The Mixed Lineage Leukemia (MLL) Protein Involved in 11 q23 Translocations Contains a Domain that Binds Cruciform DNA and Scaffold Attachment Region (SAR) DNA

Abstract

Translocations involving chromosome band Ilq23, found in acute lymphoid and myeloid leukemias, disrupt the MLL gene. This gene encodes a putative transcription factor with regions of homology to several other proteins including the zinc fingers and other domains of the Drosophila trithorax gene product, and the "AT-hook" DNA-binding motif of high mobility group proteins. We have previously demonstrated that MLL contains transcriptional activation and repression domains using a GAL4 fusion protein system (21). The repression domain, which is capable of repressing transcription 3-5-fold, is located centromeric to the breakpoint region of MLL. The activation domain, located telomeric to the breakpoint region, activated transcription from a variety of promoters including ones containing only basal promoter elements. The level of activation was very high, ranging from IO-fold to more than 300-fold, depending on the promoter and cell line used for transient transfection.In translocations involving MLL, the protein produced from the der(ll) chromosome which contains the critical junction for leukemogenesis includes the AT-hook domain and the repression domain. We assessed the DNA binding capability of the MLL AT-hook domain using bacterially expressed and purified AT-hook protein. In a gel mobility shift assay, the MLL AT-hook domain could bind cruciform DNA, recognizing structure rather than sequence of the target DNA. This binding could be specifically competed with Hoechst 33258 dye and with distamycin. In a nitrocellulose protein-DNA binding assay, the MLL AT-hook domain could bind to AT-rich SARs, but not to non-SAR DNA fragments. The role that the AT-hook binding to DNA may play in vivo is unclear, but it is likely that DNA binding could affect downstream gene regulation. The AT-hook domain retained on the der(ll) would potentially recognize a different DNA target than the one normally recognized by the intact MLL protein. Furthermore, loss of an activation domain while retaining a repression domain on the der( 11) chromosome could alter the expression of various downstream target genes, suggesting potential mechanisms of action for MLL in leukemia.

Characteristic sequence motifs located at the genomic breakpoints of the translocation t(X;18) in synovial sarcomas

“…These results suggest a strong association of topoisomerase II and the translocation breakpoints. Similar findings have been reported in liposarcomas and, more commonly, in leukemias (Broeker et al, 1996;Kanoe et al, 1999;Zhang et al, 2002). Recently, near-precise interchromosomal recombination and functional DNA topoisomerase II cleavage sites were found at MLL and AF-4 genomic breakpoints in treatment-related acute lymphoblastic leukemia (Lovett et al, 2001).…”

“…The IG/TCR recombination system has been shown to be involved in some lymphoid neoplasms, while in some other tumor types, various promoting sequences have been suggested to play a role in the mechanism that mediates chromosomal translocation. These promoting sequences include consensus sequences, such as Translin, topoisomerase I an II, chi, Alu sequences, palindromic sequences, alternating purine/pyrimidine and polypurine or polypyrimidine sequences (Bullock et al, 1985;Krowezynska et al, 1986;Nicholls et al, 1987;Boehm et al, 1989;Chen et al, 1989;Sperry et al, 1989;Camerini-Otero and Hsieh, 1993;Aoki et al, 1995;Broeker et al, 1996).…”

“…Analyses of DNA sequences located in the vicinity of chromosomal translocation breakpoints, in some kinds of tumors, have suggested that homologous recombination and/or site-specific recombinogenic sequences may be involved. These include, for example, heptamer/ nonamer sequences, Translin consensus sequences, topoisomerase I and II clevage sequences, chi-consensus sequences, Alu repeats, palindromic sequences, alternating purine/pyrimidine and polypurine or polypyrimidine sequences (Bullock et al, 1985;Finger et al, 1986;Krowezynska et al, 1986;Nicholls et al, 1987;Boehm et al, 1989;Chen et al, 1989;Sperry et al, 1989;Camerini-Otero and Hsieh, 1993;Aoki et al, 1995;Broeker et al, 1996).…”

The amino terminus targets the mixed lineage leukemia (MLL) protein to the nucleolus, nuclear matrix and mitotic chromosomal scaffolds

“…57 AT-hook motifs bind the A/T-rich DNA of SARs via minor groove contacts, presumably by recognizing the bent secondary structure characteristic of these sequences. 29,55 Since the amino terminal fragment of MLL contains three AT-hook motifs, which appear to bind to SARbut not non-SAR-containing DNA, 58 we propose that MLL binding to the SARs of metaphase chromosomes, mediated by the amino terminal AT-hook domains, affect chromatin structure and thereby exert epigenetic control over gene expression.…”

HRX Leukemic Fusion Proteins Form a Heterocomplex with the Leukemia-associated Protein SET and Protein Phosphatase 2A

“…HRX A⅐T hooks also bind DNA. To date, HRX has been shown to bind cruciform DNA and scaffold attachment region (SAR) 1 DNA (24,25). This capacity for DNA binding suggests that both HRX and HRX fusion proteins function in the cell in part through direct protein-DNA interactions, possibly in a manner similar to HMG-I(Y) proteins.…”