Transcriptional Regulation of an Evolutionary Conserved Intergenic Region of CDT2-INTS7

In the mammalian genome, a substantial number of gene pairs (approximately 10%) are arranged head-to-head on opposite strands within 1,000 base pairs, and separated by a bidirectional promoter(s) that generally drives the co-expression of both genes and results in functional coupling. The significance of unique genomic configuration remains elusive.Here we report on the identification of an intergenic region of non-homologous genes, CDT2, a regulator of DNA replication, and an integrator complex subunit 7 (INTS7), an interactor of the largest subunit of RNA polymerase II. The CDT2-INTS7 intergenic region is 246 and 245 base pairs long in human and mouse respectively and is evolutionary well-conserved among several mammalian species. By measuring the luciferase activity in A549 cells, the intergenic human sequence was shown to be able to drive the reporter gene expression in either direction and notably, among transcription factors E2F, E2F1 approximately E2F4, but not E2F5 and E2F6, this sequence clearly up-regulated the reporter gene expression exclusively in the direction of the CDT2 gene. In contrast, B-Myb, c-Myb, and p53 down-regulated the reporter gene expression in the transcriptional direction of the INTS7 gene. Overexpression of E2F1 by adenoviral-mediated gene transfer resulted in an increased CDT2, but not INTS7, mRNA level. Real-time polymerase transcription (RT-PCR) analyses of the expression pattern for CDT2 and INTS7 mRNA in human adult and fetal tissues and cell lines revealed that transcription of these two genes are asymmetrically regulated. Moreover, the abundance of mRNA between mouse and rat tissues was similar, but these patterns were quite different from the results obtained from human tissues.These findings add a unique example and help to understand the mechanistic insights into the regulation of gene expression through an evolutionary conserved intergenic region of the mammalian genome

<i> In silico</i> analysis of the CDT2-INTS7 intergenic region.

<p>(A) Nucleotide sequence of the intergenic region of CDT2 and INTS7 genes. The sequences of seven mammalian genes are aligned and conserved nucleotides are marked with asterisks under the alignment. The bent arrows indicate the transcription start sites and direction of human genes (5′-CGATA— and 5′-AGCGC— for CDT2 and INTS7, respectively), canine genes, (5′-TCAGT— and 5′-AACAG— for CDT2 and INTS7, respectively), and mouse genes (5′-GGCGG— and 5′-CGCGG— for CDT2 and INTS7, respectively). The bent arrows positioned on the sequences are for CDT2. The bent arrows positioned under the sequences are for INTS7. Transfac software (threshold >80) predicts four E2F consensus sites (E2F A∼D, marked with dotted boxes), and Sp1 (5′-GAGGCGGGGA), NF-Y (5′-AAGCCAATCAG), CREB (5′-TGACGTCA), and Myb (5′-CCAAACTGAC) transcription factor-binding sites (marked by arrows with dotted lines). (B) Computer predicted threshold (Transfac software) of E2F A∼D were summarized for seven mammalian genes.</p

Deletion analyses of the human CDT2 promoter to identify the E2F responsive site.

<p>(A) Structure of the human CDT2 gene and location of a series of deleted constructs. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) The levels of luciferase expression of human CDT2 deleted promoter constructs in A549 cells were tested with E2F1 coexpression, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3; asterisk, <i>P</i><0.05 for pcDNA3 versus E2F1).</p

RT-PCR analysis of the CDT2 and INTS7 genes in various human (A), mouse (B), and rat (C) tissues and cell lines.

<p>The lower panels in each part show the G3PDH bands of the ethidium bromide-stained gels as a control. The source of the cDNA is indicated at the top. RT-PCR products were derived from amplifications in the log range.</p

Promoter analysis of the CDT2-INTS7 intergenic region.

<p>(A) The structure of human, mouse, and canine CDT2 and INTS7 genes in the head-to-head orientation. Translation start codons (represented by ATG) of the CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate the E2F consensus sites (threshold >85). Arrows with numbers were the region and direction used for the luciferase (Luc) assay. (B) Luciferase expression of pGL3-human −363/+1, pGL3-mouse −335/+32, and pGL3-canine −312/+54 constructs in A549 cells are shown as fold induction with respect to the pGL3-Basic vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3). (C) Luciferase expression of pGL3-human −363/+1 (hereafter denoted as ABCD), pGL3-mouse −335/+32, and pGL3-canine −312/+54 constructs in A549 cells were up-regulated by co-expressing the E2F1, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3).</p

The human CDT2-INTS7 intergenic region acts as a bidirectional promoter.

<p>(A) Structure of human CDT2 and INTS7 genes in the head-to-head orientation. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) Luciferase expression of pGL3 constructs are summarized in (A) in A549 cells and are shown as fold induction with respect to the pGL3-Basic vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3).</p

The <i>Drosophila</i> Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of <i>spookier</i>

<div><p>Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C₂H₂-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly <i>Drosophila melanogaster</i>. Expression of <i>ouib</i> is predominantly limited to the PG, and genetic null mutants of <i>ouib</i> result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, <i>ouib</i> mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, <i>spookier</i>. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of <i>spok</i> by binding to a specific ~15 bp response element in the <i>spok</i> PG enhancer element. Most remarkable, the developmental arrest phenotype of <i>ouib</i> mutants is rescued by over-expression of a functionally-equivalent paralog of <i>spookier</i>. These observations imply that the main biological function of Ouib is to specifically regulate <i>spookier</i> transcription during <i>Drosophila</i> development.</p></div

Expression analysis of <i>ouib</i> in <i>Drosophila</i> larva and embryo.

<p>(A, B) RNA <i>in situ</i> hybridization of stage 14 (A) and stage 16 (B) embryos with the <i>ouib</i> antisense RNA probe. Dorsal views are shown. <i>ouib</i> signal was detected in the primordia of PG cells (arrows). An image with sense RNA probe is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005712#pgen.1005712.s004" target="_blank">S1 Fig</a> (C, D) <i>in situ</i> hybridization of third instar larval brain-ring gland complexes with the <i>ouib</i> antisense (C) and sense (D) RNA probes. <i>ouib</i> signal is detected in the ring gland including the PG cells (arrow). (E) The expression levels of <i>ouib</i> in several tissues quantified by qRT-PCR (N = 3). Total RNA was prepared from wandering third instar larvae. BR, brain; RG, ring gland; ID, imaginal disc; IN, intestine; FB, fat body; SG, salivary gland. Error bars indicate the s.e.m.</p