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A human genomic library enriched in transcriptionally active sequences (aDNA library)
A L Pelling1, A W Thorne, C Crane-Robinson
1Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, Faculty of Sciences, University of Portsmouth, Portsmouth PO1 2DT, UK.
Genome Research
|June 16, 2000
Summary
Histone H4 hyperacetylation marks active gene promoters. Researchers created an active DNA (aDNA) library from K562 cells using antibodies targeting acetylated H4, yielding a gene-rich resource for promoter discovery.
Area of Science:
- Epigenetics and Gene Regulation
- Molecular Biology
- Genomics
Background:
- Core histone hyperacetylation, particularly of histone H4, is a hallmark of active gene promoters and can extend locuswide.
- Identifying and isolating promoter regions of active genes is crucial for understanding gene regulation.
Purpose of the Study:
- To develop a method for enriching and isolating DNA sequences associated with active gene promoters.
- To construct and characterize a genomic library enriched for active gene sequences (aDNA) from K562 cells.
Main Methods:
- Utilized antibodies against hyperacetylated H4 to immunoprecipitate dinucleosomal chromatin from K562 cells.
- Generated a genomic library from the immunoprecipitated DNA (aDNA library).
- Sequenced clones from the aDNA library and compared them to nucleic acid and protein databases; employed Northern blot and RT-PCR to assess expression.
Main Results:
- Of 146 sequenced clones, 16 were assigned to known genes, with 12 being tissue-specific and predominantly located 5' to coding sequences.
- 33 clones showed high sequence identity to expressed sequence tags (dbEST).
- RT-PCR confirmed that at least 30% of tested clones represented sequences present in the mRNA population, indicating the library is gene-rich and not biased towards highly expressed genes.
Conclusions:
- The aDNA library is a valuable resource for identifying active gene promoter sequences, including those lacking CpG islands.
- This approach successfully enriches for promoter-associated DNA, offering a novel strategy for promoter discovery.