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Distinct isoforms of the Drosophila Brd4 homologue are present at enhancers, promoters and insulator sites.

Kellner WA, Van Bortle K, Li L, Ramos E, Takenaka N, Corces VG - Nucleic Acids Res. (2013)

Bottom Line: Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph.Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present.The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Emory University, Atlanta, GA 30322, USA and Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

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Fs(1)h-L interacts with insulator proteins. (A) Co-IP experiments with a protein lysate from Kc167 cells (Input) were performed using either IgG (control) or antibody against Fs(1)h-L (guinea pig). Western analysis was performed on all samples using antibodies designated on the right side of the panel at 1:10 volume of input to IP. (B) Co-IP experiments were performed as described earlier in the text with only one immunoprecipitation from the lysate using antibodies against each of the indicated insulator proteins followed by western blotting with Fs(1)h antibodies that recognize both isoforms. (C) Organization of the Fs(1)h locus. Amino acids 1169 through 1389 were placed in an expression vector with a GST tag at the N-terminus and Hisx6 tag at the C-terminus for expression in E. coli followed by purification to 1.3 µg/µl using the Hisx6 and GST tags. (D) The Fs(1)h-L specific peptide was incubated with a Kc cell protein lysate, precipitated with glutathione-agarose beads and analyzed by western blot to test for interaction with each of the insulator proteins. Results suggest that GAF, Su(Hw), CP190 and Mod(mdg4) interact, directly or indirectly, with the CTM domain of Fs(1)h-L.
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gkt722-F6: Fs(1)h-L interacts with insulator proteins. (A) Co-IP experiments with a protein lysate from Kc167 cells (Input) were performed using either IgG (control) or antibody against Fs(1)h-L (guinea pig). Western analysis was performed on all samples using antibodies designated on the right side of the panel at 1:10 volume of input to IP. (B) Co-IP experiments were performed as described earlier in the text with only one immunoprecipitation from the lysate using antibodies against each of the indicated insulator proteins followed by western blotting with Fs(1)h antibodies that recognize both isoforms. (C) Organization of the Fs(1)h locus. Amino acids 1169 through 1389 were placed in an expression vector with a GST tag at the N-terminus and Hisx6 tag at the C-terminus for expression in E. coli followed by purification to 1.3 µg/µl using the Hisx6 and GST tags. (D) The Fs(1)h-L specific peptide was incubated with a Kc cell protein lysate, precipitated with glutathione-agarose beads and analyzed by western blot to test for interaction with each of the insulator proteins. Results suggest that GAF, Su(Hw), CP190 and Mod(mdg4) interact, directly or indirectly, with the CTM domain of Fs(1)h-L.

Mentions: Fs(1)h-L&S rabbit polyclonal antibody was a kind gift from Drs D. Huang and I. Dawid (3). Antibodies to Fs(1)h-L were prepared by expression of a 220 amino acid fragment from the carboxy-terminal domain specific to this isoform (see Figure 6A) in Escherichia coli, followed by purification and immunization of rabbits and guinea pigs. For immunoprecipitation experiments, 2 × 106 Kc167 cells were lysed in cell lysis buffer [5 mM PIPES (pH 8.0), 85 mM KCl, 0.5% TX-100], nuclei were spun down, incubated in 1 ml of RIPA buffer 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin containing complete, EDTA-free protease inhibitor cocktail tablets (Roche 04 693 159 001) and briefly sonicated to ensure lysis. Nuclear lysates were treated with 5 μl of DNase I (Sigma D5319) plus 6 mM MgCl2 at 25°C for 20 min to obtain single nucleosomes. Fs(1)h-L antibodies were incubated in lysate at 4°C for 6 h and precipitated with 50/50 mixture of Protein A and Protein G beads (GE Healthcare 17-1279-01 and 17-0618-01). All immunoprecipitated materials were washed four times in 1 ml of RIPA buffer and bound proteins were eluted in 1× Laemmli buffer and analyzed by western blots. For GST pull-down experiments, 1 × 107 Kc167 cells were lysed in 1 ml of RIPA buffer containing protease inhibitors (Roche 04 693 159 001) and briefly sonicated to ensure lysis. Lysates were treated with 5 μl of DNase I (Sigma D5319) plus 6 mM MgCl2 at 25°C for 20 min to obtain single nucleosomes. Three micoliters of the Glutathione S-transferase (GST)-Fs(1)h-L specific peptide were incubated in lysate at 4°C for 6 h and precipitated with 50 µl of glutathione-agarose beads (Novagen #70541). After four washes in RIPA buffer, bound proteins were eluted in 1x Laemmeli buffer and analyzed by western blots using antibodies to each of the insulator proteins.


Distinct isoforms of the Drosophila Brd4 homologue are present at enhancers, promoters and insulator sites.

Kellner WA, Van Bortle K, Li L, Ramos E, Takenaka N, Corces VG - Nucleic Acids Res. (2013)

Fs(1)h-L interacts with insulator proteins. (A) Co-IP experiments with a protein lysate from Kc167 cells (Input) were performed using either IgG (control) or antibody against Fs(1)h-L (guinea pig). Western analysis was performed on all samples using antibodies designated on the right side of the panel at 1:10 volume of input to IP. (B) Co-IP experiments were performed as described earlier in the text with only one immunoprecipitation from the lysate using antibodies against each of the indicated insulator proteins followed by western blotting with Fs(1)h antibodies that recognize both isoforms. (C) Organization of the Fs(1)h locus. Amino acids 1169 through 1389 were placed in an expression vector with a GST tag at the N-terminus and Hisx6 tag at the C-terminus for expression in E. coli followed by purification to 1.3 µg/µl using the Hisx6 and GST tags. (D) The Fs(1)h-L specific peptide was incubated with a Kc cell protein lysate, precipitated with glutathione-agarose beads and analyzed by western blot to test for interaction with each of the insulator proteins. Results suggest that GAF, Su(Hw), CP190 and Mod(mdg4) interact, directly or indirectly, with the CTM domain of Fs(1)h-L.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814382&req=5

gkt722-F6: Fs(1)h-L interacts with insulator proteins. (A) Co-IP experiments with a protein lysate from Kc167 cells (Input) were performed using either IgG (control) or antibody against Fs(1)h-L (guinea pig). Western analysis was performed on all samples using antibodies designated on the right side of the panel at 1:10 volume of input to IP. (B) Co-IP experiments were performed as described earlier in the text with only one immunoprecipitation from the lysate using antibodies against each of the indicated insulator proteins followed by western blotting with Fs(1)h antibodies that recognize both isoforms. (C) Organization of the Fs(1)h locus. Amino acids 1169 through 1389 were placed in an expression vector with a GST tag at the N-terminus and Hisx6 tag at the C-terminus for expression in E. coli followed by purification to 1.3 µg/µl using the Hisx6 and GST tags. (D) The Fs(1)h-L specific peptide was incubated with a Kc cell protein lysate, precipitated with glutathione-agarose beads and analyzed by western blot to test for interaction with each of the insulator proteins. Results suggest that GAF, Su(Hw), CP190 and Mod(mdg4) interact, directly or indirectly, with the CTM domain of Fs(1)h-L.
Mentions: Fs(1)h-L&S rabbit polyclonal antibody was a kind gift from Drs D. Huang and I. Dawid (3). Antibodies to Fs(1)h-L were prepared by expression of a 220 amino acid fragment from the carboxy-terminal domain specific to this isoform (see Figure 6A) in Escherichia coli, followed by purification and immunization of rabbits and guinea pigs. For immunoprecipitation experiments, 2 × 106 Kc167 cells were lysed in cell lysis buffer [5 mM PIPES (pH 8.0), 85 mM KCl, 0.5% TX-100], nuclei were spun down, incubated in 1 ml of RIPA buffer 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin containing complete, EDTA-free protease inhibitor cocktail tablets (Roche 04 693 159 001) and briefly sonicated to ensure lysis. Nuclear lysates were treated with 5 μl of DNase I (Sigma D5319) plus 6 mM MgCl2 at 25°C for 20 min to obtain single nucleosomes. Fs(1)h-L antibodies were incubated in lysate at 4°C for 6 h and precipitated with 50/50 mixture of Protein A and Protein G beads (GE Healthcare 17-1279-01 and 17-0618-01). All immunoprecipitated materials were washed four times in 1 ml of RIPA buffer and bound proteins were eluted in 1× Laemmli buffer and analyzed by western blots. For GST pull-down experiments, 1 × 107 Kc167 cells were lysed in 1 ml of RIPA buffer containing protease inhibitors (Roche 04 693 159 001) and briefly sonicated to ensure lysis. Lysates were treated with 5 μl of DNase I (Sigma D5319) plus 6 mM MgCl2 at 25°C for 20 min to obtain single nucleosomes. Three micoliters of the Glutathione S-transferase (GST)-Fs(1)h-L specific peptide were incubated in lysate at 4°C for 6 h and precipitated with 50 µl of glutathione-agarose beads (Novagen #70541). After four washes in RIPA buffer, bound proteins were eluted in 1x Laemmeli buffer and analyzed by western blots using antibodies to each of the insulator proteins.

Bottom Line: Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph.Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present.The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Emory University, Atlanta, GA 30322, USA and Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA 30322, USA.

ABSTRACT
Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

Show MeSH
Related in: MedlinePlus