Journal: Frontiers in Genetics

Article Title: Chrysin Modulates Aberrant Epigenetic Variations and Hampers Migratory Behavior of Human Cervical (HeLa) Cells

doi: 10.3389/fgene.2021.768130

Figure Lengend Snippet: Relative expression of TSGs and genes related to migration and metastasis. The values are taken as mean of three experiments ±SD ( p ≤ 0.05).

Article Snippet: MTA2 , Hs00191018_m1 , metastasis associated 2 , 0.28 , 0.14.

Techniques: Expressing, Migration

Journal: Journal of Biological Chemistry

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes

doi: 10.1074/jbc.m113.538777

Figure Lengend Snippet: FIGURE 8. PLZF-RAR represses transcription of CDKN1A epigenetically by histone deacetylation and DNA methylation. A, structure of the human CDKN1A gene promoter. The arrows indicate the locations of the qChIP-PCR primer binding sites. B, qChIP assays showing PLZF-RAR binding at the endog- enous CDKN1A proximal promoter using an antibody against RAR. Cells were transfected with the PLZF-RAR expression vector and immunoprecipitated (IP) with an anti-RAR antibody. IgG, control ChIP antibody. C and D, qChIP-PCR assays showing histone modifications at the endogenous CDKN1A proximal promoter. Cells were transfected with the PLZF-RAR expression vector and lysates were immunoprecipitated with the indicated antibodies (IgG, Ac-H3, Ac-H4,H3K4-Me3,orH3K9-Me3).E,Me-DIP(methylatedDNAChIP)assaysshowingincreasedDNAmethylationattheendogenousCDKN1Aproximalpromoter following ectopic PLZF-RAR expression. HEK293 cells were transfected with a PLZF-RAR expression vector, lysates were immunoprecipitated (IP) with the antibody recognizing methylated DNA, and precipitated DNA was amplified using the primers indicated in A. F, bisulfite sequencing analysis of the methylated CDKN1Apromoter.TheproximalpromotersequencesofCDKN1A,withpotentiallymethylatedCpGsitesareshowningrayovals,andtheSp1bindingGC-boxes shown above. Open ovals, unmethylated CpG; filled ovals, methylated CpG. G, co-immunoprecipitation of PLZF-RAR, MBD3, NuRD (MTA2), DNMT1, DNMT3b, and HP1. Cell lysates from either HEK293 cells transfected with a pcDNA3 vector or a PLZF-RAR expression vector were immunoprecipitated (IP) using anti-PLZF, anti-RAR or anti-IgG antibodies and analyzed by Western blot (WB) with the indicated antibodies. H, qChIP assays showing the proximal promoter binding of PLZF-RAR, MBD3, the NuRD-HDAC3 complex, DNMT1/3b and HP1 proteins in HEK293 cells transfected with the PLZF-RAR expression vector. *, p 0.05; N.S., not significant; t test.

Article Snippet: Antibodies against p21, p53, HDAC1, HDAC3, MDM2, PLZF, RAR , Sp1, GAPDH, Myc tag, Ac-H3, Ac-H4, H3K4-Me3, H3K9Me3, MBD3, HP1, MTA2, DNMT1, DNMT3b, mSin3A, NCoR, and SMRT were purchased from Upstate, Chemicon, Cell Signaling Technology, Abcam, Calbiochem, and Santa Cruz Biotechnology.

Techniques: DNA Methylation Assay, Binding Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Control, Methylation, Amplification, Methylation Sequencing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes

doi: 10.1074/jbc.m113.538777

Figure Lengend Snippet: FIGURE 9. PLZF-RAR stimulates cell proliferation and represses CDKN1A transcription in HL-60 cells through inhibitory histone modifications and DNA methylation. A, MTT assay of cell proliferation. HL-60 cells transfected with either pcDNA3 or pSG5-PLZF-RAR plasmid were grown for 1–4 days and analyzed for MTT to formazan conversion using colorimetry at 540–600 nm. B and C, Western blot (WB) and RT-qPCR analysis of protein and mRNA levels of the HL-60cellstransfectedwiththePLZF-RARexpressionvectororacontrolplasmid.D–F,qChIPassaysofmodifiedhistonesAc-H3,Ac-H4,H3K4-Me3,H3K9-Me3, and PLZF-RAR binding at the proximal CDKN1A promoter. HL-60 cells were transfected with the PLZF-RAR expression vector and analyzed for changes in Ac-H3, Ac-H4, H3K4-Me3, and H3K9-Me3 levels at the indicated regions (Fig. 8A) using the indicated antibodies. G, Me-DIP assays to assess DNA methylation of the endogenous CDKN1A proximal promoter in HL-60 cells transfected with PLZF-RAR expression vector. Alpha X1, positive control. H–K, qChIP assays of MBD3, NuRD (MTA2), DNMT1 and -3b, and HP1 binding at the proximal CDKN1A promoter in HL-60 cells transfected with a PLZF-RAR expression vector. *, p 0.05; N.C., negative control; P.C., positive control; N.S., not significant; t test.

Article Snippet: Antibodies against p21, p53, HDAC1, HDAC3, MDM2, PLZF, RAR , Sp1, GAPDH, Myc tag, Ac-H3, Ac-H4, H3K4-Me3, H3K9Me3, MBD3, HP1, MTA2, DNMT1, DNMT3b, mSin3A, NCoR, and SMRT were purchased from Upstate, Chemicon, Cell Signaling Technology, Abcam, Calbiochem, and Santa Cruz Biotechnology.

Techniques: DNA Methylation Assay, MTT Assay, Transfection, Plasmid Preparation, Colorimetric Assay, Western Blot, Quantitative RT-PCR, Binding Assay, Expressing, Positive Control, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Transcription suppression is mediated by the HDAC1–Sin3 complex in Xenopus nucleoplasmic extract

doi: 10.1016/j.jbc.2022.102578

Figure Lengend Snippet: The Sin3 deacetylase complex promotes transcription suppression. A , sequential IP schematic. B , mock, HDAC1, or HDAC2 IPs were performed in NPE. The supernatants from HDAC1 or HDAC2 IPs were then used for a second round of IPs using the converse antibody. Isolated proteins were then analyzed by Western blot with the indicated antibodies (n = 3). 10% of total reaction sample (IN), supernatant (S), pellet (P). C , NPE was immunodepleted using preimmune (ΔMock) or MTA2 (ΔMTA2) antibodies. Depleted extracts were analyzed by Western blot using the indicated antibodies. D , pActin was incubated in mock- or MTA2-depleted extract. RNA was isolated and quantified after 120 min (n = 2). E , NPE was immunodepleted using preimmune (ΔMock) or Sin3a (ΔSin3a) antibodies. Depleted extracts were analyzed by Western blot using the indicated antibodies. F , pActin was incubated in mock- or Sin3a-depleted extract. RNA was isolated and quantified after 120 min (n = 2). G , NPE was immunodepleted using preimmune (ΔMock) or HDAC1 (ΔHDAC1) antibodies. HDAC1-depleted extract was then supplemented with immunoprecipitated proteins recovered by preimmune (+Mock IP) or Sin3a (+Sin3a IP) IP. pActin was incubated in each extract and RNA was isolated and quantified after 120 min (n = 2). Student t test: p -value < 0.05 (∗), p -value < 0.01 (∗∗), p -value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. IP, immunoprecipitation; NPE, nucleoplasmic extract.

Article Snippet: Commercial antibodies were used to detect Histone H3 (ThermoFisher PA5-16183), H4K5ac (Abclonal A15233), H4K8ac (Abclonal A7258), H4K16ac (Abclonal A5280), H3K27ac (Abclonal A7253), MTA2 (Novus Biologicals NB100-56483SS), CoREST (Millipore 07-455), H2AK5ac (Thermofisher 720070), H2BK20ac (Millipore Sigma 07-347), H3K9/14ac (ThermoFisher 49-1010), H3K23ac (Cell Signaling 8848), and RNAPII (Bethyl Laboratories A300-653A).

Techniques: Histone Deacetylase Assay, Isolation, Western Blot, Incubation, Immunoprecipitation

Journal: Biology of Reproduction

Article Title: Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.110.086397

Figure Lengend Snippet: Mta RNA and protein expression patterns in oocyte and preimplantation embryos. A) Temporal pattern of expression of Mta1, Mta2, and Mta3. The experiment was conducted twice, and the data are expressed as mean ± range and are expressed relative to the value obtained for full-grown oocytes. The amount of Mta RNA was normalized to Gfp mRNA that was added as an external control prior to RNA isolation. B) Immunoblot analysis of MTA1, MTA2, and MTA3 expression. One hundred oocytes/embryos were loaded per lane, and beta-tubulin (TUBB) was used as a loading control. The experiment was conducted three times, and similar results were obtained in each case; a representative experiment is shown. C) Immunocytochemical analysis of MTA1, MTA2, and MTA3 expression. All samples for a given MTA were processed for immunocytochemistry together, and all images were taken at the same laser power, thereby enabling direct comparison of signal intensities. The experiment was conducted three times, and at least 25 oocytes/embryos were analyzed for each sample. Shown are representative examples. INC, incompetent oocyte; GV, fully grown oocyte; MII, metaphase II; 1C, one-cell embryo; 2C, two-cell embryo; 4C, four-cell embryo; 8C, eight-cell embryo; BL, blastocyst. Original magnification x80.

Article Snippet: The qRT-PCR analysis was performed with the ABI Taqman Assay-on-demand probe/primer sets for Mta1 (Mm00475337_m1), Mta2 (Mm00488671_m1), and Mta3 (Mm00475365_m1).

Techniques: Expressing, Isolation, Western Blot, Immunocytochemistry

Journal: Biology of Reproduction

Article Title: Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.110.086397

Figure Lengend Snippet: Effects of RNAi-mediated knockdown of Mta2 on Mta1, Mta2, and Mta3 expression, and localization and amount of MTA1, MTA2, and MTA3. A) One-cell embryos were injected with either Gfp dsRNA (control) or Mta2 dsRNA and then cultured for 24, 48, or 72 h, at which time the relative abundance of Mta1, Mta2, and Mta3 transcripts was assayed by qRT-PCR and expressed relative to controls. The experiment was performed three times, and the data are expressed as mean ± SEM. B) The experiment was performed as described in A, and the samples were processed for immunocytochemical detection of MTA 1, 2, or 3 at the indicated times. The experiment was conducted three times, and at least 25 oocytes/embryos were analyzed for each sample. Original magnification x80. C) The experiment was performed as described in A, and the relative amount of MTA1 and MTA2 was determined by immunoblot analysis after 72 h of culture. The experiment was performed twice, and similar results were obtained. One hundred embryos were used for immunoblot analysis.

Article Snippet: The qRT-PCR analysis was performed with the ABI Taqman Assay-on-demand probe/primer sets for Mta1 (Mm00475337_m1), Mta2 (Mm00488671_m1), and Mta3 (Mm00475365_m1).

Techniques: Expressing, Injection, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: Biology of Reproduction

Article Title: Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.110.086397

Figure Lengend Snippet: Effect of Mta2 knockdown on expression of other components of the NuRD complex. A) One-cell embryos were injected with either Gfp dsRNA (control) or Mta2 dsRNA and then cultured 96 h in vitro. Equal numbers of dsGfp- and dsMta2-injected embryos were collected for immunoblot analysis; 100 embryos were loaded per lane, and TUBB was used as a loading control. The experiment was performed twice, and similar results were obtained. B) dsGfp- or dsMta2-injected embryos were processed for immunocytochemical detection of histone H4 acetylation state. At least 12 control and experimental embryos were analyzed, and the experiment was conducted four times. Shown are representative images. C) dsGfp- or dsMta2-injected embryos were processed for immunocytochemical detection of histone POU5F1 and NANOG. At least 12 control and experimental embryos were analyzed, and the experiment was conducted four times. Shown are representative images. Original magnification x80.

Article Snippet: The qRT-PCR analysis was performed with the ABI Taqman Assay-on-demand probe/primer sets for Mta1 (Mm00475337_m1), Mta2 (Mm00488671_m1), and Mta3 (Mm00475365_m1).

Techniques: Expressing, Injection, Cell Culture, In Vitro, Western Blot

Journal: Biology of Reproduction

Article Title: Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development 1

doi: 10.1095/biolreprod.110.086397

Figure Lengend Snippet: Loss of H19 paternal DMR methylation in MTA2-depleted blastocysts. One-cell embryos were injected with either Gfp dsRNA or Mta2 dsRNA, cultured to the blastocyst stage, and collected in pools of 2–12 blastocysts that were then subjected to bisulfite mutagenesis. A) H19, paternal DNA stands are shown. B) Snrpn, maternal strands are shown. C) Peg3, maternal strands are shown. The H19 maternal and the Snprn and Peg3 paternal strands were unmethylated in all samples. Each line of circles represents a single DNA strand, with the number to the left of the line corresponding to the number of times this pattern was seen. Each circle represents a single CpG. If the CpG was methylated, the circle is filled. Those strands with less than half of the CpGs methylated are considered hypomethylated.

Article Snippet: The qRT-PCR analysis was performed with the ABI Taqman Assay-on-demand probe/primer sets for Mta1 (Mm00475337_m1), Mta2 (Mm00488671_m1), and Mta3 (Mm00475365_m1).

Techniques: Methylation, Injection, Cell Culture, Mutagenesis

Journal: Cell Reports

Article Title: SIN3A histone deacetylase action counteracts MUS81 to promote stalled fork stability

doi: 10.1016/j.celrep.2024.113778

Figure Lengend Snippet: Sin3A prevents fork breakage in stressed conditions (A) Images of cells immunostained for chr-bound RAD51 (green) and FANCD2 (red) proteins. DNA stained with DAPI (blue). Scale bar, 10 μm. siRNAs and HU as indicated. Plots show number of FANCD2 (left) or RAD51 (right) foci per cell. Mean in black. Data are pooled from 3 different assays. >1,500 cells scored per condition. ∗∗∗ p < 0.0001; two-tailed Mann-Whitney test. Immunoblot detection of Sin3A. H3, loading control. (B) Images of cells immunostained for γH2AX (red) and chr-bound RPA (green) proteins. DNA stained with DAPI (blue). Scale bar, 25 μm. Treatment as in (A). Histograms show the percentage (mean + SD) of γH2AX (top), chr-bound RPA-positive cells (left), and double-positive cells. n = 3. >400 cells scored per condition and assay. Negative staining determined in untreated control cells. ∗ p = 0.0346 (top) and p = 0.0231 (bottom left); ∗∗ p = 0.0037; unpaired two-tailed Student’s t test. (C) Images of U2OS SEC-C (cells stably expressing Cas9) cells immunostained for γH2AX (red) protein. DNA stained with DAPI (blue). Scale bar, 10 μm. RNA guides (72 h) and HU (3 mM, 4 h) as indicated. Immunoblot detection of Sin3A in indicated samples. GAPDH, loading control. Plot shows distribution of γH2AX intensity values. Median in black. Data are pooled from 2 different assays. >140 cells scored per condition. ∗∗∗ p < 0.0001; two-tailed Mann-Whitney test. (D) Plot shows distribution of γH2AX intensity values in cells treated (4 h) with HU (3 mM) combined with sodium butyrate (NaB, 5 mM), trichostatin A (TSA, 250 nM), and romidepsin (50 nM). Median in black. Data are pooled from 4, 2, and 3 different assays, respectively. >1,100 cells scored per condition. ∗∗∗ p < 0.0001; two-tailed Mann-Whitney test. (E) Images of cells immunostained for chr-bound 53BP1 (green) protein. DNA was stained with DAPI (blue). Scale bar, 10 μm. siRNAs and HU as indicated. Plot shows number of 53BP1 foci per cell. Data are pooled from 3 different assays. >1,400 cells were scored per condition. ∗∗∗ p < 0.0001; two-tailed Mann-Whitney test. (F) Representative images of comet assay. Scale bar, 100 μm. siRNAs and HU as indicated. Histogram shows tail moment (mean + SD). n = 3. ∗∗ p = 0.0067; two-tailed unpaired Student’s t test. (G) Same as in (F) in indicated samples. Scale bar, 100 μm. Histogram shows tail moment (mean + SD). n = 4. n.s. p = 0.1087; two-tailed unpaired Student’s t test. Immunoblot detection of MTA2. GAPDH, loading control. siMTA2, MTA2 siRNA-transfected cells. HU (3 mM, 24 h) except for (C) and (D). siRNA transfection (72 h). All replicates are biological replicates. See also Figure S2 .

Article Snippet: Rabbit anti MTA2 , Atlas Antibodies , HPA006214; RRID: AB_1079421.

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, Western Blot, Control, Negative Staining, Stable Transfection, Expressing, Single Cell Gel Electrophoresis, Transfection

Journal: Cell Reports

Article Title: SIN3A histone deacetylase action counteracts MUS81 to promote stalled fork stability

doi: 10.1016/j.celrep.2024.113778

Figure Lengend Snippet:

Article Snippet: Rabbit anti MTA2 , Atlas Antibodies , HPA006214; RRID: AB_1079421.

Techniques: Recombinant, Control, Protease Inhibitor, Imaging, Reverse Transcription, Plasmid Preparation, Software, Magnetic Beads, Blocking Assay, In Situ, Western Blot, Membrane