ECB-ART-39697BMC Biol 2006 Feb 09;4:4. doi: 10.1186/1741-7007-4-4.
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CBFbeta is a facultative Runx partner in the sea urchin embryo.
BACKGROUND: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. RESULTS: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. CONCLUSION: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation.
PubMed ID: 16469111
PMC ID: PMC1395345
Article link: BMC Biol
Species referenced: Echinodermata
Genes referenced: arid3a cbfb CyIIIa impact LOC100887844 LOC115919910 LOC583082 pkcl2 runx2 SLBP
Antibodies: SLBP Ab1 cbfb Ab1 runx2 Ab1
Morpholinos: cbfb MO1
Article Images: [+] show captions
|Figure 1. SpCBFβ structure and heterodimerization with SpRunt-1. (A) Alignment of CBFβ sequences from S. purpuratus (Sp) zebrafish (Dr), Homo sapiens (Hs, variant 2 of the two mammalian splice variants), mouse (Mm, variant 1 of the two mammalian splice variants), Drosophila (two genes, DmBro and DmBgb), and C. elegans. Amino acid residues that are identical between SpCBFβ and at least one other homologue are highlighted in black, and conserved substitutions are highlighted in grey. Amino acid pairs of which the respective codons are separated by introns in each gene are underlined; the four introns conserved between urchin and vertebrates are indicated by arrowheads. (B) EMSA analysis using recombinant Runt domain (RD) or full-length SpRunt-1 (FL) either alone or in the presence of recombinant SpCBFβ. Note that the full-length SpRunt-1 does not bind DNA very effectively by itself, suggesting the presence of domains that inhibit DNA binding within the full length protein. SpCBFβ complexes with both the Runt domain and full-length SpRunt-1, as indicated by a “supershift”, but does not bind DNA by itself. (C) Immunoblot of SpCBFβ from nuclear extract (NE), and nuclear extract immundepleted (ID) and immunoprecipitated (IP) with anti-SpRunt-1 or nonspecific IgG. Molecular weight markers (in kilodaltons) are shown on the right.|
|Figure 2. Temporal expression of SpCBFβ. (A) Northern blot of total RNA from egg, morula, blastula, and gastrula stage embryos probed with SpCBFβ. The ethidium bromide stained rRNA bands from the same gel are shown as a loading control. (B) Temporal expression of SpCBFβ as measured by RT-PCR. (C) Immunoblot of equivalent amounts of total protein from egg (E), morula (M), early blastula (EB), mesenchyme blastula (MB), early gastrula (EG), late gastrula (LG) and pluteus stage (P), probed with antibodies to either SpCBFβ or SLBP (a positive control for the presence of intact protein in the egg and early embryo extracts).|
|Figure 3. Spatial expression of SpCBFβ. Whole mount in situ hybridization of (A-G) Two day old gastrula stage embryos showing global expression, with enrichment in endoderm (e) and parts of the oral ectoderm (oe), and (H-K) four day old pluteus showing expression localized to midgut (m) and hindgut (h) endoderm, and some expression in the ciliated band of the anal arm buds (K, arrows).|
|Figure 4. Comparison of SpCBFβ and SpRunt-1 morphant phenotypes. DIC images of (A) 24 h and (B) 72 h control MASO injected embryos; arrow in (B) points to skeletal rod (spicule); (C) 24 h and (D) 72 h SpRunt-1 MASO injected embryos; and (E) 24 h and (F) 72 h SpCBFβ MASO injected embryos; black arrow in (F) points to tripartite gut, and white arrowhead points to a "ventrolateral" cluster of skeletogenic mesenchyme cells. Scale bars = 20 μm; embryos in (A), (C), (D) and (E) are to the same scale. (G) Immunoblot of total blastula-stage embryo extract showing that the SpCBFβ MASO is effective in depleting endogenous SpCBFβ (arrow).|
|Figure 5. SpCBFβ does not contribute to the anti-apoptotic function of SpRunt-1. (A) Gastrula stage SpRunt-1 morphant fluorescently imaged with TUNEL (green, indicating apoptosis) and DAPI (blue, indicating DNA). (B) Gastrula stage SpCBFβ morphant fluorescently imaged with TUNEL and DAPI as in (A). (C) EMSA of recombinant wild-type and mutant (G115R) Runt domain (α) from SpRunt-1, in the presence or absence of recombinant SpCBFβ. Note that while the G115R mutant Runt domain binds DNA, it does not heterodimerize with SpCBFβ. (D) Gastrula stage SpRunt-1 morphant that has been co-injected with full-length mRNA encoding SpRunt-1-G115R, fluorescently imaged with TUNEL and DAPI as in (A). Note that while cell survival is rescued, as indicated by a lack of TUNEL signal, other aspects of development such as skeletogenesis are not.|
|Figure 6. SpCBFβ is not involved in Runx-mediated regulation of SpPKC1. (A) Relative expression levels of SpPKC1, CyIIIa, SpCyclinD and SpDri as determined by RT-PCR analysis of RNA isolated from gastrula stage SpRunt-1-knockdown and SpCBFβ-knockdown embryos. Each bar represents the average +/- standard deviation in the level of expression with respect to controls from two or more microinjection experiments. The dashed line indicates a 3-fold reduction of expression, which is taken here to be the minimum level of reduction considered to be significant. Note that whereas the expression of CyIIIa, SpCyclinD and SpDri are similarly affected by depletion of either SpRunt-1 or SpCBFβ, expression of SpPKC1 is much more strongly affected by SpRunt-1 depletion than by SpCBFβ depletion (asterisk). (B) ChIP of CyIIIa, SpPKC1, SpCyclinD and SpCdk4 using anti-SpRunt-1 and anti-SpCBFβ antibodies and chromatin prepared from late gastrula stage (48 h) embryos. A product of the expected size was obtained from the input DNA with each primer set, confirming the efficacy of the primers and PCR conditions (not shown). SpCdk4, which was not recovered by ChIP with either antibody, was chosen as a specificity control because its expression levels are not affected by knockdown of either SpRunt-1 or SpCBFβ.|
References [+] :
Adya, Function of CBFbeta/Bro proteins. 2001, Pubmed