Journal: Cell reports

Article Title: The PP2A regulatory subunit PPP2R2A controls NAD + biosynthesis to regulate T cell subset differentiation in systemic autoimmunity

doi: 10.1016/j.celrep.2024.114379

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit antibody against H1.2 (JU43-48) , Novus Biologicals , NBP2-75932; RRID:AB_2810088.

Techniques: Flow Cytometry, Control, Recombinant, Western Blot, Cell Isolation, Quantitation Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Software

Journal: Upsala Journal of Medical Sciences

Article Title: Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine

doi: 10.3109/03009734.2014.996720

Figure Lengend Snippet: Decrease in phosphate in phosphoramidate phosphorylated histone H1 (▪) and 30 kDa polylysine (⧫) during incubation with PHPT1. The concentration was 1 mg/mL of phosphohistone and 2 mg/mL of phosphopolylysine. An amount of 5 pmol PHPT1 was added per 51 µL incubation volume. The reaction was performed at pH 7.5 and 30°C during indicated times and was interrupted by centrifugation of 50 µL of the reaction mixture through a spin column containing 200 µL of DEAE-Sepharose equilibrated in 25 mM Tris/HCl pH 8.5. The protein-bound, acid-labile phosphate in the final eluate was analyzed as described under Methods. Each time point was analyzed in duplicate.

Article Snippet: In order to control that the phosphorylation of histone H1 really occurred on lysine residues, the calf thymus histone H1 obtained from SignalChem was phosphorylated to 1.5 mol phosphate/mol protein by phosphoramidate as described above and subjected to MS-sequencing after trypsination.

Techniques: Incubation, Concentration Assay, Centrifugation

Journal: Upsala Journal of Medical Sciences

Article Title: Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine

doi: 10.3109/03009734.2014.996720

Figure Lengend Snippet: Amino acid sequence and phosphorylated sites of bovine histone H1.2 as determined by mass spectrometry. Lysine residues are marked red, and those identified as targets for the phosphorylation by phosphoramidate (i.e. seven residues) are highlighted. Out of the 305 peptides that were used to identify the protein, 14 peptides were reported to contain phospholysine.

Article Snippet: In order to control that the phosphorylation of histone H1 really occurred on lysine residues, the calf thymus histone H1 obtained from SignalChem was phosphorylated to 1.5 mol phosphate/mol protein by phosphoramidate as described above and subjected to MS-sequencing after trypsination.

Techniques: Sequencing, Mass Spectrometry, Phospho-proteomics

Journal: Upsala Journal of Medical Sciences

Article Title: Phosphohistidine phosphatase 1 (PHPT1) also dephosphorylates phospholysine of chemically phosphorylated histone H1 and polylysine

doi: 10.3109/03009734.2014.996720

Figure Lengend Snippet: MS/MS spectrum showing the fragmentation pattern of one of the peptides obtained after trypsin treatment of histone H1 from SignalChem. The y-ion series is shown in red, and the b-ions series is in blue. Also shown is y- and b-ions fitting with the loss of 18 Da in violet and turquoise, respectively. Other observed but unspecified mached ions are grey. The small inset at the top shows only the y- and b-ions, with the length of the staples representing the intensity of the ions.

Article Snippet: In order to control that the phosphorylation of histone H1 really occurred on lysine residues, the calf thymus histone H1 obtained from SignalChem was phosphorylated to 1.5 mol phosphate/mol protein by phosphoramidate as described above and subjected to MS-sequencing after trypsination.

Techniques: Tandem Mass Spectroscopy

Journal: GigaScience

Article Title: Comparative genomic analysis of SET domain family reveals the origin, expansion, and putative function of the arthropod-specific SmydA genes as histone modifiers in insects

doi: 10.1093/gigascience/gix031

Figure Lengend Snippet: Function approval of SmydA-2 genes through experimental evidence. (A) In vitro methyltransferase assay of histone H3 of SmydA-2 in locusts. Anti-pan methyl lysine antibody recognizes histone H3 in vitro methylated with SmydA-2 . Anti-histone H3 serves as endogenous control for protein samples. The analyses were carried out in three replicates. ** P < 0.01. (B) Expression evidence of SmydA-2 in the brain and cuticle of locusts via fluorescence in situ hybridization analysis. Green signals indicate the expression of SmydA-2 /control, and blue signals indicate nuclear staining with Hoechst. (C) Relative gene expression of SmydA-2 in the different tissues. mRNA levels are quantified using the SYBR Green expression assays on a LightCycler 480 instrument. The qPCR data are shown as the mean ± SEM ( n = 6). (D) Survival analysis of the locusts after SmydA-2 double-strand RNA injection. Data are analyzed through the Kaplan–Meier survival curve comparison of the dsSmydA-2 and dsGFP groups for three replicates.

Article Snippet: For in vitro methyltransferase assay, 2 mg of unmodified histone H3 peptides (Sino Biological) were incubated with 1 mg of recombinant protein and 0.1 mM S-adenosyl-methionine (SAM, NEB) in a reaction buffer containing 50 mM Tris-HCl (pH 8.0), 10% glycerol, 20 mM KCl, 5 mM MgCl 2 , 1 mM DTT, and 1 mM PMSF at 30°C for two hours.

Techniques: In Vitro, Methylation, Expressing, Fluorescence, In Situ Hybridization, Staining, SYBR Green Assay, Injection

Journal: The Journal of Biological Chemistry

Article Title: Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells *

doi: 10.1074/jbc.M116.764233

Figure Lengend Snippet: HMGN2 promotes histone H1 loss to regulate STAT5 binding and gene transcription. A, efficiency of H1.2 knockdown. T47D cells were transiently transfected with siRNA targeting H1.2 using two different sequences (siH1.2 #1 and #2) or a nonspecific control (siCTL). Whole cell lysates were analyzed by Western blotting and probed with antibodies against H1.2 or tubulin (loading control). B, H1 knockdown rescues gene expression following HMGN2 knockdown. T47D shCTL and shHMGN2 cells were transiently transfected with the siRNA constructs in A and treated with or without PRL for 1 h. RNA was isolated, and cDNA was synthesized by RT-PCR and analyzed by qPCR. -Fold change was calculated relative to shCTL and siCTL with no PRL treatment. Results are presented as the mean ± S.E. (error bars), n ≥ 3 independent experiments. Statistical significance was determined by a two-sided ratio paired t test. C, H1 knockdown rescues STAT5 binding following HMGN2 knockdown. Cells were treated as in (B) and were analyzed by ChIP-qPCR for STAT5 at CISH following 45 min of PRL treatment. Recovered DNA was normalized to input and calculated as -fold enrichment compared with shHMGN2 and siCTL. Results are presented as the mean ± S.E. of three independent experiments. Statistical significance was determined by a two-sided t test assuming equal sample variance. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: RNA Interference Stable shRNA-mediated knockdown of HMGN2 via retroviral infection has been described previously ( 30 , 42 ), using the retroviral vector pRFP-C-RS expressing shRNA targeting HMGN2 (5′-GTGTCAGGCAATCTGGACTTTCCAGTGAT-3′; catalogue no. TF319505, Origene, Rockville, MD). siRNA targeting H1.2 (catalogue no. SR302039B (siH1.2-1) and SR302039A (siH1.2-2)) and non-targeting control (catalogue no. SR30004 (siCTL)) were purchased from Origene.

Techniques: Binding Assay, Transfection, Western Blot, Expressing, Construct, Isolation, Synthesized, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells *

doi: 10.1074/jbc.M116.764233

Figure Lengend Snippet: H1 knockdown enhances gene expression and breast cancer cell proliferation in response to reduced STAT5 activation. A, H1 knockdown rescues gene expression following partial STAT5 inhibition. T47D cells were transfected with siCTL, siH1-1, or siH1-2 (Fig. 8A). Transfectants were pretreated with the STAT5 inhibitor (200 μm) or DMSO VC for 1 h, followed by PRL treatment for 2 h. RNA was isolated, and cDNA was synthesized by RT-PCR and analyzed by qPCR. -Fold change was calculated relative to VC, siCTL with no PRL treatment. Results are presented as the mean ± S.E. (error bars), n ≥ 3 independent experiments. Statistical significance was determined by a two-sided ratio paired t test. B, H1 knockdown rescues cell proliferation in response to intermediate levels of STAT5 inhibition. T47D cells were transfected with siCTL or siH1 (pooled 1 and 2). Transfectants were treated with the indicated concentrations of STAT5 inhibitor, with or without PRL, for 3 days. BrdU incorporation was measured by absorbance as an indication of cell proliferation. Results are presented as the mean ± S.E. of three independent experiments. Within each individual experiment, each set of treatment conditions was carried out in triplicate. Statistical significance was determined by two-sided t test assuming equal sample variance. Statistical significance shown in the figure is comparing siCTL + PRL versus siH1 + PRL at the indicated concentration of STAT5 inhibitor. Other statistically significant comparisons are as follows: siCTL No PRL versus siH1 No PRL (p ≤ 0.01 at 0 inhibitor; p ≤ 0.05 at 25 μm); siH1 No PRL versus siH1 + PRL (p ≤ 0.05 at 25 μm; p = 0.052 at 50 μm). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: RNA Interference Stable shRNA-mediated knockdown of HMGN2 via retroviral infection has been described previously ( 30 , 42 ), using the retroviral vector pRFP-C-RS expressing shRNA targeting HMGN2 (5′-GTGTCAGGCAATCTGGACTTTCCAGTGAT-3′; catalogue no. TF319505, Origene, Rockville, MD). siRNA targeting H1.2 (catalogue no. SR302039B (siH1.2-1) and SR302039A (siH1.2-2)) and non-targeting control (catalogue no. SR30004 (siCTL)) were purchased from Origene.

Techniques: Expressing, Activation Assay, Inhibition, Transfection, Isolation, Synthesized, Reverse Transcription Polymerase Chain Reaction, BrdU Incorporation Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells *

doi: 10.1074/jbc.M116.764233

Figure Lengend Snippet: Genes regulated by H1 are enriched for STAT signaling pathways. A and B, genes found to be up-regulated by H1.2 knockdown in MCF7 cells by Kim et al. (40) were analyzed by the Enrichr enrichment analysis tool (50, 51). Significantly enriched gene sets of interest are plotted with their adjusted p values (q values), which were calculated using the Benjamini-Hochberg method for correction for multiple-hypothesis testing. KEGG pathways are shown in A along with the associated KEGG identifiers. ChIP enrichment analysis through ChEA 2015 is shown in B, with numbers indicating the publication PMID for each study.

Article Snippet: RNA Interference Stable shRNA-mediated knockdown of HMGN2 via retroviral infection has been described previously ( 30 , 42 ), using the retroviral vector pRFP-C-RS expressing shRNA targeting HMGN2 (5′-GTGTCAGGCAATCTGGACTTTCCAGTGAT-3′; catalogue no. TF319505, Origene, Rockville, MD). siRNA targeting H1.2 (catalogue no. SR302039B (siH1.2-1) and SR302039A (siH1.2-2)) and non-targeting control (catalogue no. SR30004 (siCTL)) were purchased from Origene.

Techniques: