Transcription factor RFX4 binding to the testis-specific histone H1t promoter in spermatocytes may be important for regulation of H1t gene transcription during spermatogenesis.

The X-box binding protein RFX4 is highly expressed in testis in contrast with other tissues, but its function there is unknown. Another family member abundant in testis, RFX2, has been shown to bind to the X-Box elements in the promoter of the testis specific histone H1t, which is expressed only in pachytene spermatocytes. RFX proteins are known to dimerize, and there is the possibility that the abundant testis RFX4, which is also expressed in pachytene spermatocytes as shown by RT-PCR and Western blotting, may interact with RFX2 in these cells. In EMSA anti-RFX2 polyclonal antibodies produce a supershifted complex with testis extracts and an X-Box probe. On the other hand, RFX4 polyclonal antibodies do not supershift the complex but appear to enhance formation of the complex. RFX4 appears to co-precipitate with RFX2 in immunoprecipitation, and to co-purify with RFX2 in an affinity purification using a biotinylated X-box affinity probe. In ChIP assays RFX4 also binds to the H1t promoter in vivo. These data suggest a possible regulatory role for RFX4 in transcription of the histone H1t gene during spermatogenesis.