The runt-related transcription factors (RUNX/Cbfa/AML) are essential for cellular differentiation and fetal development. C-terminal truncations of RUNX factors that eliminate the targeting of these factors to subnuclear foci result in lethal hematopoietic and skeletal phenotypes. Here we demonstrate that in living cells the RUNX C-terminus is necessary for the dynamic association of RUNX into stable subnuclear domains. Time-lapse fluorescence microscopy shows that RUNX1 and RUNX2 localize to punctate foci that remain stationary in the nuclear space. By fluorescence recovery after photobleaching assays, both proteins are shown to dynamically associate at these subnuclear foci, with a 10 second half-time of recovery. A truncation of RUNX2, removing its intranuclear targeting signal (NMTS), increases its mobility by an order of magnitude, resulting in a half-time of recovery equivalent to that of EGFP alone. We propose that the dynamic shuttling of RUNX factors in living cells to positionally stabilized foci, which is dependent on the C-terminus, is a component of the mechanism for gene regulation in vivo.

Promyelocytic leukemia (PML) nuclear bodies are important components of nuclear architecture that are functionally linked to aberrant gene expression and disease. To understand the mechanisms that modify subnuclear distribution and regulatory activities of PML domains in leukemia, we performed immunofluorescence microscopy with a panel of normal diploid cells and established cell lines. We analyzed the representation and intranuclear distribution of PML domains. We find that multiple biological parameters contribute to heterogeneity in the subnuclear organization of PML domains in a broad spectrum of cell types. The subnuclear organization of PML domains was also evaluated following transient transfection with a series of vectors expressing normal hematopoietic and leukemia-related transcription factors. Our results show that expression of a chimeric transcription factor encoded by the tumor related chromosomal translocation (8;21) involving the AML1 and ETO loci is sufficient to cause reorganization of PML domains. This finding increases our understanding of the mechanisms by which the AML1/ETO protein may contribute to modified gene expression linked to the onset and progression of t(8;21) related acute myelogenous leukemia.

Evidence is accruing that the architectural organization of nucleic acids and regulatory proteins within the cell nucleus support functional interrelationships between nuclear structure and gene expression. The punctate distribution of several transcription factors has provided several paradigms for pursuing mechanisms that direct these regulatory proteins to subnuclear sites where gene activation or suppression occurs. Sequences that are necessary and sufficient to direct regulatory proteins to transcriptionally active nuclear domains have been identified. Mutations that disrupt intranuclear targeting signals lead to modified subnuclear distribution of transcription factors and aberrant expression in tumor cells. J. Cell. Biochem. Suppls. 32/33:24-31, 1999.

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.

Mechanisms that coordinate the spatial organization of genes and regulatory proteins within the three-dimensional context of nuclear architecture contribute to the sorting of regulatory information as well as the assembly and activity of sites within the nucleus that support gene expression. In this article we will present an overview of experimental approaches that provide insight into the trafficking of the hematopoietic and bone-specific AML/CBF family of regulatory factors to transcriptionally active subnuclear sites.

Components of nuclear architecture are functionally interrelated with control of gene expression. There is growing appreciation that multiple levels of nuclear organization integrate the regulatory cues that support activation and suppression of genes as well as the processing of gene transcripts. The linear representation of genes and promoter elements provide the potential for responsiveness to physiological regulatory signals. Parameters of chromatin structure and nucleosome organization support synergism between activities at independent regulatory sequences and render promoter elements accessible or refractory to transcription factors. Association of genes, transcription factors, and the machinery for transcript processing with the nuclear matrix facilitates fidelity of gene expression within the three-dimensional context of nuclear architecture. Mechanisms must be defined that couple nuclear morphology with enzymatic parameters of gene expression. The recent characterization of factors that mediate chromatin remodeling and identification of intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs link genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue-specific control occurs in tumor cells and in neurological disorders. Compromises in nuclear structure-function interrelationships can occur as a consequence of microgravity-mediated perturbations in cellular architecture.

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. There is growing appreciation that multiple levels of nuclear organization integrate the regulatory cues that support activation and suppression of genes as well as the processing of gene transcripts. The linear organization of genes and promoter elements provide the potential for responsiveness to physiological regulatory signals. Parameters of chromatin structure and nucleosome organization support synergism between activities at independent regulatory sequences and render promoter elements accessible or refractory to transcription factors. Association of genes, transcription factors, and the machinery for transcript processing with the nuclear matrix facilitates fidelity of gene expression within the three-dimensional context of nuclear architecture. Mechanisms must be defined that couple nuclear morphology with enzymatic parameters of gene expression. The recent characterization of factors that mediate chromatin remodeling and intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs, reflect linkage of genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue-specific control that occurs in tumor cells and in neurological disorders provides a basis for high resolution diagnostic and targeted therapy.

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. In this article we focus on the concept that association of genes and cognate transcription factors with the nuclear matrix may support the formation and/or activities of nuclear domains that facilitate transcriptional regulation. Several lines of evidence are consistent with the concept that association of transcription factors with the nuclear matrix may be obligatory for fidelity of gene expression and maximal transcriptional activity. The identification of specific regions of transcription factors that are responsible for intranuclear trafficking to nuclear matrix-associated sites that support transcription, reinforces the linkage of nuclear structure to regulation of genes. CBFA2/AML-1 and CBFA1/AML-3 provide paradigms for directing gene regulatory factors to RNA polymerase II containing foci within the nucleus. The implications of modifications in the intranuclear trafficking of transcription factors for developmental and tissue-specific control, as well as for aberrations in gene expression that are associated with cancer and neurological disorders, are addressed.

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.

The multifunctional transcription factor YY1 is associated with the nuclear matrix. In osteoblasts, the interaction of several nuclear matrix-associated transcription factors with the bone specific osteocalcin gene contributes to tissue-specific and steroid hormone-mediated transcription. A canonical nuclear matrix targeting signal (NMTS) is present in all members of the AML/CBFbeta transcription factor family, but not in other transcription factors. Therefore, we defined sequences that direct YY1 (414 amino acids) to the nuclear matrix. A series of epitope tagged deletion constructs were expressed in HeLa S3 and in human Saos-2 osteosarcoma cells. Subcellular distribution was determined in whole cells and nuclear matrices in situ by immunofluorescence. We demonstrated that amino acids 257-341 in the C-terminal domain of YY1 are necessary for nuclear matrix association. We also observed that sequences within the N-terminal domain of YY1 permit weak nuclear matrix binding. Our data further suggest that the Gal4 epitope tag contains sequences that affect subcellular localization, but not targeting to the nuclear matrix. The targeted association of YY1 with the nuclear matrix provides an additional level of functional regulation for this transcription factor that can exhibit positive and negative control.