Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1/ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1/ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias.

The AML/CBFalpha runt transcription factors are key regulators of hematopoietic and bone tissue-specific gene expression. These factors contain a 31-amino acid nuclear matrix targeting signal that supports association with the nuclear matrix. We determined that the AML/CBFalpha factors must bind to the nuclear matrix to exert control of transcription. Fusing the nuclear matrix targeting signal to the GAL4 DNA binding domain transactivates a genomically integrated GAL4 responsive reporter gene. These data suggest that AML/CBFalpha must associate with the nuclear matrix to effect transcription. We used fluorescence labeling of epitope-tagged AML-1B (CBFA2) to show it colocalizes with a subset of hyperphosphorylated RNA polymerase II molecules concentrated in foci and linked to the nuclear matrix. This association of AML-1B with RNA polymerase II requires active transcription and a functional DNA binding domain. The nuclear matrix domains that contain AML-1B are distinct from SC35 RNA processing domains. Our results suggest two of the requirements for AML-dependent transcription initiation by RNA polymerase II are association of AML-1B with the nuclear matrix together with specific binding of AML to gene promoters.

Transcription factors of the AML (core binding factor-alpha/polyoma enhancer binding protein 2) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. Alternative splicing of the AML-1 gene results in two major AML variants, AML-1 and AML-1B. We show here that the transcriptionally active AML-1B binds to the nuclear matrix, and the inactive AML-1 does not. The association of AML-1B with the nuclear matrix is independent of DNA binding and requires a nuclear matrix targeting signal (NMTS), a 31 amino acid segment near the C terminus that is distinct from nuclear localization signals. A similar NMTS is present in AML-2 and the bone-related AML-3 transcription factors. Fusion of the AML-1B NMTS to the heterologous GAL4-(1-147) protein directs GAL4 to the nuclear matrix. Thus, the NMTS is necessary and sufficient to target the transcriptionally active AML-1B to the nuclear matrix. The loss of the C-terminal domain of AML-1B is a frequent consequence of the leukemia-related t(8;21) and t(3;21) translocations. Our results suggest this loss may be functionally linked to the modified interrelationships between nuclear structure and gene expression characteristic of cancer cells.