The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

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.

Early asymmetric cues triggering the dorsal/ventral gene regulatory network of the sea urchin embryo

“…Reverse-transcription and qPCR analysis were performed as described ( Cavalieri et al, 2009b , 2011 , 2013 ). Briefly, total RNA from batches of unfertilized eggs and embryos grown at the desired stage was extracted by using the Power SYBR Green Cells-to-CT kit (Ambion, Italy) and reverse transcribed following the manufacturer's recommendations.…”

Dynamic changes in the interchromosomal interaction of early histone gene loci during development of sea urchin

“…The 30-bp cis-regulatory element (M30) and the transcription factor MBF-1 are required for the activation of the H2A gene (Alessandro et al, 2002;Cavalieri et al, 2009). The sns5 insulator element and its binding factor, COMPASS-like, regulate the H1 gene expression level (Cavalieri et al, 2009(Cavalieri et al, , 2013. These promoter and insulator regions showed H3K9 acetylation at the morula stage, when the transcriptional activity is at its maximum level, while H3K9 dimethylation was observed at the gastrula stage when transcription has already been silenced (Di Caro et al, 2007).…”

Diversification of spatiotemporal expression and copy number variation of the echinoid hbox12/pmar1/micro1 multigene family

“…lividus more precisely, we performed qPCR using a universal primer pair that completely matched the sequence of all the known family members (S1 Table), and specific primers for the otp and cmpl genes [29,30], used as single copy controls. As a template for this assay, we utilized the genomes derived from sperm of seven randomly selected individuals and, much to our surprise, we found that the number of hbox12/pmar1/micro1 genes varied noticeably among individuals, ranging from 6 to 22 copies (Fig 2).…”

“…As known single copy controls we used the otp and cmpl genes [29,30]. The number of hbox12 , otp and cmpl amplicons was determined using standard curves with different dosages of plasmid DNA containing the mentioned amplicon sequences, and gene ratios were eventually calculated.…”