Looping back to leap forward: transcription enters a new era

Abstract

Comparative genome analyses reveal that organismal complexity scales not with gene number but with gene regulation. Recent efforts indicate that the human genome likely contains hundreds of thousands of enhancers, with a typical gene embedded in a milieu of tens of enhancers. Proliferation of cis-regulatory DNAs is accompanied by increased complexity and functional diversification of transcriptional machineries recognizing distal enhancers and core promoters and by the high-order spatial organization of genetic elements. We review progress in unraveling one of the outstanding mysteries of modern biology: the dynamic communication of remote enhancers with target promoters in the specification of cellular identity.

Figure 1. Organization of cis-regulatory DNAs in metazoan genomes

Metazoan genes are regulated by multiple enhancers. (A) Organization of the even-skipped (eve) locus in the Drosophila genome. The eve gene is just 3 kb in length, but is regulated by individual stripe enhancers (E) located in both 5′ and 3′ flanking regions. The eve stripe enhancers function in an additive fashion to produce 7 stripes of gene expression in the early Drosophila embryo (micrograph by Mike Perry and Michael Levine, personal communication). (B) Evolution of pelvic fins in stickleback fish. The Pitx1 gene is regulated in different tissues by a series of enhancers located in both 5′ and 3′ flanking regions. Deletion of the hindlimb enhancer results in reduced development of the pelvic fins (red) in freshwater populations (adapted from (Shapiro et al., 2004)). (C) Organization of the HoxD complex in mice. The complex is regulated by a series of flanking enhancers (purple and green ovals) located in two neighboring topological association domains (TADs). The telomeric TAD (T-DOM) regulates linked HoxD genes in the developing arm and forearm, while the centromeric TAD (C-DOM) regulates expression in the hand and the digits (adapted from Andrey et al., 2013).

Figure 2. Specialized transcription machineries

(A) A diversified set of preinitiation complexes (PICs), co-activators and chromatin remodelers orchestrates cell-specific transcription programs. In embryonic stem cells (ESCs), the XPC trimeric complex works as an OCT4/SOX2 stem cell co-activator (SCC) at distal enhancer sites (DE) to sustain the expression of pluripotency and self-renewal genes. Upon formation of embryoid bodies (EBs), TBP-associated factor TAF3 is required for endodermal lineage differentiation, mediating DNA looping between DEs and core promoters (TATA) of endoderm-specificating genes in concert with CTCF. In testis, TAF4B directs a transcription program required to preserve the germ-cell compartment; farther down the differentiation path, in round spermatids, TAF4B is replaced by a core-promoter complex comprised of the TAF7 homologue TAF7L, TBP-related factor TRF2 and TFIIA, which promotes spermatogenesis instead. TAF7L also regulates adipogenesis by associating with TBP as a component of TFIID at promoters and with PPARγ-RXR as a cofactor at enhancers on adipocyte-specific genes. In neurons, a specialized BAF chromatin-remodeling complex exists (nBAF) that includes neural specific subunits (BAF53b, BAF45b, BAF45c, CREST) and facilitates transcription of genes involved in dendrite outgrowth. (B) A yet uncharacterized, TFIID-independent PIC assembles at the TCT motif (polypyrimidine initiator) encompassing the transcription start site of ribosomal protein genes in Drosophila cells. In Drosophila S2 cells, non-canonical PICs made of TRF2/TFIIA and TBP/TFIIA are responsible for the cell cycle-restricted expression of H1 and H2B/A histone genes, respectively. TBP/TFIIA, and possibly TRF2/TFIIA, are pre-loaded on the histone locus in the G1-phase of the cell cycle, but only activate transcription when cells enter S-phase. Abbreviations: PE, proximal enhancer; PPRE, PPARγ response element; TF, sequence-specific transcription factor.

Figure 3. Structural dynamics of transcription machineries

(A) Binding of PIC components to promoter induces dramatic turns of the DNA template, as revealed by EM structure of human TFIID and TFIIA bound to a super-core promoter (adapted from (Cianfrocco et al., 2013)). (B) Different activators (p53, c-JUN, Sp1) target distinct sites and induce localized as well as common conformational changes within TFIID, as evaluated by EM structural studies (adapted from (Liu et al., 2009)). (C) ARC/CRSP mediator undergoes dramatic and distinct conformational changes when bound to VP16 versus SREBP-1a activators, as resolved by EM (adapted from (Taatjes et al., 2002)).

Figure 4. A model for the sequential activation of gene expression

Diagram of a hypothetical gene regulated by several distal enhancers located both 5′ and 3′ of the transcription unit. (A) Gene X is silent; all enhancers are inactive and contain “repressive marks” – H3K27me3 – mediated by Polycomb silencers (e.g., (Voigt et al., 2013)). (B) A pioneer factor (PF) binds to specific sites in Enhancer 1. This leads to the appearance of flanking DnaseI hypersensitive sites and, presumably, the recruitment of chromatin remodeling complexes (e.g., BAF) and histone modifying complexes (e.g., Hu et al., 2013). (C) Following changes in chromatin state, the regulatory region becomes condensed, thereby bringing Enhancer 1 into proximity with the Gene X promoter. In some cases, the promoter acquires paused Pol II prior to induction. (D) Upon binding of inductive sequence-specific transcription factors (TF), the Enhancer engages the promoter and leads to the recruitment of the PIC or release of paused Pol II to trigger expression. Cohesin has been implicated in stabilizing Enhancer-Promoter interactions (e.g., Guo et al., 2012).

Figure 5. Emerging imaging technologies

(A) Single molecule super-resolution imaging of RNA Pol II (Ignacio Izeddin, Ibrahim Cisse, Maxime Dahan and Xavier Darzacq, personal communication). Three-dimensional density map of Pol II localization in fixed nuclei (left) highlights spatial Pol II clustering, while single particle tracking in live cells (right) identifies distinct Pol II dynamic behaviors. Data were collected from an engineered cell line stably expressing the Pol II catalytic subunit (RPB1) labeled with the photo-convertible fluorescent protein Dendra2 (Cisse et al., 2013). (B) Promoter-specific transcription initiation directed by a reconstituted human Pol II system at single molecule resolution using TIRF video-microscopy. Cy5-labeled DNA templates containing a consensus Pol II promoter are immobilized on a surface, and nascent transcripts are detected based on colocalization of fluorescent probes and template signals. The two DNA templates contain (red) or lack (green) the target sequence for the transcript probe to control for specificity (adapted from (Revyakin et al., 2012)).