ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2018, Vol. 44, No. 3, pp. 362–365. © Pleiades Publishing, Ltd., 2018. Original Russian Text © N.Y. Martynova, F.M. Eroshkin, A.G. Zaraisky, 2018, published in Bioorganicheskaya Khimiya, 2018, Vol. 44, No. 3, pp. 353–356.
LETTERS TO THE EDITOR
Effect of a Heterodimeric Complex of the Transcription Factors SoxD (Sox15) and Xanf1 on the Formation of the Xanf1 Gene Expression Zone during the Early Development of the Forebrain in the Spur-Toed Frog N. Y. Martynovaa, 1, F. M. Eroshkina, and A. G. Zaraiskya aShemyakin–Ovchinnikov
Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia Received October 26, 2017; in final form, October 31, 2017
Abstract⎯The effect of a protein complex consisting of the homeodomain transcription factor Xanf1 and the HMG-containing transcription factor SoxD (homologue Sox15 in human and mouse, group G of the Sox family) on the formation of a spatial pattern of the Xanf1 gene expression has been studied. The Xanf1 factor determines the early development of the forebrain, and the disturbance of its expression results in the reduction of this part of the brain; the pan-neural factor SoxD is necessary for the formation of the neuroectoderm at the earliest stages of development. The study of the functioning of a complex of these factors is important for understanding the molecular bases of the formation of the nervous system. The functional role of the formation of the SoxD–Xanf1 complex directly in developing embryos of the spur-toed frog Xenopus laevis has been studied using the method of in situ hybridization with probes to Xanf1 mRNA. The analysis of changes in the Xanf1 gene expression by the action of the heterodimeric complex suggests that the inhibitory effect of the repressor Xanf1 on the transcription of its own gene is neutralized in the anterior zone at the neurula stage where it is functionally necessary for the stabilization of the cellular status as the anterior forebrain neuroectoderm.
Keywords: Xenopus, Xanf1, SoxD, in situ hybridization DOI: 10.1134/S106816201803010X
Investigations into the features of the formation and functioning of complexes formed by proteins regulating the gene expression during tissue differentiation are very important for the understanding of the molecular basis of development. These interactions have been characterized in great detail for HMGdomain Sox factors. It has been shown that these factors exhibit activity by binding to DNA with high specificity only if they form a heterodimeric complex with another transcription factor [1–3]. It is assumed that this partnership provides the tissue specificity of Sox factors, and this high specificity of action determines their dominant role in cell differentiation. In this study, using the in situ hybridization on intact Xenopus laevis embryos, we have shown the biological role of the interaction between the pan-neural (expressed throughout the neural plate) factor SoxD of the Sox family and the Xanf1 factor whose expres1 Corresponding
author: phone: +7 (495) 336-86-11; fax: +7 (495) 336-36-22; e-mail:
[email protected]. Abbreviations: HMG box, high-mobility group box.
sion is limited by the anterior zone of the neural plate. The effect of SoxD and Xanf1 factors and of their complex on the expression of the Xanf1 gene was estimated from changes in the region of the distribution of its endogenous mRNA. Enhanced expression of the factors in embryonal cells was provided by the translation of synthetic mRNAs microinjected into embryos. We have shown earlier that the expression of the Xanf1 gene is necessary for the normal development of the forebrain; in addition, the regulatory elements of the promoter of Xanf1 were identified of which one contained a potential binding site for the transcription factors of the Sox family [4], and a partner of Xanf1 from this family, factor SoxD, was found. It was established that factors Xanf1 and SoxD form a complex upon the interaction of their DNA-binding domains: the C-terminal region of the HMG domain of SoxD and the homeodomain of Xanf1 are involved in the binding. It was shown that Xanf1 and SoxD factors and a complex of Xanf1 and SoxD factors are capable of binding to the regulatory elements of the promoter of the Xanf1 gene [8].
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(a) Results of in situ hybridization after the microinjection of mRNA SoxD
Control
C part SoxD
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HMG SoxD
SoxD + Xanf1
Xanf1
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C
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B'
C'
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Region of distribution of injected material, FLD fluorescence
6 *
Activation of the reporter pGL3 of Xanf1 (10 ng) after mRNA microinjections SoxD 60ng HMG-SoxD 60 ng C-SoxD 60 ng *p < 0.05 * Xanf1 15 ng Xanf1 + SoxD 15 + 60
5 4 3
Scheme of the reporter vector
2
*
1
Early gastrula
C SoxD HMG-SoxD C-SoxD Xanf1 Xanf1 + SoxD
–2200 C SoxD HMG-SoxD C-SoxD Xanf1 Xanf1 + SoxD
Activity of reporter, rel. unit
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TATA Luc
Promotor of the Xanf1 gene 1-2200 mRNA
Neurula
Fig. 1. Effect of the SoxD and Xanf1 factors and of their complex on the expression of the Xanf1 gene in Xenopus laevis embryos, as indicated by the results of in situ hydridization experiments with the use of probes to mRNA (a) and the analysis of the activation of the luciferase reporter with the full-size promoter of the Xanf1 gene (b). At the upper panel are the photos of intact embryos at the neurula stage viewed from the dorsal side (a, A–F); a dark region on an embryo corresponds to the zone of staining of endogenous mRNA of the Xanf1 gene by RNA probes complementary to the coding frame of the Xanf1 gene (а, B–D) or its 3'-noncoding region (а, E, F). At the bottom panel are the photos of embryos in ultraviolet light as a control of the zone of occurrence of injected material, obtained with the use of fluorescein lysine dextran (FLD) (а, B'–F'). White arrows show the regions of changes in the Xanf1 expression zone in response to microinjections of synthetic mRNAs of SoxD and its deletion variants. The results of the analysis of the activation of the luciferase reporter with the full-size promoter of the Xanf1 gene in response to microinjections of Xanf1 and SoxD mRNAs at the gastrula and neurula stages are shown in the form of diagrams (b) with indication of the composition and amount of mRNAs used. Dashes indicate a standard deviation; the significance of differences was verified using the Student’s t-test and was *p < 0.5. C (control) is the activity of the luciferase reporter in embryos without the addition of mRNA. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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To study the effect of a complex of Xanf1 and SoxD factors on the expression of the Xanf1 gene immediately in embryos, microinjections of mRNAs encoding these factors were made followed by in situ hybridization with probes to Xanf1 mRNA. The zone of Xanf1 expression was detected using two different probes; for embryos microinjected with mRNA of SoxD and its deletion mutants, a probe to the coding region of Xanf1 mRNA was used, and for embryos injected with Xanf1 mRNA, a probe to the 3'-terminal noncoding region of the Xanf1 gene was used to exclude the cross staining with the probe of the exogenous Xanf1 mRNA. The microinjections of mRNA into one of blastomeres at the two-blastomere stage were carried out by the method described earlier [9]. Thus, the second blastomere remained intact; as a result of further cleavage, the embryo had a control half, which made it possible to record changes in the Xanf1 expression zone compared to the control immediately in one organism. The fixation of embryos was performed at stage 14 of development (neurula) and stained as described in [6]. To study the role of single domains of SoxD, two deletion mutants of the SoxD gene in vector p35T (pSp64) were created, which encode the HMG domain and the С-terminal domain of this factor (the creation of the constructs is described in [8]). An analysis of the results of in situ hybridization showed that a microinjection of full-size SoxD mRNA has a dual effect on the distribution of Xanf1 mRNA; on the one hand, the intensity of specific staining falls compared with the control (without the addition of exogenous mRNA; Fig. 1a, A), but the expression zone itself expands in this case in the posterior–ventral direction (Fig. 1a, B). The injection of mRNA encoding the С-terminal region of SoxD does not affect the expression of Xanf1 (Fig. 1a, С). A microinjection of mRNA encoding the HMG domain of SoxD, which interacts with the homeodomain of Xanf1 [8], induces an insignificant posterior expansion of the Xanf1 expression zone (Fig. 1a, D). A microinjection of Xanf1 mRNA has a strong suppressing effect on the expression of its own gene (Fig. 1a, Е). Microinjections of a mixture of Xanf1 and SoxD mRNAs lead to the restoration of the Xanf1 expression pattern (Fig. 1a, F). Thus, under the conditions of in situ hybridization experiments, the exogenous factors SoxD and Xanf1 after the combined administration of mRNAs encoding them support the expression of the Xanf1 gene. The restoration of the Xanf1 expression zone after the combined administration of Xanf1 and SoxD mRNAs occurs in 18 of 24 embryos. To confirm the results by an independent method, we performed experiments with a reporter containing
the luciferase gene under the control of a full-size promoter of the Xanf1 gene, which was constructed on the basis of vector pGl3 [4]. The experiments were carried out by the method developed earlier [10], which consists in microinjections of the reporter construct in combination with mRNAs of the factors under study into Xenopus laevis embryos at the two-blastomere stage followed by measurements of the luciferase activity in the lysate of embryos incubated to the stage of gastrula or neurula. The results of measurements were normalized using the vector pCMV-β-GAL, which carries the β-galactosidase gene under the control of the nonspecific promoter CMV. The results of the study indicated that: (1) The Xanf1 factor suppresses the activity of the promoter of Xanf1 at the stages of early gastrula and neurula and, consequently, is a repressor for the own gene (Fig. 1b). The data are consistent with the results obtained earlier [8]. (2) The full-size factor SoxD and its C-terminal and НMG domains have no statistically significant effect on the expression of the luciferase reporter at the stages of early gastrula and neurula (Fig. 1b). (3) The coexpression of factors Xanf1 + SoxD differently affects the activity of the reporter with the fullsize promoter of Xanf1 depending on the stage of development. It was shown that, at the stage of early gastrula (stages 10 and 11 of development), with a weak activation of the reporter, the coexpression of the factors does not induce substantial changes in activity; and at the stage of neurula (stage 14 of development), when the activity of the promoter is high, the formation of the Xanf1 + SoxD complex leads to additional activation of the reporter (Fig. 1b). Because during neurulation, neutralizing the inhibition by the Xanf1 factor of its own gene is necessary, the formation of the complex of Xanf1 with the SoxD factor should occur precisely at the neurula stage, which is just observed in a model system of luciferase reporters. ACKNOWLEDGMENTS Experiments on the activation of luciferase reporters were supported by the Russian Foundation for Basic Research (project no. 18-04-00674). Experiments on in situ hybridization were supported by a grant from the Russian Scientific Foundation (project no. 14-14-00557). REFERENCES 1. Kamachi, Y., Uchikawa, M., and Kondoh, H., Trends Genet., 2000, vol. 16, pp. 182–187.
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Translated by S. Sidorova
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