Journal: bioRxiv

Article Title: Kinome-wide RNAi screen uncovers role of Ballchen in maintenance of gene activation by trithorax group in Drosophila

doi: 10.1101/2020.11.26.399824

Figure Lengend Snippet: (A, B) Ballchen mutant flies ( ball 2 ) were crossed with two different alleles of Pc ( Pc 1 and Pc XL5 ), while Pc alleles ( Pc 1 and Pc XL5 ) crossed with w 1118 flies were used as control. Heterozygous Pc/+ males, Pc 1 /+ (A) and Pc XL5 /+ (B), from control crosses exhibit strong extra sex combs phenotype. In contrast, ball 2 strongly suppressed both the Pc alleles to none or few extra sex combs in ball 2 /Pc 1 (A) as well as ball 2 /Pc XL5 (B) flies. 200 male flies were analyzed for each cross and data shown represents two independent experiments. Based on strength of phenotype, male flies were categorized according to the severity of extra sex combs phenotype. These categories are: -, no extra sex combs; +, 1-2 hairs on 2 nd leg; ++, more than 3 hairs on 2 nd leg; +++, more than 3 hairs on 2 nd leg and 1-2 hairs on 3 rd leg; ++++, strong sex combs on both 2 nd and 3 rd pairs of legs. (C) ball 2 mutant flies were crossed to two different alleles of trx ( trx 1 and trx E2 ), and ball 2 /trx males in F1 were scored for loss of pigmentation in A5 abdominal segment (A5 to A4 transformation, marked with asterisk). The trx alleles ( trx 1 and trx E2 ) crossed with w 1118 flies i.e. trx 1 /+ and trx E2 /+ , show A5 to A4 transformation when compared to wild type flies. Strong enhancement of A5 to A4 transformation can be seen in ball 2 /trx double mutants when compared to trx/+ . Representative images for wild-type phenotype (top right) and A5 to A4 transformation (marked by white asterisk) are presented for trx/ball male flies (bottom right). All crosses were done in triplicates and independent t-tests were performed for analyzing each category (* p ≤ 0.05, ** p ≤ 0.01, *** p≤ 0.001 or **** p≤ 0.0001). (D) Significantly low levels of abd-A , Abd-B and pnt expression was detected through qRT-PCR in homozygous ball 2 embryos when compared with w 1118 embryos. Independent t-tests were performed for each gene analysis (* p ≤ 0.05, ** p ≤ 0.01, *** p≤ 0.001 or **** p≤ 0.0001). (E-H) Immunostaining of stage 15 embryos with anti-Abd-B is shown in w 1118 (E, F) as well as homozygous ball 2 (G, H) embryos. As compared to w 1118 (F), ball 2 embryos showed strongly diminished Abd-B (H) expression.

Article Snippet: Following antibodies were used during this study: mouse anti-Abd-B (DSHB, 1A2E9, IF: 1:40), rabbit anti-TRX (gift from R. Paro, IF: 1:20), rabbit anti-PC (Santa Cruz, D220, IF: 1:20), rabbit anti-BALL (Gift from A. Herzig, IF 1:20, ChIP: 2μl) mouse anti-GFP (Roche, 11814460001, IF:1:50), mouse anti-Tubulin (Abcam, ab44928, WB: 1:2000), mouse anti-FLAG M2 (Sigma Aldrich, WB: 1:2000, ChIP: 5μl), mouse anti-H2A-ubi (Millipore, 05-678, ChIP: 5μl).

Techniques: Mutagenesis, Transformation Assay, Expressing, Quantitative RT-PCR, Immunostaining

Journal: bioRxiv

Article Title: Kinome-wide RNAi screen uncovers role of Ballchen in maintenance of gene activation by trithorax group in Drosophila

doi: 10.1101/2020.11.26.399824

Figure Lengend Snippet: (A) Venn diagram analysis showed 85% co-occupancy of BALL on known TRX binding sites. Heat map illustration of BALL binding sites across genome show enrichment of BALL within ± 1kb of TSSs of genes. (B) Genome browser view of BALL association compared with patterns of TRX, PC, H3K27ac and H3K4me3 association at pnt and pnr genes. (C) Genome browser view of ChIP-seq data showing binding profile of BALL and its comparison with TRX, PC, H3K27ac and H3K4me3 association across the bithorax complex (BX-C). Highly enriched BALL and TRX correlates with high levels of both H3K27ac and H3K4me3 and absence of PC at CG14906 present just downstream of Ubx. In contrast, BX-C genes i.e. Ubx , abd-A and Abd-B which are silent in S2 correlates with highly enriched PC all across BX-C. Both BALL and TRX are present at few overlapping binding sites in BX-C.

Article Snippet: Following antibodies were used during this study: mouse anti-Abd-B (DSHB, 1A2E9, IF: 1:40), rabbit anti-TRX (gift from R. Paro, IF: 1:20), rabbit anti-PC (Santa Cruz, D220, IF: 1:20), rabbit anti-BALL (Gift from A. Herzig, IF 1:20, ChIP: 2μl) mouse anti-GFP (Roche, 11814460001, IF:1:50), mouse anti-Tubulin (Abcam, ab44928, WB: 1:2000), mouse anti-FLAG M2 (Sigma Aldrich, WB: 1:2000, ChIP: 5μl), mouse anti-H2A-ubi (Millipore, 05-678, ChIP: 5μl).

Techniques: Binding Assay, ChIP-sequencing

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Transcriptional control of the B3GALT5 gene by a retroviral promoter and methylation of distant regulatory elements.

doi: 10.1096/fj.13-236273

Figure Lengend Snippet: Figure 1. Detection of transcription factors HNF1/ and Cdx1/2 in human colon cancers and cell lines expressing various amounts of B3GALT5 LTR transcript. A) RNA extracted from cancers and cell lines was reverse transcribed, and normalized amounts of the resultant first-strand cDNAs were mixed with the indicated amounts of competitor (truncated) cDNAs and subjected to PCR (25 and 35 cycles for -actin and B3GALT5 LTR, respectively), using primers specific to -actin and B3GALT5 LTR transcript, respectively. Note the different amounts of B3GALT5 LTR competitor cDNA used in the case of colon cancers, cell lines (present figure), and matched pairs of colon biopsies (Supplemental Fig. S1A). Twenty percent of each amplification reaction was electrophoresed in a 1% agarose gel and visualized by ethidium bromide staining. The target doublet corresponded to the alternative splicing that had been reported (10). Representative gels are shown. Samples were not all run on the same gel, as indicated by the vertical white spaces between the gels. B) Densitometric scanning of gel images was performed to quantitate the amounts of B3GALT5 LTR transcript, which were calculated from a standard curve and normalized to the amounts calculated for -actin. Results are means sd of 3 determinations. Note the different scales used for colon cancer and the cell lines. C) Nuclear extracts (5–10 g of protein) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane that was blotted with anti-HNF1 antibody (recognizing both HNF1 and HNF1), or anti-Cdx1, anti-Cdx2, or anti-histone H3 antibodies, followed by an HRP-labeled secondary antibody and chemiluminescence detection. Longer exposures were necessary to detect a visible spot for Cdx1 and Cdx2. For comparative quantitation, see Supplemental Fig. S1B.

Article Snippet: Aliquots of nuclear extracts (5–10 g of protein) were separated by 10% SDS-PAGE; transferred to a nitrocellulose membrane (Trans-Blot SD Semi-Dry Transfer Cell; Bio-Rad Laboratories S.r.l., Milan, Italy); and blotted with rabbit polyclonal anti-HNF1 / (sc-8986, 1:200; Santa Cruz Biotechnology Italia, Milan, Italy), rabbit polyclonal anti-H3 (D2B12 4620, 1:500; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-Cdx1 (PAB4713, 1:200; Abnova, Taipei, Taiwan), or mouse monoclonal antiCdx2 (M01, 1:200; Abnova) antibodies, according to our published protocol (18).

Techniques: Expressing, Reverse Transcription, Agarose Gel Electrophoresis, Staining, Alternative Splicing, SDS Page, Membrane, Labeling, Quantitation Assay