PARENT SESSION
SLIDE SESSION 9: SPERMATOGENESIS
Chairs: Deborah O'Brien, John Herr, Cynthia Shirley (Trainee)
Univ Ottawa-UCU Auditorium
1:30 PM-3:30 PM

EVIDENCE THAT A TRANSCRIPTIONAL INSULATOR ENSURES PROGRAMMED GENE EXPRESSION OF SP-10 DURING SPERMATOGENESIS.

Reddi, Prabhakara¹, Shore, Amy ¹, Acharya, Kshitish¹, Herr, John¹, ¹

ABSTRACT- Spermatogenesis provides an excellent model to study developmental stage- and cell type-specific regulation of gene expression. Transcription of the acrosomal protein SP-10 gene was shown to be initiated in the step 1 round spermatids and its mRNA temporally restricted to stages I through VI of the mouse seminiferous cycle. Promoter analysis in transgenic mice indicated that the 294-bp proximal promoter (-266/+28) of SP-10 was sufficient to direct spermatid-specific expression of a GFP reporter. No ectopic GFP expression was observed in transgenic mice. To test the hypothesis that the proximal promoter of SP-10 shielded the transgene from position effects, a CMV enhancer was placed upstream of the SP-10 promoter plus reporter gene to mimic integration of the transgene adjacent to a ubiquitous enhancer. In COS and other cell lines, the -408/+28 SP-10 promoter or deletions thereof (but not stuffer fragments) blocked the CMV enhancer activity in a direction and position dependent manner, indicating that the SP-10 promoter functioned as a transcriptional insulator. Furthermore in transgenic mice harboring the CMV enhancer upstream of the -408/+28 SP-10 promoter, GFP expression was evident in round spermatids but not in somatic tissues suggesting that the CMV enhancer was rendered inactive in vivo. Taken together, these data suggest that the proximal promoter of the SP-10 gene could function as an activator in spermatids and insulator in other cells. The question arises as to the relevance of insulator activity at the natural genomic site for SP-10. The human genome map of the q22-23 region of chromosome 11 (the SP-10 gene locus) showed two ubiquitously expressed genes coding for integral membrane protein (Itm1) and checkpoint protein (Chk1) located within 100 kb of the SP-10 locus. The Chk1 gene, which is expressed during meiosis in spermatocytes, encompasses the entire SP-10 gene. However the SP-10 gene, programmed to be transcribed in the round spermatids, must remain silent in the spermatocytes and other tissues where Itm1 and Chk1 are activated. We propose that the SP-10 insulator forms a chromatin boundary that protects the gene from the influence of neighboring enhancers. The insulator binding proteins must then be modulated in spermatids to allow transcription of SP-10. NIH-HD 36239 (PPR), HD29099 (JCH), Fogarty fellowship to KKA.

KEY WORDS: spermatogenesis, transcription, insulator, SP-10