Sequence-specific binding of a c-myc nuclear-matrix-associated region shows increased nuclear matrix retention after leukemic cell (HL-60) differentiation.

HL-60 cells, a human promyelocytic leukemia cell line, contain amplified c-myc DNA sequences and mRNA transcripts. These cells can be induced to undergo macrophage differentiation by phorbol esters, which results in suppression of c-myc expression and cessation of cell proliferation. The nuclear matrix (NM), a nuclear skeleton resistant to DNase I digestion and high salt extraction, is proposed to be involved in DNA replication, gene regulation, and the correct distribution of DNA at mitosis. We have previously identified a nuclear-matrix-associated region (MAR) of the c-myc protooncogene to reside in a 1.4-kb region between Cla I and Eco RI restriction sites at the 3'-end of the gene. A 172-bp Dra I/Dra I subfragment of the 1.4-kb region was shown to be a major component of the MAR (myc-MAR), and this subfragment was demonstrated to be recognized by a nuclear protein (p25). In this report we demonstrate that phi X174 DNA, or the synthetic copolymers poly[d(G.C)] and poly[d(A.T)], are not effective suppressors of the binding of the myc-MAR to isolated NM, indicating that the binding sequence(s) are unique. We find that the addition of partially purified protein p25 increases the relative affinity of the myc-MAR for HL-60 NM in an in vitro assay system. NM isolated from HL-60 macrophages induced by phorbol esters retains significantly more myc-MAR DNA fragment in the presence of an excess amount of competitor DNA than does NM from untreated HL-60 cells. These data suggest that a change of the myc-MAR association with the NM occurs after monocytic differentiation of HL-60 cells.

Identification of a nuclear matrix-associated region of the c-myc protooncogene and its recognition by a nuclear protein in the human leukemia HL-60 cell line.

A nuclear matrix (NM)-associated region (MAR) of the protooncogene c-myc is identified in a human leukemia cell line (HL-60). A binding assay between isolated NM and 32P-end-labeled c-myc fragments in the presence of unlabeled competitors was used, and a 3'-end DraI/DraI fragment of 172 base pairs containing the first of the two polyadenylation [poly(A)] signals was identified as an in vitro MAR. Direct detection of endogenous c-myc fragments remaining NM bound after restriction digestion was used, and an in vivo MAR has been identified as the ClaI/EcoRI 1.4-kilobase pair fragment containing the 172-base pair in vitro MAR fragment. In addition, a nuclear protein (Mr = 25,000, p25) demonstrating preferential binding to the 172-base pair c-myc MAR has been identified and partially purified. This protein is diminished in the nuclei of the cells induced by phorbol ester to undergo macrophage differentiation. Footprint analysis shows that p25 binds to two regions of the 172-base pair fragment. One contains the first of two poly(A) addition signals and a topo II box-like sequence, and the other (AATTTCAATCCTAGTA) is 17 base pairs downstream of the first poly(A) signal.

Reassembly of c-myc and relaxation of c-fos nucleosomes during differentiation of human leukemic (HL-60) cells.

Human promyelocytic leukemic (HL-60) cells have amplified c-myc protooncogene sequences which lead to an elevated level of c-myc gene expression. Induction of HL-60 cells by phorbol esters to undergo monocytic differentiation results in the suppression of c-myc, but the activation of c-fos gene transcription. Chromatin structures of c-myc and c-fos were compared by measuring their sequences in nucleosome-associated DNA fragments. These nucleosomal particles were released from chromatin by micrococcal nuclease digestion and subsequently analyzed with two dimensional gel electrophoresis. C-myc related sequences were detected in nucleosomal DNA fragments of differentiated cells only, while the c-fos related sequences were found in nucleosomal DNAs of noninduced HL-60 cells. Since the enzyme preferentially digests relaxed DNAs, these results suggest that nucleosomal subunits of c-myc and c-fos chromatin are relaxed during the state of active transcription, and reassembled once their transcription is repressed.