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Blood First Edition Paper, prepublished online February 18, 2015; DOI 10.1182/blood-2014-10-607309
Article title: Functions of BET proteins in erythroid gene expression Short title: BET proteins in erythroid gene expression
Authors and affiliations: Aaron J. Stonestrom12, Sarah C. Hsu12, Kristen S. Jahn1, Peng Huang1, Cheryl A. Keller3, Belinda M. Giardine3, Stephan Kadauke12, Amy E. Campbell12, Perry Evans1, Ross C. Hardison3, and Gerd A. Blobel12*
1
Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia PA
2
Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA
3
Department of Biochemistry and Molecular Biology, Pennsylvania State University,
University Park, PA *
Correspondence: Gerd A. Blobel, Children’s Hospital of Philadelphia, Abramson 316H,
3615 Civic Center, Philadelphia, PA 19104-4318 email:[email protected], phone:215-590-3988, fax:215-590-4834.
Scientific category: Red Cells, Iron, and Erythropoiesis
1
Copyright © 2015 American Society of Hematology
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Key Points (1) BETs promote GATA1 chromatin occupancy and subsequently activate transcription. They are generally not required for repression. (2) BRD2 and BRD4 are essential for full GATA1 activity while BRD3 function overlaps with BRD2. Abstract Inhibitors of Bromodomain and Extra-Terminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1 regulated genes, with BRD3 binding the greatest number of GATA1 occupied
sites.
Pharmacologic
BET
inhibition
impaired
GATA1-mediated
transcriptional activation, but not repression, genome-wide. Mechanistically, BETs both promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR/CAS9-mediated genomic engineering and shRNA approaches we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.
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Introduction The mammalian Bromodomain and Extra-Terminal motif proteins (BETs) have drawn widespread interest as pharmacologic targets for the treatment of various diseases, including hematological malignancies and solid tumors1-4. Within the BET family BRD2, BRD3 and BRD4 are ubiquitously expressed in mammalian tissues, while BRDT is testis-specific. BETs contain two tandem bromodomains that mediate association with chromatin by binding to acetylated histones and transcription factors5-9. BETs function in regulatory complexes that impact mRNA production at multiple steps of the transcription cycle, such as modifying and remodeling chromatin and promoting transcription elongation 10-17. Both BRD2 and BRD4 are essential for normal development18-20. A BRD3 knockout mouse has not been reported. Promising results obtained with pharmacologic BET inhibitors in animal models of malignancy have sparked clinical trials and intensified efforts to better understand BET function 1,2,4,21. Given the widespread expression and essential functions of BETs, it was initially surprising that BET inhibitors like JQ1 elicit celland gene-specific responses. These inhibitors block the acetyl-lysine binding pockets specifically of BET family bromodomains triggering their release from acetylated lysine residues on histones and transcription factors16,22,23. JQ1 does not distinguish between BET family members, and the development of additional BET inhibitors with distinct specificities remains an important goal22,24,25. Functional similarity among BETs is suggested by strict conservation of their bromodomains, association with many of the same regulatory complexes16, and overlapping
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genomic binding profiles 26,27. Additionally, chromosomal translocation of either BRD3 or BRD4 with NUT cause histopathologically indistinguishable carcinoma 28. Despite these shared characteristics, distinct phenotypes result from depletion of individual BET family members4,18-20,27,29-34. The molecular basis for functional distinctions between BETs remains unclear, and large gaps remain in our understanding of their individual roles. Erythroid maturation is a developmental process driven in part by the erythroid master transcription factor GATA1, which activates essentially all erythroid-specific genes and silences genes associated with the immature proliferative state35,36. Mice lacking GATA1 die in utero due to failure to form mature erythroid cells37, and several types of congenital anemias in humans are associated with GATA1 mutations38,39. GATA1 is acetylated near its zinc finger DNA binding domain40, and mutations of acetylated lysines impair the ability of GATA1 to associate with chromatin in vivo41. Both BRD3 and BRD4 bind to acetylated GATA1, and chromatin immunoprecipitation (ChIP) studies suggest BRD3 in particular is present at most GATA1-occupied sites6. Exposure of erythroid cells to BET inhibitors diminishes GATA1 occupancy at a subset of target genes and prevents their activation6. However, the roles of individual BETs in GATA1 function have not been directly evaluated. Here we defined distinct mechanisms through which BETs support GATA1regulated gene expression and direct erythroid maturation. We characterized the relationship of BETs with GATA1 on a genome-wide scale and demonstrate that BETs facilitate GATA1-mediated transcriptional activation but are largely
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dispensable for repression. We further found that BETs are required not only for initial GATA1 chromatin occupancy, but also for subsequent transcription activation. GATA1-induced erythroid maturation is highly sensitive to reduced levels of BRD2 or BRD4. Unexpectedly, despite the presence of BRD3 at the great majority of GATA1 occupied sites, BRD3 is not required for normal GATA1activated transcription. However, BRD3 deficiency exacerbates transcriptional defects associated with BRD2 loss. Moreover, forced expression of BRD3 partially restores defects associated with BRD2 loss, suggesting redundant functions among these two BETs. Together these studies reveal that BETs have overlapping roles, and function at distinct steps of the transcription program controlled by GATA1, which are important considerations when interpreting the functions of chemical BET inhibitors.
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Materials and methods Cell culture Culture of GATA1- erythroblast (G1E) cells and G1E cells expressing a conditionallyactive estrogen receptor-GATA1 fusion protein has been described42. GATA1 was activated in G1E GATA1-ER by addition of 100nM estradiol for 24 hours (denoted +GATA1). Retroviral creation and infection was performed as described43. shRNA hairpins were cloned into the vector LMP(Open Biosystems). Supplemental methods contain hairpin sequences. Chromatin immunoprecipitation (ChIP) ChIP was performed as described44. Antibodies were: GATA1 (sc265-N6; Santa Cruz), BRD2 (A302-583A; Bethyl), BRD3 sera6, BRD4 (A301-985A; Bethyl), HA (12CA5). Quantitative PCR (qPCR) was run on ViiA7 System (Life Technologies) using Power SYBR Green (Invitrogen). Supplementary methods contain primers. High-throughput sequencing was performed on an Illumina Hi-seq2000 as described36. Reads were mapped to mouse genome assembly mm9. Analysis was performed using Bioconductor45, bedtools46, and the Cistrome47 Galaxy48 analysis platform. mRNA expression analyses RNA was isolated using Trizol (Life Technologies). Reverse transcriptase (RT)-qPCR was performed with iScript (BIO-RAD) and Power SYBR Green (Invitrogen). Supplementary methods contain primers. In microarrays, ERCC RNA Spike-In Mix (Ambion #4456740) was added to Trizol-homogenized samples in proportion to cell number. Mouse Gene 2.0ST arrays (Affymetrix) were hybridized per manufacturer’s
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protocols. Analysis was performed using the Robust Multi-array Analysis (RMA) method and normalized to spike-in controls49. Cas9-mediated gene knockout CAS9/GFP and guide RNA/mCherry plasmids were transiently co-transfected into G1E cells using an Amaxa II electroporator (Lonza) with program G-016 and Kit R. Single transfected cells were sorted into individual wells in a 96-well plate using a FACSAria II (BD Biosciences), expanded, and screened by DNA sequencing and immunoblot. Supplementary methods list guide RNAs. Public data access ChIP-seq and microarray data are accessible via Gene Expression Omnibus50 #GSE62737.
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Results BRD2, BRD3, and BRD4 recruitment to GATA1 activated genes We previously demonstrated that BRD3 and BRD4 directly interact with acetylated GATA1, and BRD3 is recruited to several GATA1 binding sites in a GATA1dependent manner6. To comprehensively define the role of BETs in erythroid maturation, we performed chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) for BRD2, BRD3 and BRD4 in GATA1-null erythroblast (G1E) cells in the absence and presence of an activated estradiol-inducible fusion of GATA1 to the estrogen receptor (GATA1-ER)42. G1E cells recapitulate normal terminal erythroid maturation as verified by gene expression analyses and GATA1 occupancy profiles35,51. Although we had previously generated a BRD3 ChIP-seq data set 6, we repeated this experiment to improve sequencing depth and compare BET profiles generated on the same platform. BRD2 was included in the present study as well, since although early microarray data35 suggested BRD3 and BRD4 were the only BETs expressed in G1E cells, recent genome-wide data36,52 indicated BRD2 was also expressed. This was confirmed by initial ChIP-qPCR experiments showing JQ1-sensitive BRD2 recruitment to a subset of GATA1 OS, (Figure S1A). In addition, ChIP-qPCR of exogenous HA-tagged BRD2 produced similar results (Figure S1B). When extended to a genome-wide scale (ChIP-seq) we found that BRD2, BRD3, and BRD4 occupied GATA1-bound loci, including the β -globin locus Hbb in a GATA1-dependent manner (Figure 1A, S2). Patterns of BET association with DNA did not fall into discrete peaks, but instead appear to be broader than proteins that bind directly to DNA. Given these patterns, we focused our analysis on
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quantitative description of signal intensity at particular sites rather than analysis based on peak calling. In contrast, the sharp binding profiles of GATA1 across the genome readily lent themselves to peak calling using MACS. We generated a set of high-confidence GATA1 occupied sites (OSs) by intersecting GATA1 peaks called in two independent GATA1 ChIP-seq experiments, resulting in 5259 peaks. BRD3 was present at the greatest number of GATA1 OS, with BRD4 and BRD2 being less frequently associated with GATA1 OS (Figure 1B). We quantified this by calculating ChIP-seq signal density for each BET at GATA1 sites and defining occupancy as a read density greater than two standard deviations beyond occupancy at random genomic regions. By this definition, BRD3 was present at 74% of GATA1 sites, BRD4 at 53%, and BRD2 at 27%. For validation, we also examined signals obtained with ectopic HA-tagged forms of BRD2 and BRD3. While overexpression led to a broadening of the peaks, the results are overall very similar (Figure S2, S3A). Interestingly, when ranked by H3K27ac (Dogan et al. submitted), BRD4 occupancy correlated strongly with H3K27ac in the vicinity of GATA1 OS (Figure S3B). However, the zenith of BRD4 signal localized directly over GATA1 peaks between the twin peaks of maximal H3K27ac, a pattern that likely reflects acetylated nucleosomes flanking GATA1 binding sites. GATA1 OS with high H3K27ac were more likely to be at gene promoters and more likely to be co-occupied by TAL1 (Figure S3C). We subsequently divided BET binding at GATA1 sites as binding at candidate enhancers and promoters (Figure 1C). BRD2 signal was higher at GATA1 sites than background sites, but had a similar distribution in the absence and presence of GATA1.
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In contrast, BRD3 occupancy increased dramatically at both GATA1 bound promoters and enhancers upon GATA1 activation. BRD4 occupancy at GATA1 OS was substantial in the absence of GATA1, and BRD4 signal increased further at these sites following GATA1 activation. These occupancy patterns suggest that BRD3 recruitment is to a large extent influenced by GATA1 whereas BRD2, and BRD4 recruitment are regulated by additional factors.
BETs are required for efficient GATA1-dependent transcriptional activation but not repression Pharmacologic inhibition of BETs impaired activation of several GATA1-target genes6. To evaluate the contribution of BETs to GATA1-induced gene expression changes genome wide, we performed microarray analysis on G1E cells treated with 250nM JQ1 or DMSO control concurrent with GATA1 activation or in its absence for 24 hours in biological triplicate. As dramatic alterations in cell size and RNA content occur during erythroid maturation, we added external spike-in RNA controls to each sample in proportion to cell number for normalization 49,53. Focusing first on GATA1’s impact on gene expression, we plotted all transcripts from most repressed to most activated (Figure 2A). Following GATA1 addition 5,094 transcripts decreased while only 220 increased using stringent differential expression criteria (2fold change and Bonferroni-corrected p75% retained 0-‐25%
0! 0
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Figure 6 - Stonestrom et al. !
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Prepublished online February 18, 2015; doi:10.1182/blood-2014-10-607309
Functions of BET proteins in erythroid gene expression Aaron J. Stonestrom, Sarah C. Hsu, Kristen S. Jahn, Peng Huang, Cheryl A. Keller, Belinda M. Giardine, Stephan Kadauke, Amy E. Campbell, Perry Evans, Ross C. Hardison and Gerd A. Blobel
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Blood First Edition Paper, prepublished online February 18, 2015; DOI 10.1182/blood-2014-10-607309
Article title: Functions of BET proteins in erythroid gene expression Short title: BET proteins in erythroid gene expression
Authors and affiliations: Aaron J. Stonestrom12, Sarah C. Hsu12, Kristen S. Jahn1, Peng Huang1, Cheryl A. Keller3, Belinda M. Giardine3, Stephan Kadauke12, Amy E. Campbell12, Perry Evans1, Ross C. Hardison3, and Gerd A. Blobel12*
1
Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia PA
2
Perelman School of Medicine at the University of Pennsylvania, Philadelphia PA
3
Department of Biochemistry and Molecular Biology, Pennsylvania State University,
University Park, PA *
Correspondence: Gerd A. Blobel, Children’s Hospital of Philadelphia, Abramson 316H,
3615 Civic Center, Philadelphia, PA 19104-4318 email:[email protected], phone:215-590-3988, fax:215-590-4834.
Scientific category: Red Cells, Iron, and Erythropoiesis
1
Copyright © 2015 American Society of Hematology
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Key Points (1) BETs promote GATA1 chromatin occupancy and subsequently activate transcription. They are generally not required for repression. (2) BRD2 and BRD4 are essential for full GATA1 activity while BRD3 function overlaps with BRD2. Abstract Inhibitors of Bromodomain and Extra-Terminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1 regulated genes, with BRD3 binding the greatest number of GATA1 occupied
sites.
Pharmacologic
BET
inhibition
impaired
GATA1-mediated
transcriptional activation, but not repression, genome-wide. Mechanistically, BETs both promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR/CAS9-mediated genomic engineering and shRNA approaches we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.
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Introduction The mammalian Bromodomain and Extra-Terminal motif proteins (BETs) have drawn widespread interest as pharmacologic targets for the treatment of various diseases, including hematological malignancies and solid tumors1-4. Within the BET family BRD2, BRD3 and BRD4 are ubiquitously expressed in mammalian tissues, while BRDT is testis-specific. BETs contain two tandem bromodomains that mediate association with chromatin by binding to acetylated histones and transcription factors5-9. BETs function in regulatory complexes that impact mRNA production at multiple steps of the transcription cycle, such as modifying and remodeling chromatin and promoting transcription elongation 10-17. Both BRD2 and BRD4 are essential for normal development18-20. A BRD3 knockout mouse has not been reported. Promising results obtained with pharmacologic BET inhibitors in animal models of malignancy have sparked clinical trials and intensified efforts to better understand BET function 1,2,4,21. Given the widespread expression and essential functions of BETs, it was initially surprising that BET inhibitors like JQ1 elicit celland gene-specific responses. These inhibitors block the acetyl-lysine binding pockets specifically of BET family bromodomains triggering their release from acetylated lysine residues on histones and transcription factors16,22,23. JQ1 does not distinguish between BET family members, and the development of additional BET inhibitors with distinct specificities remains an important goal22,24,25. Functional similarity among BETs is suggested by strict conservation of their bromodomains, association with many of the same regulatory complexes16, and overlapping
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genomic binding profiles 26,27. Additionally, chromosomal translocation of either BRD3 or BRD4 with NUT cause histopathologically indistinguishable carcinoma 28. Despite these shared characteristics, distinct phenotypes result from depletion of individual BET family members4,18-20,27,29-34. The molecular basis for functional distinctions between BETs remains unclear, and large gaps remain in our understanding of their individual roles. Erythroid maturation is a developmental process driven in part by the erythroid master transcription factor GATA1, which activates essentially all erythroid-specific genes and silences genes associated with the immature proliferative state35,36. Mice lacking GATA1 die in utero due to failure to form mature erythroid cells37, and several types of congenital anemias in humans are associated with GATA1 mutations38,39. GATA1 is acetylated near its zinc finger DNA binding domain40, and mutations of acetylated lysines impair the ability of GATA1 to associate with chromatin in vivo41. Both BRD3 and BRD4 bind to acetylated GATA1, and chromatin immunoprecipitation (ChIP) studies suggest BRD3 in particular is present at most GATA1-occupied sites6. Exposure of erythroid cells to BET inhibitors diminishes GATA1 occupancy at a subset of target genes and prevents their activation6. However, the roles of individual BETs in GATA1 function have not been directly evaluated. Here we defined distinct mechanisms through which BETs support GATA1regulated gene expression and direct erythroid maturation. We characterized the relationship of BETs with GATA1 on a genome-wide scale and demonstrate that BETs facilitate GATA1-mediated transcriptional activation but are largely
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dispensable for repression. We further found that BETs are required not only for initial GATA1 chromatin occupancy, but also for subsequent transcription activation. GATA1-induced erythroid maturation is highly sensitive to reduced levels of BRD2 or BRD4. Unexpectedly, despite the presence of BRD3 at the great majority of GATA1 occupied sites, BRD3 is not required for normal GATA1activated transcription. However, BRD3 deficiency exacerbates transcriptional defects associated with BRD2 loss. Moreover, forced expression of BRD3 partially restores defects associated with BRD2 loss, suggesting redundant functions among these two BETs. Together these studies reveal that BETs have overlapping roles, and function at distinct steps of the transcription program controlled by GATA1, which are important considerations when interpreting the functions of chemical BET inhibitors.
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Materials and methods Cell culture Culture of GATA1- erythroblast (G1E) cells and G1E cells expressing a conditionallyactive estrogen receptor-GATA1 fusion protein has been described42. GATA1 was activated in G1E GATA1-ER by addition of 100nM estradiol for 24 hours (denoted +GATA1). Retroviral creation and infection was performed as described43. shRNA hairpins were cloned into the vector LMP(Open Biosystems). Supplemental methods contain hairpin sequences. Chromatin immunoprecipitation (ChIP) ChIP was performed as described44. Antibodies were: GATA1 (sc265-N6; Santa Cruz), BRD2 (A302-583A; Bethyl), BRD3 sera6, BRD4 (A301-985A; Bethyl), HA (12CA5). Quantitative PCR (qPCR) was run on ViiA7 System (Life Technologies) using Power SYBR Green (Invitrogen). Supplementary methods contain primers. High-throughput sequencing was performed on an Illumina Hi-seq2000 as described36. Reads were mapped to mouse genome assembly mm9. Analysis was performed using Bioconductor45, bedtools46, and the Cistrome47 Galaxy48 analysis platform. mRNA expression analyses RNA was isolated using Trizol (Life Technologies). Reverse transcriptase (RT)-qPCR was performed with iScript (BIO-RAD) and Power SYBR Green (Invitrogen). Supplementary methods contain primers. In microarrays, ERCC RNA Spike-In Mix (Ambion #4456740) was added to Trizol-homogenized samples in proportion to cell number. Mouse Gene 2.0ST arrays (Affymetrix) were hybridized per manufacturer’s
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protocols. Analysis was performed using the Robust Multi-array Analysis (RMA) method and normalized to spike-in controls49. Cas9-mediated gene knockout CAS9/GFP and guide RNA/mCherry plasmids were transiently co-transfected into G1E cells using an Amaxa II electroporator (Lonza) with program G-016 and Kit R. Single transfected cells were sorted into individual wells in a 96-well plate using a FACSAria II (BD Biosciences), expanded, and screened by DNA sequencing and immunoblot. Supplementary methods list guide RNAs. Public data access ChIP-seq and microarray data are accessible via Gene Expression Omnibus50 #GSE62737.
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Results BRD2, BRD3, and BRD4 recruitment to GATA1 activated genes We previously demonstrated that BRD3 and BRD4 directly interact with acetylated GATA1, and BRD3 is recruited to several GATA1 binding sites in a GATA1dependent manner6. To comprehensively define the role of BETs in erythroid maturation, we performed chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) for BRD2, BRD3 and BRD4 in GATA1-null erythroblast (G1E) cells in the absence and presence of an activated estradiol-inducible fusion of GATA1 to the estrogen receptor (GATA1-ER)42. G1E cells recapitulate normal terminal erythroid maturation as verified by gene expression analyses and GATA1 occupancy profiles35,51. Although we had previously generated a BRD3 ChIP-seq data set 6, we repeated this experiment to improve sequencing depth and compare BET profiles generated on the same platform. BRD2 was included in the present study as well, since although early microarray data35 suggested BRD3 and BRD4 were the only BETs expressed in G1E cells, recent genome-wide data36,52 indicated BRD2 was also expressed. This was confirmed by initial ChIP-qPCR experiments showing JQ1-sensitive BRD2 recruitment to a subset of GATA1 OS, (Figure S1A). In addition, ChIP-qPCR of exogenous HA-tagged BRD2 produced similar results (Figure S1B). When extended to a genome-wide scale (ChIP-seq) we found that BRD2, BRD3, and BRD4 occupied GATA1-bound loci, including the β -globin locus Hbb in a GATA1-dependent manner (Figure 1A, S2). Patterns of BET association with DNA did not fall into discrete peaks, but instead appear to be broader than proteins that bind directly to DNA. Given these patterns, we focused our analysis on
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quantitative description of signal intensity at particular sites rather than analysis based on peak calling. In contrast, the sharp binding profiles of GATA1 across the genome readily lent themselves to peak calling using MACS. We generated a set of high-confidence GATA1 occupied sites (OSs) by intersecting GATA1 peaks called in two independent GATA1 ChIP-seq experiments, resulting in 5259 peaks. BRD3 was present at the greatest number of GATA1 OS, with BRD4 and BRD2 being less frequently associated with GATA1 OS (Figure 1B). We quantified this by calculating ChIP-seq signal density for each BET at GATA1 sites and defining occupancy as a read density greater than two standard deviations beyond occupancy at random genomic regions. By this definition, BRD3 was present at 74% of GATA1 sites, BRD4 at 53%, and BRD2 at 27%. For validation, we also examined signals obtained with ectopic HA-tagged forms of BRD2 and BRD3. While overexpression led to a broadening of the peaks, the results are overall very similar (Figure S2, S3A). Interestingly, when ranked by H3K27ac (Dogan et al. submitted), BRD4 occupancy correlated strongly with H3K27ac in the vicinity of GATA1 OS (Figure S3B). However, the zenith of BRD4 signal localized directly over GATA1 peaks between the twin peaks of maximal H3K27ac, a pattern that likely reflects acetylated nucleosomes flanking GATA1 binding sites. GATA1 OS with high H3K27ac were more likely to be at gene promoters and more likely to be co-occupied by TAL1 (Figure S3C). We subsequently divided BET binding at GATA1 sites as binding at candidate enhancers and promoters (Figure 1C). BRD2 signal was higher at GATA1 sites than background sites, but had a similar distribution in the absence and presence of GATA1.
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In contrast, BRD3 occupancy increased dramatically at both GATA1 bound promoters and enhancers upon GATA1 activation. BRD4 occupancy at GATA1 OS was substantial in the absence of GATA1, and BRD4 signal increased further at these sites following GATA1 activation. These occupancy patterns suggest that BRD3 recruitment is to a large extent influenced by GATA1 whereas BRD2, and BRD4 recruitment are regulated by additional factors.
BETs are required for efficient GATA1-dependent transcriptional activation but not repression Pharmacologic inhibition of BETs impaired activation of several GATA1-target genes6. To evaluate the contribution of BETs to GATA1-induced gene expression changes genome wide, we performed microarray analysis on G1E cells treated with 250nM JQ1 or DMSO control concurrent with GATA1 activation or in its absence for 24 hours in biological triplicate. As dramatic alterations in cell size and RNA content occur during erythroid maturation, we added external spike-in RNA controls to each sample in proportion to cell number for normalization 49,53. Focusing first on GATA1’s impact on gene expression, we plotted all transcripts from most repressed to most activated (Figure 2A). Following GATA1 addition 5,094 transcripts decreased while only 220 increased using stringent differential expression criteria (2fold change and Bonferroni-corrected p75% retained 0-‐25%
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JC4JC4 HA−Brd2 D HA−Brd2 JC4JC4 HA−Brd3 D HA−Brd3 DJC4 HA−Brd2 D HA−Brd3 D JC4 DJC4 DJC4 HA−Brd2 D HA−Brd2 HA−Brd2 JC4 HA−Brd3 D HA−Brd3 HA−Brd3
cells cells cells
JC4JC4 HA−Brd2 D HA−Brd2 JC4JC4 HA−Brd3 D HA−Brd3 DJC4 HA−Brd2 D HA−Brd3 D JC4 DJC4 DJC4 HA−Brd2 D HA−Brd2 HA−Brd2 JC4 HA−Brd3 D HA−Brd3 HA−Brd3
Figure 6 - Stonestrom et al. !
From www.bloodjournal.org by guest on June 7, 2015. For personal use only.
Prepublished online February 18, 2015; doi:10.1182/blood-2014-10-607309
Functions of BET proteins in erythroid gene expression Aaron J. Stonestrom, Sarah C. Hsu, Kristen S. Jahn, Peng Huang, Cheryl A. Keller, Belinda M. Giardine, Stephan Kadauke, Amy E. Campbell, Perry Evans, Ross C. Hardison and Gerd A. Blobel
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