Journal: BMC Molecular Biology

Article Title: Core histone genes of Giardia intestinalis : genomic organization, promoter structure, and expression

doi: 10.1186/1471-2199-8-26

Figure Lengend Snippet: Determination of the transcription start sites for the G. intestinalis core histone genes . A , 5'RACE analysis of the four core histone genes. The numbers at the top indicate the nucleotide positions in the DNA sequences relative to the translation start codon ( overlined ) for each gene. The him sequences are indicated in the grey boxes; the AT-rich sequences are indicated in the open boxes ; the g-CAB elements are underlined , and the bent arrows indicate the start of transcription for each gene. B , Primer extension analysis of the H4 gene. The extension products were generated from 5'end-labeled H4/PE-1b oligonucleotide and total Giardia RNA, and the DNA sequence ladder was generated using the same end-labeled oligonucleotide as a primer. The positions of the major and minor extension products are indicated by arrows in the (+)DNA strand shown on the left.

Article Snippet: An oligonucleotide, H4/PE-1b (5' GAT GGC GGG CTT CGT GAT GCC 3') with a sequence complementary to codons 25 – 31 of the G. intestinalis histone H4 gene was 5' end-radiolabeled with 33 P using T4 polynucleotide kinase (New England Biolabs) and annealed to 10 μg of Giardia total RNA.

Techniques: Generated, Labeling, Sequencing

Journal: BMC Molecular Biology

Article Title: Core histone genes of Giardia intestinalis : genomic organization, promoter structure, and expression

doi: 10.1186/1471-2199-8-26

Figure Lengend Snippet: Promoter analysis of the G. Intestinalis core histone genes . Luciferase activities were determined from Giardia transfections with a dual-luciferase reporter system. Each Giardia sample was co-transfected with an experimental plasmid and the control plasmid, and assayed sequentially for firefly and Renilla luciferase activities. The firefly luciferase activity was divided by the Renilla luciferase activity to obtain the F/R-LUC ratio. Percentages of relative luciferase activity were calculated by comparing the F/R-LUC ratio obtained from Giardia transfected with each construct relative to the ratio obtained upon transfection of the control plasmid in each experiment. A , Identification of the histone H4 promoter. Experimental plasmids contained incremental deletions of the upstream region of the H4 gene to drive the expression of the firefly luciferase (F-LUC) gene. The composition of the experimental constructs are represented by: white bar , 5' noncoding region of the H4 gene; grey bar , him sequence; black bar , AT-rich sequence; open box , firefly luciferase coding region. The numbers proximal to the white bars indicate the length of 5' noncoding region of the H4 gene remaining within each plasmid. B , Mutational analysis of the histone H4 promoter. Experimental plasmids contained mutations within the 50 bp promoter region of the histone H4 gene to drive the expression of the firefly luciferase (F-LUC) gene. In the wild-type H4 promoter sequence presented on the top line, the him is indicated by the grey box ; the AT-rich sequence is indicated by the open box ; the g-CAB elements are underlined; and the transcriptional start site is indicated by the bent arrow . C , Comparison of the four core histone promoters. The minimal 5' noncoding sequence of each core histone gene that contain the him sequence was used to drive the expression of the firefly luciferase gene in the experimental plasmid constructs. The him sequences are indicated by grey boxes ; the AT-rich sequences are indicated by open boxes; and the g-CAB elements are underlined .

Article Snippet: An oligonucleotide, H4/PE-1b (5' GAT GGC GGG CTT CGT GAT GCC 3') with a sequence complementary to codons 25 – 31 of the G. intestinalis histone H4 gene was 5' end-radiolabeled with 33 P using T4 polynucleotide kinase (New England Biolabs) and annealed to 10 μg of Giardia total RNA.

Techniques: Luciferase, Transfection, Plasmid Preparation, Activity Assay, Construct, Expressing, Sequencing

Journal: BMC Molecular Biology

Article Title: Core histone genes of Giardia intestinalis : genomic organization, promoter structure, and expression

doi: 10.1186/1471-2199-8-26

Figure Lengend Snippet: Relative expression of Giardia intestinalis core histone genes

Article Snippet: An oligonucleotide, H4/PE-1b (5' GAT GGC GGG CTT CGT GAT GCC 3') with a sequence complementary to codons 25 – 31 of the G. intestinalis histone H4 gene was 5' end-radiolabeled with 33 P using T4 polynucleotide kinase (New England Biolabs) and annealed to 10 μg of Giardia total RNA.

Techniques: Expressing

Journal: BMC Research Notes

Article Title: Tousled kinase TLK1B counteracts the effect of Asf1 in inhibition of histone H3–H4 tetramer formation

doi: 10.1186/1756-0500-2-128

Figure Lengend Snippet: A. Crosslinking of H3–H4 . Histones H3 and H4 were incubated for the indicated minutes and then crosslinked with formaldehyde before separation on a 15% SDS/PAGE. The blot was probed separately with anti-H3 and anti-H4. B. Effect of Asf1 and TLK1B on H3–H4 dimer and tetramer formation. Reactions containing the indicated combinations of H3, H4, Asf1, and TLK1B (all in equivalent amount) were crosslinked as described in Methods and immunoblotted for H4 or H3. C. Western blots for Asf1 and TLK1B. The indicated reactions as in panel B were run in duplicate lanes and immunoblotted for Asf1, H3, or TLK1B (the gel was run for a longer time than in panel B to separate the larger proteins). For antibody controls, lane 1 contained only Asf1, and lane 7 contained only TLK1B. The positions of the cross-linked complexes identified by mobility and immunoreactivity are indicated. D. TLK1B and Asf1 bind each other stoichiometrically. Reactions contained 5 μg of GST-TLK1B and varying amounts of Asf1, as indicated. After 10 min at room temperature, the samples were adsorbed on GSH-Sepharose and analyzed for bound and unbound fractions after separation on a 10% SDS/PAGE, which was stained with Coomassie blue. E. Interaction of TLK1B and Asf1 and the effect of ATP. GST-TLK1B and Asf1 (1 μg each) were incubated for 10 min at room temperature with and without 10 or 100 μM ATP, before analysis by GSH-Sepharose pull-down. The 10% SDS/PAGE gel was stained with Coomassie blue. F. MNase digestion of pBluescript assembled into pseudo-nucleosomes. In the left panel, naked supercoiled plasmid was digested with MNase for the indicated time. In the right panel, the plasmid was first incubated with equimolar H3 and H4 in high salt and then step-dialyzed as described in , before MNase digestion. The DNA was re-extracted from the reactions with Geneclean and run on a 1.5% agarose/TAE gel. The resulting ~120 bp ladder (a bit shorter than the repetitive 146 bp of nucleosomal DNA) is indicative of formation of a chromatinized template. The positions of the bands of a 100-bp ladder (GenRuler, Fermentas) are indicated.

Article Snippet: Recombinant human histone H4 was purchased from NE Biolabs and bovine H3 from Roche.

Techniques: Incubation, SDS Page, Western Blot, Staining, Plasmid Preparation

Journal: Cell reports

Article Title: Analysis of estrogen-regulated enhancer RNAs identifies a functional motif required for enhancer assembly and gene expression

doi: 10.1016/j.celrep.2022.110944

Figure Lengend Snippet: (A) Schematics of the pipeline for the discovery of FERM element within E2-regulated eRNAs using multiple em for motif elicitation (MEME) and find individual motif occurrences (FIMO) software. (B) Graphical representation of the position of the FERM element (yellow) in the 254 E2-regulated eRNAs less than 3 kb that contain at least one FERM (273 eRNAs contain at least one FERM). Each eRNA is colored blue. The sequence for the FERM element is provided at the bottom. (C) Predicted secondary structures of the PRRX2 eRNA (left) using RNAfold and the UBE2E2 eRNA (right) using Scanfold. The location of the FERM within the eRNA is marked with a red box. (D) Targeting the FERM element to the PRRX2 enhancer is sufficient to modulate target gene expression. RT-qPCR analysis of PRRX2 mRNA expression levels normalized to GAPDH mRNA levels in dCas9-expressing MCF-7 cells with the PRRX2 sgRNA fused to the FERM element or the cognate PRRX2 eRNA. Each point represents the mean ± standard error of the mean (n = 4 biological/technical replicates). The asterisks indicate significant differences from the sgRNA control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01, ****p < 0.0001). (E) ChIP-qPCR assays for ERα (left) and H3K27ac (right) at the PRRX2 enhancer with dCas9-expressing MCF-7 cells and various sgRNA/eRNA constructs. The cells were stimulated with E2 for the indicated time. Each point represents the mean ± standard error of the mean (n = 3 biological/technical replicates). The asterisks indicate significant differences from the corresponding control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01). (F) Targeting the FERM element to the UBE2E2 enhancer is sufficient to modulate target gene expression. RT-qPCR analysis of the UBE2E2 mRNA expression level normalized to GAPDH mRNA levels in dCas9-expressing MCF-7 cells with UBE2E2 sgRNA fused to the FERM element or the cognate UBE2E2 eRNA. Each point represents the mean ± standard error of the mean (n = 4 biological/technical replicates). The asterisks indicate significant differences from the sgRNA control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01, ****p < 0.0001). (G) ChIP-qPCR assays for ERα (left) and H3K27ac (right) at the UBE2E2 enhancer with dCas9-expressing MCF-7 cells and various sgRNA/eRNA constructs. The cells were stimulated with E2 for the indicated time. Each point represents the mean ± standard error of the mean (n = 3 biological/technical replicates). The asterisks indicate significant differences from the corresponding control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01). (H) Mutation of the PRRX2 eRNA FERM abolishes the stimulatory effect of eRNA on target gene expression. RT-qPCR analysis of the PRRX2 mRNA expression level normalized to GAPDH mRNA levels in dCas9-expressing MCF-7 cells with PRRX2 sgRNA fused to wild-type, mutant, or antisense wild-type PRRX2 eRNA. The cells were stimulated with E2 for the indicated time. Each point represents the mean ± standard error of the mean (n = 3 biological/technical replicates). The asterisks indicate significant differences from the corresponding control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01). (I) Boxplot analysis of ERα (left), and H3K27ac ChIP-seq (right) of ERBS containing eRNAs with or without FERM (±4 kb). Reads were normalized to the read depth of the libraries used. The boxes represent the 25th–75th percentile (band at median), with whiskers at 1.5 × interquartile range. Bars marked with different letters are significantly different—n (without FERM) = 940, n (with FERM) = 339 (one-way ANOVA, Fisher’s least significant difference post hoc test, p < 0.05). (J and K) In vitro histone acetyltransferase (HAT) assay using recombinant histone H3.1/H4 tetramer, purified p300, and (J) chemically synthesized FERM or (K) in vitro transcribed wild-type or mutant PRRX2 eRNA. The level of H3K27ac normalized to H3 was detected by Western blot. Each experiment was performed three independent times. See also .

Article Snippet: Human H3.1/H4 tetramer , NEB , Cat. No.: M2509.

Techniques: Software, Sequencing, Expressing, Quantitative RT-PCR, Construct, Mutagenesis, ChIP-sequencing, In Vitro, HAT Assay, Recombinant, Purification, Synthesized, Western Blot

Journal: Cell reports

Article Title: Analysis of estrogen-regulated enhancer RNAs identifies a functional motif required for enhancer assembly and gene expression

doi: 10.1016/j.celrep.2022.110944

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human H3.1/H4 tetramer , NEB , Cat. No.: M2509.

Techniques: Recombinant, Molecular Cloning, Software

Journal: Cell reports

Article Title: Analysis of estrogen-regulated enhancer RNAs identifies a functional motif required for enhancer assembly and gene expression

doi: 10.1016/j.celrep.2022.110944

Figure Lengend Snippet: (A) Schematics of the pipeline for the discovery of FERM element within E2-regulated eRNAs using multiple em for motif elicitation (MEME) and find individual motif occurrences (FIMO) software. (B) Graphical representation of the position of the FERM element (yellow) in the 254 E2-regulated eRNAs less than 3 kb that contain at least one FERM (273 eRNAs contain at least one FERM). Each eRNA is colored blue. The sequence for the FERM element is provided at the bottom. (C) Predicted secondary structures of the PRRX2 eRNA (left) using RNAfold and the UBE2E2 eRNA (right) using Scanfold. The location of the FERM within the eRNA is marked with a red box. (D) Targeting the FERM element to the PRRX2 enhancer is sufficient to modulate target gene expression. RT-qPCR analysis of PRRX2 mRNA expression levels normalized to GAPDH mRNA levels in dCas9-expressing MCF-7 cells with the PRRX2 sgRNA fused to the FERM element or the cognate PRRX2 eRNA. Each point represents the mean ± standard error of the mean (n = 4 biological/technical replicates). The asterisks indicate significant differences from the sgRNA control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01, ****p < 0.0001). (E) ChIP-qPCR assays for ERα (left) and H3K27ac (right) at the PRRX2 enhancer with dCas9-expressing MCF-7 cells and various sgRNA/eRNA constructs. The cells were stimulated with E2 for the indicated time. Each point represents the mean ± standard error of the mean (n = 3 biological/technical replicates). The asterisks indicate significant differences from the corresponding control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01). (F) Targeting the FERM element to the UBE2E2 enhancer is sufficient to modulate target gene expression. RT-qPCR analysis of the UBE2E2 mRNA expression level normalized to GAPDH mRNA levels in dCas9-expressing MCF-7 cells with UBE2E2 sgRNA fused to the FERM element or the cognate UBE2E2 eRNA. Each point represents the mean ± standard error of the mean (n = 4 biological/technical replicates). The asterisks indicate significant differences from the sgRNA control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01, ****p < 0.0001). (G) ChIP-qPCR assays for ERα (left) and H3K27ac (right) at the UBE2E2 enhancer with dCas9-expressing MCF-7 cells and various sgRNA/eRNA constructs. The cells were stimulated with E2 for the indicated time. Each point represents the mean ± standard error of the mean (n = 3 biological/technical replicates). The asterisks indicate significant differences from the corresponding control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01). (H) Mutation of the PRRX2 eRNA FERM abolishes the stimulatory effect of eRNA on target gene expression. RT-qPCR analysis of the PRRX2 mRNA expression level normalized to GAPDH mRNA levels in dCas9-expressing MCF-7 cells with PRRX2 sgRNA fused to wild-type, mutant, or antisense wild-type PRRX2 eRNA. The cells were stimulated with E2 for the indicated time. Each point represents the mean ± standard error of the mean (n = 3 biological/technical replicates). The asterisks indicate significant differences from the corresponding control (two-way ANOVA, Fisher’s least significant difference post hoc test, *p < 0.05, **p < 0.01). (I) Boxplot analysis of ERα (left), and H3K27ac ChIP-seq (right) of ERBS containing eRNAs with or without FERM (±4 kb). Reads were normalized to the read depth of the libraries used. The boxes represent the 25th–75th percentile (band at median), with whiskers at 1.5 × interquartile range. Bars marked with different letters are significantly different—n (without FERM) = 940, n (with FERM) = 339 (one-way ANOVA, Fisher’s least significant difference post hoc test, p < 0.05). (J and K) In vitro histone acetyltransferase (HAT) assay using recombinant histone H3.1/H4 tetramer, purified p300, and (J) chemically synthesized FERM or (K) in vitro transcribed wild-type or mutant PRRX2 eRNA. The level of H3K27ac normalized to H3 was detected by Western blot. Each experiment was performed three independent times. See also .

Article Snippet: The HAT reaction was assembled with the following components: 200 nM (~53 ng) human recombinant histone H3.1/H4 tetramer (NEB, M2509), 15 ng purified human full-length p300 , 15 μM acetyl-CoA, 0.1 nM folded RNA, 5% glycerol, 1 mM DTT, 1× complete protease inhibitor cocktail, 0.02 U/μL SUPERase inhibitor, and HAT Buffer (500 mM Tris-HCl pH 7.5, 10 mM MgCl 2 ).

Techniques: Software, Sequencing, Expressing, Quantitative RT-PCR, Construct, Mutagenesis, ChIP-sequencing, In Vitro, HAT Assay, Recombinant, Purification, Synthesized, Western Blot

Journal: Cell reports

Article Title: Analysis of estrogen-regulated enhancer RNAs identifies a functional motif required for enhancer assembly and gene expression

doi: 10.1016/j.celrep.2022.110944

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The HAT reaction was assembled with the following components: 200 nM (~53 ng) human recombinant histone H3.1/H4 tetramer (NEB, M2509), 15 ng purified human full-length p300 , 15 μM acetyl-CoA, 0.1 nM folded RNA, 5% glycerol, 1 mM DTT, 1× complete protease inhibitor cocktail, 0.02 U/μL SUPERase inhibitor, and HAT Buffer (500 mM Tris-HCl pH 7.5, 10 mM MgCl 2 ).

Techniques: Recombinant, Molecular Cloning, Software