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Shining light on dark matter in the genome
Tuesday, 2019/12/17 | 08:04:13
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Dongyin Guan and Mitchell A. Lazar; PNAS December 10, 2019 116 (50) 24919-24921
The complexity of multicellular organisms requires the genome to be transcribed in a cell-type–dependent manner that is responsive to signals, such as hormones, from the internal environment. This is mediated by the epigenome, which decorates and organizes the genome in a web of modified histone proteins functioning in nucleosomes and chemical modifications to genomic DNA arranged 3-dimensionally in the cell nucleus. Functional features of the epigenome such as acetylation of histone lysine residues are “read” by specialized proteins such as those containing bromodomains (1). Likewise, the genome itself is read by proteins known as sequence-specific transcription factors (TFs), which recognize and bind to specific motifs in genomic DNA. The totality of these sites for a given transcription factor in a given cell is known as its “cistrome” (2). Most of these binding sites occur in the ∼99% of the genome that does not encode for proteins. At some sites, the TFs control the expression of protein-encoding genes, in large part by further modifying the epigenome. These sites, known as enhancers, represent only a fraction of the cistrome, and the mechanisms determining which binding sites are functional are not well understood. In PNAS Fei et al. (3) outline the forward path by reporting a general method to screen for sequences that function as enhancers responsive to a given TF, with a focus on 2 specific TFs that provide great insight into the question of what determines a functional binding site.
Fei et al. (3) first determined the functional binding sites of forkhead box protein A1 (FOXA1), which is a pioneering TF that initiates the transition of enhancer elements from closed chromatin to a primed or poised state (4⇓–6). This allows signal-dependent TFs to gain access to DNA and established nucleosome-free regions and recruit coregulators such as histone acetyltransferases (7) as well as chromatin organization regulators (such as Mediator and Cohesin proteins) that facilitate the formation of the enhancer–promoter loops for engagement of RNA polymerase II, leading to target gene transcription (7, 8). FOXA1 is known to be particularly important for genomic recruitment of steroid hormone receptors in hormone-dependent cancers, such as estrogen receptor (ER) in breast cancer (4, 9, 10) and androgen receptor (AR) in prostate cancer (11, 12). Interestingly, unlike differentiated somatic cells which have identical genomes, DNA mutations are frequently one of the drivers for carcinogenesis and progression, and some of these may cause loss or gain of enhancer function.
Methodologically, Fei et al. (3) used a CRISPR/Cas9-based strategy to delete over 10,000 sites previously determined to be bound by FOXA1 in T47D breast cancer cells or LNCaP prostate cancer cells (Fig. 1A). The functional screen was based on changes in cancer cell survival upon binding-site deletion, and they identified 72 FOXA1 binding sites that are essential for either breast cancer T47D cells or LNCaP prostate cancer cells to survive. Interestingly, some of these binding sites were cell-type specific, indicating the functional plasticity of enhancers and their cell specificity, in part related to differential effects on ER in breast cancer and AR in prostate cancer. Indeed, an advantage of this approach is that it allows functional dissection of different binding sites for the same TF, which cannot be distinguished by the deletion of the TF itself (Fig. 1B).
See more: https://www.pnas.org/content/116/50/24919
Figure: Illuminating our understanding of functional transcription factor binding sites and enhancers. (A) A CRISPR screen designed by Fei et al. (3) to identify TF binding sites that are essential for cancer cell survival. (B–D) This screen provided several mechanistic insights, including the following: (B) identifying which specific TF binding sites function as enhancers, (C) determining which genes are targeted by functional binding sites/enhancers, and (D) predicting which disease-associated SNPs regulate the activity of functional enhancers. |
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