A Family Of G-Rich Genomic Sequences Iinteract Specifically With The Pu27 Silencer Element Of The c-Myc Promoter

Abstract

IntroductionA substantial proportion (∼80%) of c-Myc expression is regulated by the NHEIII1 element in the c-myc P1 promoter. The NHEIII1 element contains a G/C rich negative regulatory sequence (Pu27), which can form an intramolecular DNA quadruplex structure which results in c-myc silencing. Quadruplex DNA is a four-stranded structure which is quite stable and can form either “intramolecular” (one DNA strand) quadruplex, or “intermolecular” (two or four DNA strands) quadruplex. These quadruplex-forming sequences are very commonly present in the promoters of eukaryotic genes and generally are negative regulators of transcription. The equilibrium between quadruplex (transcriptionally inactive) and duplex (transcriptionally active) DNA structure is influenced by protein binding, salt concentration and supercoiling torsion (as well as other factors). Constitutive overexpression of the c-myc P0-noncoding transcript, containing the Pu27 sequence, inhibits tumorigenesis and growth of tumor cells. Oligonucleotides encoding the Pu27 sequence bind specifically to the Pu27 sequence in the c-myc promoter, inhibit c-myc expression and result in decreased cellular proliferation and cell death. This most likely reflects the formation of intermolecular quadruplex between the oligonucleotide and target sequence. In addition to the Pu27 sequence in the c-myc promoter, we have recently identified a family of 13 nearly identical Pu27 sequences in the noncoding human genome. MethodsPu27 family members were identified by a BLAT search from the UCSC Genome Browser. Circular dichroism was used to document quadruplex formation by each of the 14 Pu27 family members. Specific interaction of each Pu27 “family member” with the “parent” Pu27 sequence was documented by electrophoretic mobility shift (EMS) studies. Growth inhibition was measured for the U937, Raji, K562 and HL60 leukemic cell lines. C-Myc expression was measured by RT-PCR and Western blot analysis. ResultsWe have identified fourteen nearly identical copies of the Pu27 sequence in the noncoding genome. They are located on Chromosomes 8 (c-Myc promoter), 1, 3 (2), 5, 7, 9, 10 (2), 11, 16, 17, 20, and X. Other than the c-Myc Pu27 sequence, these sequences do not appear to be located in promoter regions. Using gel shift analysis, we demonstrated that each of the Pu27 family member sequences binds in a sequence specific manner to the “parent” sequence in the c-myc promoter. Other genomic quadruplex-forming sequences (from the K-ras, Bcl2 and VEGF promoters) do not bind to the c-myc Pu27 promoter sequence. We have previously shown that treatment of leukemic cell lines with an oligonucleotide containing the Pu27 sequence results in downregulation of c-myc transcription and inhibition of cell proliferation with subsequent cell death. The Pu27 oligonucleotide does not affect the growth of nontransformed cell lines. Each of the Pu27 family members also inhibits the growth of all of the leukemic cell lines tested, with varying efficacy. This occurs with an IC50 as low as <200 nM in all of tumor cell lines tested. Analyzing data from the Human Cancer Genome project we have shown that one of the Pu27 family members (located on chromosome 5) is frequently deleted in leukemic cells of patients with AML (data from Human Cancer Genome Project). ConclusionsThis novel family of genomic sequences interacts specifically with the Pu27 c-Myc silencer sequence and down regulates c-myc expression and cellular proliferation. This suggests that they likely play a role in regulating c-Myc expression either through direct DNA-DNA interaction or sequence-specific interaction with transcripts of one or more of the Pu27 sequences. We hypothesize that DNA-DNA interactions between these sequences may also stabilize quadruplex formation by the c-myc promoter sequence, locking it in the “off” (transcriptionally inactive) position. Disruption of this interaction may play an important role in leukemia pathogenesis. Disclosures:No relevant conflicts of interest to declare.