sp1 sp3  (Galectin Therapeutics)

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A). (Left) schematic of truncation mutants, deletion mutants, and binding site mutants of the ORF75 promoter used to make luciferase constructs. (Right) promoter luciferase activity of these constructs in 293T cells. Numbers on the top of each bar indicates average fold change relative to empty vector pGL3 control. B) Schematic showing the location and sequence of the proximal Sp1 element and ARE elements in the ORG75 promoter and the mutants. C) Sequences of the Sp consensus element of ORF75 promoter probe and the mutant probes used in EMSA. The promoter probe is a 50 nt long dsDNA labelled with 5’ IR dye CW700. The mutated Sp1 element is in purple and underlined. The terminal ATG is the start codon of ORF75 protein. D) EMSA showing binding of Sp1, Sp3, and Sp4 proteins with the double-stranded DNA (dsDNA) IR-probe of ORF75 promoter in a 8% native PAGE gel. Equal concentration of HEK293T lysates over-expressing the various Sp proteins were used in the assay. E) Same as D), but with competitive specific and Sp1 element mutated probes. F) ChIP assay analysing Sp1 and Sp3 binding regions of ORF75 promoters in latent iSLK-BAC16 cells. Binding was analysed by chromatin immunoprecipitation followed by qPCR. The schematic on the top shows the location of the various primers used to detect Sp protein abundance on the ORF75 promoter. H3 was used as a positive ChIP control and IGG antibody as isotype control. A 68 bp long DHFR gene promoter region located at -400 bp from transcription start site of the human DHFR gene was used as a positive control for Sp protein binding. Shown are the means ± standard deviations of at least 3 separate experiments. P -values (* p ≤ 0.05 , ** p ≤ 0.01, ns not significant) are calculated using two-sided paired t -test. Beta-galactosidase was used for transfection normalization. ChIP data was analysed using fold induction normalised to IgG. ChIP primer sequence in .

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Binding Assay, Luciferase, Construct, Activity Assay, Plasmid Preparation, Control, Sequencing, Mutagenesis, Clear Native PAGE, Concentration Assay, Expressing, Chromatin Immunoprecipitation, Quantitative Proteomics, Positive Control, Protein Binding, Transfection

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A ) EMSA showing binding of Sp. proteins with dsDNA probe of ORF75 promoter along with a positive control Sp1 probe (Li-COR, P/N: 829-07926) in a 8% native PAGE gel. B) WB: HEK293T whole cell lysates was probed for Sp1, Sp3 and Sp4 protein abundance. See for full blots.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Binding Assay, Positive Control, Clear Native PAGE, Quantitative Proteomics

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A) Promoter luciferase assay of ORF75 promoter constructs along with over-expression of various Sp proteins in HepG2 cells. Sp proteins were expressed from a CMV-driven pN3 vector. ORF75 full-length (p75) and truncated (p75-T2) promoters are shown. Fold change is normalized to respective pGL3 for each Sp. protein. Assayed at 72h post transfection. B) WB of Sp proteins from BCBL1 (PEL) cells and HUVEC (endothelial) cells. Blots stripped and reprobed, see for more information. C) Sp1-specific inhibitor mithramycin A (MA) treatment during ORF75 promoter (p75-T2) luciferase assay in 293T cells. D) Supershift assay. Equal amounts of HEK293T lysates (30ug) were preincubated with 1, 1.5 and 2µg of anti-Sp1 (21962-1-, Proteintech) or anti-Sp3 (26584-1-AP, Proteintech) before incubation with ORF75 promoter probe (p75 Sp1 probe) followed by gel shift assay. 6% native TBE gel. E) Promoter luciferase assay of ORF75 promoter in Schneider Drosophila line 2 (SL2) with or without exogenous human Sp1 expression. Sp1 protein expressed from pPAC vector driven by ACTIN 5C promoter. pPAC0 is the empty vector. Fold change is normalized to pGL3 with pPAC0. Assayed at 72h post nucleofection. Data are presented as mean ± SD of three individually nucleofected samples of one experiment. F) Same as in E), except with ORF75 p75-T4 truncated and Sp1 element-deleted promoter constructs. Fold change is normalized to p75 promoter with pPAC0. G) Promoter luciferase assay of ORF75 promoter and its mutants with or without sodium butyrate (SB) treatment in HEK293T cells. SB treatment 24h post transfection. Assayed at 48h post transfection. Numbers on the top of each bar represents average fold change normalized to untreated pGL3 set as 1. For A, C, F and G, shown are the means ± standard deviations of 3 separate experiments. P -values (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns not significant) are calculated using two-sided paired t -test. Beta-galactosidase was used for transfection normalization.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Luciferase, Construct, Over Expression, Plasmid Preparation, Transfection, Incubation, Gel Shift, Expressing

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A) Promoter luciferase assay of ORF75 full length promoter in TIVE (endothelial cell line) and BJAB (B-cell line) cells. Results shown are the fold change over the respective pGL3 empty vector for each cell type. Data shown are ± standard deviations of 3 separate experiments. P -values (** p ≤ 0.01) are calculated using two-sided paired t -test B) Western blot analysis of Sp1, Sp3 and Sp4 protein levels from different cell lines using whole cell lysates. Black and red arrow indicates full-length and alternate SP1 forms in W.B, respectively. C) EMSA and WB analysis. Nuclear lysates of various KSHV-infected and uninfected cell lines were analysed for their Sp1 binding activity to the proximal Sp1 element of ORF75 promoter through EMSA. The lower three panels represent parallel WB of the nuclear lysates. D) Promoter luciferase assay of various mutant ORF75 promoters in Schneider Drosophila line 2 (SL2) with exogenous human Sp1 expression. Presence of various elements are shown in the promoter schematic as different shapes. Absence of a shape in the promoter schematic indicates corresponding element mutation. Data shown are presented as mean ± SD of individually nucleofected samples of one experiment. Assayed at 72h post nucleofection. P -values (**** p ≤ 0.0001, ns not significant) are calculated using ordinary one way ANOVA. E) Same as A), except four different ORF75 promoters were used for the promoter luciferase assay in BJAB and TIVE cells. Assayed at 72h post transfection. Numbers on the top of each bar indicates average fold upregulation relative to empty vector pGL3 for each cell type set as 1. Shown are the means ± standard deviations of 3 separate experiments. P -values (** p ≤ 0.01, **** p ≤ 0.0001, ns not significant) are calculated using two-sided unpaired t -test. Blots stripped and reprobed, see for more information.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Luciferase, Plasmid Preparation, Western Blot, Infection, Binding Assay, Activity Assay, Mutagenesis, Expressing, Transfection

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A) WB of whole cell lysate (RIPA buffer) of early passage cell lines showing alternate forms of Sp1 protein in KSHV infected B-cell lines (4-12% BIS-TRIS gel, MES buffer). Sp1, Sp4 and ACTIN are same blot stripped and reprobed. Sp3 blot was run parallel. B) WB of BCBL-1 cells at different time points of lytic reactivation blotted with anti-Sp1 antibody. ORF45 and vIL6 were used here as control for lytic reactivation (10% BIS-TRIS gel, MES buffer). Sp1, ORF45 and ACTIN are same blot stripped and reprobed. vIL6 blot was run separately with the same samples. C) EMSA and western blot analysis. 10 µg of whole cell lysates (M-PER, Invitrogen) of BCBL-1 cells at different time points of lytic reactivation were analysed for their Sp1 protein binding activity to the proximal Sp1 element of ORF75 promoter through EMSA in a 8% TBE gel. The bottom three panel represent parallel WBs showing accumulation pattern of full-length Sp1 and 45 kDa Sp1 protein isoform during NaB induced lytic induction (10% BIS-TRIS gel, MES buffer). Black and red triangle indicates full-length Sp1 and 45 kDa Sp1 form binding to the proximal Sp1 DNA element of the ORF75 promoter, respectively. Sp1 and ACTIN are same blot stripped and reprobed. D) WB of uninfected TIME and infected TIME.219 cell line at different time points of lytic reactivation using TPA. LANA was used as control for infected TIME.219 cells and vIL6 was used as a control for lytic reactivation (8% Bolt BIS-TRIS gel, MES buffer). Black and red arrow indicates full-length and alternate Sp1 forms in WBs, respectively. SP1, vIL6, and ACTIN were on one blot, which was stripped and reprobed, while SP3 and LANA were on a parallel blot, which was also stripped and reprobed. See for full blots and ACTIN probe of parallel run blots.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Infection, Control, Western Blot, Protein Binding, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: A) Table showing multiple consensus Sp1 element position throughout KSHV genome. Only complete consensus sequence is shown here. Consensus sequence KGGGCGGRRY, where K stands for G or T and R stands for G or A. B) Promoter luciferase assay of ORF75-T2 promoter in HEK293T cells along with co-expression of increasing amount of RTA and LANA protein. X and 2X indicate 1:2 and 1:4 ratios of ORF75 promoter to protein expression plasmid, respectively. Assayed at 72h post transfection. Shown are the means ± standard deviations of 3 separate experiments. P -values (*** p ≤ 0.001, ns not significant) are calculated using two-sided unpaired t -test. C) Schematic diagram of various KSHV gene promoters used in this study. D) Promoter luciferase assay of vIL6 promoters along with co-expression of F-ORF75. Error bar indicate ±SD, N=3. GL: genomic location as per NC_009333 KSHV reference genome.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Sequencing, Luciferase, Expressing, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: The Elevated Expression of ORF75, a Lytic KSHV Gene, in Kaposi Sarcoma Lesions is Driven by a GC-rich DNA cis Element in its Promoter Region

doi: 10.1101/2024.09.26.615194

Figure Lengend Snippet: Simplified summary schema showing the regulation of ORF75 promoter’s basal transcription activity. The activity is primarily regulated by the binding of the Sp1 protein to Sp1 elements. There is evidence that in all cell lines, the Sp1 complex interacts with the proximal Sp1 element, the Sp1-like elements, and the CCAAT boxes to activate this transcription. Also, a repressor, whose identity is unknown but acts via the distal region of the ORF75 promoter, variably inhibits basal transcription. This repressor’s effect determines the promoter’s activity across different cell lines, even in the presence of Sp1 activation. In B-cells, the distal region of the ORF75 promoter strongly represses its activity, leading to lower ORF75 transcription during latency. By contrast, endothelial and epithelial cells experience less repression and more activation of the ORF75 promoter due to the proximal Sp1 element. This results in higher ORF75 transcript levels, even though there is no lytic viral DNA replication. This summary Fig is based on the promoter activity of different mutant promoters as shown in & E and 4A.

Article Snippet: 10 μg of Sp1 antibody (Proteintech, 21962-1-Ap), Sp3 antibody (Proteintech, 26584-1-Ap), IgG antibody (CST), and H3 antibody (Abcam, ChIP grade) were incubated with the sheared chromatin overnight at 4°C in a rotary shaker.

Techniques: Activity Assay, Binding Assay, Activation Assay, Mutagenesis

Journal: Cancer Science

Article Title: Kaposi's sarcoma–associated herpesvirus replication and transcription activator protein activates CD274/PD‐L1 gene promoter

doi: 10.1111/cas.15673

Figure Lengend Snippet: SP1 and K‐RTA–induced CD274/PD‐L1 promoter activation. A, Dose‐dependent increase of K‐RTA–mediated CD274/PD‐L1 promoter (−47 to +33) activation by SP1. HEK293 cells were cotransfected with increasing amounts of SP1 or SP3 expression plasmid (0, 83, 166, and 333 ng), 333 ng of K‐RTA–expressing plasmid, 166 ng of CD274/PD‐L1 promoter reporter plasmid, and 33 ng of pRL‐CMV vector (internal control). Luciferase activity was measured 2 d later. Results are presented as the mean ± SD ( n = 3). Dunnett's test; * p < 0.01 (K‐RTA only vs +SP1 or + SP3). B, Specific SP1 binding was tested using three divided CD274/PD‐L1 promoter DNA probes and immunopurified SP1‐HA on EMSA. Specific protein and DNA complex is shown by arrows. C, Co‐operative binding of K‐RTA and SP1 on the CD274/PD‐L1 promoter. Immunopurified FLAG‐K‐RTA (aa 1‐531) and SP1‐HA proteins from HEK293 cells were incubated with biotin‐labeled CD274/PD‐L1 promoter probe (fragment from nucleotide −47 to +43) in the indicated combination, and the specific protein‐DNA complex was analyzed by EMSA. Gray arrows indicate the protein‐DNA complex. A representative result is shown

Article Snippet: SP1 and SP3 shRNA‐expressing plasmids (sc‐29487‐SH and sc‐29490‐SH) were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Binding Assay, Incubation, Labeling