ubn1 antibody  (Proteintech)

Journal: Stem Cell Research & Therapy

Article Title: Histone chaperone HIRA complex regulates retrotransposons in embryonic stem cells

doi: 10.1186/s13287-022-02814-2

Figure Lengend Snippet: Depletion of HIRA complex members in ESCs. a qPCR analysis of the expression of Hira , Ubn2/1 , Asf1a, and Cabin1 after transfected with control shRNA and shRNAs against Hira , Ubn2/1 , Asf1a, and Cabin1 . The results were normalized to Gapdh . Data are represented as mean ± s.e.m. ( n = 3 independent experiments). *** p < 0.001 in Student’s t test. b Western blot analysis of the indicated proteins in HIRA members and Asf1a -depleted ESCs. Actin was included as a loading control. c – g qPCR analysis of the expression level of pluripotency genes ( Oct4 , Sox2, and Nanog ) in HIRA members Hira ( c ), Ubn2 ( d ), Ubn1 ( e ), Asf1a ( f ), and Cabin1 ( g )-depleted ESCs. The results were normalized to Gapdh . Data are represented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, * p < 0.05 in Student’s t test. h Western blot analysis of the expression level of pluripotency genes ( Oct4 , Sox2, and Nanog ) in HIRA members and Asf1a -depleted ESCs. Actin was included as a loading control

Article Snippet: Antibodies used for western blot were as follows: anti-Flag (1:5000, F1804, Sigma), anti-HA (1:5000, 30701ES60, Yeasen), anti-H3 (1:5000, 17168-1-AP, Proteintech), anti- β -Tubulin (1:5000, KM9003T, Sungene Biotech), anti- β -Actin (1:100,000, AC026, ABclonal), anti-H3K9me2 (1:5000, ab176882, Abcam), anti-H3k9me3 (1:100,000, ab176916, Abcam), anti-Hira (1:1000, A8461, ABclonal), anti-Ubn2 (1:1000, A10516, ABclonal), anti-Asf1a (1:1000, A6528, ABclonal), anti-Ubn1 (1:1000, sc-515340, Santa Cruz), anti-Cabin1 (1:1000, sc-514269, Santa Cruz), anti-Oct4 (1:2000, sc-5279, Santa Cruz), anti-Sox2 (1:2000, sc-365964, Santa Cruz), anti-Nanog (1:2000, sc-293121, Santa Cruz).

Techniques: Expressing, Transfection, shRNA, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Histone chaperone HIRA complex regulates retrotransposons in embryonic stem cells

doi: 10.1186/s13287-022-02814-2

Figure Lengend Snippet: HIRA complex members regulate TE transcription. a – e qPCR analysis of different subfamilies endogenous retroviruses in Hira ( a ), Ubn2 ( b ), Ubn1 ( c ), Asf1a ( d ), and Cabin1 ( e )-depleted ESCs. The results in ( a ) to ( e ) were normalized to Gapdh . Data are represented as mean ± s.e.m. ( n = 3 independent experiments) for the above qPCR results. ns: non-significant, * p < 0.05; ** p < 0.01; *** p < 0.001 in Student’s t test

Article Snippet: Antibodies used for western blot were as follows: anti-Flag (1:5000, F1804, Sigma), anti-HA (1:5000, 30701ES60, Yeasen), anti-H3 (1:5000, 17168-1-AP, Proteintech), anti- β -Tubulin (1:5000, KM9003T, Sungene Biotech), anti- β -Actin (1:100,000, AC026, ABclonal), anti-H3K9me2 (1:5000, ab176882, Abcam), anti-H3k9me3 (1:100,000, ab176916, Abcam), anti-Hira (1:1000, A8461, ABclonal), anti-Ubn2 (1:1000, A10516, ABclonal), anti-Asf1a (1:1000, A6528, ABclonal), anti-Ubn1 (1:1000, sc-515340, Santa Cruz), anti-Cabin1 (1:1000, sc-514269, Santa Cruz), anti-Oct4 (1:2000, sc-5279, Santa Cruz), anti-Sox2 (1:2000, sc-365964, Santa Cruz), anti-Nanog (1:2000, sc-293121, Santa Cruz).

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Histone chaperone HIRA complex regulates retrotransposons in embryonic stem cells

doi: 10.1186/s13287-022-02814-2

Figure Lengend Snippet: Overexpression of Hira or Ubn2 rescues the expression of retrotransposons. a The expression levels of Hira after transfected with Hira shRNA in control OE ESCs and Hira OE ESCs, as measured by RT-qPCR and normalized to Gapdh level; *** p < 0.001 in Student’s t test. b The expression levels of Ubn2 after transfected with Ubn2 shRNA in control OE ESCs and Ubn2 OE ESCs, as measured by RT-qPCR and normalized to Gapdh level; *** p < 0.001 in Student’s t test. c The expression levels of Ubn1 after transfected with Ubn1 shRNA in control OE ESCs and Ubn1 OE ESCs, as measured by RT-qPCR and normalized to Gapdh level; *** p < 0.001 in Student’s t test. d – f Immunoblot analysis of the expression of Hira ( d ), Ubn2 ( e ), or Ubn1 ( f ) after overexpression of Hira, Ubn2, or Ubn1. Actin was included as a loading control. g – i qPCR analysis of ERVs expression in Hira ( g ), Ubn2 ( h ) and Ubn1 ( i ) rescue ESCs lines. The results were normalized to Gapdh . Data are represented as mean ± s.e.m. ( n = 3 independent experiments) for the above qPCR results. ns: non-significant, ** p < 0.01; *** p < 0.001 in Student’s t test

Article Snippet: Antibodies used for western blot were as follows: anti-Flag (1:5000, F1804, Sigma), anti-HA (1:5000, 30701ES60, Yeasen), anti-H3 (1:5000, 17168-1-AP, Proteintech), anti- β -Tubulin (1:5000, KM9003T, Sungene Biotech), anti- β -Actin (1:100,000, AC026, ABclonal), anti-H3K9me2 (1:5000, ab176882, Abcam), anti-H3k9me3 (1:100,000, ab176916, Abcam), anti-Hira (1:1000, A8461, ABclonal), anti-Ubn2 (1:1000, A10516, ABclonal), anti-Asf1a (1:1000, A6528, ABclonal), anti-Ubn1 (1:1000, sc-515340, Santa Cruz), anti-Cabin1 (1:1000, sc-514269, Santa Cruz), anti-Oct4 (1:2000, sc-5279, Santa Cruz), anti-Sox2 (1:2000, sc-365964, Santa Cruz), anti-Nanog (1:2000, sc-293121, Santa Cruz).

Techniques: Over Expression, Expressing, Transfection, shRNA, Quantitative RT-PCR, Western Blot

Journal: Stem Cell Research & Therapy

Article Title: Histone chaperone HIRA complex regulates retrotransposons in embryonic stem cells

doi: 10.1186/s13287-022-02814-2

Figure Lengend Snippet: Hira and Ubn2 cooperatively regulate downstream genes in ESCs. a , b The volcano plot of gene expression in Hira ( a ) or Ubn2 ( b )-depleted ESCs versus control ESCs. Significantly upregulated genes were labeled in red and significantly downregulated genes were labeled in blue. Horizontal blue dash line marked adjusted P value (Wald test) 0.05 and vertical lines marked expression fold change 1.5. Triangles of ( a ) represent TEs with -log10 (adjusted P value) > 50. Triangles of ( b ) represent TEs with -log10 (adjusted P value) > 70. c , d Venn diagrams illustrated the numbers of upregulated ( c ) and downregulated ( d ) differentially expressed genes either shared or unique at all in Hira , Ubn1, and Ubn2 -depleted ESCs. e , f Gene ontology analysis of biological processes related to co-upregulated genes ( e ) and co-downregulated genes ( f ) in Hira or Ubn2 -depleted ESCs versus control ESCs. GO analysis was done with DAVID. g , h Gene ontology analysis of uniquely upregulated genes after the depletion of Hira ( g ) or Ubn2 ( h ). i , j Gene ontology analysis of uniquely downregulated genes after the depletion of Hira ( i ) or Ubn2 ( j )

Article Snippet: Antibodies used for western blot were as follows: anti-Flag (1:5000, F1804, Sigma), anti-HA (1:5000, 30701ES60, Yeasen), anti-H3 (1:5000, 17168-1-AP, Proteintech), anti- β -Tubulin (1:5000, KM9003T, Sungene Biotech), anti- β -Actin (1:100,000, AC026, ABclonal), anti-H3K9me2 (1:5000, ab176882, Abcam), anti-H3k9me3 (1:100,000, ab176916, Abcam), anti-Hira (1:1000, A8461, ABclonal), anti-Ubn2 (1:1000, A10516, ABclonal), anti-Asf1a (1:1000, A6528, ABclonal), anti-Ubn1 (1:1000, sc-515340, Santa Cruz), anti-Cabin1 (1:1000, sc-514269, Santa Cruz), anti-Oct4 (1:2000, sc-5279, Santa Cruz), anti-Sox2 (1:2000, sc-365964, Santa Cruz), anti-Nanog (1:2000, sc-293121, Santa Cruz).

Techniques: Expressing, Labeling

Journal: Stem Cell Research & Therapy

Article Title: Histone chaperone HIRA complex regulates retrotransposons in embryonic stem cells

doi: 10.1186/s13287-022-02814-2

Figure Lengend Snippet: HIRA complex members regulate TE transcription genome-widely. a – c Scatter diagrams show transcriptome analysis of TE expression change after the depletion of Hira ( a ) or Ubn2 ( b ) or Ubn1 ( c ) by shRNAs. TE transcript results were used to plot these diagrams. Colored dots indicate retroelements with significant expression change (Wald test, FDR adjusted P value < 0.05). d Heatmap of RNA-Seq expression Z-scores computed for genes that are differentially expressed in Hira , Ubn1, and Ubn2 depletion by shRNAs in ESCs versus control ESCs. Upregulated and downregulated genes are represented with red and blue colors respectively. Each column corresponds to a sample and each row corresponds to a specific gene

Article Snippet: Antibodies used for western blot were as follows: anti-Flag (1:5000, F1804, Sigma), anti-HA (1:5000, 30701ES60, Yeasen), anti-H3 (1:5000, 17168-1-AP, Proteintech), anti- β -Tubulin (1:5000, KM9003T, Sungene Biotech), anti- β -Actin (1:100,000, AC026, ABclonal), anti-H3K9me2 (1:5000, ab176882, Abcam), anti-H3k9me3 (1:100,000, ab176916, Abcam), anti-Hira (1:1000, A8461, ABclonal), anti-Ubn2 (1:1000, A10516, ABclonal), anti-Asf1a (1:1000, A6528, ABclonal), anti-Ubn1 (1:1000, sc-515340, Santa Cruz), anti-Cabin1 (1:1000, sc-514269, Santa Cruz), anti-Oct4 (1:2000, sc-5279, Santa Cruz), anti-Sox2 (1:2000, sc-365964, Santa Cruz), anti-Nanog (1:2000, sc-293121, Santa Cruz).

Techniques: Expressing, RNA Sequencing Assay

Journal: Stem Cell Research & Therapy

Article Title: Histone chaperone HIRA complex regulates retrotransposons in embryonic stem cells

doi: 10.1186/s13287-022-02814-2

Figure Lengend Snippet: The reduction of H3K9me2/3 after Hira or Ubn2 depletion. a Heatmap of RNA-Seq expression Z-scores computed for genes that are differentially expressed between WT ESCs and H3.3 knockout ESCs. The left side shows the genes regulated by Hira and Ubn2 respectively. The upregulated and downregulated genes are represented with red and blue colors, respectively. Each column corresponds to a sample and each row corresponds to a specific gene. b ChIP-seq enrichment of H3.3 around the center of IAPLTR2b or RLTR10D locus in WT ESCs (blue) and Hira −/− ESCs (green). The ChIP-seq signal was calculated as the log2 ratio of the normalized number of reads relative to the input. The published ChIP-seq data is from GEO: GSE117034. c ChIP-seq enrichment of H3.3 around the center of MMERGLN_LTR or MT2 locus in WT ESCs (blue) and Ubn2 −/− ESCs (red). The ChIP-seq signal was calculated as the log2 ratio of the normalized number of reads relative to the input. The data of ChIP-seq are available at GEO: GSE117034. d Western blot analysis of H3, β -Tubulin, H3K9me2 and H3K9me3 protein levels in ESCs transfected with shRNAs against target genes ( Hira , Ubn2 , and Ubn1 ) or control shRNA respectively. β -Tubulin was included as a loading control. e The relative protein level of H3K9me2 and H3K9me3 was obtained according to western blot band in ( d ). Gray scanning analysis was normalized to that of β -Tubulin. Data are represented as mean ± s.e.m. ( n = 3 independent experiments). ns: non-significant, * p < 0.05; *** p < 0.001 in Student’s t test. f Schematic of Hira and Ubn2 function in the repression of retrotransposons. In WT ESCs, Hira and Ubn2 mediate the placement of H3.3 and H3K9 methylation, and thus repress the expression of retrotransposons. In the absence of Ubn2 or Hira , retrotransposon-associated H3.3 and H3K9me2/3 reduce, and thus activate the expression of retrotransposons

Article Snippet: Antibodies used for western blot were as follows: anti-Flag (1:5000, F1804, Sigma), anti-HA (1:5000, 30701ES60, Yeasen), anti-H3 (1:5000, 17168-1-AP, Proteintech), anti- β -Tubulin (1:5000, KM9003T, Sungene Biotech), anti- β -Actin (1:100,000, AC026, ABclonal), anti-H3K9me2 (1:5000, ab176882, Abcam), anti-H3k9me3 (1:100,000, ab176916, Abcam), anti-Hira (1:1000, A8461, ABclonal), anti-Ubn2 (1:1000, A10516, ABclonal), anti-Asf1a (1:1000, A6528, ABclonal), anti-Ubn1 (1:1000, sc-515340, Santa Cruz), anti-Cabin1 (1:1000, sc-514269, Santa Cruz), anti-Oct4 (1:2000, sc-5279, Santa Cruz), anti-Sox2 (1:2000, sc-365964, Santa Cruz), anti-Nanog (1:2000, sc-293121, Santa Cruz).

Techniques: RNA Sequencing Assay, Expressing, Knock-Out, ChIP-sequencing, Western Blot, Transfection, shRNA, Methylation

Journal: Nucleic Acids Research

Article Title: HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs

doi: 10.1093/nar/gkab1221

Figure Lengend Snippet: Defect of HIRA complex results in H2A.Z decrease on genome wide. ( A ) Schematic diagram of genome editing in defective cell lines. Hira-, Ubn2- and H3.3-knockout cell lines and Ubn1/2 double mutants were generated using CRISPR-Cas9 system. For subsequent experiments, 6 × HA tag was added to the N-terminus of Srcap in R1 wild type and above defective cell lines. ( B ) Western blot respectively shows the proteins levels of Hira (a), Ubn1&Ubn2 (b), and H3.3 (c) in the defective cell lines. ( C ) Meta-analysis (left) and heatmap (right) show H2A.Z reads density around summit (upper) and TSS (below) in R1 WT , Hira-KO , Ubn2-KO/Ubn1-KD , Ubn1/2-DM and H3.3-KO cells within a ±3 kb window. ( D ) Venn diagram shows the overlap of H2A.Z down-regulated peaks between the above defective cell lines. ( E ) The reduced H2A.Z peaks in either Hira-KO or Ubn2-KO/Ubn1-KD cell lines are defined as HIRA complex regulated (HR) peaks (23 530 peaks). The 5426 HR peaks overlapping with Ubn1/2-DM cell line is defined as HIRA-regulated and H3.3-dependent (HIRA-H3.3D). The other 18 104 HR peaks are HIRA-regulated and H3.3-independent (HIRA-H3.3ID).

Article Snippet: Antibodies and beads used for immunoprecipitation and western blot were as follows: H2A.Z antibody (1:5,000, Active Motif, 39113); Ubn1 antibody (1:3,000, Abcam, ab84953); H2B antibody (1:10,000, Abcam, ab1790); Hira antibody (1:500, Millipore, WC119); HA antibody (1:2,000, CWBIO, CW01245); Ubn1 antibody (1:3,000, Santacruz, SC-515340); Ubn1 antibody (in-house antibody); Ubn2 antibody (1:3,000, in-house antibody); Ino80 antibody (1:2,000, Proteintech, 18810-1-AP); Daxx antibody (1:3,000, Santa cruz, sc-7152); mouse IgG antibody (Santa cruz, sc-2025); Srcap antibody (1:750, Kerafast, ESL103); Flag-M2 (1:3,000, Sigma, F3165); anti-HA agarose beads (Sigma, 104M4753V); Flag antibody (1:5,000, rabbit, huaxingbio, HX1819); GAPDH antibody (1:10,000, Abclonal, AC033).

Techniques: Genome Wide, Knock-Out, Generated, CRISPR, Western Blot

Journal: Nucleic Acids Research

Article Title: HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs

doi: 10.1093/nar/gkab1221

Figure Lengend Snippet: HIRA collaborates with SRCAP to deposit H2A.Z on genome. ( A ) Meta-analysis (left) and heatmap (right) show Srcap reads density around summit (upper) and TSS (below) within a ±3 kb window. ( B ) Representative loci on Slc25a42 gene show the enrichment of H2A.Z and Srcap. ( C ) ChIP-qPCR validation of H2A.Z and Srcap enrichment at promoter of Slc35a42 gene. Error bars represent data from three independent experiments. ( D ) Venn diagram shows the overlap between H2A.Z, Hira and Srcap peaks. ( E ) Western blot shows the interaction among endogenous HA-Srcap, Hira and Ubn1 from nuclear lysates. Ino80, Daxx, H2A.Z and H3.3 are also detected. ( F ) Top panel showed schematic presentation of full length and truncation mutants of Hira subunit. Bottom panel showed the biochemical interactions between Hira truncations and SRCAP complex are analyzed by GST pull-down coupled with western blot analyses. ( G ) Heatmaps (upper) show H2A.Z reads density around H2A.Z peak summits (upper) in R1 WT , Hira-KO and Hira-truncation mutant expressing cells within a ±3 kb window. Representative loci (bottom) show the enrichment of H2A.Z. (H) ChIP-qPCR of H2A.Z, Srcap and Flag at representative genes Nrg2 and Spata4 in the rescued overexpressed cell lines. Error bars represent data from three independent experiments.

Article Snippet: Antibodies and beads used for immunoprecipitation and western blot were as follows: H2A.Z antibody (1:5,000, Active Motif, 39113); Ubn1 antibody (1:3,000, Abcam, ab84953); H2B antibody (1:10,000, Abcam, ab1790); Hira antibody (1:500, Millipore, WC119); HA antibody (1:2,000, CWBIO, CW01245); Ubn1 antibody (1:3,000, Santacruz, SC-515340); Ubn1 antibody (in-house antibody); Ubn2 antibody (1:3,000, in-house antibody); Ino80 antibody (1:2,000, Proteintech, 18810-1-AP); Daxx antibody (1:3,000, Santa cruz, sc-7152); mouse IgG antibody (Santa cruz, sc-2025); Srcap antibody (1:750, Kerafast, ESL103); Flag-M2 (1:3,000, Sigma, F3165); anti-HA agarose beads (Sigma, 104M4753V); Flag antibody (1:5,000, rabbit, huaxingbio, HX1819); GAPDH antibody (1:10,000, Abclonal, AC033).

Techniques: Western Blot, Mutagenesis, Expressing

Journal: Nucleic Acids Research

Article Title: HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs

doi: 10.1093/nar/gkab1221

Figure Lengend Snippet: Defect of HIRA complex results in H2A.Z decrease on genome wide. ( A ) Schematic diagram of genome editing in defective cell lines. Hira-, Ubn2- and H3.3-knockout cell lines and Ubn1/2 double mutants were generated using CRISPR-Cas9 system. For subsequent experiments, 6 × HA tag was added to the N-terminus of Srcap in R1 wild type and above defective cell lines. ( B ) Western blot respectively shows the proteins levels of Hira (a), Ubn1&Ubn2 (b), and H3.3 (c) in the defective cell lines. ( C ) Meta-analysis (left) and heatmap (right) show H2A.Z reads density around summit (upper) and TSS (below) in R1 WT , Hira-KO , Ubn2-KO/Ubn1-KD , Ubn1/2-DM and H3.3-KO cells within a ±3 kb window. ( D ) Venn diagram shows the overlap of H2A.Z down-regulated peaks between the above defective cell lines. ( E ) The reduced H2A.Z peaks in either Hira-KO or Ubn2-KO/Ubn1-KD cell lines are defined as HIRA complex regulated (HR) peaks (23 530 peaks). The 5426 HR peaks overlapping with Ubn1/2-DM cell line is defined as HIRA-regulated and H3.3-dependent (HIRA-H3.3D). The other 18 104 HR peaks are HIRA-regulated and H3.3-independent (HIRA-H3.3ID).

Article Snippet: Antibodies and beads used for immunoprecipitation and western blot were as follows: H2A.Z antibody (1:5,000, Active Motif, 39113); Ubn1 antibody (1:3,000, Abcam, ab84953); H2B antibody (1:10,000, Abcam, ab1790); Hira antibody (1:500, Millipore, WC119); HA antibody (1:2,000, CWBIO, CW01245); Ubn1 antibody (1:3,000, Santacruz, SC-515340); Ubn1 antibody (in-house antibody); Ubn2 antibody (1:3,000, in-house antibody); Ino80 antibody (1:2,000, Proteintech, 18810-1-AP); Daxx antibody (1:3,000, Santa cruz, sc-7152); mouse IgG antibody (Santa cruz, sc-2025); Srcap antibody (1:750, Kerafast, ESL103); Flag-M2 (1:3,000, Sigma, F3165); anti-HA agarose beads (Sigma, 104M4753V); Flag antibody (1:5,000, rabbit, huaxingbio, HX1819); GAPDH antibody (1:10,000, Abclonal, AC033).

Techniques: Genome Wide, Knock-Out, Generated, CRISPR, Western Blot

Journal: Nucleic Acids Research

Article Title: HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs

doi: 10.1093/nar/gkab1221

Figure Lengend Snippet: HIRA collaborates with SRCAP to deposit H2A.Z on genome. ( A ) Meta-analysis (left) and heatmap (right) show Srcap reads density around summit (upper) and TSS (below) within a ±3 kb window. ( B ) Representative loci on Slc25a42 gene show the enrichment of H2A.Z and Srcap. ( C ) ChIP-qPCR validation of H2A.Z and Srcap enrichment at promoter of Slc35a42 gene. Error bars represent data from three independent experiments. ( D ) Venn diagram shows the overlap between H2A.Z, Hira and Srcap peaks. ( E ) Western blot shows the interaction among endogenous HA-Srcap, Hira and Ubn1 from nuclear lysates. Ino80, Daxx, H2A.Z and H3.3 are also detected. ( F ) Top panel showed schematic presentation of full length and truncation mutants of Hira subunit. Bottom panel showed the biochemical interactions between Hira truncations and SRCAP complex are analyzed by GST pull-down coupled with western blot analyses. ( G ) Heatmaps (upper) show H2A.Z reads density around H2A.Z peak summits (upper) in R1 WT , Hira-KO and Hira-truncation mutant expressing cells within a ±3 kb window. Representative loci (bottom) show the enrichment of H2A.Z. (H) ChIP-qPCR of H2A.Z, Srcap and Flag at representative genes Nrg2 and Spata4 in the rescued overexpressed cell lines. Error bars represent data from three independent experiments.

Article Snippet: Antibodies and beads used for immunoprecipitation and western blot were as follows: H2A.Z antibody (1:5,000, Active Motif, 39113); Ubn1 antibody (1:3,000, Abcam, ab84953); H2B antibody (1:10,000, Abcam, ab1790); Hira antibody (1:500, Millipore, WC119); HA antibody (1:2,000, CWBIO, CW01245); Ubn1 antibody (1:3,000, Santacruz, SC-515340); Ubn1 antibody (in-house antibody); Ubn2 antibody (1:3,000, in-house antibody); Ino80 antibody (1:2,000, Proteintech, 18810-1-AP); Daxx antibody (1:3,000, Santa cruz, sc-7152); mouse IgG antibody (Santa cruz, sc-2025); Srcap antibody (1:750, Kerafast, ESL103); Flag-M2 (1:3,000, Sigma, F3165); anti-HA agarose beads (Sigma, 104M4753V); Flag antibody (1:5,000, rabbit, huaxingbio, HX1819); GAPDH antibody (1:10,000, Abclonal, AC033).

Techniques: Western Blot, Mutagenesis, Expressing

Journal: Nucleic Acids Research

Article Title: HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs

doi: 10.1093/nar/gkab1221

Figure Lengend Snippet: Defect of HIRA complex results in H2A.Z decrease on genome wide. ( A ) Schematic diagram of genome editing in defective cell lines. Hira-, Ubn2- and H3.3-knockout cell lines and Ubn1/2 double mutants were generated using CRISPR-Cas9 system. For subsequent experiments, 6 × HA tag was added to the N-terminus of Srcap in R1 wild type and above defective cell lines. ( B ) Western blot respectively shows the proteins levels of Hira (a), Ubn1&Ubn2 (b), and H3.3 (c) in the defective cell lines. ( C ) Meta-analysis (left) and heatmap (right) show H2A.Z reads density around summit (upper) and TSS (below) in R1 WT , Hira-KO , Ubn2-KO/Ubn1-KD , Ubn1/2-DM and H3.3-KO cells within a ±3 kb window. ( D ) Venn diagram shows the overlap of H2A.Z down-regulated peaks between the above defective cell lines. ( E ) The reduced H2A.Z peaks in either Hira-KO or Ubn2-KO/Ubn1-KD cell lines are defined as HIRA complex regulated (HR) peaks (23 530 peaks). The 5426 HR peaks overlapping with Ubn1/2-DM cell line is defined as HIRA-regulated and H3.3-dependent (HIRA-H3.3D). The other 18 104 HR peaks are HIRA-regulated and H3.3-independent (HIRA-H3.3ID).

Article Snippet: Antibodies and beads used for immunoprecipitation and western blot were as follows: H2A.Z antibody (1:5,000, Active Motif, 39113); Ubn1 antibody (1:3,000, Abcam, ab84953); H2B antibody (1:10,000, Abcam, ab1790); Hira antibody (1:500, Millipore, WC119); HA antibody (1:2,000, CWBIO, CW01245); Ubn1 antibody (1:3,000, Santacruz, SC-515340); Ubn1 antibody (in-house antibody); Ubn2 antibody (1:3,000, in-house antibody); Ino80 antibody (1:2,000, Proteintech, 18810-1-AP); Daxx antibody (1:3,000, Santa cruz, sc-7152); mouse IgG antibody (Santa cruz, sc-2025); Srcap antibody (1:750, Kerafast, ESL103); Flag-M2 (1:3,000, Sigma, F3165); anti-HA agarose beads (Sigma, 104M4753V); Flag antibody (1:5,000, rabbit, huaxingbio, HX1819); GAPDH antibody (1:10,000, Abclonal, AC033).

Techniques: Genome Wide, Knock-Out, Generated, CRISPR, Western Blot

Journal: Nucleic Acids Research

Article Title: HIRA complex presets transcriptional potential through coordinating depositions of the histone variants H3.3 and H2A.Z on the poised genes in mESCs

doi: 10.1093/nar/gkab1221

Figure Lengend Snippet: HIRA collaborates with SRCAP to deposit H2A.Z on genome. ( A ) Meta-analysis (left) and heatmap (right) show Srcap reads density around summit (upper) and TSS (below) within a ±3 kb window. ( B ) Representative loci on Slc25a42 gene show the enrichment of H2A.Z and Srcap. ( C ) ChIP-qPCR validation of H2A.Z and Srcap enrichment at promoter of Slc35a42 gene. Error bars represent data from three independent experiments. ( D ) Venn diagram shows the overlap between H2A.Z, Hira and Srcap peaks. ( E ) Western blot shows the interaction among endogenous HA-Srcap, Hira and Ubn1 from nuclear lysates. Ino80, Daxx, H2A.Z and H3.3 are also detected. ( F ) Top panel showed schematic presentation of full length and truncation mutants of Hira subunit. Bottom panel showed the biochemical interactions between Hira truncations and SRCAP complex are analyzed by GST pull-down coupled with western blot analyses. ( G ) Heatmaps (upper) show H2A.Z reads density around H2A.Z peak summits (upper) in R1 WT , Hira-KO and Hira-truncation mutant expressing cells within a ±3 kb window. Representative loci (bottom) show the enrichment of H2A.Z. (H) ChIP-qPCR of H2A.Z, Srcap and Flag at representative genes Nrg2 and Spata4 in the rescued overexpressed cell lines. Error bars represent data from three independent experiments.

Article Snippet: Antibodies and beads used for immunoprecipitation and western blot were as follows: H2A.Z antibody (1:5,000, Active Motif, 39113); Ubn1 antibody (1:3,000, Abcam, ab84953); H2B antibody (1:10,000, Abcam, ab1790); Hira antibody (1:500, Millipore, WC119); HA antibody (1:2,000, CWBIO, CW01245); Ubn1 antibody (1:3,000, Santacruz, SC-515340); Ubn1 antibody (in-house antibody); Ubn2 antibody (1:3,000, in-house antibody); Ino80 antibody (1:2,000, Proteintech, 18810-1-AP); Daxx antibody (1:3,000, Santa cruz, sc-7152); mouse IgG antibody (Santa cruz, sc-2025); Srcap antibody (1:750, Kerafast, ESL103); Flag-M2 (1:3,000, Sigma, F3165); anti-HA agarose beads (Sigma, 104M4753V); Flag antibody (1:5,000, rabbit, huaxingbio, HX1819); GAPDH antibody (1:10,000, Abclonal, AC033).

Techniques: Western Blot, Mutagenesis, Expressing