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Direct binding between PU.1 and the triggering receptor expressed on myeloid cells-2 (TREM2) promoter or enhancer in vitro and in vivo and the regulation of the promoter activity of TREM2 The transcription factor PU.1 promoted TREM2 transcription. (A) Schematic representation of the TREM2 promoter sequence, which shows the sequences and positions of the four putative PU.1 binding sites highlighted in frame, used to study the binding site of the transcription factor PU.1 in the TREM2 promoter sequence. (B,C) A chromatin <t>immunoprecipitation</t> <t>(ChIP)</t> assay of microglial BV2 cells was performed using Abs against the PU.1 protein to immunoprecipitate chromatin from the cells. DNA fragments were analyzed by Quantitative real-time PCr (qRT–PCR) and are represented as percentages (%) input DNA (C) ; a PAGE gel graph (B) of the qRT–PCR results after the ChIP assay for different binding sites from (A) is shown. (D) The 3.0 kb DNA sequence upstream of the transcription start site (TSS) was cloned and inserted into the pGL4.1 basic vector. The TREM2 promoter plasmid or TREM2 promoter containing UTR fragment constructs were transfected alone or cotransfected with the PU.1 expression plasmid into HEK293 cells via Lipofectamine 3000, and the pRL-TK Renilla vector expression vector pRL-SV40 was used for normalization. After 48 h of transfection, luciferase activity was measured with a dual-luciferase reporter assay system. The data are expressed as the relative luciferase units (RLUs) of three separate experiments. *** p < 0.001.
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Direct binding between PU.1 and the triggering receptor expressed on myeloid cells-2 (TREM2) promoter or enhancer in vitro and in vivo and the regulation of the promoter activity of TREM2 The transcription factor PU.1 promoted TREM2 transcription. (A) Schematic representation of the TREM2 promoter sequence, which shows the sequences and positions of the four putative PU.1 binding sites highlighted in frame, used to study the binding site of the transcription factor PU.1 in the TREM2 promoter sequence. (B,C) A chromatin <t>immunoprecipitation</t> <t>(ChIP)</t> assay of microglial BV2 cells was performed using Abs against the PU.1 protein to immunoprecipitate chromatin from the cells. DNA fragments were analyzed by Quantitative real-time PCr (qRT–PCR) and are represented as percentages (%) input DNA (C) ; a PAGE gel graph (B) of the qRT–PCR results after the ChIP assay for different binding sites from (A) is shown. (D) The 3.0 kb DNA sequence upstream of the transcription start site (TSS) was cloned and inserted into the pGL4.1 basic vector. The TREM2 promoter plasmid or TREM2 promoter containing UTR fragment constructs were transfected alone or cotransfected with the PU.1 expression plasmid into HEK293 cells via Lipofectamine 3000, and the pRL-TK Renilla vector expression vector pRL-SV40 was used for normalization. After 48 h of transfection, luciferase activity was measured with a dual-luciferase reporter assay system. The data are expressed as the relative luciferase units (RLUs) of three separate experiments. *** p < 0.001.
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Direct binding between PU.1 and the triggering receptor expressed on myeloid cells-2 (TREM2) promoter or enhancer in vitro and in vivo and the regulation of the promoter activity of TREM2 The transcription factor PU.1 promoted TREM2 transcription. (A) Schematic representation of the TREM2 promoter sequence, which shows the sequences and positions of the four putative PU.1 binding sites highlighted in frame, used to study the binding site of the transcription factor PU.1 in the TREM2 promoter sequence. (B,C) A chromatin <t>immunoprecipitation</t> <t>(ChIP)</t> assay of microglial BV2 cells was performed using Abs against the PU.1 protein to immunoprecipitate chromatin from the cells. DNA fragments were analyzed by Quantitative real-time PCr (qRT–PCR) and are represented as percentages (%) input DNA (C) ; a PAGE gel graph (B) of the qRT–PCR results after the ChIP assay for different binding sites from (A) is shown. (D) The 3.0 kb DNA sequence upstream of the transcription start site (TSS) was cloned and inserted into the pGL4.1 basic vector. The TREM2 promoter plasmid or TREM2 promoter containing UTR fragment constructs were transfected alone or cotransfected with the PU.1 expression plasmid into HEK293 cells via Lipofectamine 3000, and the pRL-TK Renilla vector expression vector pRL-SV40 was used for normalization. After 48 h of transfection, luciferase activity was measured with a dual-luciferase reporter assay system. The data are expressed as the relative luciferase units (RLUs) of three separate experiments. *** p < 0.001.
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Direct binding between PU.1 and the triggering receptor expressed on myeloid cells-2 (TREM2) promoter or enhancer in vitro and in vivo and the regulation of the promoter activity of TREM2 The transcription factor PU.1 promoted TREM2 transcription. (A) Schematic representation of the TREM2 promoter sequence, which shows the sequences and positions of the four putative PU.1 binding sites highlighted in frame, used to study the binding site of the transcription factor PU.1 in the TREM2 promoter sequence. (B,C) A chromatin <t>immunoprecipitation</t> <t>(ChIP)</t> assay of microglial BV2 cells was performed using Abs against the PU.1 protein to immunoprecipitate chromatin from the cells. DNA fragments were analyzed by Quantitative real-time PCr (qRT–PCR) and are represented as percentages (%) input DNA (C) ; a PAGE gel graph (B) of the qRT–PCR results after the ChIP assay for different binding sites from (A) is shown. (D) The 3.0 kb DNA sequence upstream of the transcription start site (TSS) was cloned and inserted into the pGL4.1 basic vector. The TREM2 promoter plasmid or TREM2 promoter containing UTR fragment constructs were transfected alone or cotransfected with the PU.1 expression plasmid into HEK293 cells via Lipofectamine 3000, and the pRL-TK Renilla vector expression vector pRL-SV40 was used for normalization. After 48 h of transfection, luciferase activity was measured with a dual-luciferase reporter assay system. The data are expressed as the relative luciferase units (RLUs) of three separate experiments. *** p < 0.001.
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Direct binding between PU.1 and the triggering receptor expressed on myeloid cells-2 (TREM2) promoter or enhancer in vitro and in vivo and the regulation of the promoter activity of TREM2 The transcription factor PU.1 promoted TREM2 transcription. (A) Schematic representation of the TREM2 promoter sequence, which shows the sequences and positions of the four putative PU.1 binding sites highlighted in frame, used to study the binding site of the transcription factor PU.1 in the TREM2 promoter sequence. (B,C) A chromatin <t>immunoprecipitation</t> <t>(ChIP)</t> assay of microglial BV2 cells was performed using Abs against the PU.1 protein to immunoprecipitate chromatin from the cells. DNA fragments were analyzed by Quantitative real-time PCr (qRT–PCR) and are represented as percentages (%) input DNA (C) ; a PAGE gel graph (B) of the qRT–PCR results after the ChIP assay for different binding sites from (A) is shown. (D) The 3.0 kb DNA sequence upstream of the transcription start site (TSS) was cloned and inserted into the pGL4.1 basic vector. The TREM2 promoter plasmid or TREM2 promoter containing UTR fragment constructs were transfected alone or cotransfected with the PU.1 expression plasmid into HEK293 cells via Lipofectamine 3000, and the pRL-TK Renilla vector expression vector pRL-SV40 was used for normalization. After 48 h of transfection, luciferase activity was measured with a dual-luciferase reporter assay system. The data are expressed as the relative luciferase units (RLUs) of three separate experiments. *** p < 0.001.
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Direct binding between PU.1 and the triggering receptor expressed on myeloid cells-2 (TREM2) promoter or enhancer in vitro and in vivo and the regulation of the promoter activity of TREM2 The transcription factor PU.1 promoted TREM2 transcription. (A) Schematic representation of the TREM2 promoter sequence, which shows the sequences and positions of the four putative PU.1 binding sites highlighted in frame, used to study the binding site of the transcription factor PU.1 in the TREM2 promoter sequence. (B,C) A chromatin <t>immunoprecipitation</t> <t>(ChIP)</t> assay of microglial BV2 cells was performed using Abs against the PU.1 protein to immunoprecipitate chromatin from the cells. DNA fragments were analyzed by Quantitative real-time PCr (qRT–PCR) and are represented as percentages (%) input DNA (C) ; a PAGE gel graph (B) of the qRT–PCR results after the ChIP assay for different binding sites from (A) is shown. (D) The 3.0 kb DNA sequence upstream of the transcription start site (TSS) was cloned and inserted into the pGL4.1 basic vector. The TREM2 promoter plasmid or TREM2 promoter containing UTR fragment constructs were transfected alone or cotransfected with the PU.1 expression plasmid into HEK293 cells via Lipofectamine 3000, and the pRL-TK Renilla vector expression vector pRL-SV40 was used for normalization. After 48 h of transfection, luciferase activity was measured with a dual-luciferase reporter assay system. The data are expressed as the relative luciferase units (RLUs) of three separate experiments. *** p < 0.001.
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Direct binding between PU.1 and the triggering receptor expressed on myeloid cells-2 (TREM2) promoter or enhancer in vitro and in vivo and the regulation of the promoter activity of TREM2 The transcription factor PU.1 promoted TREM2 transcription. (A) Schematic representation of the TREM2 promoter sequence, which shows the sequences and positions of the four putative PU.1 binding sites highlighted in frame, used to study the binding site of the transcription factor PU.1 in the TREM2 promoter sequence. (B,C) A chromatin immunoprecipitation (ChIP) assay of microglial BV2 cells was performed using Abs against the PU.1 protein to immunoprecipitate chromatin from the cells. DNA fragments were analyzed by Quantitative real-time PCr (qRT–PCR) and are represented as percentages (%) input DNA (C) ; a PAGE gel graph (B) of the qRT–PCR results after the ChIP assay for different binding sites from (A) is shown. (D) The 3.0 kb DNA sequence upstream of the transcription start site (TSS) was cloned and inserted into the pGL4.1 basic vector. The TREM2 promoter plasmid or TREM2 promoter containing UTR fragment constructs were transfected alone or cotransfected with the PU.1 expression plasmid into HEK293 cells via Lipofectamine 3000, and the pRL-TK Renilla vector expression vector pRL-SV40 was used for normalization. After 48 h of transfection, luciferase activity was measured with a dual-luciferase reporter assay system. The data are expressed as the relative luciferase units (RLUs) of three separate experiments. *** p < 0.001.

Journal: Frontiers in Aging Neuroscience

Article Title: PU.1 dictates β-amyloid-induced TREM2 expression upregulation in microglia in a transgenic model of Alzheimer’s disease

doi: 10.3389/fnagi.2025.1537388

Figure Lengend Snippet: Direct binding between PU.1 and the triggering receptor expressed on myeloid cells-2 (TREM2) promoter or enhancer in vitro and in vivo and the regulation of the promoter activity of TREM2 The transcription factor PU.1 promoted TREM2 transcription. (A) Schematic representation of the TREM2 promoter sequence, which shows the sequences and positions of the four putative PU.1 binding sites highlighted in frame, used to study the binding site of the transcription factor PU.1 in the TREM2 promoter sequence. (B,C) A chromatin immunoprecipitation (ChIP) assay of microglial BV2 cells was performed using Abs against the PU.1 protein to immunoprecipitate chromatin from the cells. DNA fragments were analyzed by Quantitative real-time PCr (qRT–PCR) and are represented as percentages (%) input DNA (C) ; a PAGE gel graph (B) of the qRT–PCR results after the ChIP assay for different binding sites from (A) is shown. (D) The 3.0 kb DNA sequence upstream of the transcription start site (TSS) was cloned and inserted into the pGL4.1 basic vector. The TREM2 promoter plasmid or TREM2 promoter containing UTR fragment constructs were transfected alone or cotransfected with the PU.1 expression plasmid into HEK293 cells via Lipofectamine 3000, and the pRL-TK Renilla vector expression vector pRL-SV40 was used for normalization. After 48 h of transfection, luciferase activity was measured with a dual-luciferase reporter assay system. The data are expressed as the relative luciferase units (RLUs) of three separate experiments. *** p < 0.001.

Article Snippet: Microglial BV2 cells were prepared for chromatin immunoprecipitation (ChIP) following the protocol established by Millipore (Temecula, CA, United States).

Techniques: Binding Assay, In Vitro, In Vivo, Activity Assay, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Clone Assay, Plasmid Preparation, Construct, Transfection, Expressing, Luciferase, Reporter Assay