Saritas-Yildirim B et al. (2015), Identification of REST targets in the Xenopus t...

XB-ART-50675

BMC Genomics 2015 May 14;16:380. doi: 10.1186/s12864-015-1591-4.

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Identification of REST targets in the Xenopus tropicalis genome.

Saritas-Yildirim B , Childers CP , Elsik CG , Silva EM .

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A major role of REST (repressor element-1 silencing transcription factor) is to inhibit the expression of neuronal genes in neural stem cells and non-neuronal cells by binding to a 21 bp consensus sequence and recruiting epigenetic and regulatory cofactors to gene regulatory regions. In neural stem cells, REST silences differentiation-promoting genes to prevent their premature expression and is central to the regulation of neurogenesis and the balance of neural stem cells and neurons. To understand the role of REST in vertebrate neurogenesis, we performed a genome-wide screen for REST targets in Xenopus tropicalis. We identified 742 neuron-restrictive silencer elements (NRSE) associated with 1396 genes that are enriched in neuronal function. Comparative analyses revealed that characteristics of NRSE motifs in frog are similar to those in mammals in terms of the distance to target genes, frequency of motifs and the repertoire of putative target genes. In addition, we identified four F-box ubiquitin ligases as putative REST targets and determined that they are expressed in neuronal tissues during Xenopus development. We identified a conserved core of putative target genes in human, mouse and frog that may be fundamental to REST function in vertebrates. We demonstrate that NRSE sites are associated with both protein-coding genes and lncRNAs in the human genome. Furthermore, we demonstrate that REST binding sites are abundant in low gene-occupancy regions of the human genome but this is not due to an increased association with non-coding RNAs. Our findings identify novel targets of REST and broaden the known mechanism of REST-mediated silencing in neurogenesis.

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Species referenced: Xenopus tropicalis
Genes referenced: actl6a fbxl7 fbxo16 fbxo41 hdac1 kdm2b sin3a sin3b

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	Figure 1 In X. tropicalis, human, and mouse, the consensus NRSE motifs share sequence similarity and the majority of the NRSE sites are farther than 20 kb from a gene, with a single copy per gene. A. Sequence logos of the consensus NRSE motif derived from 32 bona fide mouse REST target genes ([26], top panel), from 1301 human NRSE motifs ([25], middle panel), and from 742 X. tropicalis NRSE motifs (bottom panel). Arrowheads mark the differences between the Xenopus and human consensus motifs. B. In all three vertebrate genomes, ~ 65% of the motifs are located farther than 20 kb from a gene. C. 80% to 90% of the putative REST target genes in three organisms have a single NRSE motif within 100 kb of a gene.
	Figure 2 The species-specific features of X. tropicalis NRSEs. A. The most abundant 20 motifs and copy number in the X. tropicalis genome. The asterisk marks the most abundant motif in the humans genome. The green highlight marks the linker region (positions 10 and 11) of the NRSE motif. The red âCâ is the only difference between the most abundant NRSE motifs in Xenopus and humans. B. The number of NRSE motif permutations in common between the three vertebrate genomes. C. The consensus motifs derived from the 78 NRSEs in common between humans and X. tropicalis, and the 236 X. tropicalis specific motifs. Arrowheads show the deviations from the X. tropicalis consensus derived from all motifs.
	Figure 3 Human NRSEs are associated with lncRNAs genome-wide but not in gene-distant regions. A. 17-24% of NRSE motifs in the human, mouse and frog genomes are located in gene-distant regions (GD). B. In the human genome, NRSEs within 100 kb of ncRNAs are associated with lncRNAs (chi-square, p = 0.00102547). In the figure, <100 kb denotes the NRSEs that are within 100 kb of a protein-coding gene and >100 kb is for NRSEs that are located in GD.
	Figure 4 Gene Ontology classification of putative NRSE target genes. A. Genes were linked to 14 functional groups or âothersâ subgroup based on GO descriptions. B. The 111 common NRSE target genes in human, mouse, and frog genomes are enriched in neuronal functions. All categories are statistically significant with single and double asterisks showing p-values <0.05 and <0.01, respectively.
	Figure 5 In vivo validation of the Xenopus NRSE screen. Four F-box ubiquitin ligases identified in the NRSE screen are expressed in the neuronal tissues of X. tropicalis. A. The genomic localization of the genes with respect to NRSE motifs and B. their mRNA expression hybridization during X. tropicalis development. Gastrula embryos are ventral view with dorsal to the top. Neurula embryos are dorso-lateral view with anterior to the right. Early tailbud embryos are dorsal view with anterior to the right. Tailbud heads are lateral view with anterior to the right. The arrows point the direction of genes. Intervening genes are in turquoise. Cartoons are not to scale.
	Figure 6 REST mediated repression of coding and non-coding gene expression. In neural progenitors and non-neuronal cells, REST facilitates silencing of expression by binding to highly conserved NRSE elements and recruiting co-repressors and chromatin remodeling agent to convert the topology of the local DNA to heterochromatin. During neurogenesis, REST transcription is down regulated and the protein is degraded. The absence of REST in neuronal cells allows the expression of neuron specific protein-coding genes and non-coding RNAs including micro and long non-coding RNAs.
	fbxo16 (F-box protein 16) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 11, vegetal view dorsal up.
	fbxo16 (F-box protein 16) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior right.
	fbxo16 (F-box protein 16) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.
	fbxo41 (F-box protein 41) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 11 vegetal view, dorsal up.
	fbxo41 (F-box protein 41) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior right.
	fbxo41 (F-box protein 41) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.
	fbxl7 (F-box and leucine-rich repeat protein 7) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 11, vegetal view, dorsal up.
	fbxl7 (F-box and leucine-rich repeat protein 7) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 22, dorsal view, anterior right, dorsal up.
	fbxl7 (F-box and leucine-rich repeat protein 7) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.
	Figure 1. In X. tropicalis, human, and mouse, the consensus NRSE motifs share sequence similarity and the majority of the NRSE sites are farther than 20 kb from a gene, with a single copy per gene. A. Sequence logos of the consensus NRSE motif derived from 32 bona fide mouse REST target genes ([26], top panel), from 1301 human NRSE motifs ([25], middle panel), and from 742 X. tropicalis NRSE motifs (bottom panel). Arrowheads mark the differences between the Xenopus and human consensus motifs. B. In all three vertebrate genomes, ~ 65% of the motifs are located farther than 20 kb from a gene. C. 80% to 90% of the putative REST target genes in three organisms have a single NRSE motif within 100 kb of a gene.
	Figure 2. The species-specific features of X. tropicalis NRSEs. A. The most abundant 20 motifs and copy number in the X. tropicalis genome. The asterisk marks the most abundant motif in the humans genome. The green highlight marks the linker region (positions 10 and 11) of the NRSE motif. The red âCâ is the only difference between the most abundant NRSE motifs in Xenopus and humans. B. The number of NRSE motif permutations in common between the three vertebrate genomes. C. The consensus motifs derived from the 78 NRSEs in common between humans and X. tropicalis, and the 236 X. tropicalis specific motifs. Arrowheads show the deviations from the X. tropicalis consensus derived from all motifs.
	Figure 3. Human NRSEs are associated with lncRNAs genome-wide but not in gene-distant regions. A. 17-24% of NRSE motifs in the human, mouse and frog genomes are located in gene-distant regions (GD). B. In the human genome, NRSEs within 100 kb of ncRNAs are associated with lncRNAs (chi-square, pâ=â0.00102547). In the figure, <100 kb denotes the NRSEs that are within 100 kb of a protein-coding gene and >100 kb is for NRSEs that are located in GD.
	Figure 4. Gene Ontology classification of putative NRSE target genes. A. Genes were linked to 14 functional groups or âothersâ subgroup based on GO descriptions. B. The 111 common NRSE target genes in human, mouse, and frog genomes are enriched in neuronal functions. All categories are statistically significant with single and double asterisks showing p-values <0.05 and <0.01, respectively.
	Figure 5. In vivo validation of the Xenopus NRSE screen. Four F-box ubiquitin ligases identified in the NRSE screen are expressed in the neuronal tissues of X. tropicalis. A. The genomic localization of the genes with respect to NRSE motifs and B. their mRNA expression hybridization during X. tropicalis development. Gastrula embryos are ventral view with dorsal to the top. Neurula embryos are dorso-lateral view with anterior to the right. Early tailbud embryos are dorsal view with anterior to the right. Tailbud heads are lateral view with anterior to the right. The arrows point the direction of genes. Intervening genes are in turquoise. Cartoons are not to scale.
	Figure 6. REST mediated repression of coding and non-coding gene expression. In neural progenitors and non-neuronal cells, REST facilitates silencing of expression by binding to highly conserved NRSE elements and recruiting co-repressors and chromatin remodeling agent to convert the topology of the local DNA to heterochromatin. During neurogenesis, REST transcription is down regulated and the protein is degraded. The absence of REST in neuronal cells allows the expression of neuron specific protein-coding genes and non-coding RNAs including micro and long non-coding RNAs.

References [+] :

Andrés, CoREST: a functional corepressor required for regulation of neural-specific gene expression. 1999, Pubmed