Journal: Molecular Genetics & Genomic Medicine

Article Title: Regulatory variant in FZD6 gene contributes to nonsyndromic cleft lip and palate in an African-American family

doi: 10.1002/mgg3.155

Figure Lengend Snippet: FZD6 rs138557689/C creates an allele-specific protein-binding complex and decreases FZD6 promoter activity. (A) Electrophoretic mobility shift assays (EMSA) were performed using nuclear extract from Cos7 cells. Samples were incubated with P 32 -labeled oligonucleotides containing either the ancestral A or alternate C alleles, or with unlabeled ancestral A or alternate C serving as specific competitors. Poly-dCdG was used as a nonspecific competitor. Negative controls were run using labeled probes without the nuclear extract. Bands were observed with the C allele only and the alternate band was competed out with C competitor only. (B) Hek293T cells (100,000 cells/well) were seeded for 24 h and cotransfected with ancestral or alternate promoter construct and Renilla reporter construct. Luciferase activities were normalized to the internal Renilla control. Data represent mean values ± SD from three independent experiments. Alternate C allele showed significant decrease in activity (* P < 0.001, unpaired t -test).

Article Snippet: The FZD6 putative promoter sequence was identified using information from GeneCopeia (Rockville, MD) and SwitchGear Genomics (Menlo Park, CA) and included the 2277 bp region upstream of the transcriptional start site.

Techniques: Protein Binding, Activity Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Construct, Luciferase, Control

Journal: Molecular Genetics & Genomic Medicine

Article Title: Regulatory variant in FZD6 gene contributes to nonsyndromic cleft lip and palate in an African-American family

doi: 10.1002/mgg3.155

Figure Lengend Snippet: Knockdown and overexpression of FZD6 protein in zebrafish results in craniofacial defects. Both nonoverlapping morpholinos caused the same spectrum of craniofacial abnormalities. Only MO1-injected embryos are shown as an example. (A, D, G, J, and M) are uninjected control (UIC) embryos. (B, E, H, K, and N) show the phenotypes associated with knockdown of fzd6 . (C, F, I, L, and O) show phenotypes associated with overexpression of fzd6 . (A) UIC embryos at 24 hpf. (B) fzd6 MO-injected embryos at 24 hpf. (C) fzd6 mRNA-injected embryos at 24 hpf. (D) UIC embryos at 6 dpf. (E) fzd6 MO-injected embryos at 6 dpf. (F) fzd6 mRNA-injected embryos at 6 dpf. G-O show Alcian blue and alizarin red-stained embryos at 6 dpf. Jaw and palatal abnormalities due to fzd6 knockdown are shown (red arrows) in (K and N), respectively. Specifically in (K), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (N), arrows point to a reduced ethmoid palatal plate. Jaw and palatal abnormalities due to fzd6 overexpression are shown (red arrows) in (L and O), respectively. Specifically in (L), arrows point to abnormal Meckel’s and ceratohyal cartilage in the lower jaw. In (O), arrow points to a loss of the ethmoid plate in the palate.

Article Snippet: The FZD6 putative promoter sequence was identified using information from GeneCopeia (Rockville, MD) and SwitchGear Genomics (Menlo Park, CA) and included the 2277 bp region upstream of the transcriptional start site.

Techniques: Knockdown, Over Expression, Injection, Control, Staining

Journal: Translational Neurodegeneration

Article Title: Inflammation-induced PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons

doi: 10.1186/s40035-020-00211-4

Figure Lengend Snippet: PINCH is transcriptionally regulated by MEF2A under inflammatory conditions. a Quantification of TNFα production by neurons exposed to Tat at different time points. b Quantification of PINCH mRNA levels in neurons untreated or exposed to Tat or TNFα for 48 h. c Bioinformatic analysis of lims1/pinch promoter sequence predicted a conserved putative binding site for different transcription factors (TF). Inset: Sequences show conserved binding sites for MEF2A, Cc-FOS, Cc-Jun, and AP-1. d ChIP-assay was performed in neurons untreated or exposed to Tat or TNFα. Antibodies specific for MEF2A, c-Jun, Foxd1, and HoxA9 were used to immunoprecipitate the chromatin and the fold enrichment of lims1/pinch promoter relative to the matched input control was quantified by qPCR. e Representative Western blot of lysates from neurons untreated or exposed to Tat or TNFα and probed with antibodies specific for phospho-CamkII, CamkII, phospho-P38, P38, phospho-MEF2A, MEF2A, PINCH and GAPDH. f-i Quantification of relative protein abundance of phospho-CamkII/CamkII ( f ), phospho-P38/P38 ( g ), phospho-MEF2A/MEF2A ( h ), and PINCH/GAPDH ( i ) from ( e ). j Schematic representation of the lims1/pinch promoter-luciferase constructs. The MEF2A consensus response element at − 169-175 base pairs (bp) (TATTATA) is shown in the oval. k Neurons transfected with control and lims1/pinch luciferase constructs were untreated or exposed to Tat or TNFα for 48 h and luciferase activity was measured. Data represent mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001; n = 3–5 (one-way ANOVA)

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with lims1/pinch (PINCH) (NM_004987) promoter sequence (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for the luciferase assay.

Techniques: Sequencing, Binding Assay, Control, Western Blot, Quantitative Proteomics, Luciferase, Construct, Transfection, Activity Assay

Journal: Gene Therapy

Article Title: Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells

doi: 10.1038/s41434-021-00227-z

Figure Lengend Snippet: A Bioinformatic design of first-generation Ple155, second-generation Ple265, base pair modification to produce third-generation Ple318, and cutdown to produce third-generation Ple341. ENCODE segments that potentially regulate the expression of the PCP2 gene are highlighted horizontally. Vertically highlighted genomic regions correspond to their color-matched segments included in the MiniPromoter designs and are numbered as promoter(s) (P) and regulatory region(s) (RR). B Postnatal day 4 intravenous injection of Ple265-EmGFP, harvested 4 weeks later, led to robust expression in the inner nuclear layer (INL). Scale bar, 100 µm. (For ubiquitous smCBA promoter see Fig. .) C Adult subretinal injection of Ple265-EmGFP, harvested 4 weeks later, was quantified for epifluorescence intensity in PCP2-positive cells, and showed significantly lower (~1/2×) expression compared to smCBA ( p < 0.05). (For smCBA see Fig. .) D Adult subretinal injection of Ple265-EmGFP, harvested 4 weeks later, led to robust expression in ON bipolar cells, as indicated by co-staining with the ON bipolar cell marker PCP2. Scale bar, 10 µm. (For smCBA see Fig. .) EmGFP emerald green fluorescent protein, GCL ganglion cell layer, gen generation, Inr initiator element, IPL inner plexiform layer, N number of cells counted, ONL outer nuclear layer, OPL outer plexiform layer, TF transcription factor, TSS transcription start site. Green, anti-GFP; blue, Hoechst; red, anti-PCP2; yellow, merge.

Article Snippet: MiniPromoter design sequences were ordered for direct DNA synthesis (GenScript, Inc., Piscataway, NJ), or isolated from previous Pleiades Promoter plasmids [ – ], and cloned into the multiple cloning site ( Avr II, Fse I, Mlu I, and Asc I) of our “plug and play” rAAV2 backbone plasmid version 2 (pEMS2131) [ ].

Techniques: Modification, Expressing, Injection, Staining, Marker

Journal: Gene Therapy

Article Title: Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells

doi: 10.1038/s41434-021-00227-z

Figure Lengend Snippet: A Bioinformatic design of Ple347. ENCODE segments that potentially regulate the expression of the GNGT2 gene are highlighted horizontally. Vertically highlighted genomic regions correspond to their color-matched segments included in the MiniPromoter design and are numbered as promoter(s) (P) and regulatory region(s) (RR). B Postnatal day 0 intravenous injection of Ple347-EmGFP, harvested 4 weeks later, led to robust expression in the outer nuclear layer (ONL). Scale bar, 100 µm. (For ubiquitous smCBA promoter see Fig. ). C Adult intravitreal injection of Ple347-EmGP and Ple349 ( PDE6H )-EmGFP, harvested 4 weeks later, was quantified for epifluorescence intensity in opsin-positive cells, and showed that Ple347 ( GNGT2 ) expression was significantly increased (~3×) compared to smCBA ( p < 0.001). In addition, Ple349 ( PDE6H ) expression was significantly increased compared to both smCBA (~4×) ( p < 0.001) and Ple347 (~1.5×) ( p < 0.05). (For smCBA see Fig. .) D Adult intravitreal injection of Ple347-EmGFP, harvested 4 weeks later, led to robust expression in cone cells, as indicated by co-staining with the cone cell marker opsin where EmGFP inner segments align to anti-opsin outer segments. Scale bar, 20 µm. (For smCBA see Fig. .) EmGFP emerald green fluorescent protein, GCL ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, N number of cells counted, OPL outer plexiform layer, TF transcription factor, TSS transcription start site. Green, anti-GFP; blue, Hoechst; red, anti-opsin.

Article Snippet: MiniPromoter design sequences were ordered for direct DNA synthesis (GenScript, Inc., Piscataway, NJ), or isolated from previous Pleiades Promoter plasmids [ – ], and cloned into the multiple cloning site ( Avr II, Fse I, Mlu I, and Asc I) of our “plug and play” rAAV2 backbone plasmid version 2 (pEMS2131) [ ].

Techniques: Expressing, Injection, Staining, Marker

Journal: Gene Therapy

Article Title: Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells

doi: 10.1038/s41434-021-00227-z

Figure Lengend Snippet: A Bioinformatic design of Ple348 and Ple349. ENCODE segments that potentially regulate the expression of the PDE6H gene are highlighted horizontally. Vertically highlighted genomic regions correspond to their color-matched segments included in the MiniPromoter design and are numbered as promoter(s) (P) and regulatory region(s) (RR). B Postnatal day 0 intravenous injection of Ple349-EmGFP, harvested 4 weeks later, led to robust expression in the outer nuclear layer (ONL). Scale bar, 100 µm. (For ubiquitous smCBA promoter see Fig. .) C Adult intravitreal injection of Ple349-EmGFP, harvested 4 weeks later, led to robust expression in cone cells, as indicated by co-staining with the cone cell marker opsin, where EmGFP inner segments align to anti-opsin outer segments. Scale bar, 20 µm. (For smCBA see Fig. .) EmGFP emerald green fluorescent protein, GCL ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, OPL outer plexiform layer, TF transcription factor, TSS transcription start site. Green, anti-GFP; blue, Hoechst; red, anti-opsin.

Article Snippet: MiniPromoter design sequences were ordered for direct DNA synthesis (GenScript, Inc., Piscataway, NJ), or isolated from previous Pleiades Promoter plasmids [ – ], and cloned into the multiple cloning site ( Avr II, Fse I, Mlu I, and Asc I) of our “plug and play” rAAV2 backbone plasmid version 2 (pEMS2131) [ ].

Techniques: Expressing, Injection, Staining, Marker

Journal: Gene Therapy

Article Title: Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells

doi: 10.1038/s41434-021-00227-z

Figure Lengend Snippet: A Bioinformatic design of Ple253. ENCODE segments that potentially regulate the expression of the PITX3 gene are highlighted horizontally. Vertically highlighted genomic regions correspond to their color-matched segments included in the MiniPromoter design and are numbered as promoter (P). B Postnatal day 0 intravenous injection of Ple253-EmGFP, harvested 4 weeks later, led to robust expression in the stromal layer (Str) of the cornea, as determined from Hoechst staining and cell morphology. Scale bar, 100 µm. (For ubiquitous smCBA promoter see Fig. ). C Adult intrastromal injection of Ple253-EmGFP, harvested 4 weeks later, led to robust expression in the stromal layer, as determined by cell morphology. Scale bar, 50 µm. (For smCBA see Fig. .) D Adult intrastromal injection of Ple253-EmGFP, harvested 4 weeks later, was quantified for epifluorescence intensity in stromal cells, determined by cell morphology, was and showed significantly lower (~1/5×) expression compared to smCBA ( p < 0.001). (For smCBA see Fig. .) EmGFP emerald green fluorescent protein, End endothelial layer, Epi epithelial layer, N number of cells counted, TF transcription factor, TSS transcription start site. Green, anti-GFP; blue, Hoechst.

Article Snippet: MiniPromoter design sequences were ordered for direct DNA synthesis (GenScript, Inc., Piscataway, NJ), or isolated from previous Pleiades Promoter plasmids [ – ], and cloned into the multiple cloning site ( Avr II, Fse I, Mlu I, and Asc I) of our “plug and play” rAAV2 backbone plasmid version 2 (pEMS2131) [ ].

Techniques: Expressing, Injection, Staining

Journal: Gene Therapy

Article Title: Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells

doi: 10.1038/s41434-021-00227-z

Figure Lengend Snippet: A Bioinformatic design of first-generation Ple32, and redesign to produce second- and third-generation Ple261, Ple338, Ple339, and Ple340. ENCODE segments that potentially regulate the expression of the CLDN5 gene are highlighted horizontally. Vertically highlighted genomic regions correspond to their color-matched segments included in the MiniPromoter designs and are numbered as promoter(s) (P) and regulatory region(s) (RR). B Postnatal day 4 intravenous injection of Ple32-EmGFP, harvested 1 week later, led to robust expression in the blood vessels, as determined by morphology. Scale bar, 100 µm. (For ubiquitous smCBA promoter see Fig. .) C Adult intravenous injection of Ple32-EmGFP, harvested 4 weeks later, and visualized by whole mount retina, led to robust expression in the blood vessels, as determined by morphology. Scale bar, 100 µm (For smCBA see Fig. .) D Adult intravenous injection of Ple32-EmGFP, harvested 4 weeks later, was quantified for epifluorescence intensity in CD31-positive cells, and showed significantly higher (~2×) expression compared to smCBA ( p < 0.01). (For smCBA see Fig. ). E Adult intravenous injection of Ple32-EmGFP, harvested 4 weeks later, led to robust expression in the endothelial cells of the blood–retina barrier, as indicated by co-staining with the endothelial cell marker CD31. Scale bar, 20 µm. (For smCBA see Fig. .) EmGFP emerald green fluorescent protein, GCL ganglion cell layer, INL inner nuclear layer, IPL inner plexiform layer, N number of cells counted, ONL outer nuclear layer, OPL outer plexiform layer, TF transcription factor, TSS transcription start site. Green, anti-GFP; blue, Hoechst; red, anti-CD31; yellow, merge.

Article Snippet: MiniPromoter design sequences were ordered for direct DNA synthesis (GenScript, Inc., Piscataway, NJ), or isolated from previous Pleiades Promoter plasmids [ – ], and cloned into the multiple cloning site ( Avr II, Fse I, Mlu I, and Asc I) of our “plug and play” rAAV2 backbone plasmid version 2 (pEMS2131) [ ].

Techniques: Expressing, Injection, Staining, Marker

Journal: Gene Therapy

Article Title: Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells

doi: 10.1038/s41434-021-00227-z

Figure Lengend Snippet: A Bioinformatic design of first-generation Ple264, cutdown to produce second-generation Ple315, and redesign to produce second-generation Ple316. ENCODE segments that potentially regulate the expression of the NR2E1 gene are highlighted horizontally. Vertically highlighted genomic regions correspond to their color-matched segments included in the MiniPromoter designs and are numbered as promoter(s) (P) and regulatory region(s) (RR). B Postnatal day 4 intravenous injection of Ple316-EmGFP, harvested 4 weeks later, led to robust expression with cell bodies in the inner nuclear layer (INL) and processes that span the retina. Scale bar, 100 µm. (For ubiquitous smCBA promoter see Fig. .) C Adult intravitreal injection of Ple316-EmGFP, harvested 4 weeks later, was quantified for epifluorescence intensity in CRALBP-positive cells, and showed no significant difference when compared to smCBA. (For smCBA see Fig. ). D Adult intravitreal injection of Ple316-EmGFP, harvested 4 weeks later, led to robust expression in the Müller glia cells, as indicated by co-staining with Müller glia cell marker CRALBP. Scale bar, 20 µm. (For smCBA see Fig. .) EmGFP emerald green fluorescent protein, GCL ganglion cell layer, IPL inner plexiform layer, N number of cells counted, ONL outer nuclear layer, OPL outer plexiform layer, TF transcription factor, TSS transcription start site. Green, anti-GFP; blue, Hoechst; red, anti-CRALBP; yellow, merge.

Article Snippet: MiniPromoter design sequences were ordered for direct DNA synthesis (GenScript, Inc., Piscataway, NJ), or isolated from previous Pleiades Promoter plasmids [ – ], and cloned into the multiple cloning site ( Avr II, Fse I, Mlu I, and Asc I) of our “plug and play” rAAV2 backbone plasmid version 2 (pEMS2131) [ ].

Techniques: Expressing, Injection, Staining, Marker

Journal: Gene Therapy

Article Title: Human MiniPromoters for ocular-rAAV expression in ON bipolar, cone, corneal, endothelial, Müller glial, and PAX6 cells

doi: 10.1038/s41434-021-00227-z

Figure Lengend Snippet: A Bioinformatic design of first-generation Ple255 and Ple259, which were combined to produce the second-generation redesigns Ple328 and Ple329, also bioinformatic design of SIMO element Ple330 and base pair modified Ple331. ENCODE segments that potentially regulate the expression of the PAX6 gene are highlighted horizontally. Vertically highlighted genomic regions correspond to their color-matched segments included in the MiniPromoter designs and are numbered as promoter(s) (P) and regulatory region(s) (RR). B Postnatal day 0 intravenous injection of Ple331-EmGFP, harvested 4 weeks later, led to robust expression with cell bodies in the ganglion cell layer (GCL), and the inner nuclear layer (INL). Scale bar, 100 µm. (For ubiquitous smCBA promoter see Fig. ). C Postnatal day 4 intravenous injection of Ple331-EmGFP, harvested 4 weeks later, led to robust expression with cell bodies in the GCL, INL, and processes that span the retina. Scale bar, 100 µm. (For smCBA see Fig. .) D Adult subretinal injection of Ple331-EmGFP, harvested 4 weeks later, was quantified for epifluorescence intensity in RBPMS-positive ganglion cells, and showed significantly lower (~30%) ganglion cell expression when compared to smCBA ( p < 0.01). (For smCBA see Fig. .) E Adult subretinal injection of Ple331-EmGFP, harvested 4 weeks later, led to robust expression in the PAX6-positive cells, ganglion, amacrine, horizontal, and Müller glia, as indicated by co-staining with anti-PAX6. Scale bar, 20 µm. (For smCBA see Fig. .) EmGFP emerald green fluorescent protein, IPL inner plexiform layer, N number of cells counted, ONL outer nuclear layer, OPL outer plexiform layer, TF transcription factor, TSS transcription start site. Green, anti-GFP; blue, Hoechst; red, anti-Pax6; yellow, merge.

Article Snippet: MiniPromoter design sequences were ordered for direct DNA synthesis (GenScript, Inc., Piscataway, NJ), or isolated from previous Pleiades Promoter plasmids [ – ], and cloned into the multiple cloning site ( Avr II, Fse I, Mlu I, and Asc I) of our “plug and play” rAAV2 backbone plasmid version 2 (pEMS2131) [ ].

Techniques: Modification, Expressing, Injection, Staining

Journal: bioRxiv

Article Title: Functional analysis of recurrent non-coding variants in human melanoma

doi: 10.1101/2022.06.30.498319

Figure Lengend Snippet: (A) The CDC20 promoter hotspot. All variants within the hotspot are denoted by name, # of donors with given mutation, FunSeq2 score, and GERP score. Co-occurring GG528AA double mutant is depicted above. Variants with colored text were validated by luciferase assay. (B) Altered CDC20 promoter activity for variants as assayed by luciferase reporter assays in melanoma (A375, SK-MEL5) and primary melanocytes. Boxplots depict normalized (to WT) luciferase assay results in these 3 different cell lines. The numbers below each boxplot indicate the number of donors in BRAF (left) or NRAS (right) tumors with the corresponding variant.

Article Snippet: To make the luciferase vectors, we synthesized a 170 bp sequence containing the WT CDC20 promoter sequence (chr1:43,824,464-43,824,633) (GenScript).

Techniques: Mutagenesis, Luciferase, Activity Assay, Variant Assay

Journal: bioRxiv

Article Title: Functional analysis of recurrent non-coding variants in human melanoma

doi: 10.1101/2022.06.30.498319

Figure Lengend Snippet: A) Mosaic plot of the number of donors with either BRAF and/or NRAS mutations and WT and/or mutant CDC20 promoter. (B) Variant allele frequencies of the CDC20 promoter variants (each labelled), BRAF V600E (BRAF), NRAS Q61K and NRAS Q61R (NRAS), TERT G228A and G250A (TERT) that are detected in lymph node metastases or distant metastases. (C) Each bar represents the number of specimens with the corresponding variant within the corresponding tumor subtype divided by the total number of specimens with the corresponding variant across all subtypes. Absolute counts for each subtype are labeled within the bar. Total counts across all subtypes are labeled within the legend. The total number of samples at each stage that contain one of the specified mutations are in parentheses below the row label; the number that follows is equal to the total number of samples in that tumor stage.

Article Snippet: To make the luciferase vectors, we synthesized a 170 bp sequence containing the WT CDC20 promoter sequence (chr1:43,824,464-43,824,633) (GenScript).

Techniques: Mutagenesis, Variant Assay, Labeling

Journal: bioRxiv

Article Title: Functional analysis of recurrent non-coding variants in human melanoma

doi: 10.1101/2022.06.30.498319

Figure Lengend Snippet: (A) Table summarizing motifBreakR results. All transcription factors listed have strong and significant motif altering predictions. ETS transcription factors are colored in pink. (B) Heatmap of Pearson correlation values between TF (column) and samples with WT CDC20 promoters (top row) or mutant CDC20 promoters (bottom row.)

Article Snippet: To make the luciferase vectors, we synthesized a 170 bp sequence containing the WT CDC20 promoter sequence (chr1:43,824,464-43,824,633) (GenScript).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Functional analysis of recurrent non-coding variants in human melanoma

doi: 10.1101/2022.06.30.498319

Figure Lengend Snippet: (A) Variant allele frequencies of the CDC20 promoter variants (each labelled), BRAF V600E (BRAF), NRAS Q61K and NRAS Q61R (NRAS), TERT G228A and G250A (TERT) that are detected in primary melanomas. C520T and C541T are not detected in primary melanomas and have no data points. There are no statistically significant differences between G528A, G529A and other variants. (B) CDC20 expression in CDC20-Low, CDC20-Medium, and CDC20-High nevus or melanoma samples from Kunz et al. Each data point represents the log 2 DESeq2-normalized read count of CDC20 . No nevi are classified as CDC20-high. (C and D) Gene set enrichment analysis of results for the Winnepenninckx Melanoma Metastasis Up gene set (C) and the Ehlers Aneuploidy Up gene set (D) . Each point represents a gene, ranked by expression, at the current running-sum statistic. Negative scores indicate enrichment in CDC20-high samples (as seen in C). Positive scores indicate enrichment in CDC20-low samples (as seen D). (E) Heatmap depicting z-score normalized expression patterns of 20 key neural crest transcription factors. Samples and genes are hierarchically clustered with orange and blue indicating relatively higher and lower gene expression, respectively, across samples. All columns are annotated by CDC20 expression (top row of boxes, log 2 DESeq2-normalized read count), CDC20 expression group (second row, for low, medium, or high), and sample type (nevus in orange or melanoma in dark brown).

Article Snippet: To make the luciferase vectors, we synthesized a 170 bp sequence containing the WT CDC20 promoter sequence (chr1:43,824,464-43,824,633) (GenScript).

Techniques: Variant Assay, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Functional analysis of recurrent non-coding variants in human melanoma

doi: 10.1101/2022.06.30.498319

Figure Lengend Snippet: (A) Sequence alignment of the CDC20 promoter between hg19, WT A375, A3, and A10. Arrows denoting positions of G525A, G528A, and G529A. The ETS core motif is boxed. The last nucleotide of the sequence is 37 bp upstream of the TSS of CDC20 . Nucleotides are color-coded and dashes indicate deletions. (B) Plot depicting log 2 transformed DESeq2-normalized read counts of CDC20 in WT A375 and CDC20 promoter indel strains, A3 and A10, with decreased CDC20 expression. Each point represents CDC20 expression in one sample. (C) Principal component analysis of read counts normalized by regularized log transformation using the top 500 most variable genes. The horizontal axis, PC1, explains 58% of the variance associated across all samples and separates out WT from CDC20 promoter indel cell lines. The vertical axis, PC2, explains 28% of the variance and separated A3 from A10. (D) Gene set enrichment analysis of results for the Winnepenninckx Melanoma Metastasis Up gene set. Each point represents a gene, ranked by expression, at the current running-sum statistic. Negative scores indicate enrichment in WT A375 samples as compared to the engineered indel lines A3 and A10. (E) CDC20 indel lines A3 and A10 show decreased migration capabilities compared to WT A375 cell lines. Images of scratch migration assay from day 0 (immediately after scratch) and day 1 (24 hours post-scratch). (F) Heatmap depicting z-score normalized expression patterns of 9 differentially expressed neural crest transcription factors. Samples and genes are hierarchically clustered with orange and blue indicating relatively higher or lower expression, respectively, of genes across samples. All columns are annotated by CDC20 expression (log 2 DESeq2-normalized read count), and sample type (WT or mutant). SOX5 , TFAP2B , SOX10 , MYC , and RXRG are expressed at relatively higher levels in the CDC20 promoter indel-containing A3 and A10 cell lines. FOXD1 , ETS1 , TFAP2C , and TFAP2A are higher in WT (CDC20-high) A375 cell lines. (G) Model of CDC20 expression and neural crest transcription factor signature over melanoma onset and progression. CDC20 levels increase as melanoma progresses. Neural crest transcription factors that correlate with CDC20 expression are more prevalent in migrating neural crest cells, whereas those that are relatively higher in CDC20-low settings are more prevalent in the melanocytic/pre-migratory neural crest states.

Article Snippet: To make the luciferase vectors, we synthesized a 170 bp sequence containing the WT CDC20 promoter sequence (chr1:43,824,464-43,824,633) (GenScript).

Techniques: Sequencing, Transformation Assay, Expressing, Migration, Mutagenesis

Journal: bioRxiv

Article Title: Functional analysis of recurrent non-coding variants in human melanoma

doi: 10.1101/2022.06.30.498319

Figure Lengend Snippet: (A) Heatmap depicting relative expression of 20 neural crest transcription factors using the average of all samples classified as CDC20-high, medium, or low. The median log 2 -normalized CDC20 count is listed below each column of every heatmap. Orange indicates higher expression relative to other samples for the same gene. Genes in green are upregulated in WT A375 compared to CDC20 promoter indel cell lines. Genes in red are upregulated in CDC20 promoter indel cell lines. (B) Table summarizing whether a cohort has a relative gene level that matches or does not match the gene level seen in WT or CDC20 promoter indel cell lines. For a gene to agree, it needs to have relatively higher expression in the WT lines (green genes) or relatively higher expression in the CDC20 promoter indel lines (red genes). Cohorts that have an asterisk neither completely agree or disagree (e.g. relatively higher in CDC20-medium samples or relatively high in CDC20-low and CDC20-high, see SOX10 in TCGA).

Article Snippet: To make the luciferase vectors, we synthesized a 170 bp sequence containing the WT CDC20 promoter sequence (chr1:43,824,464-43,824,633) (GenScript).

Techniques: Expressing

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Sequences of the oligonucleotides used in this study.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Sequencing, Amplification, Control

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Cell cycle dependent bi-directional activities of human BRCA2 gene promoter . (A) Genomic context of human BRCA2 gene bi-directional (BD) promoter studied. The numbers shown are with respect to the transcription start site of human BRCA2 gene. (B) Maps of the reporter constructs in pRL-Null vector used in the study. (i) The single reporter constructs: pRL-FP (forward construct) and pRL-RP (reverse construct); (ii) the dual reporter construct. URS: upstream regulatory sequence; Ex-1: exon 1; Int-1: intron 1; Rluc: Renilla luciferase; Fluc: firefly luciferase; ORF: open reading frame. (C) Activities of the BRCA2 (forward) and the ZAR2 (reverse) promoters in different lines of human breast cancer cells at G0/G1 and S/G2 phases of their cell cycles. Results are mean ± SE (n = 6). The differences between the G0/G1 and S/G2 phase cells were statistically significant (shown by '*'; p < 0.001).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Construct, Plasmid Preparation, Sequencing, Luciferase

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Relative activities of the forward and the reverse promoters of BRCA2 gene in different unsynchronized human breast cancer cells at 95% confluency.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Ratio of the forward (BRCA2) and the reverse (ZAR2) activity in the G0/G1 and S/G2 growth phases of different breast cancer cells using transient transfection with the dual reporter/promoter construct (see Fig. 1B).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Activity Assay, Transfection, Construct

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: The transcription start sites of the reverse transcript from BRCA2 gene bi-directional promoter . (A) GeneRacer amplification product for ZAR2. See Materials and methods for details. (B) Nucleotide sequence of human ZAR2/BRCA2 bi-directional promoter. The transcriptional start sites (TSSs), as determined by GeneRacer technique, are shown. The segment in green color is the sequence complementary to part of the intron 1 sequence of human BRCA2 gene, the segment in red color is from exon 1 and the blue part is from the upstream sequence of BRCA2 gene. The E-box sequence essential for BRCA2 gene expression [ , ] is underlined. The splice donor site at the ZAR2 gene exon 1/intron 1 junction is indicated by a downward arrow. The 'G' residue at the SNP site at -26 from BRCA2 gene transcription start site is shown by a red *. (C) Cartoon showing the human BRCA2 (upper panel) and ZAR2 (lower panel) gene promoter studied. The identities of ZAR2 exon1 (Ex-1) and intron 1 (Int-1) were experimentally determined in this study. TSS: transcription start site (designated as +1); URS: upstream regulatory sequence.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Amplification, Sequencing, Gene Expression, Residue

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Conservation of the BRCA2/ZAR2 genetic arrangements in vertebrates . (A) Relative chromosomal locations of BRCA2 and ZAR2 genes in different vertebrates. The maps were obtained from NCBI site for Entrez genes http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&term=BRCA2+ Not drawn to the scale. (B) Dendrogram with branch lengths for the ZAR2 proteins from different vertebrates. The putative ZAR2 protein amino acid sequences were mined from the NCBI Entrez database and dendrogram with branch length was analyzed by CLUSTALW program http://align.genome.jp/ .

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques:

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: The exon-intron structure and mRNA sequence of human ZAR2 gene . (A) Cartoon showing the exon-intron structure of human ZAR2 gene. The first exon of ZAR2 overlaps with the exon 1 of the BRCA2 gene (not drawn to the scale). (B) Nucleotide sequence of human ZAR2 mature mRNA. The 5'-UTR sequence was experimentally determined (see text for details). The putative protein coding sequence (ORF) is shown in blue and highlighted in gray. The upstream AUG (uAUG) codons at the 5'-UTR are highlighted: out-of-frame uAUGs are in yellow shades; in-frame uAUGs are in green shades. The 5'-UTR sequence overlapping with BRCA2 mRNA sequences are shaded yellow. Rest of the 5'-UTR sequence of ZAR2 mRNA is derived from the newly identified exon 1 and is shown in red.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Sequencing, Derivative Assay

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Relative expressions of BRCA2 and ZAR2 mRNAs at different cell cycle stages of human breast cells . (A) RT-PCR analysis showing the expressions of BRCA2 and ZAR2 mRNAs in the unsynchronized (mostly dividing) cells. β-Actin mRNA was used as a loading control. (B) Real-time RT-PCR evaluation of the relative levels of BRCA2 and ZAR2 mRNAs in different human breast cancer cells at G0/G1 and S/G2 phases. The differences between the G0/G1 and S/G2 phase cells were statistically significant (shown by '*'; p < 0.001). (C) Immunofluorescence confocal microscopy showing growth phase-dependent localization of N-terminal FLAG-tagged ZAR2 protein in the synchronized MCF7 cells. Anti-FLAG M2 antibody was used for the detection of FLAG-tagged ZAR2 protein in the cells. The cells were transiently transfected with the expression plasmid constructs and thus not all cells are expressing the recombinant protein.

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Quantitative RT-PCR, Immunofluorescence, Confocal Microscopy, Transfection, Expressing, Plasmid Preparation, Construct, Recombinant

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: In vivo binding of ZAR2 protein to the BRCA2/ZAR2 gene promoter . (A) PCR amplification of the immunoprecipitated chromatin DNA fragment pulled down with FLAG antibody from synchronized MCF7 cells over-expressing C-terminal FLAG-tagged ZAR2 protein at the G0/G1 and S/G2 phases. Input DNA (5% was used as control. Chromatin DNA fragments mock precipitated with mouse IgG did not significantly amplified any detectable DNA. BRCA2 gene promoter specific primers were used for PCR amplifications. (B) Quantitative ChIP analysis of ZAR2 recruitment to BRCA2/ZAR2 bi-directional promoter in MCF7 cells at G0/G1 and S/G2 phases. qChIP-PCR analyses were performed with chromatin extracts harvested from cells over expressing C-terminal FLAG-tagged ZAR2. The mean values from triplicate data points are plotted and error bars indicate ± SE. The amplification values are normalized by subtraction with IgG control antibody and then division with 1% input DNA. Data shown were representative of three independent experiments (mean + SE) and the difference between the G0/G1 phase and the S/G2 phase cells was statistically significant (shown by '*'; p < 0.001).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: In Vivo, Binding Assay, Amplification, Immunoprecipitation, Expressing, Control

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Effects of over-expression (OVEX) of the C-terminal FLAG-tagged ZAR2 in synchronized MCF7 cells on the BRCA2 and ZAR2 mRNA levels (A) and on the activities of BRCA2 and ZAR2 gene promoters (B) at the S/G2 phase . MCF7 cells were stably transfected with C-terminally FLAG-tagged ZAR2 and evaluated for their ZAR2 over expression. Levels of the mRNAs were determined by real-time RT-PCR . Promoter activities were measured in MCF7 cells transiently transfected with the single-reporter constructs (Fig. 1B) following the dual luciferase assay protocols (Promega). pGL3-Control was used as normalization control as described in the 'Methods' section. Results are mean ± SE (n = 6). '*' indicates the difference between the corresponding control and the experimental sets is statistically significant (p < 0.001).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Over Expression, Stable Transfection, Transfection, Quantitative RT-PCR, Construct, Luciferase, Control

Journal: Molecular Cancer

Article Title: Cell cycle-dependent regulation of the bi-directional overlapping promoter of human BRCA2/ZAR2 genes in breast cancer cells

doi: 10.1186/1476-4598-9-50

Figure Lengend Snippet: Effect of knockdown of ZAR2 in synchronized MCF7 cells on the BRCA2 and ZAR2 mRNA levels (A) and on the activities of BRCA2 and ZAR2 gene promoters (B) at the G0/G1 phase . ZAR2 was knocked down in MCF7 cells with two different double-stranded stealth siRNAs (Invitrogen). Levels of the mRNAs were determined by real-time RT-PCR. Promoter activities were measured in MCF7 cells transiently transfected with the single-reporter constructs (Fig. 1B) following the dual luciferase assay protocols (Promega). pGL3-Control was used as normalization control as described in the 'Methods' section. Results are mean ± SE (n = 6). '*' indicates the difference between the corresponding control and the experimental sets is statistically significant (p < 0.001).

Article Snippet: We cloned the 497 bp promoter DNA sequence (-187 to +310) of human BRCA2 gene in front of Renilla luciferase ( Rluc ) gene in pRL-Null plasmid (Promega) to obtain clones with the insert either in the forward or in the reverse orientation with respect to the luciferase gene (Fig. ).

Techniques: Knockdown, Quantitative RT-PCR, Transfection, Construct, Luciferase, Control