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A novel class of matrix attached regions (Mars) identified by random cloning and their implications in differentiation and carcinogenesis.

MARs, i.e., the attachment points of chromatin loops to the insoluble nuclear suprastructure called nuclear matrix, define the borders between domains, are the sites of initiation of DNA replication, and display transcriptional enhancer activity. MARs play an important role in differentiation and in carcinogenesis. MAR-protein complexes were proposed to interact with proteins bound to the immediate upstream regulatory region of active genes and bring the 5' end of active genes onto the nuclear matrix where transcription factors and RNA polymerases are located. We have isolated the nuclear matrix from human K562 cells, comprising 1-2% of nuclear DNA, after removal of the bulk of nuclear polynucleosomes with micrococcal nuclease. The MAR DNA was cloned in E. coli. We report here the sequence of a MAR fragment of 480 bp (clone Hum. MAR 19.2a). This sequence contains homopurine stretches of 5-10 nt alternating with homopyrimidine stretches of 4-17 nt, favoring formation of cruciform structures on the DNA. We have identified seven potential cruciform structures, characteristic elements of origins of replication, on the 480 bp 19.2a fragment. In addition this MAR clone contains three ATTA-type motifs representing homeodomain protein binding sites and five TG-rich (or its complementary CA-rich) stretches that we call TG boxes. TG boxes are found at recombination sites. in chromosome telomeres, and in the binding sites of a class of protein factors that regulate transcription and replication. The present study showing a multitude of potential cruciform structures characteristic of origins of replication in the Hum. MAR 19.2a clone gives further support to the idea that a subset of human MARs may function as origins of DNA replication. The relevance of these studies to carcinogenesis and differentiation are discussed.