PARENT SESSION
GENE REGULATION AND FUNCTION - A

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(382) THE SP-10 INSULATOR: INSIGHTS INTO ITS ENHANCER-BLOCKING FUNCTION.

Reddi, Prabhakara ¹, Urekar, Craig¹, Monroe, Jennifer¹, ¹ University of Virginia, Charlottesville, VA

ABSTRACT- Insulators are DNA sequences that mark the boundaries of genes. Insulators perform two important functions. First, they act as positional enhancer-blockers and prevent the enhancer of one gene from inappropriately activating the promoter of a neighboring gene. Second, insulators form a barrier between active and inactive chromatin and prevent the spread of condensed chromatin. Previously, we reported that the -408/-92 proximal promoter of the SP-10 gene, which codes for the acrosomal protein SP-10, functions as an enhancer blocker in plasmid based assays as well as in the context of native chromatin. In this study, we mapped the insulator activity to the -186/-135 region and showed that the 50-bp fragment is sufficient to prevent enhancer-promoter interaction when positioned between them. Three tandem repeats of this minimal insulator had an additive effect on enhancer-blocking. Further, the minimal insulator was able to function in the context of two separate core promoters, indicating the generic nature of the enhancer-blocking mechanism. Surprisingly though, the minimal insulator was not effective in the opposite orientation, that is, when facing away from the direction of transcription. The current models propose that insulators function by tethering DNA to subnuclear structures such that the resulting loop formation separates genes and their respective regulatory elements into functionally independent domains. It is difficult to fit the observed polarity of the SP-10 insulator into this model because tethering is unlikely to be affected by the orientation of the DNA fragment. The SP-10 insulator also differs from all other vertebrate insulators in another respect, that is, it does not contain binding sites for CTCF. Instead, it binds to TDP43 and PURalpha, transcription factors previously not implicated in insulator activity. Therefore, the SP-10 insulator is novel and its study is beginning to provide insights into additional mechanisms of insulator function.

KEY WORDS: enhancer blocking, transcription factors, insulator, spermatogenesis