EMSA with Isl1 truncations and Pou4f2.

<p>Left: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on SBRN3(W), but not with Isl1D1 and Isl1D2. Right: Pou4f2 forms complexes with Isl1D3, Isl1D4 and Isl1D5 on CBRN3(W), but not with Isl1D1 and Isl1D2. F indicates free probes.</p

Diagram of serial deletions of Pou4f2 and Isl1.

<p>Diagram of the Pou4f2 (Left) and Isl1 (right) protein structures and the truncated deletions we expressed in <i>E. Coli</i>. The amino acid residues for each deletion and their estimated molecular weights are indicated. All proteins are His-tagged at the C terminus for easy detection by Western Blotting, although the Isl1 protein and its truncates are detected by a polyclonal rabbit anti-Isl1. * indicates constructs capable of protein-protein interaction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092105#pone-0092105-g003" target="_blank"><b>Figs. 3</b></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092105#pone-0092105-g005" target="_blank"><b>5</b></a>).</p

Pou4f2 and Isl1 form a complex in vitro and in vivo.

<p>A. Top: GST-Pou4f2 fusion protein (GST-P), but not GST, can interact with Isl1, as detected by anti-Isl1. Bottom: anti-GST demonstrates the GST and GST-Pou4f2 proteins used in the pull-down assays. Smaller bands in the GST-Pou4f2 lane are degradation products. B. Top: GST-Isl1 fusion protein (GST-I), but not GST alone, can interact with Pou4f2 as detected by anti-Pou4f2. Bottom: anti-GST recognizes both GST and GST-Isl1. Like GST-Pou4f2, there was also degradation of GST-Isl1 as indicated by the smaller bands. C. Co-Immunoprecipitation (IP) of Isl1 and Pou4f2 by a rabbit anti-Isl1. Both proteins were expressed in the HEK293 cells and could be detected in the input lysate by a mouse anti-Isl1 and goat anti-Pou4f2. In the rabbit IgG control, neither Isl1 nor Pou4f2 could be detected. When rabbit anti-Isl1 was used for the IP, both Isl1 and Pou4f2 could be detected.</p

GST pull-down assays show other Lim-Homeodomain and POU-domain proteins interact with Pou4f2 and Isl1 respectively.

<p>A: GST-Isl1 (GST-I), but not GST alone, can interact with Pou3f2 and Pou4f3, but not Pou6f1. Pou4f2 was used as positive control. Top: Detection of pulled down proteins by anti-His. Middle: Input GST or GST-Isl1 by anti-GST. Bottom: Input POU-Homeodmain proteins detected by anti-His. B,C,D: GST-Pou4f2 (GST-P), but not GST alone, can interact with Isl2, Lhx2, but not Lhx1. In each panel, the top is detection of pulled down protein by the indicated antibodies, the middle is input GST or GST-Pou4f2 as detected by anti-GST, and the bottom is detection of the input Lim-Homeodomain proteins by the indicated antibodies (anti-Isl1 for Isl1 and Isl2, anti-Lhx2 for Lhx2, and anti-His for Lhx1).</p

Identification of domains within Pou4f2 and Isl1 responsible for their mutual interaction.

<p>A. Top: GST-Isl1 could interact with Pou4f2D4 and Pou4f2D5, but not Pou4f2D1, Pou4f2D2 or Pou4f2D3, as detected by anti-His. Full-length Pou4f2 serves as positive control. Middle: Input GST or GST-Isl1 detected by anti-GST. Bottom: Input Pou4f2 and its deletions could all be detected by anti-His. B: Top: GST-Pou4f2 (GST-P), but not GST, could interact with Isl1, Isl1D3, Isl1D4 and Isl1D5, but not with Isl1D1 and Isl1D2. Middle: Input GST or GST-Pou4f2 as detected by anti-GST. Bottom: Input Isl1 or its deletions as detected by a rabbit polyclonal anti-Isl1. Sizes (Kda) of protein markers are indicated on the side. Antibodies used for each blot are indicated at the bottom of each panel.</p

Pou4f2 and Isl1 bind to DNA elements individually and as a complex in EMSA.

<p>A: Wild-type (SBRN3(W)) sequence from the conserved element in the Sonic Hedgehog gene and its mutant sequence SBRN3(M) are shown on the top. Lines on top and beneath the SBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively. At Bottom, EMSA shows that either Pou4f2 or Isl1 alone can bind to SBRN3(W) as indicated by the slow migrating DNA-protein complexes, but not to SBRN3(M). When both Pou4f2 and Isl1 are present, they form a further slow-migrating DNA-protein complex with SBRN3(W), but not with SBRN3(M). B: Similarly, Pou4f2 or Isl1 alone binds to CBRN3(W), but not to CBRN3(M); Pou4f2 and Isl1 form a complex on CBRN3(W), but not on CBRN3(M). F indicates free probes. Lines on top and beneath the CBRN3(W) sequence indicate the ATTA cores and Pou4f2 binding motif respectively.</p

EMSA with Pou4f2 truncations and Isl1.

<p>Left: Isl1 forms complexes with Pou4f2D4 and Pou4f2D5 on SBRN3(W), but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3. Right: Isl1 forms complexes with Pou4f2 D4 and Pou4f2D5, but not with Pou4f2D1, Pou4f2D2 and Pou4f2D3 on CBRN(W). F is free probe. These results are consistent with those from the pull-down assays.</p

Isl1 and Pou4f2 contribute quantitatively to the full levels of expression of downstream genes.

<p>A. Luciferase assays indicating that whereas Pou4f2 and Isl1 can each activate transcription from the reporter alone, together they promote transcription to a higher level. N = 3 for all transfections. Significance of differences was evaluated by student t test. Double black asterisks indicate p<0.01 when compared with control, double red asterisks indicate p<0.01 when compared with Pou4f2, double purple asterisks indicates p<0.01 when compared with Isl1. B. In situ hybridization of three downstream genes, <i>Gap43</i>, <i>Nefl</i>, and <i>Stmn2</i>, of Isl1 and Pou4f2. Compared to the wild-type retina, although all three genes have significantly reduced levels of expression in the RGCs of both <i>Isl1</i>- and <i>Pou4f2</i>-null retinas, substantial levels of expression remain. In the <i>Isl1</i>/<i>Pou4f2</i> double null retinas, <i>Gap43</i> and <i>Nefl</i> are not detectable, but Stmn2 is still expressed. C. The expression levels of downstream genes of Isl1 and Pou4f2 vary greatly. Shown are the relative expression levels of five downstream genes as revealed by RNA-seq. Y axis is Fragments Per Kilobase of exon per Million fragments mapped (FPKM) based on the number of sequence reads and lengths of the transcripts of individual genes. D. Changes of expression levels of five downstream genes of Isl1 and Pou4f2 in the <i>Isl1</i>- and <i>Pou4f2</i>–null retinas as revealed by RNA-seq. WT: wild-type; P4f2: Pou4f2. The expression levels (Y axis) are calculated based on the FPKM of three replicates and the average values for each gene in the wild-type is set to1. N = 3 for all samples. Significance of differences was evaluated by student's t test. Single asterisk indicates p<0.05, double asterisks indicate p<0.01.</p

Conserved DNA motifs in <i>Ebf3</i> and <i>Irx6</i> are recognized by Isl1/Pou4f2 complex.

<p>A: Mouse-human comparison to identify conserved regions in <i>Ebf3</i> and <i>Irx6</i> by VISTA. Red marks conserved non-coding region, light-blue is UTR, and dark-blue indicates exons. Heights of peaks indicate percentage of identify. The regions where the potential Isl1/Pou4f2 binding sites were identified are underlined. B: EMSA with wild-type and mutant probes derived from <i>Ebf3</i>. The sequences of wild-type and mutant probes are shown on the top. Lines on top and underneath the wild-type sequences indicate the ATTA cores and Pou4f2 binding motif respectively. The bottom is the EMSA results. Both Isl1 and Pou4f2 could independently bind to the wild-type probe, but not the mutant probe. The Isl1/Pou4f2 complex can bind the wild-type probe, but not the mutant, as indicated by the slow-migrating complex. C: The same experiment as that of B, except that the wild-type and mutant probes were derived from <i>Irx6</i>. Both proteins as well as the complex could bind to the wild-type probe, but not the mutant one. D: ChIP analysis of Isl1 and Pou4f2 binding in vivo. Genomic DNA from ChIP by anti-Pou4f2 (P4f2) and anti-Isl1 were amplified by PCR with primers flanking the Isl1/Pou4f2-binding regions of <i>Ebf3</i> and <i>Isl1</i> and a control region of β-actin. Non-specific IgG was used as control in both ChIP experiments.</p