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

Journal: Cell reports

Article Title: Induction of broadly reactive influenza antibodies increases susceptibility to autoimmunity

doi: 10.1016/j.celrep.2022.110482

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: H4 (histone) , New England BioLabs , Cat# M2504S.

Techniques: Recombinant, Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Stripping Membranes, Microarray, Software, Imaging