A Novel Mammal-Specific Three Partite Enhancer Element Regulates Node and Notochord-Specific <em>Noto</em> Expression

<div><p>The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs) whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC) expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE) upstream the <em>Noto</em> promoter was necessary and sufficient for NNC expression from the endogenous <em>Noto</em> locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted <em>Noto</em> expression in the node and nascent notochord and thus regulates normal node development and function.</p> </div

NOCE responds to FOXA2, TEAD and TCF/LEF in vitro, but binding sites are dispensable in vivo.

<p>(A) Schematic view of location and sequences of transcription factor binding sites in NOCE. Red characters indicate exchanged nucleotides of mutated sites. (B) Transactivation of wild type and mutated NOCE constructs. Arabical numbers indicate different mutated binding sites: (1) FOXA2, (2) TCF/LEF, (3) TEAD. Cotransfected constructs of transcription factors are indicated below. (C) β-galactosidase staining of chimeric embryos carrying promoter-reporter transgenes with mutated FOXA2, TEAD and TCF/LEF binding sites (NOCE 3x mut) and an additional mutation in the HOX binding site (in NOCE 4x mut) in NOCE.</p

HPRT targeting strategy.

<p>Schematic view of wild type and E14tg2a <i>Hprt</i> locus, targeting construct and targeted locus. The targeting construct introduces the reporter construct and the promoter and first exon of human <i>HPRT</i> to restore <i>Hprt</i> function. Wild type and targeted allele are HAT resistant (HAT^R) whereas the E14tg2a deletion results in HAT sensitivity (HAT^S).</p

An orphan binding site together with a FOXA2 binding site are essential for NOCE function.

<p>(A) Schematic view of location and sequences of transcription factor binding sites in NOCE and the alignment of OBS to CE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047785#pone.0047785-Sawada1" target="_blank">[27]</a>. Red characters indicate exchanged nucleotides in mutated sites. (B) β-galactosidase stained chimeric embryos carrying a promoter-reporter transgene of NOCE with mutated OBS and/or FOXA2 binding sites. Transgenes are indicated at the left site and the stage at the top.</p

NOCE is a three partite enhancer element.

<p>(A) Schematic view of transgenes containing parts of NOCE. (B) 1 hr β-galactosidase staining of different promoter-reporter transgenes in chimeric embryos. (C) Over night β-galactosidase staining of chimeric embryos carrying the transgenes depicted under (A). The stage is indicated at the top and the transgene at the left site. Arrow heads point at stained notochords.</p

NOCE regulates endogenous allele-specific <i>Noto</i> expression.

<p>(A) Schematic view of the targeting strategy to delete NOCE in ES cells carrying alleles with green or red fluorescent proteins (GFP and RFP) in the <i>Noto</i> locus, respectively. (B) fluorescence of GFP and RFP in chimeric embryos with the parental heteroallelic cell line (B a, d), and cells in which NOCE was deleted from either the GFP (B b, e) or RFP allele (B c, f). (C) Fluorescence intensities of GFP and RFP and ratio of GFP/RFP of control chimeras, and of targeted/non-targeted (reference) alleles for chimeric embryos with one deleted NOCE allele (see also material and methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047785#pone.0047785.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047785#pone.0047785.s010" target="_blank">Table S3</a>).</p

NOCE is necessary and sufficient for reporter gene activity in the NNC.

<p>(A) Schematic view of <i>Noto</i> upstream regions of various tested transgenes. (B) Corresponding chimeric embryos stained for β-galactosidase activity at E7.5 or E9.5. Transgenes are indicated on the left site.</p

NASCAR workflow.

<p>(1) Seeds perfectly matching between query (i.e. enhancer) and target (e.g. genomic window) sequence (small black segments) are extended up- and downstream (red segments) using a match/mismatch scoring scheme to generate a raw motif profile. Motifs that overlap the predefined window boundaries are also taken into account and virtually extend the window (grey areas). (2) As a next step, overlapping regions of the extracted raw motifs in the target sequence are determined (grey areas) and the smaller motif truncated whenever it overlaps a larger one (2 to 3). Motifs smaller than the initial seed size after truncation are discarded in this step. (3) Same filtering procedure is repeated in the query sequence for the processed profile (3 to 4). (4) Motifs below the noise threshold (bright blue segment) are discarded and the basic similarity (“PURE”) score calculated from the fully filtered motif profile (dark blue). (5) In addition, a pattern detection method searches for co-linear arrangements in the profile (grey area). Panel shows the same motif composition as (4) but in a co-linear configuration. This time, the motif below the noise threshold (bright pink) is kept as it is contained in a pattern. The score of the full pattern (all pink motifs) is subsequently added to the previously calculated basic score, resulting in the “COMB” score. For a given enhancer, the whole process is repeated window by window until the last window in the target sequence is reached.</p

Selected NASCAR candidates.

<p>The first 8 entries correspond to the candidates that showed expression in medaka.</p><p>* = partial motif co-linearity, DF = “double flanked”, SF = “single flanked”</p><p>Selected NASCAR candidates.</p

Deletion of conserved motifs (grey area) from the predicted fish regions results in change of enhancer activity in both tested constructs.

<p><b>Schematic on the right shows the motif configuration in the human and medaka locus for hs1344 and hs865, respectively. The full grey area is deleted from the medaka enhancer and the remaining sequence tested for reporter expression. Images on the left show the reporter activity of the medaka constructs prior to and after the deletion.</b> Hs1344 ol2-1delta gains two symmetrical domains in the midbrain (red arrowheads), while hs865 ol2-1delta shows a loss of expression in the central part of the original domain.</p