Isolation And Identification Of A New Human Testis-specific Gene HMGB4 And Preliminary Functional Analysis

A novel human testis-specific gene termed HMGB4 was identified by digital differential display. Sequence analysis revealed that HMGB4 protein is a new member of HMGB family, lacking the acidic tail typically found in this family. Northern blot analysis revealed that a 0.7 kb size of HMGB4 transcripts was expressed only in adult human testis, but not in seven other tissues. Western blot analysis revealed that HMGB4 was strongly and preferentially expressed in the adult human testis, but not in six other tissues too. Moreover, HMGB4 was a germcell-specific gene by both in situ hybridization and immunohistochemical analyses. The results showed that HMGB4 was expressed strongly in spermatocytes, spermatids, spermatozoa and seminoma, not in Sertoli or Leydig cells. The positive signals in the testes of normal adults, patients with seminoma or cryptorchidism were detected. Also, there was not any signal in the testes of patients with Sertoli cell only syndrome. The green fluorescence produced by pEGFP-N3-HMGB4 was detected in the nuclei of GC-1 and HeLa cells after 24 h of transfection. RT-PCR in fifteen human cells showed that HMGB4 was expressed highly in NT-2, HBL100 cells and relatively lowly in MDA-MB468, MCF7, T47D, A549, HepG2, HepG3B, Hela cells, not in SK-MES-1, NCI-H460, MDA-MB453, SW480, HF-29, HEK293 cells. The results suggested that it might be involved in cancers. Overexpression of HMGB4 in GC-1 and HeLa cells was significantly delay the progression of G1 through S which was investigated by flow cytometric cell cycle analysis. HMGB4 drastically decreased the transcription of luciferase gene in transient transfection assays, so that it was a potent transcriptional repressor. In conclusion, HMGB4 is strongly and preferentially expressed in the adult testis, suggested that it may play important roles in testis development and spermatogenesis.