Replication-dependent regulation of the histone H2A gene via promoter-transcription factor interactions in {\it Drosophila melanogaster\/}

Date of Completion

January 1996

Keywords

Biology, Molecular|Biology, Genetics|Biology, Cell

Degree

Ph.D.

Abstract

The replication-dependent histone genes of Drosophila melanogaster are highly conserved, positioned at a single locus, and reiterated over one hundred times in a tandemly repeated unit. These features make classical genetic studies of these genes ineffective, and so a molecular approach was used to investigate promoter-protein interactions in transcriptional regulation. Conditions for culturing Drosophila Sc-2 cells were optimized in order to achieve uniform cell populations and improve substrate adherence. The cells performed best relative to these attributes in a commercially available serum-free medium, SF900. Changes in serum conditions do not improve growth or attachment.^ The intergenic spacer between the divergently transcribed H2A and H2B histone genes is 226 base pairs long, and is lacking any previously described histone-type regulatory motifs. It does, however, contain a highly conserved, centrally located CACCCACCCC motif which is flanked, and partially overlapped, by two motifs with identity to the AP-1 consensus binding sequence. These motifs together comprise a region now designated as "CHARM" (Conserved Histone Activator/Repressor Motif). The CACCCACCCC motif binds a Drosophila homolog (DmRAP) of the Saccharomyces cerevisiae Repressor-Activator Protein-1 (RAP-1), a multifunctional yeast chromatin architecture protein with complex context-specific activities of gene activation, repression, and telomeric silencing. Cell cycle transcription experiments indicate that the DmRAP binding site acts in a negative regulatory manner, and mutagenesis of this site uncouples H2A gene transcription from DNA replication. Mutagenesis of the "CHARM" region also suggests a positive role for the upstream AP-1-like motif. Electrophoretic mobility shift experiments reveal complex interactions in which a number of different proteins target this small intergenic region, either in concert with DmRAP or with adjacent, and perhaps competitive, overlapping sites. Inspection of the spacer sequence reveals two potential binding sites for the protein E2F, and one each for CREB and TFIID, in an arrangement that is conserved among Drosophila species. The spacer sequence also exhibits the potential for forming a large stem loop-type secondary structure with the DmRAP binding site located at the loop. The conservation of sequence and geometry in the spacer suggests a novel mechanism for regulating transcription, potentially making use of alterations in chromatin architecture. ^

Link to Full Text

Share

COinS