Pierre Loti'nin yüzüncü doğum yıldönümü

Taha Toros Arşivi, Dosya Adı: Pierre Lotiİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

Functional Diversification of Motor Neuron-specific <i>Isl1</i> Enhancers during Evolution

<div><p>Functional diversification of motor neurons has occurred in order to selectively control the movements of different body parts including head, trunk and limbs. Here we report that transcription of <i>Isl1</i>, a major gene necessary for motor neuron identity, is controlled by two enhancers, CREST1 (E1) and CREST2 (E2) that allow selective gene expression of <i>Isl1</i> in motor neurons. Introduction of GFP reporters into the chick neural tube revealed that E1 is active in hindbrain motor neurons and spinal cord motor neurons, whereas E2 is active in the lateral motor column (LMC) of the spinal cord, which controls the limb muscles. Genome-wide ChIP-Seq analysis combined with reporter assays showed that Phox2 and the Isl1-Lhx3 complex bind to E1 and drive hindbrain and spinal cord-specific expression of <i>Isl1</i>, respectively. Interestingly, Lhx3 alone was sufficient to activate E1, and this may contribute to the initiation of <i>Isl1</i> expression when progenitors have just developed into motor neurons. E2 was induced by onecut 1 (OC-1) factor that permits <i>Isl1</i> expression in LMCm neurons. Interestingly, the core region of E1 has been conserved in evolution, even in the lamprey, a jawless vertebrate with primitive motor neurons. All E1 sequences from lamprey to mouse responded equally well to Phox2a and the Isl1-Lhx3 complex. Conversely, E2, the enhancer for limb-innervating motor neurons, was only found in tetrapod animals. This suggests that evolutionarily-conserved enhancers permit the diversification of motor neurons.</p></div

Conservation of putative regulatory elements of the E1 enhancer in vertebrates.

<p>(A) Alignment of the E1 enhancers in several organisms using the mouse E1 sequences as base line. Basewise conservation scores (phyloP) of vertebrate genomes (Human, Chicken, Zebrafish, Fugu) with mouse was shown. Dashed lines indicate Phox2 and Isl1-Lhx3 binding sites within E1. (B) Aligned Phox2 (shaded in green) and Isl1-Lhx3 (shaded in red) motif sequences in E1 enhancer among multiple species. Asterisks indicate conserved nucleotides conserved in all 7 species. Lamprey-specific insertion sites with > 10 bp are hidden with their full length base pair number. (C) A phylogenetic tree of E1. (D-S) The E1::GFP reporters from mouse, chick and zebrafish had different activities when electroporated into the chick neural tube. Scale bars: in P, 100 μm for D, H, L, P; in S, 50 μm for E-G, I-K, M-O, Q-S.</p

The Isl1-Lhx3 complex activates the E1 enhancer in somatic motor neurons.

<p>(A) Isl1 and Lhx3 ChIP-Seq peaks around the <i>Isl1</i> locus in NIL cells. The highest peaks of both ChIP-Seq profiles lie in E1. (B) The Phox2 motif (shaded in green) and LIM-HD motif (shaded in red) are shown in E1. The point mutations introduced into the homeodomain recognition motifs are underlined. (C) Luciferase activity of various E1 reporters in the presence of ddI1L3. Error bars represent SEM. *<i>p</i> < 0.05; **<i>p</i> < 0.01; unpaired Student’s t-test (n = 3). (D-K) In ovo electroporation of E1 derivatives with Isl1 and Lhx3 constructs. MutE1-3 is not active in the dorsal spinal cord in the presence of Isl1 and Lhx3, unlike the others (brackets, D-F, H). Six tandem repeats of the E1-3 site (6xE1-3) were sufficient for ectopic reporter activity (bracket, I). Expression of Isl1 and Lhx3 was validated in adjacent sections in which E1 was electroporated (J, K). (L) GFP intensity in dorsal spinal cord was measured in each group. Error bars represent SEM. ***<i>p</i> < 0.001 vs. E1; ###<i>p</i> < 0.001 vs. E1 with Isl1+Lhx3; Kruskal-Wallis test (> 14 sections in 5 embryos in each group). Scale bar: 100 μm.</p

Phox2 regulates Isl1 expression via the E1 enhancer in bm/vm neurons.

<p>(A) Phox2a ChIP-Seq peaks around <i>Isl1</i> locus in NIP cells from two independent experiments. Note the highest peak in the E1 region. (B) Luciferase reporter activity of the E1 reporter and its derivatives in 293T cells. Error bars represent SEM. ***<i>p</i> < 0.001; unpaired Student’s t-test (n = 3). (C-K) Comparison of E1::GFP reporter derivatives by chick in ovo electroporation. Induction of GFP by Phox2a (brackets, E, G-I) in which <i>Phox2a</i> was induced (compare D vs F) was abolished in the case of mutE1-3 and mutE1-4 (J, K). (L) GFP intensity in dorsal spinal cord was measured in each group. Error bars represent SEM. **<i>p</i> < 0.01, ***<i>p</i> < 0.001 vs. E1; ###<i>p</i> < 0.001 vs. E1 with Phox2a; n.s., not significant; Kruskal-Wallis test (> 16 sections in 5 embryos in each group). (M, N) Interaction between the Phox2b and E1-4 motifs was demonstrated in gel shift assays and chromatin immunoprecipitation experiments. Asterisks are Phox2-containing protein-DNA complexes. (O, P) E1::ndGFP reporter activity is reduced in si<i>Phox2b</i>-electroporated group. CMV::RFP was co-electroporated to confirm electroporation efficiency. (Q) Quantification of E1::ndGFP labeled motor neurons in each group. Error bar represents SEM. ***<i>p</i> < 0.001; unpaired Student’s t-test (n = 34 sections; si<i>Phox2b</i>, n = 48 sections). Scale bars: in K, 100 μm for C-K; in P, 100 μm for O, P.</p

The E1 and E2 enhancers label different subsets of motor neurons.

<p>(A-H) Activity of the E1 enhancer fused with the nuclear GFP (nGFP) or destabilized nuclear GFP (ndGFP) reporter electroporated into HH stage 10 to 12 chick neural tubes. The enhancer activity in each motor column was analyzed by quadruple labeling of GFP, Lhx3, Foxp1 and Isl1 by immunohistochemistry. Images of GFP, Lhx3, Foxp1 and GFP, Isl1 and Foxp1 in each condition were obtained from identical sections. Arrowheads are migrating immature motor neurons (A, E). (I-P) Distribution of motor neurons labeled by the E2::nGFP and E2::ndGFP reporters. (B, D, F, H, J, L, N, P) Diagrams summarize the distribution of GFP reporter expression (circles) in MMC (green), LMCm (red), LMCl (blue), HMC (light green) and PGC (orange) at brachial and thoracic levels. (Q-T) The proportion of GFP-expressing cells in each column (>16 sections in 8 embryos in each group) was determined. Error bar represents SEM using three replicates. Scale bar: 50 μm.</p

Lhx3 and the Isl1-Lhx3 complex activate the E1 enhancer in somatic motor neurons, and this is repressed by Sox1 and Chx10.

<p>(A-B”‘) Activity of the E1::nGFP enhancer and expression of Olig2, Lhx3 or Isl1 in HH stage 23 and 26 chick embryos. Dotted lines mark the border of pMN domain and arrowheads mark migrating motor neurons that weakly express Isl1 (> 16 sections in 6 embryos in each group). (C) Summary diagram of transcription factors present in pMN and p2 progenitors and neurons. (D-H’) Co-electroporation of GFP reporters with Isl1, Lhx3, Isl1 and Lhx3 (Isl1+Lhx3), or ddI1L3 in chick neural tubes. Expression of Isl1 and Lhx3 and their activity to induce ectopic MNR2⁺ motor neurons or Chx10⁺ V2a interneurons were assessed by triple labeling of sections with markers indicated. Lower panels (D’-H’) show identical sections without GFP overlay. The E1 reporter is activated by Lhx3, Isl1+Lhx3 or ddI1L3 (brackets), which overlapped with Chx10 (open arrowheads, E, G) or MNR2 (arrowheads, F-H). (I-L) Ectopic Isl1 expression in the Isl2+Lhx3 group but not in the Lhx3-electroporated group. Overexpression of Isl2 and Lhx3 were confirmed as indicated. (M-U) Co-electroporation of GFP reporters with Lhx3, Olig2, Sox1 or Chx10 as indicated. Overexpression of Olig2, Sox1, Lhx3 and Chx10 were confirmed as indicated. The E1 reporter driven by Lhx3 was repressed in both dorsal and ventral spinal cords in the presence of Olig2, Sox1 and Chx10 (dotted brackets, N, Q, T). (V) GFP intensity in dorsal spinal cord of electroporated groups. Error bars represent SEM. ***<i>p</i> < 0.001 vs. vector; #<i>p</i> < 0.05, ##<i>p</i> < 0.01, ###<i>p</i> < 0.001 vs. Lhx3; Kruskal-Wallis test (> 14 sections in 6 embryos in each group). (W) Induction of the E1 luciferase reporter by various transcription factors. Error bars represent SEM. ***<i>p</i> < 0.001; unpaired Student’s t-test (n = 3). Scale bars: in A””, 20 μm for A-A”‘; in B”‘, 50 μm for B-B”‘; in U, 50 μm for D-U.</p

Additional file 9 of In silico re-identification of properties of drug target proteins

Figure S7. 10-fold and 10x10-fold cross-validations result in terms of the F-score and the standard derivation. (A) 10-fold cross-validation for SVM. (B) 10-fold cross-validation for RF. (C) 10X10 fold cross-validation for SVM. (D) 10X10 fold cross-validation for RF. (PDF 207 kb

Procedure Used to Obtain Overrepresented Oligomers Starting from the Overrepresented 12-Mers

<p>The overrepresented 12-mers were defined with the initial criteria: at least ten occurrences and at least 5-fold enrichment. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020151#s2" target="_blank">Results</a> and Methods for a detailed description.</p

LDA Classification Success Rates for Different Values of the Tuning Parameter τ

<div><p>(A) Training set derived largely, but not exclusively, from Xp22 (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020151#pgen-0020151-st003" target="_blank">Table S3</a>).</p><p>(B) Test set of Xp22 genes, with training performed on genes in (A).</p><p>(C) Test set of X genes outside of Xp22, with training performed on genes in (A).</p><p>(D) Training set of all X genes, including genes in Xp22. Dots indicate optimal values of τ (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020151#pgen-0020151-t004" target="_blank">Table 4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020151#s4" target="_blank">Methods</a>).</p></div