One-hybrid screening appears to be a powerful and useful tool to identify candidate transcription factors involved in the regulation of genes in spermatogenesis. Screening of a mouse testis cDNA library for proteins that bind the Clgn
promoter resulted in the identification of a cDNA encoding the testis-specific bHLH protein Tcfl5, which is homologous to human TCFL5 (37
). bHLH proteins are important gene regulatory factors, and contribute to control of many developmental pathways, such as neurogenesis, myogenesis, cell proliferation and differentiation, cell lineage determination, sex determination and other essential processes, in organisms ranging from yeast to mammals (38
The present observations on Tcfl5
mRNA and Tcfl5 protein expression in mouse testis are in agreement with the report on TCFL5
/TCFL5 expression in human testis (37
). In the mouse testis, we found Tcfl5
mRNA in meiotic pachytene spermatocytes and in post-meiotic round spermatids, while the Tcfl5 protein is detected in spermatocytes, from late pachytene until the second meiotic division, but not in post-meiotic round spermatids. Tcfl5 protein expression overlaps with the onset of expression of the putative target gene Clgn
in pachytene spermatocytes (9
). However, a function of calmegin during meiotic prophase is not evident. In Clgn
gene knockout mice, spermatogenesis is quite normal, but the Clgn
deficient spermatozoa do not adhere to the zona pellucida, resulting in male infertility (7
). There is impaired heterodimerization of fertilin α/β, which results in the absence of fertilin β from mature Clgn
knockout sperm (47
). Calmegin is thought to act as a chaperone for sperm surface proteins that mediate sperm–egg interaction, and expression of the Clgn
gene in spermatocytes seems to be like a kind of foresight of the meiotic cells looking ahead to post-meiotic spermiogenesis and sperm function.
In this context, it may be of interest to note that the Factor In the Germline α
in mouse and FIGLA
in human) encodes a bHLH protein expressed in the female germ line in both mouse and human (48
). In mice, Figla
mRNA is first detected in oocytes at E13.5 and persists in adults. The Figla protein forms a heterodimer with bHLH protein E12, and regulates the expression of the oocyte-specific zona pellucida genes Zp1
by binding to a conserved E-box upstream of the transcription start site (48
). Therefore, both bHLH proteins Tcfl5 and Figla are expressed during meiotic prophase, of male and female germ cell development, respectively, whereas putative or known target genes are not involved in meiosis but function even as late as fertilization. Tcfl5 and Figla belong to different groups within the bHLH protein family, but still, the analogy in function between these two bHLH transcription factors suggests that similar mechanisms may be operative in the regulation of sex-specific aspects of male and female gametogenesis.
Numerous bHLH proteins have been identified and have been classified according to different criteria. Atchley et al
) defined five subgroups (A–E) based upon evolutionary conservation of several amino acid residues and DNA-binding properties. In addition, the presence or absence of a leucine zipper domain is taken into account. Later studies have defined additional conserved functional protein domains that are present in subfamilies of bHLH proteins. An example of such a domain is the orange domain adjacent to the bHLH domain in the so-called hairy-related group of bHLH proteins (52
The basic domain of all bHLH proteins permits these proteins to bind to a consensus hexanucleotide that has been defined as the canonical E-box CANNTG. In addition, the HLH domain is important for homo- and/or heterodimerization (51
). Following the criteria of Atchley and Fitch (38
), we suggest that Tcfl5 is a member of the group B bHLH subfamily. However, the 152 bp putative Clgn
promoter sequence that was used in the present one-hybrid screening does not contain the consensus group B hexanucleotide E-box sequence CACGTG. The present results show that Tcfl5 binds the non-canonical E-box CACGCG.
Conserved amino acid residues in the basic region determine binding site specificity of bHLH proteins. The highly conserved Glu (E) at position 9 contacts the 5′ CA nucleotides of the E-box consensus binding site (53
). Studies with the group B bHLH protein Max pointed out that the conserved Arg (R) at position 13 is the key determinant for binding specificity of group B bHLH proteins. Analysis of protein–DNA co-crystals showed that R13 makes direct contact with the G nucleotide at position 4 in the group B binding site CACGTG (53
). However, this sequence is not present in the Clgn
promoter, and Tcfl5 preferentially binds the non-canonical CACGCG sequence. This sequence is also preferred by a subfamily of bHLH proteins, including Drosophila
hairy and deadpan (39
). These and other hairy-related bHLH proteins contain a conserved Pro (P) at position 6 of the basic region, but in Tcfl5 no Pro is found at this position. However, Tcfl5 contains an Arg at position 15 that is also found in hairy and deadpan (39
). The presence of this Arg, and the overall high similarity of this part of the basic region of Tcfl5 to that of hairy-related bHLH proteins, suggests that this Arg might play an important role in determining the DNA-binding specificity of Tcfl5. Tcfl5 lacks the Orange domain that is typical for the hairy-related subfamily of bHLH group proteins (52
), and it appears that Tcfl5 is an atypical member of the B group of bHLH proteins.
Database searches identified partial Tcfl5 expressed sequence tags (ESTs) from rat, bovine, pig, chicken and trout (data not shown). Most of these libraries were listed to be derived from reproductive tissue. Although the ESTs represent partial sequences, we found that the bHLH domain is almost completely conserved in all these species, and that the C-terminus is highly similar. Database searches also yielded information that the target gene Clgn is present and testis-specifically expressed in rat, bovine and pig (data not shown), and we would like to suggest an important role for Tcfl5 in the regulation of spermatogenesis in species ranging from fish and birds to mammals.
All bHLH proteins known to date function as homo- or heterodimers. In addition to Tcfl5, two other bHLH group family members have been found to be expressed in mouse spermatocytes. One is the hairy-related factor, Hey1, which interacts with a co-regulator Boip through its Orange domain (54
). The second is a more distant family member, a germ cell-specific truncated variant of sterol regulatory element binding protein 2, which is detected in mouse spermatocytes and round spermatids (55
). Future two-hybrid screens may identify these or other bHLH proteins as dimerization partners for Tcfl5. However, Tcfl5 may function as a homodimer. In the helix II domain, involved in dimerization, Tcfl5 is more similar to hairy and deadpan than to Mad and Mxi1 (Figure ), and hairy binds DNA as a homodimer (39
As discussed herein, timing of Tcfl5 expression in spermatogenesis would be in agreement with Tcfl5 acting as a transcriptional activator of the Clgn
gene. However, it is to be noted that members of the hairy-related bHLH family act as transcriptional repressors, inhibiting transcription by binding to co-repressors, mediated by a sequence in the C-terminus (52
). Tcfl5 does not contain such a co-repressor binding sequence, and we cannot predict whether Tcfl5 acts as transcriptional activator or repressor. The present finding that Tcfl5 protein is associated with transcriptionally active chromatin regions in spermatocytes would support a role as activator of transcription, possibly of several target genes.