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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

Identification of c-Myb footprints.(A) Workflow for identification of c-Myb footprints. (B) A pie chart representing the number of c-Myb footprints identified compared to the total number of footprints in K562 cells. (C) An illustration from Motiflab showing a c-Myb motif located at the start of the first intron of the gene FKBP5 that overlaps with a DNase I footprint with a high conservation value in K562 cells, illustration modified. The coordinates for the c-Myb footprint is shown above the illustration, and to the right are the signal intensity for the DNase I datasets, in addition to a conservation score. (D) The binding motif enriched in c-Myb footprints in K562 cells. (E) Graph of the presence of c-Myb footprints and the distances to the 100 most regulated genes upon KD in K562 cells (dots) or a selection of 100 random genes (squares), an average of ten repetitions. Zero base pair indicates that the c-Myb footprints are found inside the gene body (F) A c-Myb footprint at the TSS of GRSF1 gene mapped in all six cell-types analysed. Coordinates for c-Myb footprint are shown above, and to the left are the signal intensity for DNase I datasets. (G) Position of c-Myb footprints, and random selections of DNase I footprints and c-Myb motifs, respectively, around ENSEMBL annotated TSS in K562 cells. (H) Distribution of c-Myb footprints at annotated genes, promoters and intergenic regions in K562 cells. *Overlapping significantly higher with c-Myb footprints than expected by random sampling of K562 DNase I footprints (p' < 5x10-2).
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pone.0133280.g001: Identification of c-Myb footprints.(A) Workflow for identification of c-Myb footprints. (B) A pie chart representing the number of c-Myb footprints identified compared to the total number of footprints in K562 cells. (C) An illustration from Motiflab showing a c-Myb motif located at the start of the first intron of the gene FKBP5 that overlaps with a DNase I footprint with a high conservation value in K562 cells, illustration modified. The coordinates for the c-Myb footprint is shown above the illustration, and to the right are the signal intensity for the DNase I datasets, in addition to a conservation score. (D) The binding motif enriched in c-Myb footprints in K562 cells. (E) Graph of the presence of c-Myb footprints and the distances to the 100 most regulated genes upon KD in K562 cells (dots) or a selection of 100 random genes (squares), an average of ten repetitions. Zero base pair indicates that the c-Myb footprints are found inside the gene body (F) A c-Myb footprint at the TSS of GRSF1 gene mapped in all six cell-types analysed. Coordinates for c-Myb footprint are shown above, and to the left are the signal intensity for DNase I datasets. (G) Position of c-Myb footprints, and random selections of DNase I footprints and c-Myb motifs, respectively, around ENSEMBL annotated TSS in K562 cells. (H) Distribution of c-Myb footprints at annotated genes, promoters and intergenic regions in K562 cells. *Overlapping significantly higher with c-Myb footprints than expected by random sampling of K562 DNase I footprints (p' < 5x10-2).

Mentions: DGF is a powerful method to identify nucleotides protected by proteins at a genome-wide scale independent of antibodies [29–32]. To map changes in c-Myb occupancy during haematopoiesis, we used DGF to generate maps of c-Myb footprints with nucleotide resolution (Fig 1A). We selected haematopoietic cell-types where c-Myb is expressed at different levels: c-Myb is highly expressed in haematopoietic stem cells [33] and expressed at lower level in CD4+ T-helper cells [34] and B cells [35,36]. c-Myb is also highly expressed in most cases of leukaemia [10]. We collected available DNase I footprint datasets in six different human cell-types from three healthy donors (CD34+ (mobilized), CD20+ and Th1 cells), transformed B-lymphocytes (GM12865) and two cancer cell-types where c-Myb is upregulated: erythroleukaemia (K562) and promyelocytic leukaemia (NB4) [31].


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)

Identification of c-Myb footprints.(A) Workflow for identification of c-Myb footprints. (B) A pie chart representing the number of c-Myb footprints identified compared to the total number of footprints in K562 cells. (C) An illustration from Motiflab showing a c-Myb motif located at the start of the first intron of the gene FKBP5 that overlaps with a DNase I footprint with a high conservation value in K562 cells, illustration modified. The coordinates for the c-Myb footprint is shown above the illustration, and to the right are the signal intensity for the DNase I datasets, in addition to a conservation score. (D) The binding motif enriched in c-Myb footprints in K562 cells. (E) Graph of the presence of c-Myb footprints and the distances to the 100 most regulated genes upon KD in K562 cells (dots) or a selection of 100 random genes (squares), an average of ten repetitions. Zero base pair indicates that the c-Myb footprints are found inside the gene body (F) A c-Myb footprint at the TSS of GRSF1 gene mapped in all six cell-types analysed. Coordinates for c-Myb footprint are shown above, and to the left are the signal intensity for DNase I datasets. (G) Position of c-Myb footprints, and random selections of DNase I footprints and c-Myb motifs, respectively, around ENSEMBL annotated TSS in K562 cells. (H) Distribution of c-Myb footprints at annotated genes, promoters and intergenic regions in K562 cells. *Overlapping significantly higher with c-Myb footprints than expected by random sampling of K562 DNase I footprints (p' < 5x10-2).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133280.g001: Identification of c-Myb footprints.(A) Workflow for identification of c-Myb footprints. (B) A pie chart representing the number of c-Myb footprints identified compared to the total number of footprints in K562 cells. (C) An illustration from Motiflab showing a c-Myb motif located at the start of the first intron of the gene FKBP5 that overlaps with a DNase I footprint with a high conservation value in K562 cells, illustration modified. The coordinates for the c-Myb footprint is shown above the illustration, and to the right are the signal intensity for the DNase I datasets, in addition to a conservation score. (D) The binding motif enriched in c-Myb footprints in K562 cells. (E) Graph of the presence of c-Myb footprints and the distances to the 100 most regulated genes upon KD in K562 cells (dots) or a selection of 100 random genes (squares), an average of ten repetitions. Zero base pair indicates that the c-Myb footprints are found inside the gene body (F) A c-Myb footprint at the TSS of GRSF1 gene mapped in all six cell-types analysed. Coordinates for c-Myb footprint are shown above, and to the left are the signal intensity for DNase I datasets. (G) Position of c-Myb footprints, and random selections of DNase I footprints and c-Myb motifs, respectively, around ENSEMBL annotated TSS in K562 cells. (H) Distribution of c-Myb footprints at annotated genes, promoters and intergenic regions in K562 cells. *Overlapping significantly higher with c-Myb footprints than expected by random sampling of K562 DNase I footprints (p' < 5x10-2).
Mentions: DGF is a powerful method to identify nucleotides protected by proteins at a genome-wide scale independent of antibodies [29–32]. To map changes in c-Myb occupancy during haematopoiesis, we used DGF to generate maps of c-Myb footprints with nucleotide resolution (Fig 1A). We selected haematopoietic cell-types where c-Myb is expressed at different levels: c-Myb is highly expressed in haematopoietic stem cells [33] and expressed at lower level in CD4+ T-helper cells [34] and B cells [35,36]. c-Myb is also highly expressed in most cases of leukaemia [10]. We collected available DNase I footprint datasets in six different human cell-types from three healthy donors (CD34+ (mobilized), CD20+ and Th1 cells), transformed B-lymphocytes (GM12865) and two cancer cell-types where c-Myb is upregulated: erythroleukaemia (K562) and promyelocytic leukaemia (NB4) [31].

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