Journal: Nature Communications

Article Title: Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

doi: 10.1038/s41467-020-16880-8

Figure Lengend Snippet: a Quantitative PCR analysis of HBB transcription level in HEK293T cells transfected with type I–F PaeCascade 2-vector systems and crRNA targeting HBB . Left: schematic illustration of different type I–F PaeCascade VPR activators generated from 2-vector systems in Fig. . Gray: Csy1; red: Csy2; blue: Csy3; yellow: Csy4; orange flag: VPR. Different fusions of PaeCascade subunits resulted in different locations and copy numbers of VPR. HEK293T cells were transfected with PaeCascade 2-vector systems and crRNA vector. 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. b Quantitative PCR analysis of gene transcription levels in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR 2-vector system) targeting different regions upstream the transcriptional start site (TSS) of six genes ( HBB , HBG , SOX2, OCT4 , IL1B , and IL1R2 ). n.d.: not determined. c Histogram showing the normalized mean transcription activating levels of HBB , HBG , SOX2, OCT4 , IL1B , and IL1R2 induced by type I–F PaeCascade VPR (Csy3-VPR 2-vector system) transcription activator targeting different regions upstream of TSS. For normalization of data from different TSS among different genes, data in the same gene were processed by percentage normalization with 100% defined by the sum of all values in data set. The normalized values of all six genes were pooled and plotted as box & whiskers plot with min to max option. The median value is displayed as the center of the data set, and is derived using the lower and upper quartile values. The maximum and minimum values are displayed as whiskers. d The efficiency of target gene activation as a function of basal transcript levels. Data from ( b ) were plotted by fold changes comparing to negative control and relative basal transcript level of HBB, HBG, SOX2, OCT4, IL1B, and IL1R2 . Each dot represented the mean relative activation level of each crRNA from the three replications in ( b ). Ctrl: non-targeting crRNA control. Data in ( a , b ) represented three biological repeats and displayed as mean ± S.E.M. Statistical significance was calculated using one-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a file.

Article Snippet: A site for spacer cloning flanked by two Csy4 direct repeats (DR) or Cas6f direct repeats was ligated into lentiGuide-Puro (addgene #52963) between BsmBI and EcoRI restriction sites to generate pLenti-crRNA-IF or pLenti-crRNA-IFv vectors.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Generated, RNA Extraction, Derivative Assay, Activation Assay, Negative Control, Control

Journal: Nature Communications

Article Title: Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

doi: 10.1038/s41467-020-16880-8

Figure Lengend Snippet: a Quantitative PCR analysis of HBB, HBG , and SOX2 transcription levels in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR 2-vector system) with spacers in different lengths. Upper: schematic illustration of differences in Csy3 copy numbers in type I–F PaeCascade VPR (Csy3-VPR) with spacers in different lengths. Gray: Csy1; red: Csy2; blue: Csy3; yellow: Csy4; orange flag: VPR. The longer the spacer was, the more Csy3-VPR in type I–F PaeCascade. Lower: quantitative PCR analysis of HBB, HBG , and SOX2 transcription level in HEK293T cells cotransfected with type I–F PaeCascade VPR and crRNA targeting HBB, HBG , and SOX2 . 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. b Quantitative PCR analysis of HBB , HBG , SOX2, OCT4 , IL1B , and IL1R2 transcription levels in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR 2-vector system) and crRNAs targeting −100 bp (crRNA1) and −200 bp (crRNA2) upstream of TSS in Fig. . Upper: schematic illustration of enhancing transcription level by two crRNAs targeting the same gene. Lower: quantitative PCR analysis of HBB , HBG , SOX2, OCT4 , IL1B , and IL1R2 transcription levels in HEK293T cells. 2 crRNAs indicates two independent crRNA expression vectors. c Quantitative PCR analysis of HBG transcription level in HEK293T cells cotransfected with type I–F PaeCascade VPR (Csy3-VPR) and crRNA with different distances to crRNA2 (−200 bp upstream TSS in Fig. ). 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. Ctrl: non-targeting crRNA control. Data represented three biological repeats and displayed as mean ± S.E.M. Statistical significance was calculated using one-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a file.

Article Snippet: A site for spacer cloning flanked by two Csy4 direct repeats (DR) or Cas6f direct repeats was ligated into lentiGuide-Puro (addgene #52963) between BsmBI and EcoRI restriction sites to generate pLenti-crRNA-IF or pLenti-crRNA-IFv vectors.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, RNA Extraction, Expressing, Control

Journal: Nature Communications

Article Title: Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

doi: 10.1038/s41467-020-16880-8

Figure Lengend Snippet: a Schematic illustrating pre-crRNA processed by Csy4 in human cells. Tandem spacer containing premature crRNA (DR-spacer1-DR-spacer2-DR) was transcribed and processed by Csy4 into two mature crRNAs. White box: human U6 promoter (hU6); Gray box: direct repeats (DR); Red box: spacer 1; Blue box: spacer 2. b Quantitative PCR analysis of HBB , HBG and SOX2 transcription levels in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR) with two crRNA expression vectors (crRNA1 + crRNA2) or customized CRISPR arrays (CRISPR arrays 1/2) targeting −100 bp (crRNA1) and −200 bp (crRNA2) upstream of TSS in Fig. . Upper: schematic illustration of Csy4 processing customized CRISPR arrays targeting two sites on the same gene. Lower: quantitative PCR analysis of HBB , HBG , and SOX2 transcription level in HEK293T cells. HEK293T cells were transfected with PaeCascade 2-vector systems (Csy3-VPR) and crRNA expression vectors as indicated. 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. c Quantitative PCR analysis of multiplex gene activation in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR) with 2 or 3 independent crRNA vectors or customized CRISPR arrays (CRISPR array) targeting different genes. Upper: Schematic illustration of Csy4 processing customized CRISPR arrays targeting two sites on different genes. Lower: quantitative PCR analysis of multiplex activating level in HEK293T cells. HEK293T cells were transfected with PaeCascade 2-vector systems (Csy3-VPR) and crRNA expression vectors as indicated. 2 crRNAs indicates two independent crRNA expression vectors. 3 crRNAs indicates three independent crRNA expression vectors. CRISPR array, customized CRISPR array in one vector. 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. Ctrl: non-targeting crRNA control. Data represented three biological repeats and displayed as mean ± S.E.M. Statistical significance was calculated using one-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a file.

Article Snippet: A site for spacer cloning flanked by two Csy4 direct repeats (DR) or Cas6f direct repeats was ligated into lentiGuide-Puro (addgene #52963) between BsmBI and EcoRI restriction sites to generate pLenti-crRNA-IF or pLenti-crRNA-IFv vectors.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Expressing, CRISPR, Plasmid Preparation, RNA Extraction, Multiplex Assay, Activation Assay, Control

Journal: Nature Communications

Article Title: Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells

doi: 10.1038/s41467-020-16880-8

Figure Lengend Snippet: a Schematic illustration of crRNA variants containing 6-nt mismatches to the targeted site. There were five crRNA variants carrying 6-nt mismatches to the targeted DNA. Mismatched bases are highlighted in red and PAM is highlighted in green. Gray: Csy1; red: Csy2; blue: Csy3; yellow: Csy4. b Quantitative PCR analysis of HBB and HBG transcription levels in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR) with full-length crRNA or 6-nt mismatched crRNA variants in ( a ). HEK293T cells were transfected with PaeCascade 2-vector systems (Csy3-VPR) and crRNA expression vectors. 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. Ctrl: non-targeting crRNA control. c Schematic illustration of crRNA variants with single mismatches to the targeted site. There were 32 crRNA variants each carrying one single mismatch to the targeted DNA. Mismatched bases are highlighted in red and PAM is highlighted in green. d Quantitative PCR analysis of HBB and HBG transcription levels in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR) with full-length crRNA or single nucleotide mismatched crRNA variants in ( c ). HEK293T cells were transfected with PaeCascade 2-vector systems (Csy3-VPR) and crRNA expression vectors. 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. Ctrl: non-targeting crRNA control. Error bars represented three biological repeats and displayed as mean ± S.E.M. Statistical significance was calculated using one-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001). Source data are provided as a file.

Article Snippet: A site for spacer cloning flanked by two Csy4 direct repeats (DR) or Cas6f direct repeats was ligated into lentiGuide-Puro (addgene #52963) between BsmBI and EcoRI restriction sites to generate pLenti-crRNA-IF or pLenti-crRNA-IFv vectors.

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing, RNA Extraction, Control

Journal:

Article Title: A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont

doi: 10.1016/j.cell.2009.02.041

Figure Lengend Snippet: (A) 2D gel of AAL-purified proteins obtained from B. fragilis whole cell lysate. Gene numbers indicate proteins that were unequivocally identified by MS analysis. (B) Whole cell lysates of wild-type (WT) and Δgmd-fclΔfkp (ΔΔ), separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His or BF3567-His purified from E. coli. (C) Whole cell lysates of wild-type (WT) and Δgmd-fclΔfkp (ΔΔ) expressing His-tagged glycoprotein candidates, separated by SDS-PAGE, blotted and probed with antibody to the His-tag. BF3810 is an example of a negative result. (D) BF2494-His purified from B. fragilis (Bf) or E. coli (Ec), separated by SDS-PAGE and stained with Coomassie Blue or Pro-Q Emerald Glycostain (first two panels), or blotted and probed with AAL, antiserum to the glycan of BF2494, antibody to the His-tag, or anti-BF2494-His purified from E. coli. (E) Six additional His-tagged glycoproteins purified from B. fragilis and analyzed as in panel D.

Article Snippet: Reverse transcriptase (RT)-PCR B. fragilis was grown to OD 600 0.8, harvested, and total RNA was extracted with the RNeasy Mini Kit using RNAprotect with on-column digestion using the RNase-Free DNase Set (all products from Qiagen) and further DNase treatment was performed with the DNA-free kit (Ambion).

Techniques: Two-Dimensional Gel Electrophoresis, Purification, SDS Page, Expressing, Staining, Glycoproteomics

Journal:

Article Title: A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont

doi: 10.1016/j.cell.2009.02.041

Figure Lengend Snippet: (A) Cytoplasmic and periplasmic fractions of wild-type B. fragilis, separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His purified from E. coli. (B) Cytoplasmic and periplasmic fractions from B. fragilis expressing His-tagged BF2494, BF0447, BF0935 or BF2334, separated by SDS-PAGE, blotted and probed with anti-His-tag. (C) Intact B. fragilis Δtsr15M8 cells were incubated with proteinase K for the indicated times and the lysate separated by SDS-PAGE, blotted and probed with antisera to His-tagged BF3567, AapA, or BF2494 purified from E. coli. BF2494 and AapA are controls located in the periplasm or on the surface of the cell, respectively. (D) Wild-type B. fragilis expressing BF0522-His or BF3918-His were incubated with proteinase K for the indicated times, lysed, separated by SDS-PAGE, blotted and probed with anti-His-tag.

Article Snippet: Reverse transcriptase (RT)-PCR B. fragilis was grown to OD 600 0.8, harvested, and total RNA was extracted with the RNeasy Mini Kit using RNAprotect with on-column digestion using the RNase-Free DNase Set (all products from Qiagen) and further DNase treatment was performed with the DNA-free kit (Ambion).

Techniques: SDS Page, Purification, Expressing, Incubation

Journal:

Article Title: A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont

doi: 10.1016/j.cell.2009.02.041

Figure Lengend Snippet: (A) Amino acid sequence of BF2494. The signal peptide is underlined. Segments in upper case and bolded are S/T-containing tryptic peptides that were not detected in at least 2 of 6 MS analyses of purified BF2494-His (excluding the signal peptide). Boxed or underlined residues in these segments were mutated to alanine to determine whether they are glycosylated; boxed residues were found to be glycosylated and underlined residues were not. (B) Wild-type (WT) and mutant BF2494-His proteins purified from B. fragilis, separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His purified from E. coli or stained with Coomassie Blue. (C) Same as panel B except that the gel was either stained with Pro-Q Emerald Glycostain, or blotted and probed with AAL or the antiserum to the glycan of BF2494. (D) Cytoplasmic and periplasmic fractions of B. fragilis expressing His-tagged BF2494 Δ(2-18), separated by SDS-PAGE, blotted and probed with anti-His-tag. The periplasmic fraction is significantly contaminated with cytoplasmic material but the His-tagged protein is partitioned between the fractions in the proportions expected for a cytoplasmic molecule (Table S2). (E) Same as panel D except that the protein is the triple T→A BF2494-His molecule. (F) Alignment of protein sequences surrounding the three glycosylation sites of BF2494. Glycosylated residues are underlined and conserved or similar residues are bold. (G) Whole cell lysates of B. fragilis expressing wild-type and various mutant BF2494-His proteins, separated by SDS-PAGE, blotted and probed with antibody to the His-tag.

Article Snippet: Reverse transcriptase (RT)-PCR B. fragilis was grown to OD 600 0.8, harvested, and total RNA was extracted with the RNeasy Mini Kit using RNAprotect with on-column digestion using the RNase-Free DNase Set (all products from Qiagen) and further DNase treatment was performed with the DNA-free kit (Ambion).

Techniques: Sequencing, Purification, Mutagenesis, SDS Page, Staining, Glycoproteomics, Expressing

Journal:

Article Title: A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont

doi: 10.1016/j.cell.2009.02.041

Figure Lengend Snippet: (A) The region of the B. fragilis genome containing metG and genes BF4298-4306. Putative glycosyltransferase genes are hatched. (B) Phosphoimager scan of SDS-PAGE-separated whole cell lysates of wild-type, Δgmd-fclΔfkp, and Δ(BF4298-4306) grown in a medium containing 3H-fucose. (C) Whole cell lysates of wild-type and mutants separated by SDS-PAGE, blotted and probed with antiserum to BF2494-His purified from E. coli. (D) Whole cell lysates of wild-type and mutant B. fragilis expressing BF0447-His or BF0522-His, separated by SDS-PAGE, blotted and probed with anti-His-tag. (E) RT-PCR amplification of a 1142 bp region including portions of the coding regions of metG and wzx, as indicated in panel A. Reactions were performed with and without reverse transcriptase (RT).

Article Snippet: Reverse transcriptase (RT)-PCR B. fragilis was grown to OD 600 0.8, harvested, and total RNA was extracted with the RNeasy Mini Kit using RNAprotect with on-column digestion using the RNase-Free DNase Set (all products from Qiagen) and further DNase treatment was performed with the DNA-free kit (Ambion).

Techniques: SDS Page, Purification, Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Reverse Transcription

Journal:

Article Title: A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont

doi: 10.1016/j.cell.2009.02.041

Figure Lengend Snippet: (A) The lfg region of B. fragilis and the corresponding regions downstream of metG in other intestinal Bacteroides species. Putative glycosyltransferases are hatched. (B) Whole cell lysates of various Bacteroides species separated by SDS-PAGE, blotted and probed with antiserum to the glycan of BF2494. (C) Same as panel B except that the blot was probed with antiserum to BF2494 purified from E. coli, which recognizes the protein component of the molecule. (D) Alignment of segments of BF2494 and its orthologs demonstrating conservation of the glycosylation motif (bolded). The three residues that are glycosylated in B. fragilis (T87, T178 and T231), and its counterparts in the orthologous proteins are underlined. (E) Western blot analysis of whole cell lysates of various Bacteroides species expressing the B. fragilis wild-type BF2494-His protein (WT), or the B. fragilis BF2494-His triple glycosylation site mutant (T87A.T178A.T231A), probed with anti-His-tag.

Article Snippet: Reverse transcriptase (RT)-PCR B. fragilis was grown to OD 600 0.8, harvested, and total RNA was extracted with the RNeasy Mini Kit using RNAprotect with on-column digestion using the RNase-Free DNase Set (all products from Qiagen) and further DNase treatment was performed with the DNA-free kit (Ambion).

Techniques: SDS Page, Glycoproteomics, Purification, Western Blot, Expressing, Mutagenesis

Journal: Endocrinology

Article Title: FOXL2 C134W -Induced CYP19 Expression via Cooperation With SMAD3 in HGrC1 Cells

doi: 10.1210/en.2017-03207

Figure Lengend Snippet: Potential binding sites of FOX (FBE) and SMAD (SBE) in the promoter region of human CYP19. Human CYP19 promoter region was analyzed by the Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) database that hosts data generated by universal protein-binding microarray technology on the in vitro DNA binding specificities of proteins. The coding sequence is underlined.

Article Snippet: Because human FOXL2 gene is extremely rich in GC (>84% in part), we amplified several short DNA segments overlapping in the entire coding sequence of the gene by PCR using two different DNA polymerases, Phusion Hot Start Flex DNA Polymerase (catalog no. M0535S; New England BioLabs, Ipswich, MA) and Platinum SuperFi DNA Polymerase (catalog no. 12351010; Thermo Fisher Scientific), following the manufacturer’s protocols.

Techniques: Binding Assay, Generated, Protein Binding, Microarray, In Vitro, Sequencing

Journal: Cancer research

Article Title: A targeted quantitative proteomic approach assesses the reprogramming of small GTPases during melanoma metastasis

doi: 10.1158/0008-5472.CAN-17-3811

Figure Lengend Snippet: (A) Bisulfite sequencing demonstrated the methylation status of CpG sites in the promoter region of RAB38 gene in the three paired primary/metastatic cell lines, where high levels of methylation were observed for the WM-115, IGR39, WM793 and 1205Lu, but not for the WM-266-4 and IGR37 melanoma cell lines. CpG sites in the promoter region of RAB38 gene are indicated by short vertical bars, and exons are designated with black rectangles on the top. The arrow indicates the transcription start site (TSS). Each horizontal line represents one separate clone that was sequenced, and open and filled circles represent unmethylated and methylated CpG sites, respectively; (B) Increased mRNA expression levels of MITF, TBC1D16-47kDa and RAB38 after 5-aza-2′-deoxycytidine (5-Aza) treatment (96 h) in WM-115 cells; (C) Increased mRNA expression levels of MITF, TBC1D16-47kDa and RAB38 after 5-Aza treatment (96 h) in IGR39 cells; (D) Box plots representing DNA methylation in 4 primary melanoma and 33 metastatic melanoma samples (accession number: GSE44662). Metastatic melanomas contained lower RAB38 promoter methylation. The error bars in panels (B) and (C) represent mean ± SEM. The p values were calculated by using an unpaired two-tailed Student’s t test: “ns”, not significant; “*”, 0.01 < p < 0.05; “**”, 0.001 < p < 0.01; “***”, 0.0001 < p < 0.001; “****”, p < 0.0001.

Article Snippet: Approximately 5 × 10 3 cells collected from six melanoma cell lines: WM-115, WM-266-4, IGR39, IGR37, WM793 and 1205Lu, respectively, were lysed and treated with bisulfite using the EZ DNA Methylation-Direct Kit (Zymo Research, CA).

Techniques: Methylation Sequencing, Methylation, Expressing, DNA Methylation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity

doi: 10.1038/s41467-022-28747-1

Figure Lengend Snippet: a Repression of transcription by H-NS in E. coli . The genomic region encompassing ydbCD is shown. Data for H-NS occupancy are shown by the green graph . The total RNA abundance determined by RNA-seq in wild-type and Δ hns cells is shown by the red and blue graphs respectively. Transcription start sites (TSSs) were identified by cappable-seq for wild-type (pink graph) and Δ hns (mauve graph) cells. In the cappable-seq data only RNA 5′ ends are sequenced and so the upstream edge of each peak indicates a TSS. Sequence reads mapping to the top and bottom DNA strands are shown above and below the central horizontal line in each plot. The y -axis scales are identical for data obtained using wild-type and Δ hns cells for each type of experiment. Genes are shown by blue arrows. b Volcano plots illustrating differences in the distribution of signals obtained by RNA-seq (top panel) or cappable-seq (bottom panel) in the presence and absence of H-NS in E. coli . For the RNA-seq analysis, each data point represents the average signal across an individual gene. In the cappable-seq data plot, each data point represents a separate TSS. For both plots, data points are coloured to indicate DNA regions bound by (green) or free from (grey) H-NS. c The pie charts illustrate the distribution of TSSs obtained by cappable-seq from wild-type (left hand side) and Δ hns (right hand side) E. coli cells. The TSSs are further separated into those in H-NS bound (green) and H-NS free (grey) regions. For all pie charts, dark shading indicates TSSs in coding DNA whilst pale shading identifies TSSs in non-coding regions. d Repression of transcription by Rok in B. subtilis . The genomic region encompassing yydBCD is shown. Data for Rok occupancy are shown by the orange graph . Colour coding is otherwise as shown in ( a ) except that here the comparison is between wild-type and Δ rok B. subtilis cells. e Volcano plots illustrating differences signals obtained by RNA-seq (top panel) or cappable-seq (bottom panel) in the presence and absence of Rok in B. subtilis . Data points are as described for ( b ) and coloured to indicate DNA regions bound by (orange) or free from (grey) Rok. f Pie charts illustrate the distribution of TSSs obtained by cappable-seq from wild-type (left hand side) and Δ hns (right hand side) B. subtilis cells. The TSSs are further separated into those in Rok bound (orange) and Rok free (grey) regions. For all pie charts, dark shading indicates TSSs in coding DNA whilst pale shading identifies TSSs in non-coding regions.

Article Snippet: E. coli RNA polymerase was purchased from New England Biolabs and σ 70 was purified as described previously .

Techniques: RNA Sequencing Assay, Sequencing

Journal: Nature Communications

Article Title: Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity

doi: 10.1038/s41467-022-28747-1

Figure Lengend Snippet: a Rok represses transcription of the B. subtilis comK mRNA in vivo. Data for Rok occupancy (orange) , total RNA abundance (red and blue) and transcription start site (TSS) usage (pink and mauve) are shown. Sequence reads mapping to the top and bottom DNA strands are shown above and below the central horizontal line in each plot. The y -axis scales are identical for data obtained using wild-type and Δ rok cells for each type of experiment. Genes are shown by blue arrows. b Rok represses transcription of the B. subtilis comK mRNA in vivo. The schematic illustrates the comK gene and regulatory region, cloned in plasmid pSR, and used as a template for in vitro transcription. The comK TSS is shown as a bent black arrow, the comK gene is shown as a block blue arrow, and the sequence encoding the λ oop transcriptional terminator is indicated by a stem loop schematic. B. subtilis σ A RNA polymerase (0.5 μM) and Rok (0, 0.5, or 1.0 μM) were added as indicated. Note that the 696 nt comK mRNA is easily discernible and there is no evidence for transcription initiation within comK . Species of RNA over ~1000 nt in length are derived from sites elsewhere on the plasmid template. The RNAI transcript is encoded by the plasmid replication origin. The experiment was done twice with similar results. c H-NS represses transcription initiation within the E. coli agaB coding sequence in vivo. Data for H-NS occupancy are in green and otherwise as indicated in ( a ) except that wild-type and Δ hns E. coli cells are compared. d H-NS represses transcription initiation within the E. coli agaB coding sequence in vitro. The schematic illustrates a section of DNA cloned in plasmid pSR and used as a template for in vitro transcription. The expected size of the agaB mRNA is 684 nucleotides (nt). The gel image shows transcripts generated by E. coli σ 70 RNA polymerase (0.5 μM) using this DNA template. The expected position of agaB mRNA is indicated by an arrow head but is obscured by many similarly sized and smaller transcripts derived from agaB coding sequence. H-NS was added at concentrations of 0, 0.5, 1.0 or 2.0 μM. The experiment was done twice with similar results.

Article Snippet: E. coli RNA polymerase was purchased from New England Biolabs and σ 70 was purified as described previously .

Techniques: In Vivo, Sequencing, Clone Assay, Plasmid Preparation, In Vitro, Blocking Assay, Derivative Assay, Generated

Journal: Nature Communications

Article Title: Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity

doi: 10.1038/s41467-022-28747-1

Figure Lengend Snippet: a Positioning of promoter −10 elements and transcription start sites in E. coli and B. subtilis . The bar charts show the percentage of promoter −10 elements located at indicated distances upstream of transcription start sites (TSSs, +1) identified by cappable-seq for E. coli and B. subtilis . b The panel shows DNA sequence logos generated by aligning nucleic acid regions upstream of B. subtilis (left) or E. coli (right) transcription start sites. The more variable spacing between transcription start sites and promoter −10 elements in E. coli generates a motif that misrepresents the consensus −10 element sequence (5′-TATAAT-3′). There is no overall sequence preference for the promoter discriminator region in E. coli whilst an AT-rich sequence is common in B. subtilis . c DNA sequences of the B. subtilis veg promoter and derivatives with either an AT-rich or GC-rich discriminator sequence. d Results of in vitro transcription assays using DNA templates containing one of the promoter sequences shown in ( c ). Experiments were done with either the B. subtilis (Bs) σ A , or E. coli (Ec) σ 70 , RNA polymerase holoenzyme (0.5 μM). The 155 nucleotide (nt) transcript is generated from the cloned promoter and the 108 nt RNAI transcript is derived from the plasmid replication origin. The gel image shows a representative result and the bar chart shows a quantification of three independent experiments. Error bars show S.D. and the centre of the error bars is the mean. P was calculated using a two-tailed student’s T -test.

Article Snippet: E. coli RNA polymerase was purchased from New England Biolabs and σ 70 was purified as described previously .

Techniques: Sequencing, Generated, In Vitro, Clone Assay, Derivative Assay, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity

doi: 10.1038/s41467-022-28747-1

Figure Lengend Snippet: a The Venn diagram shows the distribution of TSSs identified in B. subtilis by cappable-seq in different genetic backgrounds. The teal area represents TSSs only detected upon expression of E. coli rpoD whilst the pink section represents TSSs only identified in the absence of rpoD expression. The overlap identifies those TSSs detected both with and without rpoD expression. b The pie charts show the distribution of B. subtilis TSSs identified in different genetic backgrounds and in different parts of the genome. The number of TSSs dependent on E. coli σ 70 expression is higher in horizontally acquired AT-rich sections of DNA targeted by Rok. Conversely, the number of σ A dependent TSSs is lower in these regions. c Examples of E. coli σ 70 dependent transcription initiation within horizontally acquired B. subtilis genes. Data from ChIP-seq experiments for Rok occupancy are shown by the orange graph. Transcription start sites (TSSs) were identified by cappable-seq for wild-type (pink graph), Δ rok (mauve graph) and B. subtilis cells carrying the σ 70 encoding rpoD gene (teal graph). Sequence reads mapping to the top and bottom DNA strands are shown above and below the central horizontal line in each plot. Genes are indicated with block blue arrows. d Results of in vitro transcription assays using AT-rich horizontally acquired genes targeted by Rok cloned in plasmid pSR as the DNA template. Transcription reactions were done using core RNA polymerase, from either B. subtilis or E. coli , in conjunction with either σ Α or σ 70 (0.5 μM final holoenzyme concentration). For each DNA template, bands corresponding to full-length mRNAs are indicated by coloured arrowheads. The experiment was done twice with similar results.

Article Snippet: E. coli RNA polymerase was purchased from New England Biolabs and σ 70 was purified as described previously .

Techniques: Expressing, ChIP-sequencing, Sequencing, Blocking Assay, In Vitro, Clone Assay, Plasmid Preparation, Concentration Assay

Journal: Nature Communications

Article Title: Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity

doi: 10.1038/s41467-022-28747-1

Figure Lengend Snippet: a Schematic representations of E. coli σ 70 and B. subtilis σ A . Individual domains are labelled σ 1 through σ 4 and the non-conserved region (NCR) specific to E. coli σ 70 is also shown. Sub-regions of each σ factor are labelled 1.1 through 4.2 and are separated by dashed lines where required. Side chains R157 and R486 important for the promiscuous behaviour of σ 70 are shown. Side chain D222 of σ A is in the position equivalent to that of R486 in σ 70 . b Location of R157 and R486 in E. coli σ 70 RNA polymerase bound to promoter DNA. The top and bottom images are derived from PDB accession numbers 6PSQ and 6CA0 respectively. Whilst present in the structures, RNA polymerase core enzyme has been hidden from view for clarity. Colour coding of σ 70 matches ( a ) and DNA is shown in orange. c The E. coli σ 70 Mut derivative has DNA opening properties similar to B. subtilis σ A . The gel image shows KMnO 4 reactivity patterns at the B. subtilis veg promoter due to DNA opening by B. subtilis (lanes 1–3) or E. coli (lanes 4–6) core RNA polymerase in complex with either σ A , σ 70 or σ 70 Mut as indicated (0.5 μM final holoenzyme concentration). The gel is calibrated with a Maxam-Gilbert G + A sequencing reaction. The experiment was done twice with similar results. d Results of in vitro transcription assays using B. subtilis (lanes 1–3) or E. coli (lanes 4–6) core RNA polymerase in complex with either σ A , σ 70 or σ 70 Mut as indicated (0.5 μM final holoenzyme concentration). The 155 nucleotide (nt) transcript is generated from the B. subtilis veg promoter and the 108 nt RNAI transcript is derived from the plasmid replication origin. The gel image shows a representative result and the bar chart shows a quantification of 3 independent experiments. Error bars show S.D. and the centre of the error bars is the mean. P was calculated using a two-tailed student’s T -test. e Results of in vitro transcription assays using AT-rich horizontally acquired genes targeted by Rok cloned in plasmid pSR as DNA templates. Transcription reactions were done using core RNA polymerase, from either B. subtilis or E. coli , in conjunction with either σ Α , σ 70 or σ 70 Mut (0.5 μM final holoenzyme concentration). For each DNA template, bands corresponding to full-length mRNAs are indicated by coloured arrowheads. The experiment was done twice with similar results.

Article Snippet: E. coli RNA polymerase was purchased from New England Biolabs and σ 70 was purified as described previously .

Techniques: Derivative Assay, Concentration Assay, Sequencing, In Vitro, Generated, Plasmid Preparation, Two Tailed Test, Clone Assay

Journal: Nature Communications

Article Title: Xenogeneic silencing strategies in bacteria are dictated by RNA polymerase promiscuity

doi: 10.1038/s41467-022-28747-1

Figure Lengend Snippet: a In E. coli , H-NS (green) binds to extensive tracts of DNA. Consequently, the comparatively promiscuous E. coli housekeeping RNA polymerase (blue ovals) is prevented from synthesising mRNAs, and many spurious intragenic RNAs, from sections of AT-rich horizontally acquired DNA (blue block arrows). H-NS may repress transcription by blocking access of RNA polymerase to the DNA and by trapping RNA polymerase at promoters (bent arrows). b In B. subtilis , Rok (orange) binds to shorter tracts of DNA around promoters (bent arrows) at gene 5′ ends. This stops the synthesis of mRNAs from sections of AT-rich horizontally acquired DNA (blue block arrows). The less promiscuous housekeeping RNA polymerase of B. subtilis containing σ A (red ovals) is not prone to spurious intragenic transcription initiation. Trapping of RNA polymerase early during elongation appears commonplace.

Article Snippet: E. coli RNA polymerase was purchased from New England Biolabs and σ 70 was purified as described previously .

Techniques: Blocking Assay

Journal: Cell reports

Article Title: DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

doi: 10.1016/j.celrep.2022.111089

Figure Lengend Snippet: Figure 1. The effect of PARP-1 on interactions of DDX helicases with R loops and the influence of DDX18 on reducing excessive R-loop accu- mulation via PARP-1 (A and B) Western blot showing the interaction of R loops and various helicases from U2OS cells treated with PARP-1 siRNA (si-PARP-1) or control siRNA (si-control; non-targeting pool); S9.6 antibody was used to pull down the R loops/protein complexes. Input shows the protein expression in cell lysates. Equal amounts of cells were used in both si-control and si-PARP-1 groups (representative of three independent experiments). (B) Western blot showing the interaction between R loops and DDX18 from U2OS cells incubated with DMSO or 20 mM of CPT for 20 min and from si-control or si-PARP-1 U2OS cells with the same treatment (representative of three independent experiments). (C) DRIP analysis from U2OS cells treated with si-control or DDX18 siRNA (si-DDX18) followed by no drug or 20 mM CPT treatment for 10 min with or without RNH. The samples were subjected to qPCR analysis against primers as indicated in the method details. The data are presented as mean ± SD (n = 3 biological in- dependent experiments). (D) Immunofluorescence of U2OS cells that were treated with si-control, RNH1 siRNA (si-RNH1), si-DDX18#1, siDDX18#2, or si-PARP-1 with S9.6 and anti- nucleolin antibodies. DAPI staining shows the nucleus. Graph shows the quantification of fluorescent signals. The nucleolar contribution to the signal is sub- tracted from the total nuclear signal. The data are shown as the mean ± SEM (n R 4 from three independent experiments). (E) Immunofluorescence of U2OS cells or DDX18 siRNA-resistant clones, which were treated with si-control or si-DDX18 with S9.6 and anti-nucleolin antibodies. DAPI staining shows the nucleus. Graph shows the quantification of fluorescent signals as previously described. The data are shown as the mean ± SEM (n R 5 from two independent experiments). p values were calculated using two-tailed unpaired Student’s t test with Welch’s correction, *p < 0.05; ****p < 0.0001; NS, not significant (p > 0.05).

Article Snippet: U2OS, U2OS DR (direct repeats)-GFP and HeLa cells were originally obtained from ATCC (http://www.atcc.org).

Techniques: Western Blot, Control, Expressing, Incubation, Staining, Clone Assay, Two Tailed Test

Journal: Cell reports

Article Title: DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

doi: 10.1016/j.celrep.2022.111089

Figure Lengend Snippet: Figure 2. Loss of DDX18 slows DNA replication (A) Cell proliferation of HeLa cells treated with si-control or si-DDX18 followed by MTT dye staining. The data are presented as mean ± SD (n = 6 biological independent experiments). (B) Measurement of DNA fibers. U2OS cells were transfected with si-control or si-DDX18 followed by incubation with CldU and IdU sequentially. The graph shows the quantifications of CldU-IdU contiguous tracts. The data are presented as mean ± SEM (n R 23 from two independent experiments). (C) EdU incorporation in U2OS cells transfected with si-control or si-DDX18. Representative images show the EdU (green) and nucleus (DAPI, blue). The relative signal intensities are presented as mean ± SEM (n = 3 independent experiments). (D) EdU incorporation in U2OS cells that were overexpressed with RNH1 followed by transfection with si-control or si-DDX18. The relative signal intensities are presented as mean ± SEM (n = 2 independent experiments). (E) CldU (green) and IdU (red) replication tracks with hydroxyurea treatment in U2OS cells transfected with si-control or si-DDX18. Representative DNA fiber images are shown to the bottom left, and CldU length is shown to the right. The data are presented as median with interquartile range (n R 32 from two in- dependent experiments). p values were calculated using two-tailed unpaired Student’s t test with Welch’s correction, *p < 0.05; ****p < 0.0001; NS, not significant (p > 0.05).

Article Snippet: U2OS, U2OS DR (direct repeats)-GFP and HeLa cells were originally obtained from ATCC (http://www.atcc.org).

Techniques: Control, Staining, Transfection, Incubation, Two Tailed Test

Journal: Cell reports

Article Title: DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

doi: 10.1016/j.celrep.2022.111089

Figure Lengend Snippet: Figure 3. DDX18 accumulation at laser-irradiated regions is PAR- and RNA dependent (A) Time-lapse microscopy at laser-irradiated regions of EGFP-DDX18-overexpressed U2OS cells treated with si-control, si-PARP-1, or PARG siRNA (si-PARG). Graph shows the quantification of normalized fluorescent intensity. The data are presented as mean ± SEM (n R 5 from three independent experiments). (B) Time-lapse microscopy at laser-irradiated regions of EGFP-DDX18-overexpressed U2OS cells treated with DMSO, olaparib (2 mM), or veliparib (5 mM) before Hoechst treatment. Graph shows the quantification of normalized fluorescent intensity. The data are presented as mean ± SD (n R 4 biological independent experiments). (C–E) Western blot showing protein expression in lysate or chromatin-bound fraction from U2OS cells treated with si-control, si-PARP-1, or si-PARG. Staining was done using antibodies against selected proteins as indicated (representative of two [lysate] and three [chromatin] independent experiments). (legend continued on next page)

Article Snippet: U2OS, U2OS DR (direct repeats)-GFP and HeLa cells were originally obtained from ATCC (http://www.atcc.org).

Techniques: Irradiation, Time-lapse Microscopy, Control, Western Blot, Expressing, Staining

Journal: Cell reports

Article Title: DDX18 prevents R-loop-induced DNA damage and genome instability via PARP-1.

doi: 10.1016/j.celrep.2022.111089

Figure Lengend Snippet: Figure 6. DDX18 knockdown leads to R-loop-induced DNA damage and genome instability (A) Graph shows the fold change of gH2AX foci of U2OS cells treated with si-control or si-DDX18. The data are presented as mean ± SEM (n = 3 from two in- dependent experiments). (B) Fold change of gH2AX foci of RNH-overexpressing U2OS cells treated with si-control or si-DDX18. The data are shown as the mean ± SEM (three independent experiments). (C) Graph shows the percentage of RPA32 foci of U2OS cells treated with si-control or si-DDX18 followed by 12.5 Gy irradiation. The data are presented as mean ± SEM (three [si-control and si-DDX18#1] and two [si-DDX18#2] independent experiments). (legend continued on next page)

Article Snippet: U2OS, U2OS DR (direct repeats)-GFP and HeLa cells were originally obtained from ATCC (http://www.atcc.org).

Techniques: Knockdown, Control, Irradiation