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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.
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