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	Figure 1. A single copy of the MBF-1 activator binding site enhances transgene expression. The M30-tk-CAT transgenes, bearing one, two or three copies of the 30 bp H2A modulator sequence in different location and orientation, were microinjected into sea urchin zygotes. Total RNA from 30 to 50 gastrula stage embryos, microinjected with the indicated transgenes, were hybridized with a 32P-labelled CAT antisense probe and processed for the RNase protection assay described in ‘Materials and Methods’ section. Asterisk indicates the protected RNA band for the CAT transcript.
	Figure 2. In vivo competition assay to knock-down the H2A enhancer function in transgenic embryos. (A) Annotated map of the P. lividus wild type and deletion mutants early H3, H2A and H1 histone genes, highlighting the cis-regulatory sequence elements. The horizontal black line and arrow-shaped boxes represent, respectively, the genomic DNA and coding sequences, while the bent arrows denote the putative transcription start site. (B) The P. lividus histone gene constructs, orientated as in the endogenous histone gene repeat, were co-injected with excess of the modulator binding site (M30) into S. granularis zygotes. RNase protection was carried out by hybridizing antisense 32P-labelled RNA, transcribed in vitro from H3, H2A and H1 subclones, with total RNA exctracted from 25 injected embryos at morula (Mor) and gastrula (Gas) stages. The two H2A and H3 probes were hybridized together. The protected 409, 357 and 209 nt RNA bands, respectively, for the H2A, H3 and H1 transcripts are indicated.
	Figure 3. In vivo competition assay for endogenous H3, H2A and H1 histone gene expression. (A) Relative abundance of histone mRNAs in the P. lividus embryo at morula stage. A similar prevalence is detected for the two nucleosomal H3 and H2A mRNAs, while the H1 linker histone mRNA peaks at about one-third of the value for the formers. (B) Endogenous histone gene expression analysis carried out in embryos at morula stage microinjected with excess of the M30 sequence element or the mutated M30 mut oligonucleotide as a control. Graphs show n-fold changes in mRNA expression level of histone genes based on the threshold cycle number (Ct) of injected embryos compared to that of the uncompeted control embryos. Ct numbers were normalized for the endogenous MBF-1 in the same sample. Data were derived from two independent microinjection experiments and each bar represents the average of triplicate samples from the two batches of embryos.
	Figure 4. Knock-down MBF-1 function by microinjection of a synthetic mRNA encoding for as dominant repressor (dnMBF-1). Increasing amounts (0.1–1 pg) of the chimeric RNA were injected in P. lividus zygotes and RNA extracted from embryos at morula stage. Graphs show n-fold changes in mRNA expression level of histone genes based on the threshold cycle number (Ct) of dnMBF-1 injected embryos compared to that of the uninjected control embryos. Ct numbers were normalized for the endogenous MBF-1 in the same sample, by amplifying a fragment of the coding region external to the DNA binding domain. Data were derived from two independent microinjection experiments and each bar represents the average of triplicate samples from the two batches of embryos.
	Figure 5. In vivo competition assay to inhibit the sns5 insulator function in transgenic embryos. Wild-type histone gene construct H3-H2A-H1 and the deletion mutant ΔpH3-H2A-ΔpH1 (showed in Figure 2A), were microinjected with excess of the BoxA sequence element into S. granularis zygotes. RNase protection was carried out as for Figure 2. The protected 409, 357 and 209 nt RNA bands, respectively, for the H2A, H3 and H1 transcripts are indicated.
	Figure 6. Endogenous gene expression analysis upon impairment of the sns5 insulator function by in vivo competition assay with excess of the BoxA sequence element. Graphs show n-fold changes in mRNA expression level of histone genes based on the threshold cycle number (Ct) of injected embryos compared to that of the uncompeted control embryos. Ct numbers were normalized for the endogenous MBF-1 in the same sample. 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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