Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
PLoS Genet
2013 Jan 01;99:e1003847. doi: 10.1371/journal.pgen.1003847.
Show Gene links
Show Anatomy links
The Compass-like locus, exclusive to the Ambulacrarians, encodes a chromatin insulator binding protein in the sea urchin embryo.
Cavalieri V
,
Melfi R
,
Spinelli G
.
???displayArticle.abstract???
Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts.
Figure 1. The CMPl protein family.(A) Predicted amino-acid sequence of the P. lividus CMPl, with Compass- and Homeo- domain highlighted in green and yellow, respectively. (B) Comparison of the CMPl, CMP and SATB Homeodomains among various species. Differently coloured boxes highlight similarities; dashes represent the gaps inserted for maximal alignment; position 48 is marked by a filled circle, whereas the glycine insertion in SATB sequences is pointed by a triangle. (C) Rooted neighbor-joining tree constructed from the Homeodomains of representative CMPl, CMP and SATB family members. P. lividus sequence names are in red; numbers above nodes record percent bootstrap values, while asterisks indicate outgroups. Complete taxonomic names and accession numbers of all the sequences used to elaborate the tree are listed in Supplementary Table S1.
Figure 2. The Cmp/Cmpl locus and its products.(A) Schematic drawing of the Cmp/Cmpl gene structure showing the positions of exons. Phylogenetic footprinting analysis is shown beneath the diagram. (B) Structure of the alternatively spliced mRNAs. (C) Diagrammatic representation of the CMPl and CMP protein domain organization. Red square indicates common amino acidic sequence at the N-terminal side. (D) qRT-PCR analysis of Cmpl and Cmp transcripts throughout P. lividus embryogenesis. Values at the various stages are shown as fold difference with respect to the 16-cell stage, which displays roughly equal amount of Cmpl and Cmp mRNAs. Bars are standard errors for the qPCR replicas.
Figure 3. Association of the CMPl protein to the boxB cis-element on sns5.(A) Western blot analysis to test the specificity of the anti-CMPl antibody. Nuclear extracts from morula (lanes 1, 3) and gastrula (lanes 2, 4) embryos were fractioned by SDS-PAGE, blotted on nitrocellulose membrane and incubated with anti-CMPl antibody (lanes 1, 2) or pre-immune serum (lanes 3, 4). (B) ChIP-qPCR analysis of the sns5 occupancy by CMPl. ChIP assays were performed on chromatin extracted from embryos at the indicated stages and precipitated with antiserum against CMPl or Otx, or incubated without adding antibodies (−), followed by qPCR amplification of an sns5 fragment containing the boxB sequence, or hbox12 and otp promoter fragments. As described previously, the association of the Otx transcription factor to its binding site within the hbox12 promoter correlates with the hbox12 transcriptional state [32]. Data are normalized according to the percent of input method. Bars are as in Figure 2D. (C) Scheme of the reporter and effector constructs used in the trans-activation assay. (D) Trans-activation analysis in developing sea urchin embryos observed at the mesenchyme blastula stage. DIC, epifluorescence and merged images, respectively ordered from top to bottom, are shown for each embryo. (E) GFP expression levels assessed by qPCR in transgenic embryos at the mesenchyme blastula stage. Graphs show n-fold changes in mRNA expression level of GFP based on the normalized threshold cycle numbers, with respect to the 5×boxB-pH3-GFP/VP16-CMPl co-injected embryos. Bars are as in Figure 2D.
Figure 4. Knock-down of the M30-enhancer and/or sns5-insulator functions and effect on the endogenous early histone gene transcription.(A) Annotated map of the P. lividus early H3, H2A and H1 histone genes, highlighting the cis-regulatory sequence elements. The horizontal black line represents the genomic DNA, while the bent arrows denote the putative transcription start site. (B–E) qPCR analysis of histone gene transcription carried out in embryos at morula stage injected with the VP16-CMPl/VP16-CMP transcripts (B), or with increasing amounts of the α-MBF1 (C) or α-CMPl (D) antibodies, or the chimeric dnMBF1 mRNA together with saturating dosage of the α-CMPl antibody (E). Graphs show n-fold changes in mRNA expression level of histone genes based on the normalized threshold cycle number of injected embryos compared to that of the uninjected control embryos. Data were derived from two independent microinjection experiments and each bar represents the average of triplicate samples from the two batches of embryos.
Acuto,
Functional characterization of the sea urchin sns chromatin insulator in erythroid cells.
2005, Pubmed,
Echinobase
Acuto,
Functional characterization of the sea urchin sns chromatin insulator in erythroid cells.
2005,
Pubmed
,
Echinobase
Alessandro,
Identification of the enhancer binding protein MBF-1 of the sea urchin modulator alpha-H2A histone gene.
2002,
Pubmed
,
Echinobase
Bell,
The protein CTCF is required for the enhancer blocking activity of vertebrate insulators.
1999,
Pubmed
Blanton,
Protein:protein interactions and the pairing of boundary elements in vivo.
2003,
Pubmed
Bromham,
Hemichordates and deuterostome evolution: robust molecular phylogenetic support for a hemichordate + echinoderm clade.
1999,
Pubmed
Bushey,
Chromatin insulators: regulatory mechanisms and epigenetic inheritance.
2008,
Pubmed
Bushey,
Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions.
2009,
Pubmed
Bürglin,
Homeodomain subtypes and functional diversity.
2011,
Pubmed
Bürglin,
Loss and gain of domains during evolution of cut superclass homeobox genes.
2002,
Pubmed
Castresana,
Codon reassignment and amino acid composition in hemichordate mitochondria.
1998,
Pubmed
Castresana,
The mitochondrial genome of the hemichordate Balanoglossus carnosus and the evolution of deuterostome mitochondria.
1998,
Pubmed
,
Echinobase
Cavalieri,
Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo.
2011,
Pubmed
,
Echinobase
Cavalieri,
Impairing Otp homeodomain function in oral ectoderm cells affects skeletogenesis in sea urchin embryos.
2003,
Pubmed
,
Echinobase
Cavalieri,
Regulatory sequences driving expression of the sea urchin Otp homeobox gene in oral ectoderm cells.
2007,
Pubmed
,
Echinobase
Cavalieri,
cis-Regulatory sequences driving the expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm territory of the Paracentrotus lividus sea urchin embryo.
2008,
Pubmed
,
Echinobase
Cavalieri,
Promoter activity of the sea urchin (Paracentrotus lividus) nucleosomal H3 and H2A and linker H1 {alpha}-histone genes is modulated by enhancer and chromatin insulator.
2009,
Pubmed
,
Echinobase
Cavalieri,
Functional studies of regulatory genes in the sea urchin embryo.
2009,
Pubmed
,
Echinobase
Ceska,
The X-ray structure of an atypical homeodomain present in the rat liver transcription factor LFB1/HNF1 and implications for DNA binding.
1993,
Pubmed
D'Apolito,
The sea urchin sns5 insulator protects retroviral vectors from chromosomal position effects by maintaining active chromatin structure.
2009,
Pubmed
,
Echinobase
Di Caro,
Constitutive promoter occupancy by the MBF-1 activator and chromatin modification of the developmental regulated sea urchin alpha-H2A histone gene.
2007,
Pubmed
,
Echinobase
Di Caro,
Down-regulation of early sea urchin histone H2A gene relies on cis regulative sequences located in the 5' and 3' regions and including the enhancer blocker sns.
2004,
Pubmed
,
Echinobase
Di Simone,
The sea urchin sns insulator blocks CMV enhancer following integration in human cells.
2001,
Pubmed
,
Echinobase
Dixon,
Topological domains in mammalian genomes identified by analysis of chromatin interactions.
2012,
Pubmed
Englbrecht,
Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome.
2004,
Pubmed
Furlong,
Bayesian phylogenetic analysis supports monophyly of ambulacraria and of cyclostomes.
2002,
Pubmed
Fuss,
Drosophila endoderm development requires a novel homeobox gene which is a target of Wingless and Dpp signalling.
1998,
Pubmed
Gaszner,
The Zw5 protein, a component of the scs chromatin domain boundary, is able to block enhancer-promoter interaction.
1999,
Pubmed
Gehring,
Homeodomain proteins.
1994,
Pubmed
Gerasimova,
Coordinated control of dCTCF and gypsy chromatin insulators in Drosophila.
2007,
Pubmed
Geyer,
DNA position-specific repression of transcription by a Drosophila zinc finger protein.
1992,
Pubmed
Gudynaite-Savitch,
Strategies to mitigate transgene-promoter interactions.
2009,
Pubmed
Heger,
Loss of the insulator protein CTCF during nematode evolution.
2009,
Pubmed
Hino,
Sea urchin arylsulfatase insulator exerts its anti-silencing effect without interacting with the nuclear matrix.
2006,
Pubmed
,
Echinobase
Hou,
CTCF-dependent enhancer-blocking by alternative chromatin loop formation.
2008,
Pubmed
Jung,
Identification of conserved Drosophila-specific euchromatin-restricted non-coding sequence motifs.
2010,
Pubmed
Kim,
Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome.
2007,
Pubmed
Kurukuti,
CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2.
2006,
Pubmed
Melfi,
Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences.
2000,
Pubmed
,
Echinobase
Mohan,
The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning.
2007,
Pubmed
Moon,
CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator.
2005,
Pubmed
Murrell,
Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops.
2004,
Pubmed
Palla,
Enhancer blocking activity located near the 3' end of the sea urchin early H2A histone gene.
1997,
Pubmed
,
Echinobase
Peterson,
Animal phylogeny and the ancestry of bilaterians: inferences from morphology and 18S rDNA gene sequences.
2001,
Pubmed
Peterson,
Isolation of Hox and Parahox genes in the hemichordate Ptychodera flava and the evolution of deuterostome Hox genes.
2004,
Pubmed
Phillips,
CTCF: master weaver of the genome.
2009,
Pubmed
Purbey,
PDZ domain-mediated dimerization and homeodomain-directed specificity are required for high-affinity DNA binding by SATB1.
2008,
Pubmed
Scott,
Enhancer blocking by the Drosophila gypsy insulator depends upon insulator anatomy and enhancer strength.
1999,
Pubmed
Spinelli,
The modulator is a constitutive enhancer of a developmentally regulated sea urchin histone H2A gene.
2002,
Pubmed
,
Echinobase
Splinter,
CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus.
2006,
Pubmed
Swalla,
Deciphering deuterostome phylogeny: molecular, morphological and palaeontological perspectives.
2008,
Pubmed
,
Echinobase
Takatori,
Evolution of CUT class homeobox genes: insights from the genome of the amphioxus, Branchiostoma floridae.
2008,
Pubmed
Wada,
Details of the evolutionary history from invertebrates to vertebrates, as deduced from the sequences of 18S rDNA.
1994,
Pubmed
,
Echinobase
Wang,
Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast.
1993,
Pubmed
Wang,
The structural basis for the oligomerization of the N-terminal domain of SATB1.
2012,
Pubmed
Xu,
Mapping of INS promoter interactions reveals its role in long-range regulation of SYT8 transcription.
2011,
Pubmed
Yajima,
ISWI contributes to ArsI insulator function in development of the sea urchin.
2012,
Pubmed
,
Echinobase
Yasui,
SATB1 targets chromatin remodelling to regulate genes over long distances.
2002,
Pubmed
Zhao,
Visualization of chromosomal domains with boundary element-associated factor BEAF-32.
1995,
Pubmed
