5' structural motifs and Xenopus beta globin gene activation

Area of Science:

• Molecular Biology
• Genetics
• Chromatin Structure

Background:

• The Xenopus beta globin gene's 5' flanking region exhibits distinct chromatin structures in different cell types.
• Nuclease-hypersensitive sites (HSs) are crucial regulatory elements in gene expression.
• Previous studies suggested erythroid-specific factors might be involved in HS formation.

Purpose of the Study:

• To analyze the chromatin structure of the Xenopus beta globin gene's 5' flanking region.
• To identify sequences responsible for the formation of nuclease-hypersensitive sites.
• To investigate the role of erythroid-specific factors in HS formation.

Main Methods:

• Chromatin analysis in erythroid and non-erythroid cells.
• Supercoiled plasmid and minichromosome assembly in HeLa cells.
• Nuclease digestion assays (single and double strand-specific nucleases, restriction enzymes).
• Deletional analysis of the 5' flanking region.

Main Results:

• Two erythroid chromatin-specific HSs were identified: one at the cap site and another 1000 base-pairs upstream.
• (AT)n tracts located upstream of HSs were preferentially cleaved in supercoiled plasmids, but not in chromatin.
• HS structure in HeLa cell minichromosomes mimicked that in erythroid chromatin, suggesting independence from erythroid-specific factors.
• Deletion of the upstream (AT)n tract and further deletions, including part of the HS itself, did not affect HS formation or gene transcription.
• Plasmid sequences replacing deleted regions became hypersensitive, indicating sequence-directed HS formation.

Conclusions:

• The formation of the cap site HS is not dependent on erythroid-specific factors.
• HS formation is directed by sequences downstream from position -116, acting over a distance of at least 50 base-pairs.
• The (AT)n tracts play a role in DNA structure in plasmids but not in directing HS formation within chromatin.