Long-range chromatin analysis of the human MYC locus by pulsed-field gel electrophoresis.
The identification of cis-acting regulatory elements has been greatly facilitated by the perception that nonnucleosomal regions of chromatin, including sites where transacting factors are bound, are hypersensitive to cleavage by nucleases. Hence, mapping of DNaseI-hypersensitive sites (HSs) has become particularly valuable for the detection of transcriptional control elements. The utility of this technique, however, may be constrained by the huge size of some eukaryotic gene domains or by the nonavailability of genomic probes. Apparently, both of these drawbacks hold true for the human protooncogene MYC. To overcome these limitations, we investigated the feasibility of mapping DNaseI-HSs in large restriction fragments. By using MYC-amplified cell lines, we devised a simple protocol that allowed for the detection of DNaseI-HSs at a distance of several hundred kb. In an attempt to identify additional regulatory elements required for MYC expression, we used this method to establish the long-range chromatin structure of four MYC amplicons. This method has potential benefits and applications.
Mautner
J
J
GSF, Institut für Klinische Molekularbiologie und Tumorgenetik, Munich, Federal Republic of Germany.
Bornkamm
G W
GW
Polack
A
A
D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification; D004249:DNA Damage; D003850:Deoxyribonuclease I / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D018628:Gene Dosage; D005786:Gene Expression Regulation; D016259:Genes, myc; D018922:HL-60 Cells; D006801:Humans; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012680:Sensitivity and Specificity
D002843:Chromatin; D004247:DNA; D003850:Deoxyribonuclease I