The development, differentiation and growth of cells and tissues require precisely regulated patterns of
gene expression. Enhancers work as
cis-regulatory elements to mediate both spatial and temporal control of development by turning on
transcription in specific cells and/or repressing it in other cells. Thus, the particular combination of
transcription factors and other DNA-binding proteins in a developing tissue controls which genes will be expressed in that tissue. Enhancers allow the same gene to be used in diverse processes in space and time.
Identification and characterization Traditionally, enhancers were identified by
enhancer trap techniques using a reporter gene or by comparative sequence analysis and computational genomics. In genetically tractable models such as the fruit fly
Drosophila melanogaster, for example, a reporter construct such as the
lacZ gene can be randomly integrated into the genome using a
P element transposon. If the reporter gene integrates near an enhancer, its expression will reflect the expression pattern driven by that enhancer. Thus, staining the flies for LacZ expression or activity and cloning the sequence surrounding the integration site allows the identification of the enhancer sequence. The development of genomic and epigenomic technologies, however, has dramatically changed the outlook for
cis-regulatory modules (CRM) discovery.
Next-generation sequencing (NGS) methods now enable high-throughput functional CRM discovery assays, and the vastly increasing amounts of available data, including large-scale libraries of
transcription factor-binding site (TFBS) motifs, collections of annotated, validated CRMs, and extensive
epigenetic data across many cell types, are making accurate computational CRM discovery an attainable goal. An example of NGS-based approach called
DNase-seq have enabled identification of nucleosome-depleted, or open chromatin regions, which can contain CRM. More recently techniques such as
ATAC-seq have been developed which require less starting material. Nucelosome depleted regions can be identified in vivo through expression of
Dam methylase, allowing for greater control of cell-type specific enhancer identification. Computational methods include
comparative genomics, clustering of known or predicted TF-binding sites, and supervised machine-learning approaches trained on known CRMs. All of these methods have proven effective for CRM discovery, but each has its own considerations and limitations, and each is subject to a greater or lesser number of false-positive identifications. In the
comparative genomics approach,
sequence conservation of
non-coding regions can be indicative of enhancers. Sequences from multiple species are aligned, and conserved regions are identified computationally. Identified sequences can then be attached to a reporter gene such as
green fluorescent protein or lacZ to determine the
in vivo pattern of gene expression produced by the enhancer when injected into an embryo.
mRNA expression of the reporter can be visualized by
in situ hybridization, which provides a more direct measure of enhancer activity, since it is not subjected to the complexities of
translation and
protein folding. Although much evidence has pointed to sequence conservation for critical developmental enhancers, other work has shown that the function of enhancers can be conserved with little or no primary sequence conservation. For example, the
RET enhancers in humans have very little sequence conservation to those in
zebrafish, yet both species' sequences produce nearly identical patterns of reporter gene expression in zebrafish.
In segmentation of insects The enhancers determining early
segmentation in
Drosophila melanogaster embryos are among the best characterized developmental enhancers. In the early fly embryo, the
gap gene transcription factors are responsible for activating and repressing a number of segmentation genes, such as the
pair rule genes. The gap genes are expressed in blocks along the anterior-posterior axis of the fly along with other
maternal effect transcription factors, thus creating zones within which different combinations of transcription factors are expressed. The pair-rule genes are separated from one another by non-expressing cells. Moreover, the stripes of expression for different pair-rule genes are offset by a few cell diameters from one another. Thus, unique combinations of pair-rule gene expression create spatial domains along the anterior-posterior axis to set up each of the 14 individual segments. The 480 bp enhancer responsible for driving the sharp stripe two of the pair-rule gene
even-skipped (
eve) has been well-characterized. The enhancer contains 12 different binding sites for maternal and gap gene transcription factors. Activating and repressing sites overlap in sequence.
Eve is only expressed in a narrow stripe of cells that contain high concentrations of the activators and low concentration of the repressors for this enhancer sequence. Other enhancer regions drive
eve expression in 6 other stripes in the embryo.
In vertebrate patterning Establishing body axes is a critical step in animal development. During mouse embryonic development,
Nodal, a
transforming growth factor-beta superfamily ligand, is a key gene involved in patterning both the anterior-posterior axis and the left-right axis of the early embryo. The
Nodal gene contains two enhancers: the Proximal Epiblast Enhancer (PEE) and the Asymmetric Enhancer (ASE). The PEE is upstream of the Nodal gene and drives
Nodal expression in the portion of the
primitive streak that will differentiate into the node (also referred to as the
primitive node). The PEE turns on Nodal expression in response to a combination of Wnt signaling plus a second, unknown signal; thus, a member of the LEF/TCF transcription factor family likely binds to a TCF binding site in the cells in the node. Diffusion of Nodal away from the node forms a gradient which then patterns the extending anterior-posterior axis of the embryo. The ASE is an intronic enhancer bound by the
fork head domain transcription factor Fox1. Early in development, Fox1-driven Nodal expression establishes the visceral endoderm. Later in development, Fox1 binding to the ASE drives
Nodal expression on the left side of the lateral plate
mesoderm, thus establishing left-right asymmetry necessary for asymmetric organ development in the mesoderm. Establishing three
germ layers during
gastrulation is another critical step in animal development. Each of the three germ layers has unique patterns of gene expression that promote their differentiation and development. The
endoderm is specified early in development by
Gata4 expression, and Gata4 goes on to direct gut morphogenesis later.
Gata4 expression is controlled in the early embryo by an intronic enhancer that binds another forkhead domain transcription factor, FoxA2. Initially the enhancer drives broad gene expression throughout the embryo, but the expression quickly becomes restricted to the endoderm, suggesting that other repressors may be involved in its restriction. Late in development, the same enhancer restricts expression to the tissues that will become the stomach and pancreas. An additional enhancer is responsible for maintaining
Gata4 expression in the endoderm during the intermediate stages of gut development.
Multiple enhancers promote developmental robustness Some genes involved in critical developmental processes contain multiple enhancers of overlapping function. Secondary enhancers, or "
shadow enhancers", may be found many kilobases away from the primary enhancer ("primary" usually refers to the first enhancer discovered, which is often closer to the gene it regulates). On its own, each enhancer drives nearly identical patterns of gene expression. Are the two enhancers truly redundant? Recent work has shown that multiple enhancers allow fruit flies to survive environmental perturbations, such as an increase in temperature. When raised at an elevated temperature, a single enhancer sometimes fails to drive the complete pattern of expression, whereas the presence of both enhancers permits normal gene expression. ==Evolution of developmental mechanisms==