“Accurate and fiducial-marker-free correction for three-dimensional chromatic shift in biological fluorescence microscopy.” Sci Rep. “SPEKcheck – fluorescence microscopy spectral visualisation and optimisation: a web application, javascript library, and data resource.” Wellcome Open Res. “Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy.” Proceedings of the National Academy of Sciences Feb 2019, 201810690; DOI:10.1073/pnas.1810690116 An epigenetic mechanism of resistance to targeted therapy in T cell acute lymphoblastic leukemia. BET bromodomain inhibitors block growth of pancreatic cancer cells in three-dimensional collagen.

The overwhelming predominance of gene repression upon GATA1 induction contrasts with prior studies of GATA1-mediated transcriptome changes that were based on internal standards and concluded that the number of activated and repressed genes are similar. 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. Pharmacologic inhibition of BETs impaired activation of several GATA1 target genes.

The mere presence of BETs at a given gene does not predict JQ1 response. In addition, other acetylated transcription factors might contribute to BET recruitment. BET-binding patterns are likely determined by association not only with acetylated GATA1 but also histone acetylation, which increases at many sites upon GATA1 activation. BRD3 is recruited to nearly all GATA1 sites whereas BRD4 occupies approximately one-half.

BETs are required for successful GATA1-dependent transcriptional activation nevertheless not repression

These observations do not support categorization of SEs as distinct JQ1-hypersensitive entities. Genes adjacent or overlapped by SEs were only minimally more JQ1-sensitive than all expressed genes. Both BRD4 and H3K27ac have been used to identify “superenhancers” (SEs), 29,56 This is consistent with observations that not all BET-occupied genes respond to BET inhibitors. We further confirmed this at known GATA-target genes in independent experiments by RT-qPCR (Figure 2D).

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. BETs promote GATA1 chromatin occupancy and subsequently activate transcription; they are generally not required for repression.

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.

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.

Mammalian polycomb-like Pcl2/Mtf2 is a novel regulatory component of PRC2 that can differentially modulate polycomb activity both at the Hox gene cluster and at Cdkn2a genes. Disruption of a conserved region of Xist exon 1 impairs Xist RNA localisation and X-linked gene silencing during random and imprinted X chromosome inactivation.

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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.

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To dissect the roles of individual BETs in GATA1-driven erythropoiesis, we used a loss-of-function approach combining CRISPR-Cas9-engineered gene disruption 61 Current knowledge of BET function is largely built on studies using inhibitors that do not distinguish between individual BETs. *P < .05="" comparing="" jq1="" treated="" and="" control="" samples="" (2-sample="" t="" test).="" (b)="" primary="" transcript="" rt-qpcr="" of="" indicated="" transcripts="" following="" jq1="" treatment="" in="" gata1-induced="">

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 (β-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.

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