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

Abstract

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.

S/MAR-binding properties of Sox2 and its involvement in apoptosis of human NT2 neural precursors

“…The authors showed sequence retention in the nuclear matrix fraction, but ruled out p25 as a MAR-binding protein. 19 In the present study, we confirmed the existence of the MAR within a ClaI/EcoRI fragment containing the c-myc 3 0 UTR and demonstrated that the 22 bp BUR-like core sequence, highly homologous to the SATB1-binding site in the IgH gene, was located adjacent to, but not within the 172 bp DraI/DraI fragment (Figure 1). This 22 bp sequence was clearly making physical contact with MAR-binding proteins, that is, SATB1 in Jurkat cells 6 and Sox2 in NT2 cells, and, therefore, may be regarded as the true BUR element within the c-myc MAR.…”

“…# X00364, UCSC Human genome version hg16) obtained via the UCSC Genome Browser. 25 The analysis confirmed the presence of a 370 bp S/MAR region located in the 3 0 UTR between nt 7366-7735, consistent with previous studies of Chou et al, 18,19 who reported the presence of a MAR at the 3 0 -end of the gene within a 1.4 kb ClaI and EcoRI restriction fragment (Figure 1). Furthermore, within this 370 bp S/MAR region, we identified a sequence (5 0 TTTTTTTCTA TTGTTTTTAGAA3 0 , nt 7627-7647) highly homologous to the well-characterized 25 bp BUR core element of the mouse IgH gene.…”

“…Early reports by Chou et al 18,19 indicated that the A-T-rich sequence, 5 0 AATTTC AATAATAGTA3 0 , containing the first poly(A) addition signal and located within the 172 bp DraI/DraI fragment (Figure 1), is recognized by an as yet unidentified 25 kDa (p25) nuclear protein. The authors showed sequence retention in the nuclear matrix fraction, but ruled out p25 as a MAR-binding protein.…”

“…The existence of a MAR within the c-myc gene has been known for some time; 18,19,31 however, neither the core BUR element nor any binding protein that makes physical contact with the DNA has been identified. Early reports by Chou et al 18,19 indicated that the A-T-rich sequence, 5 0 AATTTC AATAATAGTA3 0 , containing the first poly(A) addition signal and located within the 172 bp DraI/DraI fragment (Figure 1), is recognized by an as yet unidentified 25 kDa (p25) nuclear protein.…”

“…To address this question, we examined the integrity of the S/MAR region of the c-myc proto-oncogene, located within the 3 0 UTR, 18,19 in human NT2 embryonal carcinoma cells undergoing apoptosis in response to oxygen and glucose deprivation (OGD). The c-myc S/MAR sequence also contains a 22 bp BUR-like core element, a putative protein-binding site.…”