Enhancer function: new insights into the regulation of tissue-specific gene expression

Abstract

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.

Figure 1. Regulatory elements of transcription 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, interspersed with silencer and insulator elements. Recent genome-wide data have revealed that many enhancers can be defined by unique chromatin features and the binding of CBP.

CBP:	CREB (Cyclic-AMP Response Element Binding)-binding protein.
CTCF:	CCCTC-binding factor.
DPE:	Distal promoter elements.
H3K4me1/2:	histone H3 mono/di-methylation at lysine 4.
H3K4me3:	histone H3 trimethylation at lysine 4.
H3K27me3:	histone H3 trimethylation at lysine 27.
H3.3/H2A.Z:	histone variants H3.3 and H2A.Z.
LCR:	Locus control region.
RNAPII:	RNA polymerase II.
TATA:	5′-TATAAAA-3′ core DNA sequences.
TSS:	Transcription start site.

Figure 2. Many functional enhancers contain dynamic nucleosomes

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. b. In LNCaP cells, stimulation of androgen-mediated transcription programs by dihydrotestosterone (DHT) leads to the displacement of the unstable nucleosome at the enhancer by incoming transcription factors. c. Binding of E47 protein to the enhancer is facilitated by outward movement of the H3K4me2-marked nucleosomes at the transcription factor binding site.

AR:	Androgen receptor.
CD4:	Cluster of differentiation 4.
DHT:	Dihydrotestosterone.
E47:	Immunoglobulin E2-Box Binding Protein isoform 47.
FoxA1:	Foxhead box A1.
H3/H2:	histone H3 and H2.

Figure 3. Chromatin signatures at enhancers act as epigenetic signals for gene induction

a. Cell-type specific distribution of H3K4me2 marks at the enhancers allows the differential binding of FoxA1 and specific transcription factors to the chromatin sites, which results in the activation of distinct transcriptional programs. AR: androgen receptor, ER: estrogen receptor. b. In ESCs, Sox2 contributes to the establishment of the H3K4me2 mark at the λ5-VpreB1 enhancer whereas Foxd3 represses intergenic transcription. These epigenetic marks at the enhancer are required for tissue-specific expression during differentiation.

AR:	Androgen receptor.
ER:	Estrogen receptor.
FoxA1:	Foxhead box A1.
Foxd3:	Foxhead box D3.
H3K9me2:	histone H3 dimethylation at lysine 9.
Sox2:	SRY (sex determining region Y)-box 2.

Figure 4. Cohesins stabilize enhancer-promoter interactions by different strategies

a and b. CTCF/Cohesin-mediated chromatin loops bring enhancer and promoters into close proximity at different gene loci. a. In the human APO gene cluster, long-range interactions between the AC2, AR1 and AC3 insulator sites result in the formation of two chromatin loops. The C3 enhancer and C3/A4/A5 promoters reside in one loop, whereas the A1 promoter is present in a different loop. b. The IFN-γ locus is reorganized by CTCF/cohesin-mediated interactions during Th1 differentiation. c. Association of enhancers and promoters at several genes in ESCs is mediated by physical interactions between mediator and cohesin complexes.

APO:	Apolipoprotein.
CTCF:	CCCTC-binding factor.
IL26:	Interleukins 26.
IFNγ:	Interferon-gamma.
Oct4:	octamer-binding transcription factor 4.
RNAPII:	RNA polymerase II.

Figure 5. Non-coding RNAs mediate enhancer function

a. The transcription of eRNA positively correlates with the level of mRNA synthesis at nearby genes and requires the presence of an intact promoter at the gene, suggesting the presence of long-range associations between the enhancer and promoter. The type of mediator (purple diamond) and cofactors (yellow circle) involved remains to be determined. b A hypothetical model in which long ncRNA-a7 serves as a scaffold for the assembly of transcription factors (yellow circle) or other chromatin remodeling enzymes (brown oval) at the Snai1 promoter to facilitate gene activation.

Arc:	Activity-regulated cytoskeleton-associated protein
CREB:	Cyclic-AMP Response Element Binding Protein.
CBP:	CREB-binding protein.
eRNA:	enhancer RNA.
mRNA:	messenger RNA.
ncRNA:	non-coding RNA.
Snai1:	Snail homolog 1a.

Figure 6. Histone crosstalk between enhancers and promoters regulates RNAPII elongation

Serum stimulation induces PIM1-mediated phosphorylation of preacetylated H3K9S10 at the FOSL1 enhancer. Binding of 14-3-3 to H3S10ph nucleosomes recruits MOF, which in turn acetylates H4K16 (H4K16ac). In Drosophila, Elp3 and JIL-1 respectively acetylate and phosphorylate H3K9S10ph at the promoter. The modified nucleosomes act as a platform for the sequential recruitment of BRD4 and P-TEFb, which then facilitates the release of the promoter-proximal paused RNAPII. The type of mediator (purple diamond) and co-factors (yellow circle) involved remains to be determined.

Brd4:	Bromodomain containing 4
ELP-3:	Elongator complex protein 3.
FOSL1:	FOS-like antigen 1.
H3K9acS10P:	histone H3 acetylation at lysine 9 and phosphorylation at serine 10.
H4K16ac:	histone H4 acetylation at lysine 16.
MOF:	Male absent on the first, histone H4 lysine 16-specific acetyltransferase.
MSK:	Mitogen and Stress-Activated Protein Kinase.
PIM1:	Proviral integration site 1, a proto-oncogene serine/threonine-protein kinase.
P-TEFb:	positive transcription elongation factor b (P-TEFb).
RNAPII:	RNA polymerase II.