Genome-wide localization of small molecules

Anders, Lars; Guenther, Matthew G.; Qi, Jun; Fan, Zi Peng; Marineau, Jason; Rahl, Peter B.; Lovén, Jakob; Sigova, Alla A.; Smith, William B.; Lee, Tong Ihn; Bradner, James E.; Young, Richard A.

doi:10.1038/nbt.2776

Cited by 161 publications

(174 citation statements)

References 44 publications

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“…In this case, we relied on previous structureactivity data to guide the design of an IntC-JQ1 construct in which the probe was predicted to retain activity (28). Specifically, the free-acid form of JQ1 was attached via a flexible hydrophilic linker to a cargo peptide bearing a reactive cysteine (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Liszczak

Brown

Kim

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

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Section: Resultsmentioning

confidence: 99%

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Liszczak

Brown

Kim

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…22,24,25 Functional similarity among BETs is suggested by strict conservation of their bromodomains, association with many of the same regulatory complexes, 16 and overlapping genomic-binding profiles. 26,27 Additionally, chromosomal translocation of either BRD3 or BRD4 with NUT causes histopathologically indistinguishable carcinoma.28 Despite these shared characteristics, distinct phenotypes result from depletion of individual BET family members. 4,[18][19][20]27,[29][30][31][32][33][34] The molecular basis for functional distinctions between BETs remains unclear, and large gaps remain in our understanding of their individual roles.…”

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confidence: 99%

“…This is consistent with observations that not all BET-occupied genes respond to BET inhibitors. 22,26,29,32 Both BRD4 and H3K27ac have been used to identify "superenhancers" (SEs), 29,56 and BRD4 SEs have been suggested to reside near genes particularly sensitive to JQ1. We defined SEs using the script ROSE 29 which aggregates enhancer regions within 12.5 kb (supplemental Figure 6A).…”

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confidence: 99%

Functions of BET proteins in erythroid gene expression

Stonestrom

Hsu

Jahn

et al. 2015

Blood

109

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• BETs promote GATA1 chromatin occupancy and subsequently activate transcription; they are generally not required for repression.• BRD2 and BRD4 are essential for full GATA1 activity whereas BRD3 function overlaps with BRD2.Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about 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 promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPRCas9-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. (Blood. 2015;125(18):2825-2834 IntroductionThe mammalian bromodomain and extraterminal motif proteins (BETs) have drawn widespread interest as pharmacologic targets for the treatment of various diseases, including hematologic malignancies and solid tumors. [1][2][3][4] Within the BET family, BRD2, BRD3, and BRD4 are ubiquitously expressed in mammalian tissues, whereas BRDT is testis-specific. BETs contain 2 tandem bromodomains that mediate association with chromatin by binding to acetylated histones and transcription factors. [5][6][7][8][9] BETs function in regulatory complexes that impact messenger RNA (mRNA) production at multiple steps of the transcription cycle, such as modifying and remodeling chromatin and promoting transcription elongation. [10][11][12][13][14][15][16][17] Both BRD2 and BRD4 are essential for normal development.18-20 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 cell-and genespecific 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 factors....

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“…Thus, mapping the genome-wide binding profile for such small molecules would represent a significant advantage for drug development and formulating mechanistic insights. In fact, just such an approach, named "Chem-seq," was recently reported for location of bromodomain inhibitors (8).…”

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confidence: 99%

Chem-seq permits identification of genomic targets of drugs against androgen receptor regulation selected by functional phenotypic screens

Jin

Yang

Xie

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Understanding the mechanisms by which compounds discovered using cell-based phenotypic screening strategies might exert their effects would be highly augmented by new approaches exploring their potential interactions with the genome. For example, altered androgen receptor (AR) transcriptional programs, including castration resistance and subsequent chromosomal translocations, play key roles in prostate cancer pathological progression, making the quest for identification of new therapeutic agents and an understanding of their actions a continued priority. Here we report an approach that has permitted us to uncover the sites and mechanisms of action of a drug, referred to as "SD70," initially identified by phenotypic screening for inhibitors of ligand and genotoxic stress-induced translocations in prostate cancer cells. Based on synthesis of a derivatized form of SD70 that permits its application for a ChIP-sequencing-like approach, referred to as "Chem-seq," we were next able to efficiently map the genomewide binding locations of this small molecule, revealing that it largely colocalized with AR on regulatory enhancers. Based on these observations, we performed the appropriate global analyses to ascertain that SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the Jumonji domain-containing demethylase, KDM4C. Global location of candidate drugs represents a powerful strategy for new drug development by mapping genomewide location of small molecules, a powerful adjunct to contemporary drug development strategies.transcription | histone demethylase A lthough numerous small molecules functioning as drugs have been discovered through various strategies, identifying drug targets and their mechanisms of action is a challenging process (1). In fact, a full understanding of mechanisms of action is available for only some of chemical entities that are approved by US Food and Drug Administration (2). Therefore, any approaches that would augment our identification of drug mechanism would be desirable for both basic and clinical applications. The application of high-throughput sequencing technology to global genomic approaches licenses our ability to approach drug mechanisms using strategies similar to those used to investigate transcriptional regulation. ChIP sequencing (ChIPseq) had permitted to identify the location of many DNA binding transcription factors, cofactors, and other proteins in the genome (3-5), and infers that if similar approaches could be applied to chemicals identified in library screens, particularly those involving readout of transcription or other genomic regulatory events, we could obtain clues about potential genomic actions of many of these newly identified chemicals that might bind directly or indirectly to chromatin (6, 7). Thus, mapping the genome-wide binding profile for such small molecules would represent a significant advantage for drug development and formulating mechanistic insights. In fact, just such an app...

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Genome-wide localization of small molecules

Cited by 161 publications

References 44 publications

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Functions of BET proteins in erythroid gene expression

Chem-seq permits identification of genomic targets of drugs against androgen receptor regulation selected by functional phenotypic screens

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