Journal: International Journal of Biological Sciences

Article Title: Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation

doi: 10.7150/ijbs.92992

Figure Lengend Snippet: MT3 overexpression promotes proliferation of HPASMCs. A , MT3 expression levels were detected by Western blot in control (Lenti-Flag) and MT3-overexpressed (Lenti-MT3-Flag) HPASMCs. B , Representative light microscopy images of MT3-overexpressed HPASMCs cultured in normoxia/hypoxia for 0 and 48 hours (scale bar, 50 μm). C , The number of HPASMCs with or without MT3 overexpression was counted after normoxia/hypoxia treatment for 48 hours (n=6). D , CCK-8 assay was used to evaluate cell viability (n=10). E and F , EdU incorporation assay was performed to show the proliferation capacity of MT3-overexpressed (or not) HPASMCs (n=10, scale bar, 20 μm). G and H , Western blot analysis and quantification showing the protein level of the proliferation marker PCNA in HPASMCs of the indicated groups (n=4). * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The MT3 overexpression plasmid (MT3-Flag) was synthesized by Beijing Tsingke Biotech Co., Ltd, and the MTF1 overexpression plasmid (MTF1-ORF, CH835264) was purchased from Shandong Weizhen Biotechnology Co., Ltd, which were then cloned into the pHAGE-Flag plasmid.

Techniques: Over Expression, Expressing, Western Blot, Control, Light Microscopy, Cell Culture, CCK-8 Assay, Marker

Journal: International Journal of Biological Sciences

Article Title: Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation

doi: 10.7150/ijbs.92992

Figure Lengend Snippet: MT3 promotes HPASMC proliferation by regulating the Zn 2+ -MTF1 axis. A and B , Representative images showing Zn 2+ fluorescence staining of MT3 knockdown HPASMCs subjected to 48 hours of normoxia/hypoxia treatment by a Zinpyr-1 probe and quantified in B (n=15, scale bar, 10 μm). C , Quantitative analysis of the mean fluorescence intensity (MFI) of Zn 2+ in HPASMCs with the indicated treatments by flow cytometry (n=6). D and E , Representative images and quantitative analysis of Zn 2+ fluorescence staining in MT3-overexpressed HPASMCs subjected to 48 hours of normoxia/hypoxia treatment by a Zinpyr-1 probe (n=15, scale bar, 10 μm). F , Representative images and quantitative analysis of the mean fluorescence intensity (MFI) of Zn 2+ in HPASMCs with the indicated treatments by flow cytometry (n=4). Exogenous Zn 2+ was supplied by 50 μM ZnSO 4 co-incubation with HPASMCs MT3 knockdown or not. After all groups were treated with hypoxia for 48 hours, G , Cell counting assay was performed (n=6), H , CCK-8 assay was detected to assess cell viability (n=10), and EdU incorporation assay was performed to assess the proliferative capacity of the cells in I and J (n=15, scale bar, 20 μm). K , HPASMCs infected with Lenti-Flag and Lenti-MT3-Flag were subsequently reinfected with Lenti-PLKO.1 and Lenti-shMTF1, and number of cells counted after 48 hours of hypoxia. Under the same conditions as in K , CCK-8 ( L ) and EdU incorporation assays ( M and N ) were performed to determine cell viability and proliferation capacity, respectively (n=10 for CCK-8 and EdU, scale bar, 20 μm). * P <0.05, ** P <0.01, *** P <0.001, n.s indicates no significant difference.

Article Snippet: The MT3 overexpression plasmid (MT3-Flag) was synthesized by Beijing Tsingke Biotech Co., Ltd, and the MTF1 overexpression plasmid (MTF1-ORF, CH835264) was purchased from Shandong Weizhen Biotechnology Co., Ltd, which were then cloned into the pHAGE-Flag plasmid.

Techniques: Fluorescence, Staining, Knockdown, Flow Cytometry, Incubation, Cell Counting, CCK-8 Assay, Infection

Journal: International Journal of Biological Sciences

Article Title: Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation

doi: 10.7150/ijbs.92992

Figure Lengend Snippet: MT3 promotes autophagosome formation in HPASMCs by upregulating ATG5 expression. A , Gene Ontology (GO) enrichment analysis showing the biological processes enriched by DEGs between control and MT3 knockdown groups. B , Gene set enrichment analysis (GSEA) showing the decrease in autophagic activity. C - H , Autophagic flux of HPASMCs was detected by infection with GFP-mCherry-LC3-overexpressing lentivirus under the indicated treatments. Yellow dots indicate autophagosomes and red dots indicate autolysosomes. C and D , Representative images and quantitative analysis showing the autophagic flux activity of MT3 knockdown (or not) or overexpression (or not) followed by normoxic or hypoxic stimulation for 12 hours, respectively (n=10, scale bar, 10 μm). E - H , Representative images and quantitative analysis were performed under the conditions of C and D followed by treatment with 3-MA (5 μM) or CQ (10 μM) (n=10, scale bar, 10 μm). For C-H, *P<0.05 versus normoxia+Lenti-PLKO.1 or normoxia+Lenti-Flag or normoxia+Lenti-PLKO.1+3-MA/CQ or normoxia+Lenti-Flag+3-MA/CQ, # P <0.05 versus hypoxia+Lenti-PLKO.1 or hypoxia+Lenti-Flag or hypoxia+Lenti-PLKO.1+3-MA/CQ or hypoxia+Lenti-Flag+3-MA/CQ, n.s. indicates no significant difference. I and K , Western blot and quantitative analysis showed the protein expression levels of LC3 I/II under MT3 knockdown (or not) or MT3 overexpression (or not) and treated with CQ (10 μM) and normoxia/hypoxia for 12 hours (n=4). J and L , The protein expression levels of ATG5, ATG7, and p62 were detected by Western blot and quantitative analysis in HPASMCs infected with Lenti-shMT3 (or not) or Lenti-MT3-Flag (or not) and normoxia/hypoxia for 12 hours (n=4). * P <0.05, ** P <0.01, *** P <0.001, n.s. indicates no significant difference.

Article Snippet: The MT3 overexpression plasmid (MT3-Flag) was synthesized by Beijing Tsingke Biotech Co., Ltd, and the MTF1 overexpression plasmid (MTF1-ORF, CH835264) was purchased from Shandong Weizhen Biotechnology Co., Ltd, which were then cloned into the pHAGE-Flag plasmid.

Techniques: Expressing, Control, Knockdown, Activity Assay, Infection, Over Expression, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation

doi: 10.7150/ijbs.92992

Figure Lengend Snippet: MT3 promotes HPASMC proliferation via Zn 2+ -MTF1-ATG5 regulation of autophagosome formation. A and B , ATG5 mRNA levels in HPASMCs with MT3 knockdown (or not) or MT3 overexpression (or not) were detected by RT-PCR (n=6). C , Western blot and quantitative analysis showing the expression level of ATG5 in control and MT3 knockdown groups of HPASMCs treated with ZnSO 4 (50 μM) and normoxia/hypoxia for 48 hours (n=4). D , Western blot and quantification analysis showed the expression level of ATG5 in MT3-overexpressed (or not) HPASMCs associated with infection of Lenti-PLKO.1 or Lenti-shMTF1 and normoxia/hypoxia treatment for 48 hours (n=4). E , Luciferase activity of the pGL3-ATG5 promoter in HEK293T cells overexpressing (or not) MTF1 (n=6). F - I , Representative images and quantitative analysis of MT3-overexpressing (or not) HPASMCs infected with Lenti-PLKO.1 and Lenti-shMTF1 ( F and G ) or Lenti-shATG5 ( H and I ) and normoxia/hypoxia for 12 hours showed changes in autophagic flux (n=10, scale bar, 10 μm). J-M , HPASMCs first infected with Lenti-Flag or Lenti-MT3-Flag were reinfected with Lenti-PLKO.1 or Lenti-shATG5 and subjected to hypoxia for 48 hours for all groups. The number of HPASMCs was counted after the indicated treatments in J (n=6), CCK-8 assay was used to evaluate cell viability in K (n=10), and EdU incorporation assay was used to evaluate the proliferative capacity of HPASMCs in L and M (n=15, scale bar, 20 μm). * P <0.05, ** P <0.01, *** P <0.001, For G and I, * P <0.05 versus Lenti-Flag+Lenti-PLKO.1, # P <0.05 versus Lenti-MT3-Flag+Lenti-PLKO.1, n.s indicates no significant difference.

Article Snippet: The MT3 overexpression plasmid (MT3-Flag) was synthesized by Beijing Tsingke Biotech Co., Ltd, and the MTF1 overexpression plasmid (MTF1-ORF, CH835264) was purchased from Shandong Weizhen Biotechnology Co., Ltd, which were then cloned into the pHAGE-Flag plasmid.

Techniques: Knockdown, Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Control, Infection, Luciferase, Activity Assay, CCK-8 Assay

Journal: International Journal of Biological Sciences

Article Title: Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation

doi: 10.7150/ijbs.92992

Figure Lengend Snippet: Schematic summary. Our findings demonstrate that hypoxia treatment increases the expression of MT3 in HPASMCs, which can release Zn 2+ and increase the intracellular Zn 2+ concentration. Zn 2+ activates MTF1 and mediates MTF1 translocation to the nucleus. MTF1 binds directly to the promoter of the ATG5 gene to facilitate its transcription. ATG5 is a key protein for autophagy initiation and can activate autophagy by mediating autophagosome formation, which in turn promote PASMC proliferation and the development of PH. Therefore, MT3 facilitates PASMC proliferation by promoting Zn 2+ -MTF1-ATG5 axis-mediated autophagosome formation. Thus, targeted inhibition of MT3, Zn 2+ -MTF1 may be promising therapeutic strategies to inhibit pulmonary vascular remodeling and halt the development of PH.

Article Snippet: The MT3 overexpression plasmid (MT3-Flag) was synthesized by Beijing Tsingke Biotech Co., Ltd, and the MTF1 overexpression plasmid (MTF1-ORF, CH835264) was purchased from Shandong Weizhen Biotechnology Co., Ltd, which were then cloned into the pHAGE-Flag plasmid.

Techniques: Expressing, Concentration Assay, Translocation Assay, Inhibition

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: TAF15 Stabilized LINC00665 and MTF1 Played an Oncogenic Role in Glioma Cells (A) Relative expression of LINC00665 in glioma cells treated with TAF15 overexpression (n = 3, each group; ∗p < 0.05 versus TAF15 + -NC group). (B and C) An RNA-IP assay (B) and RNA pull-down assay (C) were used to identify LINC00665 in the TAF15 complex. LINC00665 enrichment was measured using quantitative real-time PCR (n = 3, each group; ∗∗p < 0.01 versus anti-IgG group). (D) Expression level of nascent LINC00665 was measured by quantitative real-time PCR (n = 3, each group; p > 0.05 versus TAF15 + -NC group). (E) The half-life of LINC00665 in the U87 glioma cells (left) and U251 glioma cells (right) treated with TAF15 overexpression. (F) MTF1 expression levels in NBTs, LGGTs, and HGGTs are shown (∗∗p < 0.01 versus NBTs group; ## p < 0.01 versus LGGTs group). (G) MTF1 expression levels in HA, U87, and U251 cell lines are shown (n = 3, each group; ∗∗p < 0.01 versus HA group). (H) A CCK-8 assay was used to measure the effect of MTF1 on the proliferation of glioma cells. (I) The apoptotic percentages of glioma cells were detected with MTF1 upregulation or downregulation. (J) A transwell assay was used to measure the effect of MTF1 on cell migration and invasion of U87 and U251 glioma cells (n = 3, each group; ∗p < 0.05 versus MTF1 + -NC group; #p < 0.05 versus MTF1 − -NC group). Scale bars represent 200 μm.

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Expressing, Over Expression, Pull Down Assay, Real-time Polymerase Chain Reaction, CCK-8 Assay, Transwell Assay, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: LINC00665 Destabilized MTF1 mRNA by Interacting with STAU1, thus Regulating Glioma Cell Malignant Progression (A and B) Quantitative real-time PCR (A) and western blot (B) were applied to test expression levels of MTF1 (∗p < 0.05 versus LINC00665 + -NC group; # p < 0.05 versus STAU1 − -NC group; ▲ p < 0.05 versus LINC00665 + group). (C) LINC00665 and MTF1 could bind to STAU1 protein, respectively. Both relative enrichment levels of LINC00665 and MTF1 were measured using quantitative real-time PCR (n = 3, each group; ∗∗p < 0.01 versus anti-IgG group). (D) The predicted LINC00665 binding site in MTF1 (MTF1-3′ UTR-WT) and the designed mutant sequence (MTF1-3′ UTR-Mut) are shown (left). Luciferase reporter gene assays of HEK293T cells are shown (right) (n = 3, each group; ∗p < 0.05 versus MTF1-3′ UTR-WT + LINC00665-NC group). (E) Remaining MTF1 mRNA (%) at the different actinomycin D treatment times in control group, LINC00665 + -NC group, and LINC00665 + group. (F) Remaining MTF1 mRNA (%) in control group, STAU1 − -NC group, and STAU1 − group. (G–I) CCK-8 (G), flow cytometry (H), and transwell assays (I) were used to assess the proliferation, apoptosis, migration, and invasion capacity of U87 and U251 glioma cells (n = 3, each group; ∗p < 0.05 versus control group; # p < 0.05 versus LINC00665 + + MTF1 + group; ▲ p < 0.05 versus LINC00665 − + MTF1 − group). Scale bars represent 200 μm.

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Binding Assay, Mutagenesis, Sequencing, Luciferase, Control, CCK-8 Assay, Flow Cytometry, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: GTSE1 Was Upregulated in Glioma Cells, and Knockdown of GTSE1 Inhibited Malignant Biological Behaviors (A) GTSE1 expression levels in NBTs, LGGTs, and HGGTs are shown (∗∗p < 0.01 versus NBTs group; ## p < 0.01 versus LGGTs group). (B) GTSE1 expression levels in HA, U87, and U251 cell lines are shown (n = 3, each group; ∗∗p < 0.01 versus HA group). (C) CCK-8 assay was performed to assess the effect of GTSE1 knockdown on the proliferation of glioma cells. (D) The ratio of apoptotic glioma cells were detected with GTSE1 downregulation. (E) Number of migrated and invaded glioma cells was counted to represent the effect of GTSE1 inhibition on cell migration and invasion (n = 3, each group; ∗p < 0.05 versus GTSE1 − -NC group). Scale bars represent 200 μm. (F) The expression level of GTSE1 mRNA in glioma cells treated with MTF1 variation. (G) The expression level of GTSE1 protein in glioma cells are shown (n = 3, each group; ∗p < 0.05 versus MTF1 + -NC group; # p < 0.05 versus MTF1 − -NC group). (H) The expression level of GTSE1 mRNA in glioma cells treated with YY2 variation. (I) The expression level of GTSE1 protein in glioma cells treated with YY2 variation (n = 3, each group; ∗p < 0.05 versus YY2 + -NC group; # p < 0.05 versus YY2 − -NC group).

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Knockdown, Expressing, CCK-8 Assay, Inhibition, Migration

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: MTF1 and YY2 Bound to the GTSE1 Promoter and Regulated the Expression Level of GTSE1 (A) Schematic representation of the human GTSE1 promoter region 2,000 bp upstream of the transcription start site (TSS), which was designated as +1. Putative MTF1 binding site was indicated. PCR was conducted with the resulting precipitated DNA. (B) The change of GTSE1 mRNA expression level caused by LINC00665 and MTF1 variation. (C) The change of GTSE1 protein expression level caused by LINC00665 and MTF1 variation (n = 3, each group; ∗p < 0.05 versus control group; # p < 0.05 versus LINC00665 + + MTF1 + group; ▲ p < 0.05 versus LINC00665 − + MTF1 − group). (D) YY2 bound to the promoter of GTSE1 in glioma cells. Three putative YY2 binding sites were indicated. PCR was conducted with the resulting precipitated DNA. (E) The change of GTSE1 mRNA expression level caused by LINC00665 and YY2 variation. (F) The change of GTSE1 protein expression level caused by LINC00665 and YY2 variation (n = 3, each group; ∗p < 0.05 versus control group; # p < 0.05 versus LINC00665 + + YY2 + group; ▲ p < 0.05 versus LINC00665 − + YY2 − group).

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Expressing, Binding Assay, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: In Vivo Study and the Schematic Picture of the Mechanisms in Our Study (A) Nude mice carrying tumors from respective groups are shown. The sample tumors from respective groups are shown. (B) Tumor volume was calculated every 4 days after injection, and tumors were excised after 44 days (∗p < 0.05 versus control group; # p < 0.05 versus TAF15 + group; ▲ p < 0.05 versus LINC00665 + group; ▪ p < 0.05 versus MTF1 + group; ● p < 0.05 versus YY2 + group). (C) The survival curves of nude mice injected into the right striatum. (D) The schematic picture of the mechanism in which TAF15 combined with LINC00665, thus enhancing the degradation of MTF1 and YY2 mRNA via the SMD pathway in glioma cells. Ter, translation termination codon.

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: In Vivo, Injection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: shRNA Sequences

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: shRNA, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: lncRNA LINC00665 Stabilized by TAF15 Impeded the Malignant Biological Behaviors of Glioma Cells via STAU1-Mediated mRNA Degradation

doi: 10.1016/j.omtn.2020.05.003

Figure Lengend Snippet: ChIP Primers

Article Snippet: TAF15 full-length plasmid TAF15 + , MTF1 full-length plasmid MTF1 + , YY2 full-length plasmid YY2 + , and their respective expression vectors (NCs) were designed and synthesized in GenScript (Nanjing, China), while LINC00665 full-length plasmid LINC00665 + and its NC were designed and synthesized in GeneChem (Shanghai, China).

Techniques: Control, Binding Assay

Journal: European Journal of Human Genetics

Article Title: An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease

doi: 10.1038/s41431-018-0221-4

Figure Lengend Snippet: Genomic context of the candidate causative variant. a UCSC Genome Browser (hg19 minus strand) view of the region near the candidate causative variant positioned 676 bp upstream of the canonical ATP7B translation start site (chr13:g.52,586,149A>G, cyan highlight and asterisk). The variant lies in a 100-way vertebrate alignment conserved element and region of high cross-species sequence conservation as computed by PhastCons and PhyloP [30]. The chromatin surrounding chr13:g.52,586,149T>C is hypersensitive to DNaseI (and, therefore, open and accessible) in HepG2 cells. The sequence cloned into the luciferase reporter vector is indicated by the black bar. b Genotype verification for chr13:g.52,586,149T>C (red asterisk) by Sanger sequencing. Representative chromatograms show the indicated number (n=x/y) of amplification products containing the reference (T) or alternate (C) allele for each individual. Box indicates a predicted binding site for MTF1. c The candidate causative variant (bolded, pink) in the Wilson Disease (WD) patient disrupts a key base in the MTF1 position weight matrix. This exact base (boxed) is unaltered in all primates and in dozens of other mammals, even as distant as Tasmanian devil (Supplementary Fig. 1b) (color figure online).

Article Snippet: For experiments involving overexpression of MTF1, the human MTF1-FLAG expression plasmid (ABclonal, Woburn, MA, USA: HG15046-CF) was transfected (200 ng per well) 24 h prior to introducing the luciferase vectors.

Techniques: Variant Assay, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Amplification, Binding Assay

Journal: European Journal of Human Genetics

Article Title: An MTF1 binding site disrupted by a homozygous variant in the promoter of ATP7B likely causes Wilson Disease

doi: 10.1038/s41431-018-0221-4

Figure Lengend Snippet: Functional interrogation of chr13:g.52,586,149T>C. a Luciferase reporter assays in HepG2 cells were performed to quantify differences in transactivation by a 379 bp fragment of the ATP7B promoter with the chr13:g.52,586,149T (Ref) or chr13:g.52,586,149C (Alt) allele, in the presence (+) or absence (−) of MTF1 overexpression (OE). Both with and without MTF1 OE, the reference allele drove significantly higher expression compared to the alternate allele (comparison 1 and 2, respectively). Both alleles experienced dramatic increases in activity with MTF1 OE (comparisons 3 and 4), but the reference allele yielded a greater increase in expression than did the alternate allele in this context (comparison 5). Bars represent mean ± SD. Two-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. b ChIP-qPCR was performed to determine the extent of MTF1 binding at the SNV locus (ATP7B) compared to a computationally predicted negative control (Neg Ctrl, in an intron of WDPCP) and to a previously published [32] experimentally validated locus (Pos Ctrl, in the 5′ UTR of SELENOH). Enrichment of DNA immunoprecipitated by MTF1-specific vs. non-specific isotype-matched control antibodies was measured in technical triplicate by quantitative PCR analysis. Bars represent mean ± SD. One-tailed p-values from unpaired t-tests for each comparison are shown above the associated bracket. c Proposed model for disease-causing mechanism of chr13:g.52,586,149T>C: (1) Excess intracellular accumulation of copper, Cu2+, increases (2) expression or nuclear translocation of MTF1 [12, 13]. In wildtype individuals, MTF1 then binds at chr13:g.52,586,149T (3) to recruit transcriptional machinery for upregulating ATP7B expression [34] and eliminating copper through serum ceruloplasmin and, ultimately, through the bile. We propose that the Wilson Disease (WD) patient’s homozygous single nucleotide promoter variant chr13:g.52,586,149T>C exhibits reduced affinity to MTF1. (4) The result is an insufficient ATP7B transcriptional response, consequent copper accumulation, and symptoms characteristic of Wilson Disease

Article Snippet: For experiments involving overexpression of MTF1, the human MTF1-FLAG expression plasmid (ABclonal, Woburn, MA, USA: HG15046-CF) was transfected (200 ng per well) 24 h prior to introducing the luciferase vectors.

Techniques: Functional Assay, Luciferase, Over Expression, Expressing, Activity Assay, Two Tailed Test, Binding Assay, Negative Control, Immunoprecipitation, Real-time Polymerase Chain Reaction, One-tailed Test, Translocation Assay, Variant Assay