To further test the idea of functional overlap between BRD2 and BRD3, we examined erythroid maturation as reflected in hemoglobinization (red coloring) following GATA1 activation (Figure 6B). Similar to results in G1E cells, activation of erythroid gene expression was impaired by JQ1 whereas gene repression occurred normally (Figure 2E, supplemental Figure 7B). BET occupancy was not a strong predictor of JQ1 sensitivity overall, however, a weak relationship between JQ1 effects and BRD4 occupancy at promoters was observed (supplemental Figure 2B). Given the widespread expression and essential functions of BETs, it was initially surprising that BET inhibitors like JQ1 elicit cell- and gene-specific responses. Within the BET family, BRD2, BRD3, and BRD4 are ubiquitously expressed in mammalian tissues, whereas BRDT is testis-specific.
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We also noted induction of the transcriptional repressor HEXIM1 upon BET inhibition as has been observed in other cell types. Only 1 of 10 genes proximal to the top SEs identified declined by >50% upon BET inhibition (supplemental Figure 6C). We further used H3K27ac signal intensity to define SEs, and found that these were even less JQ1-sensitive than those defined using BRD4 signal. and BRD4 SEs have been suggested to reside near genes particularly sensitive to JQ1.
Gambling bets are required for efficient GATA1-dependent transcriptional activation but not repression
Insights into GATA-1-mediated gene activation versus repression via genome-wide chromatin occupancy analysis. Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line. Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1.
Moreover, forced expression of BRD3 partially restores defects associated with BRD2 loss, suggesting redundant functions among these 2 BETs. We further found that BETs are required not only for initial GATA1 chromatin occupancy, but also for subsequent transcription activation. Exposure of erythroid cells to BET inhibitors diminishes GATA1 occupancy at a subset of target genes and prevents their activation. JQ1 does not distinguish between BET family members, and the development of additional BET inhibitors with distinct specificities remains an important goal.
Many reports attribute the effects of BET inhibitors to BRD4 based on experiments in which individual BETs are depleted. This implies that the combined amounts of BRD2 and BRD3 may be functionally as relevant as the levels of the individual proteins or any potentially unique features of them. Interestingly, Brd3 mRNA is approximately fourfold less abundant than Brd2 mRNA according to RNA-seq data sets in G1E cells. A requirement for BRD3 was only uncovered in the context of BRD2 depletion, indicating at least partially overlapping functions among these proteins. Despite the strong effects of JQ1 on GATA1-dependent erythroid differentiation, no evidence suggests that BET inhibition causes anemia in animals or humans.
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However, transient shRNA-mediated depletion of BRD4 significantly decreased GATA1-induced gene expression (Figure 5C) supporting its importance in this process. However, the effects of BRD2 depletion were less pronounced than those observed with JQ1 treatment implicating additional BETs in GATA1-driven erythroid gene expression. Several GATA1 targets, including Hbb-b1 (Î²-globin), Klf1, and Nfe2, were immediately repressed upon BET inhibition, suggesting a role for transcription of BETs downstream of GATA1 occupancy (Figure 4B, supplemental Figure 9B). To evaluate the contribution of BETs to GATA1-induced gene expression changes genome-wide, we performed microarray analysis on G1E cells treated with 250 nM JQ1 or dimethylsulfoxide control concurrent with GATA1 activation for 24 hours in biological triplicate.
These results are consistent with BETs functioning principally in GATA1-mediated activation and having little role in repression. BET inhibition increased mRNA levels at some genes, which could be due to repressive functions of BETs or to indirect action. In contrast, transcripts that decreased upon GATA1 induction decreased no more or less on average with concurrent JQ1 treatment. genes most activated by GATA1 were the most sensitive to JQ1. A preponderance of genes were most highly expressed in untreated G1E cells and declined slightly upon JQ1 treatment.
For validation, we also examined signals obtained with ectopic HA-tagged forms of BRD2 and BRD3. We quantified this by calculating ChIP-seq signal density for each BET at GATA1 sites and defining occupancy as a read density >2 standard deviations beyond occupancy at random genomic regions. In addition, ChIP-qPCR of exogenous HA-tagged BRD2 produced similar results (supplemental Figure 1B). suggested BRD3 and BRD4 were the only BETs expressed in G1E cells, recent genome-wide data 36,52 We previously demonstrated that BRD3 and BRD4 directly interact with acetylated GATA1, and BRD3 is recruited to several GATA1-binding sites in a GATA1-dependent manner.
This was confirmed by initial ChIP-qPCR experiments showing JQ1-sensitive BRD2 recruitment to a subset of the GATA1 occupied site (OS) (supplemental Figure 1A). G1E cells recapitulate normal terminal erythroid maturation as verified by gene expression analyses and GATA1 occupancy profiles. 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).
Yet the two books appear to be sufficiently different in spirit and subject matter to justify the publication of this manuscript; in particular, the present book includes a discussion of topological tensor products, nuclear spaces, ordered topological vector spaces, and an appendix on positive operators. It is shown that the predual of the signed weight space of a tensor product of discrete manuals is the projective (ordered) tensor product of the preduals of the signed weight spaces of the factors. A compactness condition due to Cookâ€”here calleddiscretenessâ€”is discussed and shown to be preserved under the formation of tensor products.
Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition. Formation of a tissue-specific histone acetylation pattern by the hematopoietic transcription factor GATA-1.
Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma. Brd4 and JMJD6-associated anti-pause enhancers in regulation of transcriptional pause release. Brd2 disruption in mice causes severe obesity without type 2 diabetes. Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4.
(D) Quantification of mitochondrial area over total cell area measured from multiple HGC-27 cells transfected with EGFPâ€“RAB26, EGFPâ€“RAB26T77N, EGFPâ€“RAB26Q123L and EGFP. (E) Pearson’s correlation coefficient quantification of pixel-to-pixel colocalization of multiple cells transfected with EGFPâ€“RAB26, EGFPâ€“RAB26T77N, EGFPâ€“RAB26Q123L and GFP with LAMP1 lysosomes. (E) Density gradient cell fractionation experiment with numbered density fractions analyzed for lysosomes with anti-LAMP1 antibody, EGFP-tagged RAB26 by both anti-GFP and anti-RAB26 antibodies (middle and lower panel), and endogenous RAB26 by anti-RAB26 immunoblotting (lower panel). (C) Lower magnification epifluorescence images of HGC-27 cells with more representative, low-level endogenous RAB26 (green) and LAMP1 (red). (B) Confocal immunomicroscopy of endogenous RAB26 (green) and LAMP1 (red) (N, nucleus).