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c-Myb Binding Sites in Haematopoietic Chromatin Landscapes.

Bengtsen M, Klepper K, Gundersen S, Cuervo I, Drabløs F, Hovig E, Sandve GK, Gabrielsen OS, Eskeland R - PLoS ONE (2015)

Bottom Line: Furthermore, we find that c-Myb footprints co-localise with active histone mark H3K4me3 and are significantly enriched at exons.We analysed co-localisation of c-Myb footprints with 104 chromatin regulatory factors in K562 cells, and identified nine proteins that are enriched together with c-Myb footprints on genes positively regulated by c-Myb and one protein enriched on negatively regulated genes.Our data suggest that c-Myb is a transcription factor with multifaceted target regulation depending on cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
Strict control of tissue-specific gene expression plays a pivotal role during lineage commitment. The transcription factor c-Myb has an essential role in adult haematopoiesis and functions as an oncogene when rearranged in human cancers. Here we have exploited digital genomic footprinting analysis to obtain a global picture of c-Myb occupancy in the genome of six different haematopoietic cell-types. We have biologically validated several c-Myb footprints using c-Myb knockdown data, reporter assays and DamID analysis. We show that our predicted conserved c-Myb footprints are highly dependent on the haematopoietic cell type, but that there is a group of gene targets common to all cell-types analysed. Furthermore, we find that c-Myb footprints co-localise with active histone mark H3K4me3 and are significantly enriched at exons. We analysed co-localisation of c-Myb footprints with 104 chromatin regulatory factors in K562 cells, and identified nine proteins that are enriched together with c-Myb footprints on genes positively regulated by c-Myb and one protein enriched on negatively regulated genes. Our data suggest that c-Myb is a transcription factor with multifaceted target regulation depending on cell type.

No MeSH data available.


Related in: MedlinePlus

Validation of c-Myb footprints by DAMID.(A) Expression of the Flag-c-Myb-Dam construct. K562 cells were transfected with a plasmid encoding the c-Myb-Dam together with the activator plasmid pVgRXR and treated with 2 uM of Ponasterone A. Expression of the fusion construct was detected by immunoblotting against Flag-tag. (B-J) Association of the control Dam and c-Myb-Dam at specific loci containing one or more c-Myb footprints quantified with qPCR and normalised to Dam. The upper panels show the genomic region in the UCSC browser (hg19) presenting DNase I signals, c-Myb footprints and oligos for qPCR. The coordinates for the c-Myb footprint are shown above the illustration. The values represent the average from two independent cell lines-/+ SEM.
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pone.0133280.g003: Validation of c-Myb footprints by DAMID.(A) Expression of the Flag-c-Myb-Dam construct. K562 cells were transfected with a plasmid encoding the c-Myb-Dam together with the activator plasmid pVgRXR and treated with 2 uM of Ponasterone A. Expression of the fusion construct was detected by immunoblotting against Flag-tag. (B-J) Association of the control Dam and c-Myb-Dam at specific loci containing one or more c-Myb footprints quantified with qPCR and normalised to Dam. The upper panels show the genomic region in the UCSC browser (hg19) presenting DNase I signals, c-Myb footprints and oligos for qPCR. The coordinates for the c-Myb footprint are shown above the illustration. The values represent the average from two independent cell lines-/+ SEM.

Mentions: In order to further validate the deduced c-Myb footprints, we performed a DamID analysis in K562 cells (S4A Fig) [46,47]. DNA adenine methyltransferase (Dam) was fused to full-length c-Myb, and we generated a pool of stably transfected cells that express trace amounts of Dam or c-Myb-Dam. It is critical to keep the Dam and Myb-Dam expression low to avoid too high background methylation. This precludes direct detection of the trace levels by normal Western blotting. We used an ecdysone-inducible promoter to detect the c-Myb-Dam expression and performed transient transfection together with the pVgRXR vector encoding the ecdysone receptor in K562 cells and induced expression by the ecdysone analog Ponasterone A [48]. A clear induction of the fusion protein was observed (Fig 3A). To rule out the effects of random integration of transgenes, we used two stable K562 pool cell lines for Dam and Dam-Myb derived at different time points. Finally, we used qPCR with oligos spanning selected c-Myb footprints to map c-Myb binding at these sites and compared the signals to those obtained with the Dam only cells.


c-Myb Binding Sites in Haematopoietic Chromatin Landscapes.

Bengtsen M, Klepper K, Gundersen S, Cuervo I, Drabløs F, Hovig E, Sandve GK, Gabrielsen OS, Eskeland R - PLoS ONE (2015)

Validation of c-Myb footprints by DAMID.(A) Expression of the Flag-c-Myb-Dam construct. K562 cells were transfected with a plasmid encoding the c-Myb-Dam together with the activator plasmid pVgRXR and treated with 2 uM of Ponasterone A. Expression of the fusion construct was detected by immunoblotting against Flag-tag. (B-J) Association of the control Dam and c-Myb-Dam at specific loci containing one or more c-Myb footprints quantified with qPCR and normalised to Dam. The upper panels show the genomic region in the UCSC browser (hg19) presenting DNase I signals, c-Myb footprints and oligos for qPCR. The coordinates for the c-Myb footprint are shown above the illustration. The values represent the average from two independent cell lines-/+ SEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514710&req=5

pone.0133280.g003: Validation of c-Myb footprints by DAMID.(A) Expression of the Flag-c-Myb-Dam construct. K562 cells were transfected with a plasmid encoding the c-Myb-Dam together with the activator plasmid pVgRXR and treated with 2 uM of Ponasterone A. Expression of the fusion construct was detected by immunoblotting against Flag-tag. (B-J) Association of the control Dam and c-Myb-Dam at specific loci containing one or more c-Myb footprints quantified with qPCR and normalised to Dam. The upper panels show the genomic region in the UCSC browser (hg19) presenting DNase I signals, c-Myb footprints and oligos for qPCR. The coordinates for the c-Myb footprint are shown above the illustration. The values represent the average from two independent cell lines-/+ SEM.
Mentions: In order to further validate the deduced c-Myb footprints, we performed a DamID analysis in K562 cells (S4A Fig) [46,47]. DNA adenine methyltransferase (Dam) was fused to full-length c-Myb, and we generated a pool of stably transfected cells that express trace amounts of Dam or c-Myb-Dam. It is critical to keep the Dam and Myb-Dam expression low to avoid too high background methylation. This precludes direct detection of the trace levels by normal Western blotting. We used an ecdysone-inducible promoter to detect the c-Myb-Dam expression and performed transient transfection together with the pVgRXR vector encoding the ecdysone receptor in K562 cells and induced expression by the ecdysone analog Ponasterone A [48]. A clear induction of the fusion protein was observed (Fig 3A). To rule out the effects of random integration of transgenes, we used two stable K562 pool cell lines for Dam and Dam-Myb derived at different time points. Finally, we used qPCR with oligos spanning selected c-Myb footprints to map c-Myb binding at these sites and compared the signals to those obtained with the Dam only cells.

Bottom Line: Furthermore, we find that c-Myb footprints co-localise with active histone mark H3K4me3 and are significantly enriched at exons.We analysed co-localisation of c-Myb footprints with 104 chromatin regulatory factors in K562 cells, and identified nine proteins that are enriched together with c-Myb footprints on genes positively regulated by c-Myb and one protein enriched on negatively regulated genes.Our data suggest that c-Myb is a transcription factor with multifaceted target regulation depending on cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, University of Oslo, Oslo, Norway.

ABSTRACT
Strict control of tissue-specific gene expression plays a pivotal role during lineage commitment. The transcription factor c-Myb has an essential role in adult haematopoiesis and functions as an oncogene when rearranged in human cancers. Here we have exploited digital genomic footprinting analysis to obtain a global picture of c-Myb occupancy in the genome of six different haematopoietic cell-types. We have biologically validated several c-Myb footprints using c-Myb knockdown data, reporter assays and DamID analysis. We show that our predicted conserved c-Myb footprints are highly dependent on the haematopoietic cell type, but that there is a group of gene targets common to all cell-types analysed. Furthermore, we find that c-Myb footprints co-localise with active histone mark H3K4me3 and are significantly enriched at exons. We analysed co-localisation of c-Myb footprints with 104 chromatin regulatory factors in K562 cells, and identified nine proteins that are enriched together with c-Myb footprints on genes positively regulated by c-Myb and one protein enriched on negatively regulated genes. Our data suggest that c-Myb is a transcription factor with multifaceted target regulation depending on cell type.

No MeSH data available.


Related in: MedlinePlus