<Emphasis Type="Italic">cis</Emphasis>-Decoder discovers constellations of conserved DNA sequences shared among tissue-specific enhancers

A systematic approach is described for analysis of evolutionarily conserved cis-regulatory DNA using cis-Decoder, a tool for discovery of conserved sequence elements that are shared between similarly regulated enhancers. Analysis of 2,086 conserved sequence blocks (CSBs), identified from 135 characterized enhancers, reveals most CSBs consist of shorter overlapping/adjacent elements that are either enhancer type-specific or common to enhancers with divergent regulatory behaviors. Our findings suggest that enhancers employ overlapping repertoires of highly conserved core elements.     

cis-Regulatory Complexity within a Large Non-Coding Region in the Drosophila Genome

Analysis of cis-regulatory enhancers has revealed that they consist of clustered blocks of highly conserved sequences. Although most characterized enhancers reside near their target genes, a growing number of studies have shown that enhancers located over 50 kb from their minimal promoter(s) are required for appropriate gene expression and many of these ‘long-range’ enhancers are found in genomic regions that are devoid of identified exons. To gain insight into the complexity of Drosophila cis-regulatory sequences within exon-poor regions, we have undertaken an evolutionary analysis of 39 of these regions located throughout the genome. This survey revealed that within these genomic expanses, clusters of conserved sequence blocks (CSBs) are positioned once every 1.1 kb, on average, and that a typical cluster contains multiple (5 to 30 or more) CSBs that have been maintained for at least 190 My of evolutionary divergence. As an initial step toward assessing the cis-regulatory activity of conserved clusters within gene-free genomic expanses, we have tested the in-vivo enhancer activity of 19 consecutive CSB clusters located in the middle of a 115 kb gene-poor region on the 3^rd chromosome. Our studies revealed that each cluster functions independently as a specific spatial/temporal enhancer. In total, the enhancers possess a diversity of regulatory functions, including dynamically activating expression in defined patterns within subsets of cells in discrete regions of the embryo, larvae and/or adult. We also observed that many of the enhancers are multifunctional–that is, they activate expression during multiple developmental stages. By extending these results to the rest of the Drosophila genome, which contains over 70,000 non-coding CSB clusters, we suggest that most function as enhancers.     

A systematic approach to identify functional motifs within vertebrate developmental enhancers

Uncovering the cis-regulatory logic of developmental enhancers is critical to understanding the role of non-coding DNA in development. However, it is cumbersome to identify functional motifs within enhancers, and thus few vertebrate enhancers have their core functional motifs revealed. Here we report a combined experimental and computational approach for discovering regulatory motifs in developmental enhancers. Making use of the zebrafish gene expression database, we computationally identified conserved non-coding elements (CNEs) likely to have a desired tissue-specificity based on the expression of nearby genes. Through a high throughput and robust enhancer assay, we tested the activity of ∼ 100 such CNEs and efficiently uncovered developmental enhancers with desired spatial and temporal expression patterns in the zebrafish brain. Application of de novo motif prediction algorithms on a group of forebrain enhancers identified five top-ranked motifs, all of which were experimentally validated as critical for forebrain enhancer activity. These results demonstrate a systematic approach to discover important regulatory motifs in vertebrate developmental enhancers. Moreover, this dataset provides a useful resource for further dissection of vertebrate brain development and function.     

Can a few non‐coding mutations make a human brain?

The recent finding that the human version of a neurodevelopmental enhancer of the Wnt receptor Frizzled 8 (FZD8) gene alters neural progenitor cell cycle timing and brain size is a step forward to understanding human brain evolution. The human brain is distinctive in terms of its cognitive abilities as well as its susceptibility to neurological disease. Identifying which of the millions of genomic changes that occurred during human evolution led to these and other uniquely human traits is extremely challenging. Recent studies have demonstrated that many of the fastest evolving regions of the human genome function as gene regulatory enhancers during embryonic development and that the human‐specific mutations in them might alter expression patterns. However, elucidating molecular and cellular effects of sequence or expression pattern changes is a major obstacle to discovering the genetic bases of the evolution of our species. There is much work to do before human‐specific genetic and genomic changes are linked to complex human traits. Also watch the Video Abstract .     

Different E-box regulatory sequences are functionally distinct when placed within the context of the troponin I enhancer.

Basic helix-loop-helix (bHLH) regulatory proteins are known to bind to a single DNA consensus sequence referred to as an E-box. The E-box is present in the regulatory elements of many developmentally controlled genes, including most muscle-specific genes such as troponin I (TnI). Although the E-box consensus is minimally defined as CANNTG, the adjacent nucleotides of functional E-boxes are variable for genes regulated by the bHLH proteins. In order to examine how E-box regulatory regions containing different internal and flanking nucleotides function when placed within the context of a single regulatory element, the E-box region (14 bp) present within the TnI enhancer was substituted with the corresponding E-box sequences derived from the muscle-specific M-creatine kinase (MCK) and cardiac alpha-actin regulatory elements as well as from the immunoglobulin kappa (Ig kappa) enhancer. Within the TnI enhancer, the E-box sequence derived from cardiac alpha-actin was inactive whereas the corresponding sequence from the MCK right E-box efficiently restored wild-type enhancer activity in muscle cells. Intermediate levels of gene activity were observed for TnI enhancers containing E-boxes derived from the MCK left E-box site or from the Ig kappa E2 E-box. DNA binding studies of MyoD:E12 protein complexes with each substituted TnI enhancer confirmed that DNA binding activity in vitro mimics the relative strength of the enhancers in vivo. These studies demonstrate that the specific nucleotide composition of individual E-boxes, which are contained within the regulatory elements of most if not all muscle-specific genes, contributes to the complex regulatory mechanisms governing bHLH-mediated gene expression.     

Identification of a dopaminergic enhancer indicates complexity in vertebrate dopamine neuron phenotype specification

The dopaminergic neurons of the basal ganglia play critical roles in CNS function and human disease, but specification of dopamine neuron phenotype is poorly understood in vertebrates. We performed an in vivo screen in zebrafish to identify dopaminergic neuron enhancers, in order to facilitate studies on the specification of neuronal identity, connectivity, and function in the basal ganglia. Based primarily on identification of conserved non-coding elements, we tested 54 DNA elements from four species (zebrafish, pufferfish, mouse, and rat), that included 21 genes with known or putative roles in dopaminergic neuron specification or function. Most elements failed to drive CNS expression or did not express specifically in dopaminergic neurons. However, we did isolate a discrete enhancer from the otpb gene that drove specific expression in diencephalic dopaminergic neurons, although it did not share sequence conservation with regulatory regions of otpa or other dopamine-specific genes. For the otpb enhancer, regulation of expression in dopamine neurons requires multiple elements spread across a large genomic area. In addition, we compared our in vivo testing with in silico analysis of genomic regions for genes involved in dopamine neuron function, but failed to find conserved regions that functioned as enhancers. We conclude that regulation of dopaminergic neuron phenotype in vertebrates is regulated by dispersed regulatory elements.     

Functional and mechanistic diversity of distal transcription enhancers

Biological differences among metazoans and between cell types in a given organism arise in large part due to differences in gene expression patterns. Gene-distal enhancers are key contributors to these expression patterns, exhibiting both sequence diversity and cell type specificity. Studies of long-range interactions indicate that enhancers are often important determinants of nuclear organization, contributing to a general model for enhancer function that involves direct enhancer-promoter contact. However, mechanisms for enhancer function are emerging that do not fit solely within such a model, suggesting that enhancers as a class of DNA regulatory element may be functionally and mechanistically diverse.     

A regulatory code for neurogenic gene expression in the Drosophila embryo

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permitted the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. We discuss the prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression.