A 6.86 kb rat genomic DNA fragment containing the testis-specific histone H1t gene and the histone H4t gene has been sequenced. S1-nuclease protection analyses of total cellular RNA from rat liver and testis showed that histone H1t mRNA was present only in testis. Examination of various highly enriched populations of rat testis cell types revealed that H1t mRNA was found exclusively in a fraction enriched in pachytene spermatocytes. When protein, DNA interactions within the proximal promoter region of the histone H1t gene were examined by electrophoretic mobility shift assays, only minor differences were found in mobility shift patterns of the H1t promoter in assays comparing binding of nuclear proteins from pachytene spermatocytes and early spermatids. However, major differences in binding were observed upon comparing nuclear proteins from rat pachytene spermatocytes to liver. Comparison of binding patterns of rat testis, rat hepatoma H4 cells, HeLa cells, and COS-1 cells also revealed dramatic differences. Transcriptional activity of the histone H1t promoter was examined by measuring H1t promoted chloramphenicol acetyltransferase (CAT) mRNA levels in transient expression assays in transfected rat hepatoma H4 cells, HeLa cells, and COS-1 cells. These assays revealed that the histone H1t promoted CAT gene functioned poorly in HeLa cells and COS-1 cells compared to expression with the parent SV40 promoted vector pSV2CAT. The H1t promoted CAT gene apparently did not work at all in transfected rat hepatoma H4 cells, which is consistent with testis germinal cell specific expression of the histone H1t gene.

Transcription of the mammalian testis-specific linker histone H1t gene occurs only in pachytene primary spermatocytes during spermatogenesis. Studies of the wild type (Wt) and mutant H1t promoters in transgenic mice show that transcription of the H1t gene is dependent upon the TE promoter element. We purified an 85 kDa protein from rat testis nuclear extracts using the TE1 subelement as an affinity chromatography probe and analysis revealed that the protein was RFX2. The TE1 element is essentially an X-box DNA consensus element and regulatory factor X (RFX) binds specifically to this element. Polyclonal antibodies directed against RFX2 supershift the low mobility testis nuclear protein complex formed in electrophoretic mobility shift assays (EMSA). RFX2 derived from primary spermatocytes, where the transcription factor is relatively abundant, binds with high affinity to the TE1 element. Coexpression of RFX2 together with an H1t promoter/reporter vector activates the H1t promoter in a cultured GC-2spd germinal cell line, but mutation of either the TE1 subelement or the TE2 subelements represses activity. These observations lead us to conclude that the TE1 and TE2 subelements of the testis-specific histone H1t promoter are targets of the transcription factor RFX2 and that this factor plays a key role in activating transcription of the H1t gene in primary spermatocytes. Published 2003 Wiley-Liss, Inc.