Three distinct domains in TEL-AML1 are required for transcriptional repression of the IL-3 promoter

Abstract

A cytogenetically cryptic (12;21) translocation is the most common molecular abnormality identi®ed in childhood acute lymphoblastic leukemia (ALL), and it generates a chimeric TEL-AML1 protein. Fusion of the Helix-Loop-Helix (HLH) (also called the pointed) domain of TEL to AML1 has been suggested to convert AML1 from a transcriptional activator to a repressor. To de®ne the structural features of this chimeric protein required for repression, we analysed the transcriptional activity of a series of TEL-AML1 mutants on the AML1-responsive interleukin-3 (IL-3) promoter, a potentially relevant gene target. Our results demonstrate that TEL-AML1 represses basal IL-3 promoter activity in lymphoid cells, and deletion mutant analysis identi®ed three distinct domains of TEL-AML1 that are required for repression; the HLH (pointed) motif contained in the TEL portion of TEL-AML1, and both the runt homology domain (Rhd) and the 74 amino acids downstream of the Rhd that are present in the AML1 portion of the fusion protein. Although AML1B (and a shorter AML1 isoform, AML1A) have transcriptional activating activity on the IL-3 promoter, fusion of the AML1 gene to the TEL gene generates a repressor of IL-3 expression. Consistent with this activity, freshly isolated human ALL cells that contain TEL-AML1 do not express IL-3.

TEL-AML1 preleukemic activity requires the DNA binding domain of AML1 and the dimerization and corepressor binding domains of TEL

“…In this assay, TEL-AML1 shows only weak DNA binding, the binding being more readily visualized upon supershifting of the complex using an anti-HA monoclonal antibody (mAb) (Figure 3). In agreement with previous studies Uchida et al, 1999), full length AML1 and the D214, D289 and DHLH mutants, all showed enhanced DNA binding in comparison with TEL-AML1, Figure 1 TEL-AML1 deletion mutant constructs. Depicted are the wild-type TEL and AML1 proteins, the TEL-AML1 fusion protein and various mutant TEL-AML1 proteins that lack the HLH or CR domains of TEL, the RHD of AML1, two deletions of the transactivation domain and C-terminal amino acids of AML1, or contain an RHD point mutation.…”

“…To determine which domains are required for TEL-AML1 activity in vitro and in vivo, we generated a number of deletion mutants based on previous studies (Fenrick et al, 1999;Uchida et al, 1999;Guidez et al, 2000) (Figure 1). These studies found that deletion of the runt homology domain (RHD) domain abolished TEL-AML1 binding to AML1 consensus sequences, whereas deletions of the C-terminal transactivation domain of AML1 or the HLH domain of TEL did not affect the ability of TEL-AML1 to bind to DNA (Uchida et al, 1999).…”

“…These studies found that deletion of the runt homology domain (RHD) domain abolished TEL-AML1 binding to AML1 consensus sequences, whereas deletions of the C-terminal transactivation domain of AML1 or the HLH domain of TEL did not affect the ability of TEL-AML1 to bind to DNA (Uchida et al, 1999). The complementary DNAs (cDNAs) were inserted into the murine stem cell virus (MSCV)-based retroviral vector pMSCV-IRES-EGFP upstream of the internal ribosome entry site (IRES) and a cDNA encoding enhanced green fluorescent protein (EGFP).…”

“…An RHD mutation disrupting DNA binding abrogates TEL-AML1 activity Although the RHD deletion has previously been shown to abrogate DNA binding of TEL-AML1 (Uchida et al, 1999), this domain is also responsible for binding CBFb, the non-DNA-binding heterodimerization partner of AML1 (Scandura et al, 2002). In order to determine which function of the RHD is required for TEL-AML1 activity, the colony forming ability of c-Kit þ cells transduced with the MSCV-R201Q construct was analysed.…”

“…TEL-AML1 is a chimeric transcription factor that can bind AML1 target sequences and potentially deregulate gene expression in cells harboring the translocation. TEL-AML1 has been shown to inhibit AML1-dependent transcription of several promoters in transient transfection assays and functional domains of both TEL and AML1 are required for this activity Fears et al, 1997;Fenrick et al, 1999;Uchida et al, 1999). These studies suggested that the TEL moiety of the fusion converts AML1 from an activator to a repressor of transcription.…”