Basic helix-loop-helix transcription factor Tcfl5 interacts with the Calmegin gene promoter in mouse spermatogenesis.
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.
Siep
Michel
M
Department of Reproduction and Development, Erasmus MC, PO Box 1738, NL-3000 DR Rotterdam, The Netherlands.
Sleddens-Linkels
Esther
E
Mulders
Sabine
S
van Eenennaam
Hans
H
Wassenaar
Evelyne
E
Van Cappellen
Wiggert A
WA
Hoogerbrugge
Jos
J
Grootegoed
J Anton
JA
Baarends
Willy M
WM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D037281:Calnexin / Q000235:genetics*; D018257:Helix-Loop-Helix Motifs; D008297:Male; D051379:Mice; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D013090:Spermatocytes / Q000378:metabolism; D013091:Spermatogenesis; D013737:Testis / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D002135:Calcium-Binding Proteins; D037281:Calnexin; D018832:Molecular Chaperones; D014157:Transcription Factors; C494412:Tcfl5 protein, mouse; C087341:calmegin