to decrease promoter activity. We have also shown that Amlexanox treatment sensitizes CRPC cells to Olaparib in vitro and in mouse models. Taken together, targeting IKKε with Amlexanox combined with Olaparib may lead to additional effective therapeutic strategies in the management of patients with CRPC.The progression of prostate cancer (PC) is often characterized by the development of castrate-resistant PC (CRPC). Patients with CRPC are treated with a variety of agents including new generation hormonal therapies or chemotherapy. However, as the cancer develops more resistance mechanisms, these drugs eventually become less effective and finding new therapeutic approaches is critical to improving patient outcomes. Previously, we have shown that IKKε depletion and IKKε inhibitors, BX795 and Amlexanox, decrease CRPC cell proliferation in vitro and in vivo and that IKKε inhibitors induce a senescence phenotype accompanied by increased DNA damage and genomic instability in CRPC cells. Here, we describe a new role for IKKε in DNA damage repair involving Rad51 and examine the therapeutic potential of Amlexanox combined with the PARP inhibitor Olaparib in CRPC cell lines. Combining Amlexanox with Olaparib decreased CRPC cell proliferation and enhanced DNA damage through the inhibition of Olaparib-induced Rad51 recruitment and expression in CRPC cells or IKKε-depleted PC-3 cells. We demonstrated that Rad51 promoter activity, measured by luciferase assay, was decreased with Amlexanox treatment or IKKε depletion and that Amlexanox treatment decreased the occupancy of transcription factor C/EBP-β on the Rad51 promoter. Our mouse model also showed that Amlexanox combined with Olaparib inhibited tumor growth of CRPC xenografts. Our study highlights a new role for IKKε in DNA damage repair through the regulation of Rad51 transcription and provides a rationale for the combination of Amlexanox and Olaparib in the treatment of patients with CRPC.

Alzheimer's disease is associated with disruption in thiamin transport physiology: A potential role for neuroinflammation

Alzheimer's disease (AD) is a neurodegenerative disease characterized by Amyloid-β peptide (Aβ) containing plaques and cognitive deficits. The pathophysiology of AD also involves neuroinflammation. Vitamin B1 (thiamin) is indispensable for normal cellular energy metabolism. Thiamin homeostasis is altered in AD, and its deficiency is known to aggravate AD pathology. Little, however, is known about possible alterations in level of expression of thiamin transporters-1 and -2 (THTR-1 and -2) in the brain of AD, and whether pro-inflammatory cytokines affect thiamin uptake by brain cells. We addressed these issues using brain tissue samples [prefrontal cortex (PFC) and hippocampus (HIP)] from AD patients and from 5XFAD mouse model of AD, together with cultured human neuroblastoma SH-SY5Y cells as model. Our results revealed a significantly lower expression of both THTR-1 and THTR-2 in the PFC and HIP of AD patients and 5XFAD mouse model of AD compared to appropriate normal controls. Further, we found that exposure of the SH-SY5Y cells to pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) led to a significant inhibition in thiamin uptake. Focusing on IL-1β, we found the inhibition in thiamin uptake to be time-dependent and reversible; it was also associated with a substantial reduction in expression of THTR-1 (but not THTR-2) protein and mRNA as well as a decrease in promoter activity of the SLC19A2 gene (which encodes THTR-1). Finally, using transcriptomic analysis, we found that thiamin availability in SH-SY5Y cells caused changes in the expression of genes relevant to AD pathways. These studies demonstrate, for the first time, that thiamin transport physiology/molecular biology parameters are negatively impacted in AD brain and that pro-inflammatory cytokines inhibit thiamin uptake by neuroblastoma cells. The results also support a possible role for thiamin in the pathophysiology of AD.

NF\u03baB (RelA) mediates transactivation of hnRNPD in oral cancer cells

Heterogeneous Ribonucleoprotein D (hnRNPD) is an RNA binding protein involved in post-transcriptional regulation of multiple mediators of carcinogenesis. We previously demonstrated a strong association of hnRNPD over expression with poor outcome in Oral Squamous Cell Carcinoma (OSCC). However, hitherto the precise molecular mechanism of its overexpression in oral cancer was not clear. Therefore, in an attempt to elucidate the transcriptional regulation of hnRNPD expression, we cloned 1406 bp of 5ʹ flanking region of human hnRNPD gene along with 257 bp of its first exon upstream to promoterless luciferase reporter gene in pGL3-Basic. Transfection of the resulting construct in SCC-4 cells yielded 1271 fold higher luciferase activity over parent vector. By promoter deletion analysis, we identified a canonical TATA box containing 126 bp core promoter region that retained ~ 58% activity of the full length promoter. In silico analysis revealed the presence of four putative NFκB binding motifs in the promoter. Sequential deletion of these motifs from the full-length promoter reporter construct coupled with luciferase assays revealed an 82% decrease in promoter activity after deletion of the first (−1358/−1347) motif and 99% reduction after the deletion of second motif (−1052/−1041). In-vivo binding of NFκB (RelA) to these two motifs in SCC-4 cells was confirmed by ChIP assays. Site directed mutagenesis of even one of these two motifs completely abolished promoter activity, while mutagenesis of the remaining two motifs had marginal effect on the same. Consistent with these findings, treatment of SCC-4 cells with PDTC, a known inhibitor of NFκB dramatically reduced the levels hnRNPD mRNA and protein. Finally, the expression of hnRNPD and NFκB in clinical specimen from 37 oral cancer patients was assessed and subjected to Spearmen’s Correlation analysis which revealed a strong positive correlation between the two. Thus, results of the present study for the first time convincingly demonstrate NFκB (RelA) mediated transcriptional upregulation of hnRNPD expression in oral cancer.

Transcriptional Regulation of the Synaptic Vesicle Protein Synaptogyrin-3 (SYNGR3) Gene: The Effects of NURR1 on Its Expression.

Synaptogyrin-3 (SYNGR3) is a synaptic vesicular membrane protein. Amongst four homologues (SYNGR1 to 4), SYNGR1 and 3 are especially abundant in the brain. SYNGR3 interacts with the dopamine transporter (DAT) to facilitate dopamine (DA) uptake and synaptic DA turnover in dopaminergic transmission. Perturbed SYNGR3 expression is observed in Parkinson’s disease (PD). The regulatory elements which affect SYNGR3 expression are unknown. Nuclear-receptor-related-1 protein (NURR1) can regulate dopaminergic neuronal differentiation and maintenance via binding to NGFI-B response elements (NBRE). We explored whether NURR1 can regulate SYNGR3 expression using an in silico analysis of the 5′-flanking region of the human SYNGR3 gene, reporter gene activity and an electrophoretic mobility shift assay (EMSA) of potential cis-acting sites. In silico analysis of two genomic DNA segments (1870 bp 5′-flanking region and 1870 + 159 bp of first exon) revealed one X Core Promoter Element 1 (XCPE1), two SP1, and three potential non-canonical NBRE response elements (ncNBRE) but no CAAT or TATA box. The longer segment exhibited gene promoter activity in luciferase reporter assays. Site-directed mutagenesis of XCPE1 decreased promoter activity in human neuroblastoma SH-SY5Y (↓43.2%) and human embryonic kidney HEK293 cells (↓39.7%). EMSA demonstrated NURR1 binding to these three ncNBRE. Site-directed mutagenesis of these ncNBRE reduced promoter activity by 11–17% in SH-SY5Y (neuronal) but not in HEK293 (non-neuronal) cells. C-DIM12 (Nurr1 activator) increased SYNGR3 protein expression in SH-SY5Y cells and its promoter activity using a real-time luciferase assay. As perturbed vesicular function is a feature of major neurodegenerative diseases, inducing SYNGR3 expression by NURR1 activators may be a potential therapeutic target to attenuate synaptic dysfunction in PD.

Wnt/β-catenin signaling and p68 conjointly regulate CHIP in colorectal carcinoma

Emerging evidences suggest abundant expression of Carboxy terminus of Hsc70 Interacting Protein or CHIP (alias STIP1 Homology and U–box Containing Protein 1 or STUB1) in colorectal carcinoma, but the mechanistic detail of this augmented expression pattern is unclear. The signature driver of canonical Wnt pathway, β-catenin, and its co-activator RNA helicase p68, are also overexpressed in colorectal carcinoma. In this study, we describe a novel mechanism of Wnt/β-catenin and p68 mediated transcriptional activation of CHIP gene leading to enhanced proliferation of colorectal carcinoma cells. Bioinformatic analyses reconfirmed an elevated CHIP expression level in colorectal carcinoma datasets. Wnt3A treatment and pharmacological activation of canonical Wnt signaling pathway resulted in increased nuclear translocation of β-catenin, augmenting CHIP expression. Likewise, immunoblotting and Real time PCR following overexpression and knockdown of β-catenin and p68 demonstrated upregulated and downregulated CHIP expression, respectively, at both mRNA and protein levels. p68 along with β-catenin were found to occupy Transcription Factor 4 (TCF4) binding sites on endogenous CHIP promoter and regulate its transcription. After cloning CHIP promoter, the increased and decreased promoter activities of CHIP induced by overexpression and knockdown of either β-catenin or p68 further confirmed transcriptional regulation of CHIP gene by Wnt/β-catenin signaling cascade. Finally, enhanced cellular propagation and migration of colorectal carcinoma cells induced by ‘Wnt/β-catenin-p68-CHIP’ axis established the significance of this pathway in oncogenesis. To the best of our knowledge, this is the first report elucidating the mechanistic details of transcriptional regulation of CHIP (STUB1) gene expression.

Heat and Hypoxia Exposure Mediates Circadian Rhythms Response via Methylation Modification in Apostichopus japonicas

As global warming progresses, heat and hypoxia are gradually becoming important factors threatening the survival, reproduction, and development of marine organisms. To determine the effect of heat and hypoxia on Apostichopus japonicus, whole genome methylation of the respiratory tree was determined under heat, hypoxia, and heat-hypoxia conditions [designed as heat stress treatment (HT), hypoxia treatment (LO), and heat-hypoxia combined treatment (HL) groups]. The number of differentially methylated regions (DMRs) under three treatments was determined based on the Venn diagram. The network of the DMRs associated with promoters that were co-existed under the three conditions showed that circadian rhythm was involved based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Circadian rhythm-related genes, CRY1a, CRY1b, CLC, and TIM, decreased in LO and HL groups, while CRY1a, CRY1b, and BMAL1 increased in the HT group. Bisulfite sequencing PCR (BSP) showed that the methylation levels of CpG island regions in the promoters of CRY1a and CRY1b were upregulated in HT, LO, and HL groups, leading to the decreased promoter activity of CRY1a and CRY1b. RNAi of CRY1a and CRY1b led to increased enzyme activities of two energy-related enzymes, pyruvate kinase (PK) catalyzing the rate-limiting step in glycolysis, and ATPase hydrolyzing ATP to ADP, which were also increased under the three tested conditions. Thus, it was concluded that A. japonicus may respond to the heat, hypoxia, and heat-hypoxia stresses via the DNA methylation of heat, hypoxia, and heat-hypoxia stresses via the DNA methylation of CpG islands of circadian rhythm-related genes, which increased the activity of energy-related enzymes.

Characterization of the G protein-coupled receptor kinase 6 promoter reveals a functional CREB binding site.

BackgroundG protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression.MethodsFive deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots.ResultsInvestigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression.ConclusionWe characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.

In vitro and in vivo assessment of the protective effect of sufentanil in acute lung injury.

ObjectivesTo investigate the mechanisms underlying the protective effect of sufentanil against acute lung injury (ALI).Material and MethodsRats were administered lipopolysaccharide (LPS) by endotracheal instillation to establish a model of ALI. LPS was used to stimulate BEAS-2B cells. The targets and promoter activities of IκB were assessed using a luciferase reporter assay. Apoptosis of BEAS-2B cells was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling.ResultsSufentanil treatment markedly reduced pathological changes in lung tissue, pulmonary edema and secretion of inflammatory factors associated with ALI in vivo and in vitro. In addition, sufentanil suppressed apoptosis induced by LPS and activated NF-κB both in vivo and in vitro. Furthermore, upregulation of high mobility group box protein 1 (HMGB1) protein levels and downregulation of miR-129-5p levels were observed in vivo and in vitro following sufentanil treatment. miR-129-5p targeted the 3ʹ untranslated region and its inhibition decreased promoter activities of IκB-α. miR-129-5p inhibition significantly weakened the protective effect of sufentanil on LPS-treated BEAS-2B cells.ConclusionSufentanil regulated the miR-129-5p/HMGB1 axis to enhance IκB-α expression, suggesting that sufentanil represents a candidate drug for ALI protection and providing avenues for clinical treatment.

Genetics and Genomics of SOST: Functional Analysis of Variants and Genomic Regulation in Osteoblasts.

SOST encodes the sclerostin protein, which acts as a key extracellular inhibitor of the canonical Wnt pathway in bone, playing a crucial role in skeletal development and bone homeostasis. The objective of this work was to assess the functionality of two variants previously identified (the rare variant rs570754792 and the missense variant p.Val10Ile) and to investigate the physical interactors of the SOST proximal promoter region in bone cells. Through a promoter luciferase reporter assay we show that the minor allele of rs570754792, a variant located in the extended TATA box motif, displays a significant decrease in promoter activity. Likewise, through western blot studies of extracellular and intracellular sclerostin, we observe a reduced expression of the p.Val10Ile mutant protein. Finally, using a circular chromosome conformation capture assay (4C-seq) in 3 bone cell types (MSC, hFOB, Saos-2), we have detected physical interactions between the SOST proximal promoter and the ECR5 enhancer, several additional enhancers located between EVT4 and MEOX1 and a distant region containing exon 18 of DHX8. In conclusion, SOST presents functional regulatory and missense variants that affect its expression and displays physical contacts with far reaching genomic sequences, which may play a role in its regulation within bone cells.

Transcription Factor RUNX1 Regulates Factor FXIIIA Subunit (F13A1) Expression in Megakaryocytic Cells and Platelet F13A1 Expression is Downregulated in RUNX1 Haplodeficiency

Plasma Factor XIII, a transglutaminase, is a complex of two A (gene F13A1) chains with catalytic activity and two B chains (gene F13B). FXIII induces clot stabilization and retraction via cross-linking of fibrin monomers and other proteins. Factor XIII deficiency in plasma causes a major bleeding disorder with impaired clot retraction. FXIIIA is synthesized in megakaryocytes, and is abundant in platelets. It is expressed on activated platelets and cross-links proteins on platelet surface. Little is known regarding the regulation of F13A1 in megakaryocytes and platelets. RUNX1 is a major hematopoietic transcription factor and regulates expression of numerous genes in megakaryocytes and platelets. Patients with RUNX1 haplodeficiency (RHD) with heterozygous mutations have thrombocytopenia, platelet dysfunction with impaired responses to activation, and predisposition to myeloid malignancies. Platelet expression profiling using Affymetrix microarrays of a patient (P1) with a heterozygous RUNX1 mutation (c.969-323G>T) revealed decreased F13A expression, which encodes for subunit A of FXIII: fold-change 0.30; p=0.006; patient profiled twice 10 months apart and compared to 6 healthy subjects. These findings were confirmed by qPCR With RT-PCR platelet F13A mRNA expression was decreased to ~ 20% of mean expression in 4 subjects. Platelet F13A protein expression was decreased by ~50% as assessed by corrected total cellular fluorescence imaging. Platelet F13A mRNA expression was also decreased (to less than 20% of expression in 5 controls) in two siblings (P2 and P3 ages 8 and 3 years) from an unrelated family with a heterozygous RUNX1 mutation 508+1G>A). Plasma FXIII was normal in the patient P1. We studied the regulation of F13A by RUNX1 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic transformation. In silico analyses of the 5' upstream region of F13A showed 7 RUNX1 consensus sites within 545bp from ATG. Chromatin immunoprecipitation studies performed using HEL cells and RUNX1 antibody showed that that RUNX1 binds in the region encompassing site 4 and sites 5-7. In parallel, electrophoretic mobility shift assay (EMSA) studies showed binding of nuclear protein to labeled DNA probes with sites 1, 4, 5, 6, and 7. RUNX1 antibody produced a "supershift" with addition suggesting RUNX1 binding. Site-directed mutagenesis of the RUNX1 binding sites in F13A1 promoter decreased promoter activity in luciferase reporter studies in HEL cells, providing evidence that the binding sites are functional. Further, overexpression of RUNX1 in HEL cells increased F13A1 promoter activity and protein while siRNA RUNX1 knockdown reduced F13A1 protein. We assessed clot retraction over 90 min at 37oC in the patient P1 and his daughter (also with RUNX1 mutation) in citrated whole blood following addition of tissue factor (1 pM) and CaCl2 (1 mM), and in washed platelets resuspended in platelet poor plasma from a healthy subject supplemented with added fibrinogen (0.5 mg/mL) and stimulated with 0.5 U/mL thrombin in the presence of CaCl2. These preliminary studies suggested that clot retraction was slower in the patient P1 compared to the controls, particularly in studies with platelet suspensions. Conclusions: Overall, these studies provide the first evidence that hematopoietic transcription factor RUNX1 regulates expression of F13A1 in megakaryocytic cells and that platelet expression of F13A1 is decreased in platelets from patients with RUNX1 haplodeficiency. The decreased expression of F13A1 in RHD may contribute to the platelet dysfunction in RHD. These findings are particularly interesting because they reflect regulation of a coagulation protein (FXIIIA) by hematopoietic transcription factor RUNX1. Disclosures Lambert: Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Consultancy; Educational Concepts in Medicine: Consultancy; Octapharma: Consultancy, Research Funding; Bayer: Consultancy; Platelet Disorder Support Association (PDSA): Consultancy; 22qSociety: Consultancy; ITP Australia: Consultancy; CdLS Foundation: Consultancy; RDMD ITP study: Consultancy; Sysmex: Research Funding; AstraZeneca: Research Funding; Argenix: Consultancy; ClinGen: Honoraria.

DNA Methylation as a Potential Molecular Mechanism in X-linked Dystonia-Parkinsonism.

X-linked dystonia-parkinsonism is a neurodegenerative movement disorder. The underlying molecular basis has still not been completely elucidated, but likely involves dysregulation of TAF1 expression. In X-linked dystonia-parkinsonism, 3 disease-specific single-nucleotide changes (DSCs) introduce (DSC12) or abolish (DSC2 and DSC3) CpG dinucleotides and consequently sites of putative DNA methylation. Because transcriptional regulation tightly correlates with specific epigenetic marks, we investigated the role of DNA methylation in the pathogenesis of X-linked dystonia-parkinsonism. DNA methylation at DSC12, DSC3, and DSC2 was quantified by bisulfite pyrosequencing in DNA from peripheral blood leukocytes, fibroblasts, induced pluripotent stem cell-derived cortical neurons and brain tissue from X-linked dystonia-parkinsonism patients and age- and sex-matched healthy Filipino controls in a prospective study. Compared with controls, X-linked dystonia-parkinsonism patients showed striking differences in DNA methylation at the 3 investigated CpG sites. Using methylation-sensitive luciferase reporter gene assays and immunoprecipitation, we demonstrated (1) that lack of DNA methylation because of DSC2 and DSC3 affects gene promoter activity and (2) that methylation at all 3 investigated CpG sites alters DNA-protein interaction. Interestingly, DSC3 decreased promoter activity per se compared with wild type, and promoter activity further decreased when methylation was present. Moreover, we identified specific binding of proteins to the investigated DSCs that are associated with splicing and RNA and DNA binding. We identified altered DNA methylation in X-linked dystonia-parkinsonism patients as a possible additional mechanism modulating TAF1 expression and putative novel targets for future therapies using DNA methylation-modifying agents. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.