A Simple Predictive Enhancer Syntax for Hindbrain Patterning Is Conserved in Vertebrate Genomes

<div><p>Background</p><p>Determining the function of regulatory elements is fundamental for our understanding of development, disease and evolution. However, the sequence features that mediate these functions are often unclear and the prediction of tissue-specific expression patterns from sequence alone is non-trivial. Previous functional studies have demonstrated a link between PBX-HOX and MEIS/PREP binding interactions and hindbrain enhancer activity, but the defining grammar of these sites, if any exists, has remained elusive.</p><p>Results</p><p>Here, we identify a shared sequence signature (syntax) within a heterogeneous set of conserved vertebrate hindbrain enhancers composed of spatially co-occurring PBX-HOX and MEIS/PREP transcription factor binding motifs. We use this syntax to accurately predict hindbrain enhancers in 89% of cases (67/75 predicted elements) from a set of conserved non-coding elements (CNEs). Furthermore, mutagenesis of the sites abolishes activity or generates ectopic expression, demonstrating their requirement for segmentally restricted enhancer activity in the hindbrain. We refine and use our syntax to predict over 3,000 hindbrain enhancers across the human genome. These sequences tend to be located near developmental transcription factors and are enriched in known hindbrain activating elements, demonstrating the predictive power of this simple model.</p><p>Conclusion</p><p>Our findings support the theory that hundreds of CNEs, and perhaps thousands of regions across the human genome, function to coordinate gene expression in the developing hindbrain. We speculate that deeply conserved sequences of this kind contributed to the co-option of new genes into the hindbrain gene regulatory network during early vertebrate evolution by linking patterns of <i>hox</i> expression to downstream genes involved in segmentation and patterning, and evolutionarily newer instances may have continued to contribute to lineage-specific elaboration of the hindbrain.</p></div

CNEs containing PBX-HOX motifs driving expression in hindbrain of transgenic zebrafish.

<p>Images show expression driven during transient transgenic assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130413#sec017" target="_blank">methods</a>) between 2 and 3 dpf. Insets show comparison with RFP in rhombomeres 3 and 5. <i>hoxd</i>.10479 (A) at ~42 hpf in ventral r5r6; hoxd.10482 lateral (B) and dorsal (C) views at ~56 hpf in lateral r2, r4, r6, pectoral fin and spinal cord; <i>bnc2</i>.8642 (D) at ~60 hpf in hindbrain; <i>hmx2</i>.9713 (E) at ~60 hpf in hindbrain and spinal cord; <i>dachd</i>.11206 (F) at ~72hpf in hindbrain and spinal cord; <i>foxd3</i>.327 (G) at ~72hpf in ventral r5 and r6; <i>foxd3</i>.365 throughout the central nervous system, most strongly in the ventral hindbrain, and cranial ganglia. fb: forebrain; mb: midbrain; hb: hindbrain; sc: spinal cord; pf: pectoral fin; pa: pharyngeal arches/neural crest; cg: cranial ganglia; r3 r5: rhombomeres 3 and 5.</p

Hindbrain enhancers typically contain both PBX-HOX and MEIS/PREP motifs within 100bp.

<p>Motif discovery on a set of 38 hindbrain enhancers using MEME detects two enriched motifs. The first (A) resembles PBX and HOX binding preferences (B) and the second (C) resembles MEIS and PREP binding preferences (D). Furthermore, in all but one case these motifs occur within 100bp in positive enhancers, schematically represented in (E).</p

The PBX-HOX and MEIS/PREP motifs of four enhancers are essential for their function.

<p>Histograms for four elements, <i>pax2</i>.174 (A), <i>meis2a</i>.1042 (B), <i>meis1</i>.1705 (C) and <i>foxd3</i>.327 (D), showing the number of embryos with GFP positive cells in forebrain (fb), midbrain (mb), hindbrain (hb) and spinal cord (sc) when expressing wild-type (green), MEIS/PREP site mutant (blue) or PBX-HOX site mutant (red) constructs. Annotation displays p values for one-tailed paired t tests. All mutant constructs show a significant (student's t test p = <0.05) reduction in the number of embryos positive for hindbrain. Wild-type <i>pax2</i>.174 (E) drives expression in hindbrain and lens (green), whereas mutant constructs do not drive this pattern. Wild-type <i>meis2a</i>.1042 (F) drives expression in the central nervous system particularly the anterior hindbrain. Mutant constructs fail to recapitulate this expression. Wild-type <i>meis1</i>.1705 (G) drives expression in the hindbrain and spinal cord, but mutant constructs drive expression only in spinal cord. Wild-type <i>foxd3</i>.327 (H) drives expression in posterior hindbrain (green), but expression driven by constructs where either the MEIS/PREP (blue) or the PBX-HOX (red) motif is mutated is frequently ectopic in midbrain, anterior hindbrain and spinal cord.</p

Distances between PBX-HOX and MEIS/PREP motifs across the whole human genome.

<p>Graph shows the distribution of gaps between PBX-HOX and MEIS/PREP motifs across the whole genome (black) and human conserved regions defined by GERP (blue).</p

Number of hb_40 elements per Mb in different types of sequence.

<p>GERP regions, CNEs, enhancers, positive enhancers, positive hindbrain enhancers.</p><p>* This figure is artificially low compared with the CNEs, as the Vista enhancers have long flanking regions (an average size of 1,779bp compared with 116bp for CNEs). In terms of numbers, 21/238 hb+ Vista enhancers (11.34%) have hb_40 syntax compared with 93/6691 CNEs (1.4%).</p><p>Number of hb_40 elements per Mb in different types of sequence.</p

Boletín de Segovia: Número 143 - 1846 noviembre 15

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Additional file 7: Table S2. of Complete re-sequencing of a 2Mb topological domain encompassing the FTO/IRXB genes identifies a novel obesity-associated region upstream of IRX5

All associating haplotypes (P value <0.05) from the Haploview LD block definitions with MAF >0.05. (DOCX 24 kb

Additional file 1: Table S1. of Complete re-sequencing of a 2Mb topological domain encompassing the FTO/IRXB genes identifies a novel obesity-associated region upstream of IRX5

Sample IDs, BMIs, ages and case/control classifications for the samples that were included in this study that passed quality control (n = 284). (DOCX 41 kb

Genome-wide association analyses for lung function and chronic obstructive pulmonary disease identify new loci and potential druggable targets

Chronic Obstructive Pulmonary Disease (COPD) is characterised by reduced lung function and is the third leading cause of death globally. Through genome-wide association discovery in 48,943 individuals, selected from extremes of the lung function distribution in UK Biobank, and follow-up in 95,375 individuals, we increased the yield of independent signals for lung function from 54 to 97. A genetic risk score was associated with COPD susceptibility (odds ratios per standard deviation of the risk score (~6 alleles) (95% confidence interval) 1.24 (1.20-1.27), P=5.05x10^-49) and we observed a 3.7 fold difference in COPD risk between highest and lowest genetic risk score deciles in UK Biobank. The 97 signals show enrichment in development, elastic fibres and epigenetic regulation pathways. We highlight targets for drugs and compounds in development for COPD and asthma (genes in the inositol phosphate metabolism pathway and CHRM3) and describe targets for potential drug repositioning from other clinical indications