Transcription factors of the AML (core binding factor-␣͞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.The gene encoding transcription factor AML-1 [core binding factor ␣ (CBF-␣)͞polyoma enhancer binding protein 2 (PEBP2)] is frequently the target of multiple chromosomal translocations in lymphoid and myeloid leukemias (1-10). Human AML-1 recognizes the core motif 5Ј-TGYGGT (Y ϭ C or T) and heterodimerizes with the non-DNA-binding partner [11][12][13][14]. The AML-1 class of proteins includes AML-2 and AML-3, and also the murine homologs PEBP 2␣A and BEBP 2␣B, which share a runt homology DNA-binding domain (rhd) first documented in the Drosophila runt gene (12-18). The human AML-1 gene comprises at least eight different exons and multiple polyadenylylation sites (19) and encodes multiple alternatively spliced proteins. The largest of these is AML-1B (480 aa), a transcriptional activator (14,17). The shorter AML-1 protein (250 aa) does not activate transcription (13,14). The major distinction between inactive AML-1 and transcriptionally active AML-1B is use of the alternative C-terminal exons 7B and 8 that are absent in AML-1. These exons are separated from the rhd by many leukemia-associated chromosomal translocations.AML-related factors are expressed in a variety of tissues including lymphoid, myeloid, and osteoblast lineages (1,17,18,(20)(21)(22), where they are key components of mechanisms mediating tissue-specific transcription (22)(23)(24)(25)(26)(27)(28)(29). Recent studies have shown that the nuclear matrix protein NMP-2, specific to osseous cells, is an AML-related protein (21) and that AML transcription factors activate the bone-specific osteocalcin promoter through an NMP-2 binding site (22).Regulation of gene expression is linked to organization of nuclear structure, a principal component of which is the filamentous ribonucleoprotein network known as the nuclear matrix. The nuclear matrix is i...

The AML͞CBF␣ 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 AML͞CBF␣ 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 AML͞CBF␣ must associate with the nuclear matrix to effect transcription. We used f luorescence labeling of epitopetagged 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.Many observations suggest a linkage between nuclear architecture and the regulation of gene expression. These include drastic changes in nuclear morphology often seen in differentiation and in transformation to malignancy. The filamentous ribonucleoprotein network known as the nuclear matrix is a major component of nuclear structure (1-5). It serves to localize gene sequences and may maintain the distribution of regulatory factors throughout the nuclear space (6-19).The AML͞CBF␣ transcription factors are key regulators of hematopoietic and bone-tissue-specific gene expression (20)(21)(22)(23). We have recently shown that the transcriptionally active AML factors contain a specific sequence that targets them to the nuclear matrix (24). In contrast, several inactive forms of AML that lack this targeting signal do not associate with the matrix (24). The targeting sequence resides in a 31-aa segment (nuclear matrix targeting signal, NMTS; aa 351-381) within the C-terminal domain. This NMTS is physically distinct from the nuclear localization signal and functions autonomously to direct AML-1B (CBFA2) to the nuclear matrix (24). These findings suggest that association with nuclear structure may be an important aspect of the mechanisms of gene regulation.In this report we begin to examine the significance of the AML-1B transcription factor binding to the nuclear matrix. We show that AML-1B localizes to sites where there is also active transcription. We demonstrate that the NMTS, which directs AML-1B to the nuclear matrix, functions as a transactivation domain when interacting with an appropriate promoter. Furthermore, we show that the AML-1B transcription factor is targeted to discrete sites on the nuclear matrix. These sites also contain the hyperphosphorylated active form of RNA polymerase II. Colocalization of AML...

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 (AML1͞ETO) 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 AML1͞ETO 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. M odifications in nuclear morphology are a hallmark of tumor cells and are used for diagnosis. However, there is limited knowledge of the subnuclear changes that accompany or contribute to tumor-related alterations of nuclear architecture. Biochemical evidence indicates that the protein composition of the nuclear matrix is modified in tumor cells (1-7). The nuclear matrix is a scaffold that provides a means for localizing genes and regulatory factors throughout the nuclear space (8-11). Consequently, analysis of targeting signals that direct regulatory factors to nuclear matrix-associated subnuclear sites (12) may provide insight into nuclear structure-function relationships that are compromised in cancer.The acute myelogenous leukemia (AML) transcription factor AML1 1 is a key regulator of hematopoiesis (13,14). Numerous cytogenetic abnormalities that involve genes encoding AML1 or its partner core binding factor ␤ have been identified in AML and acute lymphocytic leukemia (15-18). The frequent 8;21 translocation produces a chimeric protein (AML1͞ETO) in which the C terminus of AML1 is replaced by the unrelated ETO (MTG8) protein (19-23). The 8;21 translocation occurs in approximately 15% of AML in adult patients (24-26).The normal form of the AML1 protein has an N-terminal region containing a runt homology DNA-binding domain (27) and a multifunctional C-terminal region that supports transactivation, repression, and intranuclear targeting (28-34). Alternative splicing of the AML1 gene can generate several protein isoforms. The predominant isoform AML1B (480 aa) contains a 31-aa nuclear matrix-targeting signal (NMTS) in the C terminus. This targeting signal is necessary and sufficient to direct the factor to nuclear matrix-associated subnuclear sites that support transcription (30). Many of these AML1 sites are localized together with the hyperphosphorylated active form of RNA polymerase II o , and this association requires the presence of the runt homology domain of AML1B (35).The AML1͞ETO fusion product is expre...

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