High-throughput analyses of hnRNP H1 dissects its multi-functional aspect

<p>hnRNPs are polyvalent RNA binding proteins that have been implicated in a range of regulatory roles including splicing, mRNA decay, translation, and miRNA metabolism. A variety of genome wide studies have taken advantage of methods like CLIP and RIP to identify the targets and binding sites of RNA binding proteins. However, due to the complex nature of RNA-binding proteins, these studies are incomplete without assays that characterize the impact of RBP binding on mRNA target expression. Here we used a suite of high-throughput approaches (RIP-Seq, iCLIP, RNA-Seq and shotgun proteomics) to provide a comprehensive view of hnRNP H1s ensemble of targets and its role in splicing, mRNA decay, and translation. The combination of RIP-Seq and iCLIP allowed us to identify a set of 1,086 high confidence target transcripts. Binding site motif analysis of these targets suggests the TGGG tetramer as a prevalent component of hnRNP H1 binding motif, with particular enrichment around intronic hnRNP H1 sites. Our analysis of the target transcripts and binding sites indicates that hnRNP H1s involvement in splicing is 2-fold: it directly affects a substantial number of splicing events, but also regulates the expression of major components of the splicing machinery and other RBPs with known roles in splicing regulation. The identified mRNA targets displayed function enrichment in MAPK signaling and ubiquitin mediated proteolysis, which might be main routes by which hnRNP H1 promotes tumorigenesis.</p

The <i>Six2</i> regulatory landscape is altered in the <i>Br</i> mouse leading to reduced Six2 expression and ectopic Six3 expression in the kidney.

<p>(A) Schematic showing the X-irradiation induced breakpoints and subsequent deletion with inversion that resulted in the <i>Br</i> allele. LE = Lens enhancer (putative), PE = proximal enhancer, DE = distal enhancer. Black box between the <i>Six2</i> and <i>Six3</i> loci represents the predicted boundary. (B) Interaction matrix (top) generated by Hi-C data (Hardison lab hESC Hi-C data, <a href="http://promoter.bx.psu.edu/hi-c/view.php" target="_blank">http://promoter.bx.psu.edu/hi-c/view.php</a>). Genomic view showing Six2 ChIP-seq, CTCF-NP ChIP-seq, and 4C-seq data (bottom). The region inverted in the <i>Br</i> allele is highlighted. Dashed square indicates a predicted TAD boundary element that lies between <i>Six3</i> and <i>Six2</i> loci [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007181#pgen.1007181.ref070" target="_blank">70</a>]. (C) qPCR showing the relative expression levels of <i>Six2</i> and <i>Six3</i> in E13.5 kidneys of the indicated genotype. (D) E13.5 kidneys of the indicated genotype were sectioned and immunostained for Six2 and Six3. Arrow points to the low level Six3 expression in nephron progenitors. (E) Immunostaining for Six2, Six3, cytokeratin (CK), and DAPI in E11.5 kidneys of the indicated genotype.</p

Six2, Hoxd11, Osr1, and Wt1 binding sites are enriched near nephron progenitor specific genes and those associated with differentiation programs.

<p>(A) Scatter plots show gene expression profiles and correlation of the Six2GFP+ versus the Six2GFP- RNA-seq from E16.5 mouse kidney cortex. Specific genes for each category are highlighted in orange (Six2+) or blue (Six2-). TPM = Transcripts Per Kilobase Million. (B) Scatter plots show gene expression profiles and correlation of RNA-seq from E16.5 Cited1RFP+ cells versus P2 Six2GFP+ cells. Genes specific to each population are highlighted in red (Cited1RFP+) or green (Six2GFP+). Examples of specific genes are listed and highlighted on the plot. (C) Barplots show p-values indicating enrichment of Six2, Hoxd11, Osr1, and Wt1 binding sites, as well as regulatory hotspots (Six2/Hoxd11/Osr1/Wt1 overlapping sites) in genes that are specific to the Six2+ cortex fraction, specific to the Six2- cortex, enriched in self-renewing nephron progenitors, or enriched in differentiating nephron progenitors, respectively. The regulatory domain was defined as +/-500 kb from transcription start site. TFBS = transcription factor binding site. ‘Obs.’, number of peaks associated with genes annotated with corresponding term; ‘Exp.’, number of peaks expected to be associated with genes annotated with corresponding term by chance. Fold represents the fold enrichment or expected values. (D) Bar plots showing the percentage of total genes for each condition (x-axis) that falls into each category of 1) nephron progenitor (NP) enriched, 2) enriched in self-renewing nephron progenitors, or 3) enriched in differentiating progenitors.</p

Deletion of the <i>Six2</i> distal enhancer leads to reduction in <i>Six2</i> levels and concomitant loss of a <i>Six2</i> allele results in severe renal hypoplasia.

<p>(A) Schematic of the <i>Six2</i> locus showing the location of the proximal (PE) and distal (DE) enhancer elements. The <i>DE</i> was targeted for deletion using CRISPR/Cas9 and the resulting Cas9-mediated deletion of the <i>Six2-DE</i> is shown. (B) Brightfield images of whole urogenital systems from E16.5 embryos resulting from <i>Six2</i>^{<i>∆DE/+</i>} matings or <i>Six2</i>^<i>CE/+</i> x <i>Six2</i>^{<i>∆DE/+</i>} crosses. (C) Immunostaining for Wt1 to identify nephron progenitors and podocytes, LTL (<i>Lotus tetragonolobus</i> lectin) to mark proximal tubules, and Cdh1 to show the collecting duct network of kidneys associated with (B). (D) Immunostaining for Six2 to identify nephron progenitors in kidneys associated with (B). (E) Box plots showing results of qPCR for <i>Six2</i> and <i>Pax2</i> (normalized to <i>GAPDH</i>) from nephron progenitors (NP) and nephron progenitor-depleted cortex. Genotypes and number of samples analyzed are shown. (F) Samples from <i>Six2</i>^{<i>GCE/GCE</i>} were compared to <i>Six2</i>^{<i>∆DE/GCE</i>} collected at early stages of kidney development and immunostained with Six2 to mark the nephron progenitors, Pax8 to identify differentiating structures (Pax8 antibody appears to cross react with Pax2 as seen by expression in Ecad+ collecting duct), and Ecad to mark epithelial structures.</p

Regulatory hotspots in nephron progenitors defined by co-binding of Six2, Hoxd11, Osr1 and Wt1.

<p>(A) Heatmap shows significance of pairwise overlap between transcription factor binding sites (left, represented by binomial -log10 p-value) or between assigned target genes (right, represented by ratio). TFBS = transcription factor binding site. (B) Venn diagram shows the overlap of Six2, Hoxd11, Osr1, and Wt1 binding sites (left) and target genes (right). The 4-way overlapping sites were defined as the ‘regulatory hotspots’. The 4-way overlapping target genes were defined as ‘core targets’. (C) Barplots show result of gene ontology (GO) analysis on the ‘regulatory hotspots’ (left). Examples of ‘core targets’ known to have roles in the nephron progenitors and their differentiation are listed (right). (D) Genome browser view of Six2, Hoxd11, Osr1, and Wt1 ChIP-seq signals at the ‘regulatory hotspots’ (shadow area) near <i>Six2</i> and <i>Wnt4</i>. (E) Six2 immunoprecipitation from E16.5 kidney nuclear extracts. Western blot was probed with antibodies to Six2, Hoxd11, and Wt1 to identify protein complexes.</p