Comparative functional genomics revealed conservation and diversification of three enhancers of the isl1 gene for motor and sensory neuron-specific expression

Abstract

Islet-1 (Isl1) is a member of the Isl1 family of LIM-homeodomain transcription factors (LIM-HD) that is expressed in a defined subset of motor and sensory neurons during vertebrate embryogenesis. To investigate how this specific expression of isl1 is regulated, we searched for enhancers of the isl1 gene that are conserved in vertebrate evolution. Initially, two enhancer elements, CREST1 and CREST2, were identified downstream of the isl1 locus in the genomes of fugu, chick, mouse, and human by BLAST searching for highly similar elements to those originally identified as motor and sensory neuron-specific enhancers in the zebrafish genome. The combined action of these elements is sufficient for completely recapitulating the subtype-specific expression of the isl1 gene in motor neurons of the mouse spinal cord. Furthermore, by direct comparison of the upstream flanking regions of the zebrafish and human isl1 genes, we identified another highly conserved noncoding element, CREST3, and subsequently C3R, a similar element to CREST3 with two CDP CR1 recognition motifs, in the upstream regions of all other isl1 family members. In mouse and human, CRESTs are located as far as more than 300 kb away from the isl1 locus, while they are much closer to the isl1 locus in zebrafish. Although all of zebrafish CREST2, CREST3, and C3R activate gene expression in the sensory neurons of zebrafish, CREST2 of mouse and human does not have the sequence necessary for sensory neuron-specific expression. Our results revealed both a remarkable conservation of the regulatory elements regulating subtype-specific gene expression in motor and sensory neurons and the dynamic process of reorganization of these elements whereby each element increases the level of cell-type specificity by losing redundant functions with the other elements during vertebrate evolution.

Zebrafish Prickle1b mediates facial branchiomotor neuron migration via a farnesylation-dependent nuclear activity

“…To generate rescue constructs, sequences encoding full-length and mutant Venus-Pk1b were inserted downstream of the zCREST1 enhancer (Uemura et al, 2005) and a minimal promoter, in a plasmid containing Tol2 transposition sequences (Kawakami and Shima, 1999). DNA encoding each rescue construct (40 ng/l) and Tol2 transposase mRNA (40 ng/l), with or without Pk1bMO (2 ng/nl), were injected into Tg(zCREST1:membRFP) embryos at the 1-to 2-cell stage.…”

“…As this elevated Pk1b expression in the neurons might be crucial for proper function, we next designed a rescue approach that would recapitulate this expression pattern. Using the zCREST1 enhancer of the islet1 regulatory elements (Uemura et al, 2005), we drove expression of Venus-tagged Pk1b variants specifically in the cranial branchiomotor neurons (CBMNs; Fig. 4A).…”

Islet1 selectively promotes peripheral axon outgrowth in Rohon‐Beard primary sensory neurons

“…Isl1 is expressed in motor and retinal neurons of the central nervous system (CNS) and in various types of sensory neurons, including neurons of the dorsal root and cranial ganglia (Appel et al, 1995;Elshatory et al, 2007;Hutchinson and Eisen, 2006;Pfaff et al, 1996;Sun et al, 2008;Thaler et al, 2002Thaler et al, , 2004Tsuchida et al, 1994;Uemura et al, 2005). In mice, isl1-deficient embryos die before sensory neuron maturation (Pfaff et al, 1996).…”

“…In zebrafish, the members of the Isl family (Isl1, Isl2a, and Isl2b, which were previously known as Isl1, Isl2, and Isl3, respectively) are expressed not only in motor neurons but also in Rohon-Beard (RB) primary sensory neurons (Appel et al, 1995;Inoue et al, 1994;Miyashita et al, 2004;Segawa et al, 2001;Tokumoto et al, 1995;Uemura et al, 2005). RB neurons are multipolar neurons that extend their central axons longitudinally within the dorsal spinal cord both rostrally and caudally, and also extend their highly branched peripheral axons subcutaneously, transmitting sensory stimuli to the CNS (Bernhardt et al, 1990;Ribera and Nusslein-Volhard, 1998).…”

An Evolutionarily Conserved TNF-α–Responsive Enhancer in the Far Upstream Region of Human CCL2 Locus Influences Its Gene Expression

“…Such regulatory elements 1) are typically composed of long stretches of noncoding nucleotides (.100 bp) that share a high degree of homology between different species (.70%) and may exhibit functional conservation of TF binding sites; 2) display increased susceptibility to nuclease digestion and undergo epigenetic changes such as demethylation and altered histone patterns that are associated with gene expression (17); and 3) may directly interact with proximal promoters, and such DNA-DNA interactions may be important for optimal gene expression (18)(19)(20)(21). Recent availability of wholegenome sequences from several mammalian species has provided a tremendous resource for identification and characterization of such novel regulatory elements.…”