PARENT SESSION

(M487) GENES REGULATED BY THE PEM (RHOX5) HOMEODOMAIN PROTEIN IN SERTOLI CELLS.

Hu, Zhiying ¹, MacLean, James ¹, Wilkinson, Miles ¹, ¹ M.D. Anderson Cancer Center, University of Texas, Houston, TX

ABSTRACT- Homeodomain proteins are a large class of transcription factors that were originally discovered by virtue of their function in embryonic development. Downstream targets of homeobox genes have proven difficult to identify. Here, we describe initial studies designed to identify target genes regulated by the homeodomain gene Pem (Rhox5), a member of the Rhox homeobox gene cluster that we recently discovered on the mouse X chromosome. Rhox5 is essential for normal spermatogenesis; male mice lacking Rhox5 are subfertile and suffer from increased germ-cell apoptosis, reduced spermatid output, and spermatozoa motility defects. To understand the molecular mechanism of Rhox5 action, we used microarray analysis to identify genes differentially expressed in response to Rhox5 in both a cultured cell line in vitro and in the testis in vivo, as the latter provides an indication of physiological relevance and the former allows easy manipulation to study the regulation. We chose to identify Rhox5-regulated genes in a Sertoli-cell line, as Rhox5 expression is restricted to Sertoli cells in the testis. Microarray analysis revealed 182 transcripts whose expression was changed by 2-fold or more in response to Rhox5 expression in this Sertoli-cell line. Real-time PCR analysis confirmed the regulation of 10 out of 12 selected genes. Many of these genes were previously implicated as being involved in cell-cell interactions, oxidative-stress responses, apoptosis, cell proliferation, and tumorigenesis. To date, we have identified two genes that are regulated in the same manner in response to Rhox5 both in vitro and in vivo: Transferrin receptor (Trfr1) and carboxypeptidase X (Cpx-1). Trfr1 functions in Sertoli cells to deliver iron. By positively regulating Trfr1 expression, Rhox5 may stimulate Sertoli-cell metabolism and thereby indirectly promote germ-cell maturation. Cpx-1 is a newly discovered member of the metalcarboxypeptidase family that appears to be concentrated in the extracellular matrix. Its physiological substrates and why Rhox5 downregulates its expression remains to be determined. Studies are in progress to identify the cis elements in Trfr1 and Cpx-1 that permit them to be regulated by Rhox5.

KEY WORDS: homeobox, gene regulation, Sertoli cell