In addition to the protein-encoding region of the gene, regulatory sequences can be located on either end of the gene (or even within it). These regulatory sequences—the promoter, enhancers, and silencers—are necessary for controlling where, when, and how actively a particular gene is transcribed. When located on the same chromosome as the gene (and they usually are), they can be referred to as cis-regulatory elements. Promoters are sites where RNA polymerase II binds to the DNA sequence to initiate transcription. Promoters of genes that synthesize messenger RNAs (i.e., those genes that encode proteins6) are typically located immediately upstream from the site where RNA polymerase II initiates transcription. Most of these promoters contain a stretch of about 1000 base pairs that is rich in the sequence CG, often referred to as CpG (a C and a G connected through the normal phosphate bond). These regions are called CpG islands. The reason transcription is initiated near CpG islands is thought to involve proteins called basal transcription factors, which are present in every cell and specifically bind to the CpG-rich sites. These basal transcription factor proteins form a “saddle” that can recruit RNA polymerase II and position it appropriately for the polymerase to begin transcription. RNA polymerase II does not bind to every promoter in the genome at the same time, however. Rather, it is recruited to and stabilized on the promoters by DNA sequences called enhancers that signal where and when a promoter can be used and how much gene product to make. In other words, enhancers control the efficiency and rate of transcription from a specific promoter.
Enhancers serve as critical regulatory elements in higher eukaryotic cells. The first identification of enhancer elements was in 1981. There is a large number of enhancers in the mammalian genome. 1 Enhancer function underlies regulatory processes by which cells establish patterns of gene expression. Recent results suggest that many enhancers are specified by particular chromatin marks in pluripotent cells, which may be modified later in development to alter patterns of gene expression and cell differentiation choices. These marks may contribute to the repertoire of epigenetic mechanisms responsible for cellular memory and determine the timing of transcription factor accessibility to the enhancer. Mechanistically, cohesin and non-coding RNAs are emerging as crucial players responsible for facilitating enhancer-promoter interactions at some genes. Surprisingly, these interactions may be required not only to facilitate initiation of transcription but also to activate the release of RNA polymerase II (RNAPII) from promoter-proximal pausing. Regulation of transcription is accomplished mainly by enhancers, which are DNA sequences containing multiple binding sites for a variety of transcription factors. Enhancers can activate transcription independent of their location, distance or orientation with respect to the promoters of genes. In some cases, they can even activate transcription of genes located in a different chromosome. A key issue central to the understanding of enhancer function, is how regulatory elements that show such variability in their relationship to promoters contribute to the precise regulation of transcription. Enhancers are viewed as clusters of DNA sequences capable of binding combinations of transcription factors that then interact with components of the Mediator complex or transcription factor II D (TFIID) to help recruit RNA polymerase II (RNAPII).In order to accomplish this, enhancer-bound transcription factors loop out the intervening sequences and contact the promoter region, explaining the ability of enhancers to act in a distance-independent fashion 2
Transcriptional regulatory elements in metazoans.
The promoter is typically comprised of proximal, core and downstream elements. Transcription of a gene can be regulated by multiple enhancers that are located distantly and interspersed with silencer and insulator elements, which are bound by regulatory proteins such as CCCTC-binding factor (CTCF). Recent genome-wide data have revealed that many enhancers can be defined by unique chromatin features and the binding of cyclic AMP-responsive element-binding (CREB) protein (CBP).
Activation of eukaryotic genes requires, in addition, decompaction of the chromatin fibre. This task is also carried out by enhancer-bound transcription factors that can recruit histone modifying enzymes or ATP-dependent chromatin remodelling complexes to alter chromatin structure and increase the accessibility of the DNA to other proteins. Enhancers are sequences that may carry epigenetic information in the form of specific histone modifications. These chromatin marks are first established early in development and are modified as cells differentiate along specific lineages. Interestingly, some of these histone modifications may serve as marks for future gene expression, whereas others may play a more active part in the transcription activation process. In addition, it appears that enhancer sequences themselves are not mere binding sites for transcription factors but are also transcribed into non-coding RNAs — which, together with cohesin, may have an active role in the control of transcription by stabilizing long-range enhancer-promoter interactions. The role of these interactions is not limited to the regulation of transcription initiation; the interactions may also be required to release RNAPII from promoter-proximal pausing. These findings are beginning to clarify the mechanisms by which enhancers can activate transcription over relatively long distances in an orientation-independent manner with the exquisite precision required to orchestrate the complex process of cell differentiation during development.
Chromatin features of enhancers
The distribution of post-translational histone modifications and the presence of specific histone variants influence gene expression by orchestrating the interaction of transcription factors with the chromatin fibre. Nucleosome dynamics define transcriptional enhancers. Assembly, mobilization and disassembly of nucleosomes can influence transcription and other processes that act on eukaryotic DNA. The boundaries of cis-regulatory domains are marked by high rates of histone replacement, and transcription start sites are frequently associated with regions of low nucleosome occupancy (normally termed nucleosome-free regions). Nucleosome instability contributes to gene regulation by facilitating the access of transcription factors to promoters and other regulatory elements. The presence of highly unstable nucleosomes containing the histone variants H3.3 and H2A.Z at several well-characterized enhancers suggests that nucleosome dynamics might also be important for their regulatory functions.
Many functional enhancers contain dynamic nucleosomes.
Scenarios are illustrated for sets of enhancers at which nucleosome positioning of composition is distinct — in different cell types or before and after transcription factor binding.
a | Histone variants H3.3/H2A.Z are found at the enhancer, which is DNase I hypersensitive only in CD4+ T cells but not in HeLa cells. In HeLa cells, the canonical histones H2 and H3 are instead present at the enhancer.
b | In LNCaP cells (a human prostate cancer cell line), stimulation of androgen-mediated transcription programmes by dihydrotestosterone (DHT) leads to the displacement of the unstable nucleosome at the enhancer by the incoming transcription factors, androgen receptor (AR) and forkhead box protein A1 (FOXA1).
c | In pre-pro-B cells (precursors of fully differentiated B cells) binding of immunoglobulin E2‑box binding protein isoform 47 (E47) to the enhancer is facilitated by outward movement of the H3K4me2-marked nucleosomes at the transcription factor binding site. H3/H2, histone variants H3 or H2; H3.3/H2A.Z, histone variants H3.3 or H2A.Z; H3K4me2, histone H3 dimethylation at lysine 4.
Isolation of HeLa cell chromatin with low salt concentration, which maintains the association of unstable histone variants to DNA, has led to the discovery that H3.3/H2A.Z-containing nucleosome core particles are enriched at the nucleosome-free regions of regulatory elements across the genome. Furthermore, regions of the genome that are hypersensitive to DNase I in CD4+ T cells but not in HeLa cells contain unstable double-variant nucleosomes in the CD4+ T cells but not the HeLa cells (FIG.a). This result indicates that the presence of dynamic nucleosomes reflects the activity of the hypersensitive sites, which in turn carry histone modifications correlated with enhancer activity.
In contrast, DNA sequences called silencers can prevent promoter use and inhibit gene transcription. Transcription factors are proteins that bind DNA with precise sequence recognition for specific promoters, enhancers, or silencers. Transcription factors that bind enhancers can activate a gene by (1) recruiting enzymes (such as histone acetyltransferases) that break up the nucleosomes in the area or (2) stabilizing the transcription initiation complex. Thus, transcription factors usually work in two nonexclusive ways:
1. Once bound, transcription factors can bind cofactors that recruit nucleosome modifying proteins (such as histone methyltransferases and acetyltransferases) that make that area of the genome accessible for RNA polymerase II to bind and enable the chromatin in that vicinity to be unwound and transcribed.
2. Transcription factors can form bridges, looping the chromatin such that the transcription factors (and their histone-modifying enzymes) on enhancers can be brought into the vicinity of the promoter. In the activation of mammalian β-globin genes, such a bridge uniting the promoter and enhancer is formed by proteins that bind to transcription factors on both the enhancer and promoter sequences. These proteins recruit the nucleosome-modifying enzymes and transcription-associated factors (TAFs) that stabilize RNA polymerase II