Journal: iScience

Article Title: Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation

doi: 10.1016/j.isci.2023.106743

Figure Lengend Snippet:

Article Snippet: Mouse anti-Histone H2B , Cell Signaling Technology , Cat #2934 RRID: AB_2295301.

Techniques: Recombinant, Mutagenesis, Software, CRISPR

Journal: PLoS ONE

Article Title: Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

doi: 10.1371/journal.pone.0061447

Figure Lengend Snippet: (A) RQ-PCR analysis of MLL in SU-DHL-5 (expression level was set to unity) and in leukemia/lymphoma control cell lines. (B) RQ-PCR analysis of MLL, NKX2-1 and HEY1 in siRNA-treated SU-DHL-5 cells (left). ChIP analysis of the NKX2-1 and HEY1 promoters in SU-DHL-5 and SU-DHL-4 (for control) showed representation of particular histone H3 modifications, as shown by PCR amplification of genomic fragments (right). Untreated genomic DNA served as positive control, NTC: no template control. (C) Copy number analysis by genomic profiling indicates presence of aberrations at MLL at 11q23. Inserts show an enlargement of chromosome 11 obtained by SKY karyotyping and an enlargement of the MLL locus obtained by genomic profiling. (D) FISH analysis of the MLL locus in SU-DHL-5 (below) using BAC probes as indicated above. The results show one wild type allele and one amplified MLL locus. (E) RT-PCR analysis of MLL fusion genes in SU-DHL-5 and particular positive and negative control cell lines. TEL expression served as control, NTC: no template control. (F) Copy number analysis by genomic profiling of chromosome 6 indicates extended deletions at both arms. The HIST1 locus maps to the breakpoint region at 6p22. Genes located in deleted regions include JARID2, SOX4, HMGN3, AKAP7 and MAP3K4. Insert shows chromosomes 6 analyzed by SKY karyotyping indicating rearrangements at both chromosomes. (G) FISH analysis of the histone gene cluster HIST1 at 6p22 (below) using painting probe and BACs as indicated above. (H) RQ-PCR analysis of selected histone genes in SU-DHL-5 and SU-DHL-4 for control (left). PAGE analysis of histone proteins in three DLBCL cell lines (middle) demonstrates elevated levels in SU-DHL-5. Western blot analysis of H2B, H2Bub1 and ERK (for control) in four DLBCL cell lines (right) demonstrates elevated levels in SU-DHL-5.

Article Snippet: ChIP analysis was performed with the ChIP Assay Kit (Millipore-Upstate, Schwalbach, Germany) as described by the manufacturer, isolating genomic DNA fragments generated by sonication, using antibodies anti-NKX2-1 (EP1584Y, Abgent), anti-H2B (53H3, Cell Signaling, Danvers, MA, USA), anti-H2Bub1 (D11, Cell Signaling), anti-H3K4me3 (mAbcam1012, Abcam, Cambridge, UK) and anti-H3K27me3 (mAbcam6002, Abcam).

Techniques: Expressing, Amplification, Positive Control, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot

Journal: PLoS ONE

Article Title: Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

doi: 10.1371/journal.pone.0061447

Figure Lengend Snippet: The figure summarizes the regulations of the genes described in this study, highlighting a central position of NKX2-1. Chromosomal aberrations activate expression of MLL (11q23) and histones including H2B (6p22). MLL together with H2Bub1 generate an activatory chromatin structure at NKX2-1. This structure is reinforced by reduced expression of USP46 and E2F6 and elevated expression of RNF20/40, JMJD3 and HOPX, and mediates activation of NKX2-1. NKX2-1 in turn activates directly expression of HEY1 which performs inhibition of B-cell differentiation. Both, NKX2-1 and HEY1 contribute to the activatory chromatin structure by regulating RNF40 and E2F6, respectively. IL4/STAT3-signaling enhances expression of NKX2-1. TNFa, cGMP, cAMP and PRKCE support NFkB which activates both NKX2-1 and HEY1. Reduced expression of PDE6D and enhanced expression of NOS1 contribute to elevated cGMP. NOS1 and PRKCE are activated by NKX2-1. HEY1 mediates reduced expression of PDE4A resulting in elevated cAMP levels. Finally, NOTCH-signaling and TGFb-signaling (via SMAD) activate HEY1 expression. SMAD and NKX2-1 interact and coactivate HEY1 transcription.

Article Snippet: ChIP analysis was performed with the ChIP Assay Kit (Millipore-Upstate, Schwalbach, Germany) as described by the manufacturer, isolating genomic DNA fragments generated by sonication, using antibodies anti-NKX2-1 (EP1584Y, Abgent), anti-H2B (53H3, Cell Signaling, Danvers, MA, USA), anti-H2Bub1 (D11, Cell Signaling), anti-H3K4me3 (mAbcam1012, Abcam, Cambridge, UK) and anti-H3K27me3 (mAbcam6002, Abcam).

Techniques: Expressing, Activation Assay, Inhibition, Cell Differentiation