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...
Typically, eukaryotic nuclei contain 10–30 prominent domains (referred to here as SC-35 domains) that are concentrated in mRNA metabolic factors. Here, we show that multiple specific genes cluster around a common SC-35 domain, which contains multiple mRNAs. Nonsyntenic genes are capable of associating with a common domain, but domain “choice” appears random, even for two coordinately expressed genes. Active genes widely separated on different chromosome arms associate with the same domain frequently, assorting randomly into the 3–4 subregions of the chromosome periphery that contact a domain. Most importantly, visualization of six individual chromosome bands showed that large genomic segments (∼5 Mb) have striking differences in organization relative to domains. Certain bands showed extensive contact, often aligning with or encircling an SC-35 domain, whereas others did not. All three gene-rich reverse bands showed this more than the gene-poor Giemsa dark bands, and morphometric analyses demonstrated statistically significant differences. Similarly, late-replicating DNA generally avoids SC-35 domains. These findings suggest a functional rationale for gene clustering in chromosomal bands, which relates to nuclear clustering of genes with SC-35 domains. Rather than random reservoirs of splicing factors, or factors accumulated on an individual highly active gene, we propose a model of SC-35 domains as functional centers for a multitude of clustered genes, forming local euchromatic “neighborhoods.”
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...
Specific mammalian genes functionally and dynamically associate together within the nucleus. Yet, how an array of many genes along the chromosome sequence can be spatially organized and folded together is unknown. We investigated the 3D structure of a well-annotated, highly conserved 4.3-Mb region on mouse chromosome 14 that contains four clusters of genes separated by gene “deserts.” In nuclei, this region forms multiple, nonrandom “higher order” structures. These structures are based on the gene distribution pattern in primary sequence and are marked by preferential associations among multiple gene clusters. Associating gene clusters represent expressed chromatin, but their aggregation is not simply dependent on ongoing transcription. In chromosomes with aggregated gene clusters, gene deserts preferentially align with the nuclear periphery, providing evidence for chromosomal region architecture by specific associations with functional nuclear domains. Together, these data suggest dynamic, probabilistic 3D folding states for a contiguous megabase-scale chromosomal region, supporting the diverse activities of multiple genes and their conserved primary sequence organization.
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