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TaKaRa cytomegalovirus
Cytomegalovirus, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cytomegalovirus/product/TaKaRa
Average 92 stars, based on 66 article reviews
Price from $9.99 to $1999.99
cytomegalovirus - by Bioz Stars, 2020-09
92/100 stars

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Clone Assay:

Article Title: Evidence for the multimeric structure of ferroportin
Article Snippet: .. A mouse Fpn cDNA was cloned into the Xho1 sites of the cytomegalovirus (CMV)–containing vectors pEGFP-N1 (Clontech, Mountain View, CA), pCMV-Tag4B-FLAG (Stratagene, La Jolla, CA) or pCMV-Tag5–c-myc (Stratagene). ..

Transfection:

Article Title: Skeletal Overexpression of Connective Tissue Growth Factor Impairs Bone Formation and Causes Osteopenia
Article Snippet: .. A cytomegalovirus (CMV) directed β-galactosidase expression construct (Clontech, San Jose, CA) was used to control for transfection efficiency. .. Cells were exposed to the FuGENE-DNA mix for 16 h and transferred to serum-free medium for 8 h, treated with BMP-2, Wnt 3a or vehicle for 24 h, as indicated in the text and legends, and harvested.

Synthesized:

Article Title: Mutational Analysis of the Cytoplasmic Tail of Jaagsiekte Sheep Retrovirus Envelope Protein
Article Snippet: .. JSRV Env (pCMV3JS21ΔGP) expresses wild-type JSRV Env from a cytomegalovirus (CMV) promoter ( To create the amphipathic helix-GFP reporter plasmids (Fig. ), sense and antisense oligomers encoding the first 18 amino acids of the cytoplasmic tail were synthesized, annealed, and inserted upstream and in frame with the enhanced green fluorescent protein (EGFP) gene in pEGFP-N1 (Clontech) at BamHI and HindIII sites. .. To disrupt the amphipathic helix, oligomers were synthesized that mutated either hydrophobic residues to aspartate (hydrophilic-GFP) or hydrophilic residues to proline (hydrophobic-GFP).

Construct:

Article Title: Skeletal Overexpression of Connective Tissue Growth Factor Impairs Bone Formation and Causes Osteopenia
Article Snippet: .. A cytomegalovirus (CMV) directed β-galactosidase expression construct (Clontech, San Jose, CA) was used to control for transfection efficiency. .. Cells were exposed to the FuGENE-DNA mix for 16 h and transferred to serum-free medium for 8 h, treated with BMP-2, Wnt 3a or vehicle for 24 h, as indicated in the text and legends, and harvested.

Article Title: LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex ▿LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex ▿ †
Article Snippet: .. 99-PUR was constructed by combining an EcoRI/TthIII-blunted fragment from a modified version of vector pCEP4 (Invitrogen), lacking the cytomegalovirus (CMV) promoter, with an EcoRI-BamHI fragment from pPUR (Clontech). .. This hybrid construct contains the puromycin resistance gene.

Expressing:

Article Title: Skeletal Overexpression of Connective Tissue Growth Factor Impairs Bone Formation and Causes Osteopenia
Article Snippet: .. A cytomegalovirus (CMV) directed β-galactosidase expression construct (Clontech, San Jose, CA) was used to control for transfection efficiency. .. Cells were exposed to the FuGENE-DNA mix for 16 h and transferred to serum-free medium for 8 h, treated with BMP-2, Wnt 3a or vehicle for 24 h, as indicated in the text and legends, and harvested.

Modification:

Article Title: LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex ▿LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex ▿ †
Article Snippet: .. 99-PUR was constructed by combining an EcoRI/TthIII-blunted fragment from a modified version of vector pCEP4 (Invitrogen), lacking the cytomegalovirus (CMV) promoter, with an EcoRI-BamHI fragment from pPUR (Clontech). .. This hybrid construct contains the puromycin resistance gene.

Plasmid Preparation:

Article Title: LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex ▿LINE-1 ORF1 Protein Localizes in Stress Granules with Other RNA-Binding Proteins, Including Components of RNA Interference RNA-Induced Silencing Complex ▿ †
Article Snippet: .. 99-PUR was constructed by combining an EcoRI/TthIII-blunted fragment from a modified version of vector pCEP4 (Invitrogen), lacking the cytomegalovirus (CMV) promoter, with an EcoRI-BamHI fragment from pPUR (Clontech). .. This hybrid construct contains the puromycin resistance gene.

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  • 85
    TaKaRa cmv empty vector
    HDAC1 recruitment by YY1 is required for the repression of LTR. Cell extracts were prepared from <t>GFP-positive</t> cells cotransfected with empty <t>CMV</t> vector, Tat, Tat plus wild-type YY1, and Tat plus mutant YY1 lacking the HDAC1 interaction domain. CAT assays (upper panel) were performed to correlate the level of LTR expression with the acetylation of histone H4 and HDAC1 occupancy (middle panel) at nuc 1, as measured by ChIP assays. A YY1 mutant with a deletion of HDAC1 interaction domain was unable to repress the LTR, increase HDAC1 occupancy, or significantly decrease H4 acetylation at nuc 1.
    Cmv Empty Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cmv empty vector/product/TaKaRa
    Average 85 stars, based on 1 article reviews
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    cmv empty vector - by Bioz Stars, 2020-09
    85/100 stars
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    cmv  (TaKaRa)
    92
    TaKaRa cmv
    Construction of plasmids. The commercial plasmids pEGFP-C1 and pEGFP-N1 contain the <t>CMV</t> promoter which acts in mammalian cells, and the promoter of baculovirus <t>ie2</t> which works in insect cells derives from the plasmid pIZ-V5/His. All the recombinant plasmids contained the promoter of ie2 following the promoter of CMV except pie2/EGFP-Atg8, which had no the promoter of CMV. The EGFP after Atg8 did not express because Atg8 has a stop codon. PCR was used for site-directed mutagenesis and truncation of open reading frames. Atg8 116G has a glycine (G) residue at C terminus (the 116 th amino acid residue). Atg8 62 had 62 amino acid residues of the N terminus of Atg8. F77/79A and F60/62A indicated that tyrosine (F) residues were replaced with alanine (A) residues.
    Cmv, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 341 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cmv/product/TaKaRa
    Average 92 stars, based on 341 article reviews
    Price from $9.99 to $1999.99
    cmv - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    86
    TaKaRa hcmv miep
    The abundance of H3K4me3 and H3K27me3 at the <t>HCMV</t> <t>MIEP</t> in shControl and shEZH2 HFFs, at the pre-immediate early stage of the infection. A. Graphical illustration of the MIEP of HCMV. Open boxes represent regions probed by ChIP for association with histone H3K27me3 and histone H3K4me3. Enh stands for Enhancer and crs stands for cis-repressive sequence. B: HFFs were transduced with either the pLKO.1 empty vector or the shEZH2 lentiviral construct. A western blot of cell lysates was probed with the EZH2 antibody. C-E. HFFs transduced with a lentivirus construct of shEZH2 or with the empty vector, were infected with HCMV (MOI 0.5 PFUs/cell). ChIPs using antibodies against H3K27me3 and H3K4me3 were analysed by qPCR using primers specific for the indicated loci (see materials and methods ), at 0.5 h, 1.5 h and 3 h.p.i. The results are given as mean + SD for triplicate Real Time PCRs. The asterisks indicate statistical significance. * indicates p
    Hcmv Miep, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hcmv miep/product/TaKaRa
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    HDAC1 recruitment by YY1 is required for the repression of LTR. Cell extracts were prepared from GFP-positive cells cotransfected with empty CMV vector, Tat, Tat plus wild-type YY1, and Tat plus mutant YY1 lacking the HDAC1 interaction domain. CAT assays (upper panel) were performed to correlate the level of LTR expression with the acetylation of histone H4 and HDAC1 occupancy (middle panel) at nuc 1, as measured by ChIP assays. A YY1 mutant with a deletion of HDAC1 interaction domain was unable to repress the LTR, increase HDAC1 occupancy, or significantly decrease H4 acetylation at nuc 1.

    Journal: Molecular and Cellular Biology

    Article Title: Counterregulation of Chromatin Deacetylation and Histone Deacetylase Occupancy at the Integrated Promoter of Human Immunodeficiency Virus Type 1 (HIV-1) by the HIV-1 Repressor YY1 and HIV-1 Activator Tat

    doi: 10.1128/MCB.22.9.2965-2973.2002

    Figure Lengend Snippet: HDAC1 recruitment by YY1 is required for the repression of LTR. Cell extracts were prepared from GFP-positive cells cotransfected with empty CMV vector, Tat, Tat plus wild-type YY1, and Tat plus mutant YY1 lacking the HDAC1 interaction domain. CAT assays (upper panel) were performed to correlate the level of LTR expression with the acetylation of histone H4 and HDAC1 occupancy (middle panel) at nuc 1, as measured by ChIP assays. A YY1 mutant with a deletion of HDAC1 interaction domain was unable to repress the LTR, increase HDAC1 occupancy, or significantly decrease H4 acetylation at nuc 1.

    Article Snippet: Expression plasmids driven by the cytomegalovirus (CMV) immediate-early promoter, CMV-empty vector, CMV-green fluorescent protein (GFP) (Clontech, Palo Alto, Calif.), CMV-YY1 , and CMV-Tat were purified by using an Endofree plasmid kit (Qiagen, Valencia, Calif.) and quantified by using a spectrophotometer.

    Techniques: Plasmid Preparation, Mutagenesis, Expressing, Chromatin Immunoprecipitation

    YY1 recruits HDAC1 and inhibits histone H4 acetylation at the nuc 1 region of LTR. HeLa LTR-CAT cells were cotransfected with GFP plus empty CMV vector or with GFP plus CMV-YY1 and then sorted by FACS and ChIP assays performed with antibodies against acetylated histone H4, HDAC1, and LSF. (A) YY1 increased HDAC1 occupancy and decreased acetylation of histone H4 at nuc 1. (B) Overexpression of YY1 did not change LSF occupancy at nuc 1.

    Journal: Molecular and Cellular Biology

    Article Title: Counterregulation of Chromatin Deacetylation and Histone Deacetylase Occupancy at the Integrated Promoter of Human Immunodeficiency Virus Type 1 (HIV-1) by the HIV-1 Repressor YY1 and HIV-1 Activator Tat

    doi: 10.1128/MCB.22.9.2965-2973.2002

    Figure Lengend Snippet: YY1 recruits HDAC1 and inhibits histone H4 acetylation at the nuc 1 region of LTR. HeLa LTR-CAT cells were cotransfected with GFP plus empty CMV vector or with GFP plus CMV-YY1 and then sorted by FACS and ChIP assays performed with antibodies against acetylated histone H4, HDAC1, and LSF. (A) YY1 increased HDAC1 occupancy and decreased acetylation of histone H4 at nuc 1. (B) Overexpression of YY1 did not change LSF occupancy at nuc 1.

    Article Snippet: Expression plasmids driven by the cytomegalovirus (CMV) immediate-early promoter, CMV-empty vector, CMV-green fluorescent protein (GFP) (Clontech, Palo Alto, Calif.), CMV-YY1 , and CMV-Tat were purified by using an Endofree plasmid kit (Qiagen, Valencia, Calif.) and quantified by using a spectrophotometer.

    Techniques: Plasmid Preparation, FACS, Chromatin Immunoprecipitation, Over Expression

    Changes of histone H4 acetylation and HDAC1 occupancy at nuc 1 correlate with Tat activation of LTR. HeLa LTR-CAT cells were cotransfected with GFP and empty CMV vector or with GFP and CMV-Tat plasmids. After 24 h, transfected cells were separated into GFP-positive and -negative populations by FACS. Extracts were prepared for CAT and ChIP assays with antibodies against acetylated histone H4, HDAC1, or LSF. (A) Tat-activated integrated LTR-CAT in HeLa reporter line as shown by CAT assay. (B) A CAT assay showed cotransfection exclusively delivered Tat plasmids into GFP-positive cells. (C) ChIP assays showed increased acetylation of histone H4 and decreased HDAC1 occupancy at nuc 1 of LTR upon Tat activation in GFP-positive cells. (D) Overexpression of Tat did not affect LSF occupancy at nuc 1.

    Journal: Molecular and Cellular Biology

    Article Title: Counterregulation of Chromatin Deacetylation and Histone Deacetylase Occupancy at the Integrated Promoter of Human Immunodeficiency Virus Type 1 (HIV-1) by the HIV-1 Repressor YY1 and HIV-1 Activator Tat

    doi: 10.1128/MCB.22.9.2965-2973.2002

    Figure Lengend Snippet: Changes of histone H4 acetylation and HDAC1 occupancy at nuc 1 correlate with Tat activation of LTR. HeLa LTR-CAT cells were cotransfected with GFP and empty CMV vector or with GFP and CMV-Tat plasmids. After 24 h, transfected cells were separated into GFP-positive and -negative populations by FACS. Extracts were prepared for CAT and ChIP assays with antibodies against acetylated histone H4, HDAC1, or LSF. (A) Tat-activated integrated LTR-CAT in HeLa reporter line as shown by CAT assay. (B) A CAT assay showed cotransfection exclusively delivered Tat plasmids into GFP-positive cells. (C) ChIP assays showed increased acetylation of histone H4 and decreased HDAC1 occupancy at nuc 1 of LTR upon Tat activation in GFP-positive cells. (D) Overexpression of Tat did not affect LSF occupancy at nuc 1.

    Article Snippet: Expression plasmids driven by the cytomegalovirus (CMV) immediate-early promoter, CMV-empty vector, CMV-green fluorescent protein (GFP) (Clontech, Palo Alto, Calif.), CMV-YY1 , and CMV-Tat were purified by using an Endofree plasmid kit (Qiagen, Valencia, Calif.) and quantified by using a spectrophotometer.

    Techniques: Activation Assay, Plasmid Preparation, Transfection, FACS, Chromatin Immunoprecipitation, Cotransfection, Over Expression

    Binding of LSF to LTR correlates with HDAC1 recruitment and hypoacetylation at the nuc 1 region of LTR. HeLa LTR-CAT cells cotransfected with GFP plus empty CMV vector, GFP plus CMV-LSF, or GFP plus CMV-dnLSF were sorted by FACS and ChIP assays performed with antibodies against acetylated histone H4 or HDAC1. (A) Overexpression of dnLSF decreased LSF occupancy at nuc 1. (B) Overexpression of dnLSF increased acetylation of histone H4 (upper panel) and decreased HDAC1 occupancy (middle panel) at nuc 1.

    Journal: Molecular and Cellular Biology

    Article Title: Counterregulation of Chromatin Deacetylation and Histone Deacetylase Occupancy at the Integrated Promoter of Human Immunodeficiency Virus Type 1 (HIV-1) by the HIV-1 Repressor YY1 and HIV-1 Activator Tat

    doi: 10.1128/MCB.22.9.2965-2973.2002

    Figure Lengend Snippet: Binding of LSF to LTR correlates with HDAC1 recruitment and hypoacetylation at the nuc 1 region of LTR. HeLa LTR-CAT cells cotransfected with GFP plus empty CMV vector, GFP plus CMV-LSF, or GFP plus CMV-dnLSF were sorted by FACS and ChIP assays performed with antibodies against acetylated histone H4 or HDAC1. (A) Overexpression of dnLSF decreased LSF occupancy at nuc 1. (B) Overexpression of dnLSF increased acetylation of histone H4 (upper panel) and decreased HDAC1 occupancy (middle panel) at nuc 1.

    Article Snippet: Expression plasmids driven by the cytomegalovirus (CMV) immediate-early promoter, CMV-empty vector, CMV-green fluorescent protein (GFP) (Clontech, Palo Alto, Calif.), CMV-YY1 , and CMV-Tat were purified by using an Endofree plasmid kit (Qiagen, Valencia, Calif.) and quantified by using a spectrophotometer.

    Techniques: Binding Assay, Plasmid Preparation, FACS, Chromatin Immunoprecipitation, Over Expression

    Construction of plasmids. The commercial plasmids pEGFP-C1 and pEGFP-N1 contain the CMV promoter which acts in mammalian cells, and the promoter of baculovirus ie2 which works in insect cells derives from the plasmid pIZ-V5/His. All the recombinant plasmids contained the promoter of ie2 following the promoter of CMV except pie2/EGFP-Atg8, which had no the promoter of CMV. The EGFP after Atg8 did not express because Atg8 has a stop codon. PCR was used for site-directed mutagenesis and truncation of open reading frames. Atg8 116G has a glycine (G) residue at C terminus (the 116 th amino acid residue). Atg8 62 had 62 amino acid residues of the N terminus of Atg8. F77/79A and F60/62A indicated that tyrosine (F) residues were replaced with alanine (A) residues.

    Journal: PLoS ONE

    Article Title: Distribution, Cleavage and Lipidation of Atg8 Fusion Proteins in Spodoptera litura Sl-HP Cells

    doi: 10.1371/journal.pone.0096059

    Figure Lengend Snippet: Construction of plasmids. The commercial plasmids pEGFP-C1 and pEGFP-N1 contain the CMV promoter which acts in mammalian cells, and the promoter of baculovirus ie2 which works in insect cells derives from the plasmid pIZ-V5/His. All the recombinant plasmids contained the promoter of ie2 following the promoter of CMV except pie2/EGFP-Atg8, which had no the promoter of CMV. The EGFP after Atg8 did not express because Atg8 has a stop codon. PCR was used for site-directed mutagenesis and truncation of open reading frames. Atg8 116G has a glycine (G) residue at C terminus (the 116 th amino acid residue). Atg8 62 had 62 amino acid residues of the N terminus of Atg8. F77/79A and F60/62A indicated that tyrosine (F) residues were replaced with alanine (A) residues.

    Article Snippet: Firstly, the ie2 promotor was inserted after the promoter of CMV of the plasmid pEGFP-N1 (Clonetech, CA).

    Techniques: Plasmid Preparation, Recombinant, Polymerase Chain Reaction, Mutagenesis

    Orientation of the 3′-CMV promoter-independent gene expression. a Orientation of the 3′-CMV promoter in the CMV-KLF16-CMV construct was flipped, termed CMV-KLF16-CMV (flipped), and then expression of KLF16 protein was assessed by Western blot analysis after transfecting the plasmid including indicated comparative vectors in HEK293. Tubulin was used as a control for loaded amounts of protein. b Primers were designed to the indicated locations of the CMV-KLF16-CMV and the CMV-KLF16-CMV (flipped) constructs (see Materials and Methods )

    Journal: Molecular Biotechnology

    Article Title: Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

    doi: 10.1007/s12033-014-9738-0

    Figure Lengend Snippet: Orientation of the 3′-CMV promoter-independent gene expression. a Orientation of the 3′-CMV promoter in the CMV-KLF16-CMV construct was flipped, termed CMV-KLF16-CMV (flipped), and then expression of KLF16 protein was assessed by Western blot analysis after transfecting the plasmid including indicated comparative vectors in HEK293. Tubulin was used as a control for loaded amounts of protein. b Primers were designed to the indicated locations of the CMV-KLF16-CMV and the CMV-KLF16-CMV (flipped) constructs (see Materials and Methods )

    Article Snippet: A quantitative RT-PCR analysis showed that a detectable amount (7.35 ± 3.25) of transcripts containing KLF16 flanked by the 3′-CMV promoter was observed when CMV/AS primer (Fig. b) was used for reverse transcription in cells transfected with the CMV–CMV vector (Table ).

    Techniques: Expressing, Construct, Western Blot, Plasmid Preparation

    Construction of the C-TSC cassette. a Expression of KLF16 ( top ) or REIC/Dkk-3 ( bottom ) proteins after transfecting the indicated constructs including a vector containing the CAG promoter in HEK293 cells. Tubulin was used as a control for loaded amounts of protein. b To further improve the expression, 3 promoter sequences of hTERT, SV40, and CMV were tandemly inserted downstream of the REIC cDNA-BGH (bovine growth hormone)-polyA signal. RU5′, a part of the HTLV type 1 LTR inserted for better efficiency of transcription and translation. CMV–CMV and CMV–RU5′–hTERT–SV40–CMV were constructed on the basis of pDNR-1r and pIDT-SMART promoter-less vectors ( top ). The vectors were transfected to HEK293 cells, and levels of inserted REIC protein were determined by Western blot analysis ( top ). Tubulin was used as a control for loaded amounts of protein. Finally, we named the improved high expression cassette C-TSC ( bottom )

    Journal: Molecular Biotechnology

    Article Title: Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

    doi: 10.1007/s12033-014-9738-0

    Figure Lengend Snippet: Construction of the C-TSC cassette. a Expression of KLF16 ( top ) or REIC/Dkk-3 ( bottom ) proteins after transfecting the indicated constructs including a vector containing the CAG promoter in HEK293 cells. Tubulin was used as a control for loaded amounts of protein. b To further improve the expression, 3 promoter sequences of hTERT, SV40, and CMV were tandemly inserted downstream of the REIC cDNA-BGH (bovine growth hormone)-polyA signal. RU5′, a part of the HTLV type 1 LTR inserted for better efficiency of transcription and translation. CMV–CMV and CMV–RU5′–hTERT–SV40–CMV were constructed on the basis of pDNR-1r and pIDT-SMART promoter-less vectors ( top ). The vectors were transfected to HEK293 cells, and levels of inserted REIC protein were determined by Western blot analysis ( top ). Tubulin was used as a control for loaded amounts of protein. Finally, we named the improved high expression cassette C-TSC ( bottom )

    Article Snippet: A quantitative RT-PCR analysis showed that a detectable amount (7.35 ± 3.25) of transcripts containing KLF16 flanked by the 3′-CMV promoter was observed when CMV/AS primer (Fig. b) was used for reverse transcription in cells transfected with the CMV–CMV vector (Table ).

    Techniques: Expressing, Construct, Plasmid Preparation, Transfection, Western Blot

    The abundance of H3K4me3 and H3K27me3 at the HCMV MIEP in shControl and shEZH2 HFFs, at the pre-immediate early stage of the infection. A. Graphical illustration of the MIEP of HCMV. Open boxes represent regions probed by ChIP for association with histone H3K27me3 and histone H3K4me3. Enh stands for Enhancer and crs stands for cis-repressive sequence. B: HFFs were transduced with either the pLKO.1 empty vector or the shEZH2 lentiviral construct. A western blot of cell lysates was probed with the EZH2 antibody. C-E. HFFs transduced with a lentivirus construct of shEZH2 or with the empty vector, were infected with HCMV (MOI 0.5 PFUs/cell). ChIPs using antibodies against H3K27me3 and H3K4me3 were analysed by qPCR using primers specific for the indicated loci (see materials and methods ), at 0.5 h, 1.5 h and 3 h.p.i. The results are given as mean + SD for triplicate Real Time PCRs. The asterisks indicate statistical significance. * indicates p

    Journal: PLoS Pathogens

    Article Title: The Downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 Axis and by Human Cytomegalovirus (HCMV) Associated Factors Allows the Activation of the HCMV Major IE Promoter and the Transition to Productive Infection

    doi: 10.1371/journal.ppat.1004136

    Figure Lengend Snippet: The abundance of H3K4me3 and H3K27me3 at the HCMV MIEP in shControl and shEZH2 HFFs, at the pre-immediate early stage of the infection. A. Graphical illustration of the MIEP of HCMV. Open boxes represent regions probed by ChIP for association with histone H3K27me3 and histone H3K4me3. Enh stands for Enhancer and crs stands for cis-repressive sequence. B: HFFs were transduced with either the pLKO.1 empty vector or the shEZH2 lentiviral construct. A western blot of cell lysates was probed with the EZH2 antibody. C-E. HFFs transduced with a lentivirus construct of shEZH2 or with the empty vector, were infected with HCMV (MOI 0.5 PFUs/cell). ChIPs using antibodies against H3K27me3 and H3K4me3 were analysed by qPCR using primers specific for the indicated loci (see materials and methods ), at 0.5 h, 1.5 h and 3 h.p.i. The results are given as mean + SD for triplicate Real Time PCRs. The asterisks indicate statistical significance. * indicates p

    Article Snippet: To determine the effects of NDY1/KDM2B, EZH2 JARID2 and GFI1 on the activity of the HCMV MIEP in the absence of viral infection, a MIEP-EGFP reporter construct (pEGFP-C1) (Clontech) was transfected into HEK 293T cells or their derivatives in which NDY1/KDM2B, EZH2 or JARID2 were knocked down or GFI1 was overexpressed and the expression of EGFP was monitored by fluorescence microscopy or flow cytometry.

    Techniques: Infection, Chromatin Immunoprecipitation, Sequencing, Transduction, Plasmid Preparation, Construct, Western Blot, Real-time Polymerase Chain Reaction

    Infection by HCMV depends on the downregulation of GFI1, a repressor of immediate-early gene transcription. Model summarizing the data on the interaction between the virus and the host. (Panel A) This panel describes the infection of wild type HFFs. The incoming virus rapidly degrades GFI1 to allow the activation of the MIEP of HCMV, and viral infection. In addition, virus infection alters the expression of NDY1/KDM2B, EZH2, JARID2 and JMJD3. The solid lines from these molecules to GFI1 indicate that they actively repress GFI1 both before and after infection, although due to HCMV-induced changes in their expression, the repression is enhanced after infection. (Panel B, Left) The repression of GFI1 in uninfected cells was blocked by the knockdown of NDY1/KDM2B, EZH2 or JARID2 and by the overexpression of JMJD3, resulting in significant up-regulation of GFI1 (dotted lines). (Panel B, Right) describes the infection of HFFs in the left side of panel B. The virus continues to degrade GFI1. However, the degradation of GFI1 by the virus is insufficient to downregulate it to levels that allow the activation of the MIEP and viral infection.

    Journal: PLoS Pathogens

    Article Title: The Downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 Axis and by Human Cytomegalovirus (HCMV) Associated Factors Allows the Activation of the HCMV Major IE Promoter and the Transition to Productive Infection

    doi: 10.1371/journal.ppat.1004136

    Figure Lengend Snippet: Infection by HCMV depends on the downregulation of GFI1, a repressor of immediate-early gene transcription. Model summarizing the data on the interaction between the virus and the host. (Panel A) This panel describes the infection of wild type HFFs. The incoming virus rapidly degrades GFI1 to allow the activation of the MIEP of HCMV, and viral infection. In addition, virus infection alters the expression of NDY1/KDM2B, EZH2, JARID2 and JMJD3. The solid lines from these molecules to GFI1 indicate that they actively repress GFI1 both before and after infection, although due to HCMV-induced changes in their expression, the repression is enhanced after infection. (Panel B, Left) The repression of GFI1 in uninfected cells was blocked by the knockdown of NDY1/KDM2B, EZH2 or JARID2 and by the overexpression of JMJD3, resulting in significant up-regulation of GFI1 (dotted lines). (Panel B, Right) describes the infection of HFFs in the left side of panel B. The virus continues to degrade GFI1. However, the degradation of GFI1 by the virus is insufficient to downregulate it to levels that allow the activation of the MIEP and viral infection.

    Article Snippet: To determine the effects of NDY1/KDM2B, EZH2 JARID2 and GFI1 on the activity of the HCMV MIEP in the absence of viral infection, a MIEP-EGFP reporter construct (pEGFP-C1) (Clontech) was transfected into HEK 293T cells or their derivatives in which NDY1/KDM2B, EZH2 or JARID2 were knocked down or GFI1 was overexpressed and the expression of EGFP was monitored by fluorescence microscopy or flow cytometry.

    Techniques: Infection, Activation Assay, Expressing, Over Expression

    NDY1/KDM2B, EZH2 JARID2 and JMJD3 control the expression of GFI1, a direct repressor of the HCMV MIEP, by regulating histone H3K27 tri-methylation in the GFI1 promoter. A . (Upper panel). Schematic diagram of the major immediate-early promoter of HCMV, showing the relative location of the binding sites of the indicated transcriptional regulators (activators and repressors). (Lower panel). The expression of the indicated transcriptional regulators in HFFs in which EZH2, NDY1/KDM2B, or JARID2 were knocked down, or JMJD3 was overexpressed via transduction with the indicated constructs, was measured by real time RT-PCR. The bars show the relative expression of GFI1 (mean ± SD) in the cells transduced with these constructs. The western blot in the inset shows that the GFI1 protein, the only transcriptional regulator whose expression at the RNA level was induced by these constructs, is also upregulated. B . The knock down of NDY1/KDM2B, EZH2 or JARID2 enhances the binding of GFI1 to the HCMV promoter. HFFs were transduced with shEZH2, shNDY1/KDM2, shJARID2 or the empty lentiviral vector and they were subsequently infected with HCMV. ChIP assays addressing the binding of GFI1 on the two known GFI1 binding sites in the HCMV promoter or in exon 1 of the immediate-early region were carried out using lysates harvested from these cells 1 hour post-infection The bars show the fold increase in GFI1 binding (mean ± SD) in the shEZH2, shNDY1/KDM2B and shJARID2-transduced cells relative to the cells transduced with the empty vector. C . GFI1 is a direct repressor of the HCMV MIE promoter. HEK 293T cells transduced with the indicated lentiviral or retroviral constructs were transfected with an HCMV MIEP-EGFP reporter in which the HCMV MIEP was either wild type or mutated in the two known GFI1 binding sites. The activity of the HCMV MIEP was monitored by both fluorescence microscopy (upper panel) and fluorescence densitometric analyses (lower panel). Bars in the lower panel show the relative EGFP fluorescence in the indicated cells (mean ± SD). D . The knockdown of NDY1/KDM2B, EZH2 and JARID2 decrease the abundance of histone H3K27me3 in a negative regulatory domain of the GFI1 promoter (site # 1). ChIP analyses addressing the abundance of H3K27me3 at five different sites within the GFI1 promoter in HFFs transduced with the indicated constructs. The p16 Ink4a locus was used as the positive control. The upper panel shows the position of the five selected sites, relative to the transcription start site in the GFI1 promoter (arrow). The bars in the lower panel show the fold change in the abundance of H3K27me3 (mean ± SD) at these sites, and in the p16 Ink4a locus. NRE: Negative Regulatory Element.

    Journal: PLoS Pathogens

    Article Title: The Downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 Axis and by Human Cytomegalovirus (HCMV) Associated Factors Allows the Activation of the HCMV Major IE Promoter and the Transition to Productive Infection

    doi: 10.1371/journal.ppat.1004136

    Figure Lengend Snippet: NDY1/KDM2B, EZH2 JARID2 and JMJD3 control the expression of GFI1, a direct repressor of the HCMV MIEP, by regulating histone H3K27 tri-methylation in the GFI1 promoter. A . (Upper panel). Schematic diagram of the major immediate-early promoter of HCMV, showing the relative location of the binding sites of the indicated transcriptional regulators (activators and repressors). (Lower panel). The expression of the indicated transcriptional regulators in HFFs in which EZH2, NDY1/KDM2B, or JARID2 were knocked down, or JMJD3 was overexpressed via transduction with the indicated constructs, was measured by real time RT-PCR. The bars show the relative expression of GFI1 (mean ± SD) in the cells transduced with these constructs. The western blot in the inset shows that the GFI1 protein, the only transcriptional regulator whose expression at the RNA level was induced by these constructs, is also upregulated. B . The knock down of NDY1/KDM2B, EZH2 or JARID2 enhances the binding of GFI1 to the HCMV promoter. HFFs were transduced with shEZH2, shNDY1/KDM2, shJARID2 or the empty lentiviral vector and they were subsequently infected with HCMV. ChIP assays addressing the binding of GFI1 on the two known GFI1 binding sites in the HCMV promoter or in exon 1 of the immediate-early region were carried out using lysates harvested from these cells 1 hour post-infection The bars show the fold increase in GFI1 binding (mean ± SD) in the shEZH2, shNDY1/KDM2B and shJARID2-transduced cells relative to the cells transduced with the empty vector. C . GFI1 is a direct repressor of the HCMV MIE promoter. HEK 293T cells transduced with the indicated lentiviral or retroviral constructs were transfected with an HCMV MIEP-EGFP reporter in which the HCMV MIEP was either wild type or mutated in the two known GFI1 binding sites. The activity of the HCMV MIEP was monitored by both fluorescence microscopy (upper panel) and fluorescence densitometric analyses (lower panel). Bars in the lower panel show the relative EGFP fluorescence in the indicated cells (mean ± SD). D . The knockdown of NDY1/KDM2B, EZH2 and JARID2 decrease the abundance of histone H3K27me3 in a negative regulatory domain of the GFI1 promoter (site # 1). ChIP analyses addressing the abundance of H3K27me3 at five different sites within the GFI1 promoter in HFFs transduced with the indicated constructs. The p16 Ink4a locus was used as the positive control. The upper panel shows the position of the five selected sites, relative to the transcription start site in the GFI1 promoter (arrow). The bars in the lower panel show the fold change in the abundance of H3K27me3 (mean ± SD) at these sites, and in the p16 Ink4a locus. NRE: Negative Regulatory Element.

    Article Snippet: To determine the effects of NDY1/KDM2B, EZH2 JARID2 and GFI1 on the activity of the HCMV MIEP in the absence of viral infection, a MIEP-EGFP reporter construct (pEGFP-C1) (Clontech) was transfected into HEK 293T cells or their derivatives in which NDY1/KDM2B, EZH2 or JARID2 were knocked down or GFI1 was overexpressed and the expression of EGFP was monitored by fluorescence microscopy or flow cytometry.

    Techniques: Expressing, Methylation, Binding Assay, Transduction, Construct, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Infection, Chromatin Immunoprecipitation, Transfection, Activity Assay, Fluorescence, Microscopy, Positive Control