Studies in detection limits and selectivity of probes used for the assessment of histone acetylation at specific loci

Abstract

Highly sensitive and selective probes for screening historic acetylation at particular loci are necessary. This work describes the design of selective DNA probes for the identification of specific chromosomal regions and highlights the importance of signal amplification for probes of less than 2 kilobases (kb), and the effect that particle size used for the amplification may have on its ability to bind to the target. Single copy FISH probes were designed for the identification of target regions, and generated by PCR amplification. Probe characterization was done by nick translation labeling with biotin or digoxigenin and detection with avidin or anti-digoxigenin. The probes proved to be selective, with detection efficiencies between 50 and 90%. Different labeling approaches were evaluated for the use of 3D DNA structures (dendrimers) to amplify the scFISH probe fluorescent signal. Low signal amplification of a 2.4 kb probe with anti-biotin dendrimers suggested that steric hindrance might keep dendrimers from binding to target probes. Qualitative studies of samples immobilized in polyacrylamide by near confocal microscopy demonstrated that after incubation with dendrimers there were biotins still available for binding. Furthermore, detection of higher number of quantum dots (15 nm in diameter) bound to probe than dendrimers (90 nm diameter) suggested that particle size will have to be considered when designing a method for signal amplification.

For the detection of histone acetylation, antibodies against H4-Ac16 and H4-nonAc were generated by conjugation of the target peptide to PPD. After affinity purification, the cross-reactivity of these antibodies to the non-antigenic peptide was ∼8%. Immunofluorescence experiments with commercial and in-house antibodies on chromatin extended fibers agreed with the “zip” model of acetylation for H4, where the order of acetylation is from lysine 16 to lysine 5. It was infered from theoretical calculations and previous reports that the study of acetylation changes at regions of ∼2.4 kb would require detection of modifications in 28 histone molecules, indicating the need for signal amplification for the determination.