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.
BET inhibition suppressed surface expression of the erythroid maturation marker TER-119, but had no overt toxic effect (supplemental Figure 7A). We measured gene expression in mouse fetal liver erythroid progenitors differentiated in the presence or absence of JQ1. In contrast, activation by GATA1 strongly predicted JQ1 responsiveness (supplemental Figure 6B). We next tested the degree to which BET occupancy determines JQ1 sensitivity.
Consistent with phenotypic results, BRD3 overexpression also rescued BRD2 deficiency at most, but not all, genes examined (Figure 6C, supplemental Figure 10B). Retroviral BRD2 expression restored this defect, confirming specificity of BRD2 gene targeting. As expected, BRD3 knockdown on its own had no significant impact on gene activation.
These occupancy patterns suggest that BRD3 recruitment is to a large extent influenced by GATA1 whereas BRD2 and BRD4 recruitment is regulated by additional factors. BRD3 was present at the greatest number of GATA1 OSs, with BRD4 and BRD2 being less frequently associated with GATA1 OS (Figure 1B). When extended to a genome-wide scale (ChIP-seq), we found that BRD2, BRD3, and BRD4 occupied GATA1-bound loci, including the Î²-globin locus Hbb in a GATA1-dependent manner (Figure 1A, supplemental Figure 2). Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency.
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.
(B) Confocal section of EGFPâ€“RAB26-expressing cell (green) stained for lysosomes (anti-LAMP1 antibody, red) and mitochondria (MitoTracker, purple). (D) Confocal fluorescence microscopy of the localization of EGFPâ€“RAB26Q123L (green) in transfected HGC-27 cells immunostained with anti-LAMP1 antibody (red). (C) Confocal fluorescence microscopy of the distribution of EGFPâ€“RAB26T77N (green) in transfected HGC-27 cells immunostained with anti-LAMP1 antibody (red). (C) Epifluorescence microscopy of HGC-27 cells co-transfected with EGFPâ€“RAB26 (green) and RFP-tagged cathepsin D (CTSDâ€“RFP, red).
To this end, we used shRNAs to deplete BRD3 in BRD2-replete and BRD2-deficient cells (Figure 6A, supplemental Figure 10A). These results suggest BRD2 and BRD4 are individually required for normal GATA1-mediated transcriptional activation. As BRD3 occupies nearly all GATA1 OSs, we had speculated that it was the most relevant BET in GATA1-mediated transcription. In contrast, transcription of other genes like Hba-a1 (Î±-globin) and Uros (involved in heme synthesis) were unperturbed by short-term JQ1 treatment despite their sensitivity to long-term JQ1 exposure and proximity to BET-bound regulatory elements. Indeed, JQ1 treatment of 1 hour removed BETs from all sites examined with relatively little effect on GATA1 occupancy (Figure 4A, supplemental Figure 9A).
- The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo.
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- , the Wittstock injective tensor product of â„“Ï† with a Banach lattice X, has the Radon-Nikodym property if and only if both â„“Ï† and X have the Radon-Nikodym property and each positive continuous linear operator from hÏ†* to X is compact.
- Right panels show epifluorescence microscopy of EGFPâ€“RAB26T77N (green) co-stained for LAMP1 (red).
These inhibitors block the acetyl-lysineâ€“binding pockets specifically of BET family bromodomains triggering their release from acetylated lysine residues on histones and transcription factors. 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. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. BRD2 and BRD4 are essential for full GATA1 activity whereas BRD3 function overlaps with BRD2.
Double bromodomain-containing gene Brd2 is essential for embryonic development in mouse. The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription.
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.
BRD4 is an atypical kinase that phosphorylates serine2 of the RNA polymerase II carboxy-terminal domain. SCL and associated proteins distinguish active from repressive GATA transcription factor complexes. CREB-binding protein acetylates hematopoietic transcription factor GATA-1 at functionally important sites.
Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Brd4 is required for recovery from antimicrotubule drug-induced mitotic arrest: preservation of acetylated chromatin. The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. This work was supported by National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases grants R01-DK054937 (G.A.B.), R56-DK065806 (R.C.H., G.A.B), and T32-DK007780 (A.J.S.), National Cancer Institute grant F30-CA189553 (S.C.H.), and a generous donation by the DiGaetano family (G.A.B., P.E.).
Conformational switching of the pseudokinase domain promotes human MLKL tetramerization and cell death by necroptosis Here the authors report PARP2 activation mechanisms and its role in the formation of branched poly(ADP-ribose) chains in response to DNA damage. PARP1 and PARP2 of the PARP family enzymes are involved in DNA damage response. Here the authors use evolutionary alignments of NRPS/PKS gene clusters to guide rational design of complexes that can produce novel lactones.