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Nucleic Acids Research 2004 32(21):6425-6436; doi:10.1093/nar/gkh979
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Published online 7 December 2004

Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved

Basic helix–loop–helix transcription factor Tcfl5 interacts with the Calmegin gene promoter in mouse spermatogenesis

Michel Siep, Esther Sleddens-Linkels, Sabine Mulders1, Hans van Eenennaam1, Evelyne Wassenaar, Wiggert A. Van Cappellen, Jos Hoogerbrugge, J. Anton Grootegoed and Willy M. Baarends*

Department of Reproduction and Development, Erasmus MC, PO Box 1738, NL-3000 DR Rotterdam, The Netherlands and 1 Target Discovery Unit, NV Organon, PO Box 20, NL-5340 BH Oss, The Netherlands

* To whom correspondence should be addressed. Tel: +31 10 4087345; Fax: +31 10 4089461; Email: w.baarends{at}erasmusmc.nl

Received September 21, 2004; Revised and Accepted November 15, 2004

In mouse spermatogenesis, differentiating germ line cells initiate expression of specific genes at subsequent developmental steps. The Calmegin (Clgn) gene is first expressed in meiotic prophase, in primary spermatocytes, and encodes a protein that acts as a chaperone. To identify testis-specific transcription factors that control expression of the Clgn gene in spermatogenesis, we performed a yeast one-hybrid screening with a Clgn promoter sequence as bait DNA. This screening resulted in the identification of mouse Tcfl5 as a candidate Clgn promoter-binding protein. Tcfl5 is a member of the basic helix–loop–helix (bHLH) family of transcription factors, and mouse Tcfl5 shows 83% amino acid sequence identity with human TCFL5. Gel-shift and yeast one-hybrid experiments showed that Tcfl5 interacts with a non-canonical CACGCG site that is present in the Clgn promoter. By using northern blot, RT–PCR and in situ hybridization, mouse Tcfl5 mRNA was detected only in testis, with the highest expression level in primary spermatocytes and round spermatids. The highest level of Tcfl5 protein was found in primary spermatocytes at the diplotene stage of meiotic prophase, where the protein colocalizes with transcriptionally active chromatin.


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