Association of Runt homologs with a beta subunit and with the nuclear matrix

Each of the two human genes encoding the alpha and beta subunits of a heterodimeric transcription factor, PEBP2, has been found at the breakpoints of two characteristic chromosome translocations associated with acute myeloid leukemia, suggesting that they are candidate proto-oncogenes. Polyclonal antibodies against the alpha and beta subunits of PEBP2 were raised in rabbits and hamsters. Immunofluorescence labeling of NIH 3T3 cells transfected with PEBP2 alpha and -beta cDNAs reveals that the full-size alpha A1 and alpha B1 proteins, the products of two related but distinct genes, are located in the nucleus, while the beta subunit is localized to the cytoplasm. Deletion analysis demonstrates that there are two regions in alpha A1 responsible for nuclear accumulation of the protein: one maps to the region between amino acids 221 and 513, and the other maps to the Runt domain (amino acids 94 to 221) harboring the DNA-binding and the heterodimerizing activities. When the full-size alpha A1 and beta proteins are coexpressed in a single cell, the former is present in the nucleus and the latter still remains in the cytoplasm. However, the N- or C-terminally truncated alpha A1 proteins devoid of the region upstream or downstream of the Runt domain colocalize with the beta protein in the nucleus. In these cases, the beta protein appears to be translocated into the nucleus passively by binding to alpha A1. The chimeric protein containing the beta protein at the N-terminal region generated as a result of the inversion of chromosome 16, colocalizes to the nucleus with alpha A1 more readily than the normal beta protein (Lu, 1995).

The AML-1/ETO fusion protein is created by the (8;21) translocation, the second most frequent chromosomal abnormality associated with acute myeloid leukemia. In the fusion protein, the AML-1 runt homology domain, which is responsible for DNA binding and CBF beta interaction, is linked to ETO, a gene of unknown function. The primary sequences of the runt homology domain indicates no known DNA binding motifs, but the domain is predicted to contain six beta-strands, two alpha-helices and a nucleotide binding motif. Mutagenesis of AML-1/ETO was performed to delimit the functional domains of the chimeric protein. Most mutations in the runt homology domain that resulted in reduced CBF beta binding also inhibited DNA binding, indicating that the DNA and CBF beta binding sequences are tightly linked. However, these activities were separated by a point mutation of residue 144, within the putative ATP binding motif, which nearly eliminates DNA binding, but does not affect CBF beta binding. Random mutagenesis has identified as critical for both DNA and CBF beta binding the hydrophobic face of the amphipathic fifth beta-strand, adjacent to the putative ATP binding motif. C-terminal deletion mutants of AML-1/ETO indicate that ETO sequences are essential for interference with AML-1B-mediated transcriptional activation, and that residue 540 defines the C-terminal boundary of a potential repression domain. Thus, these mutational analyses define the regions of AML-1/ETO that regulate its function and that may be important in promoting leukemia (Lenny, 1995).

The AML1 gene encodes DNA-binding proteins that contain the runt homology domain. The gene is found at the breakpoints of t(8;21), t(3;21), and t(12;21) translocations associated with myelogenous leukemias. AML1 heterodimerizes with PEBP2beta/CBFbeta, resulting in the enhanced affinity with DNA. The runt homology domain is responsible for binding with DNA and heterodimerizing with PEBP2beta/CBFbeta. AML1 is thought to perform a pivotal role in myeloid cell differentiation, however, it can cause neoplastic transformation when overexpressed in fibroblasts. The reducing reagent, dithiothreitol (DTT) markedly enhances the DNA binding of AML1 expressed in COS7 cells. Oxidation by diamide or modification by N-ethylmaleimide of the free sulfhydryl residues inhibits the interaction of AML1 with DNA. The diamide effect is reversible with excess DTT, whereas DTT cannot restore the DNA binding of AML1 treated with N-ethylmaleimide. Site-directed mutagenesis to serine of the amino acid residue 72, a highly conserved cysteine in the runt homology domain of AML1, almost completely abolishes DNA binding without altering the interaction with PEBP2beta/CBFbeta. This substitution also impairs transactivation through the consensus DNA sequence and transformation of fibroblasts induced by AML1b. These data indicate an essential role for the conserved cysteine residue in DNA binding of AML1, and it is possible that the redox state of AML1 can contribute to the regulation of its function (Kurokawa, 1996).

A member of the polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is composed of PEBP2 alphaB1/AML1 (as the alpha subunit) and a beta subunit. It plays an essential role in definitive hematopoiesis and is frequently involved in the chromosomal abnormalities associated with leukemia. Functionally separable modular structures in PEBP2 alphaB1 are reported for DNA binding and for transcriptional activation. DNA binding through the Runt domain of PEBP2 alphaB1 is hindered by the adjacent carboxy-terminal region: this inhibition is relieved by interaction with the beta subunit. Utilizing a reporter assay system in which both the alpha and beta subunits are required to achieve strong transactivation, the presence of transcriptional activation and inhibitory domains in PEBP2 alphaB1 was uncovered that is only apparent in the presence of the beta subunit. The inhibitory domain keeps the full transactivation potential of full-length PEBP2 alphaB1 below its maximum potential. Fusion of the transactivation domain of PEBP2 alphaB1 to the yeast GAL4 DNA-binding domain confers transactivation potential, but further addition of the inhibitory domain diminishes the activity. These results suggest that the activity of the alpha subunit as a transcriptional activator is regulated intramolecularly as well as by the beta subunit. PEBP2 alphaB1 and the beta subunit are targeted to the nuclear matrix via signals distinct from the nuclear localization signal. The transactivation domain by itself is capable of associating with the nuclear matrix, which implies the existence of a relationship between transactivation and nuclear matrix attachment (Kanno, 1998).

AML1, a gene on chromosome 21 that encodes a transcription factor, is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) chromosomal translocations associated with myelogenous leukemias; as a result, chimeric proteins AML1/ETO(MTG8) and AML1/Evi-1 are generated, respectively. To clarify the roles of AML1/ETO(MTG8) and AML1/Evi-1 in leukemogenesis, subcellular localization of these chimeric proteins were determined by immunofluorescence labeling and subcellular fractionation of COS-7 cells, which express these chimeric proteins. AML1/ETO(MTG8) and AML1/Evi-1 are nuclear proteins, as is wild-type AML1. Polyomavirus enhancer binding protein (PEBP)2beta/core binding factor [CBF]beta, a heterodimerizing partner of AML1 that is located mainly in the cytoplasm, is translocated into the nucleus with dependence on the runt domain of AML1/ETO(MTG8) or AML1/Evi-1 when coexpressed with these chimeric proteins. When a comparable amount of either wild-type AML1 or the chimeric proteins is coexpressed with PEBP2beta/CBFbeta, more of the cells expressing the chimeric proteins show the nuclear accumulation of PEBP2beta/CBFbeta, as compared with the cells expressing wild-type AML1. The chimeric proteins associate with PEBP2beta/CBFbeta more effectively than wild-type AML1. These data suggest that the chimeric proteins are able to accumulate PEBP2beta/CBFbeta in the nucleus more efficiently than wild-type AML1, probably because of the higher affinities of the chimeric proteins for PEBP2beta/CBFbeta. These effects of the chimeric proteins on the cellular distribution of PEBP2beta/CBFbeta possibly cause the dominant negative properties of the chimeric proteins over wild-type AML1 and account for one of the mechanisms by which these chimeric proteins contribute to leukemogenesis (Tanaka, 1998).

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. 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). This is 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. 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. These results suggest this loss may be functionally linked to the modified interrelationships between nuclear structure and gene expression characteristic of cancer cells (Zeng, 1997).

The subnuclear location of transcription factors may functionally contribute to the regulation of gene expression. Several classes of gene regulators associate with the nuclear matrix in a cell type, cell growth, or cell cycle related-manner. To understand control of nuclear matrix-transcription factor interactions during tissue development, the subnuclear partitioning of a panel of transcription factors (including NMP-1/YY-1, NMP-2/AML, AP-1, and SP-1) were systematically analyzed during osteoblast differentiation using biochemical fractionation and gel shift analyses. Nuclear matrix association of the tissue-specific AML transcription factor NMP-2, but not the ubiquitous transcription factor YY1, is developmentally upregulated during osteoblast differentiation. There are multiple AML isoforms in mature osteoblasts, consistent with the multiplicity of AML factors that are derived from different genes and alternatively spliced cDNAs. These AML isoforms include proteins derived from the AML-3 gene and partition between distinct subcellular compartments. It is concluded that the selective partitioning of the YY1 and AML transcription factors with the nuclear matrix involves a discriminatory mechanism that targets different classes and specific isoforms of gene regulatory factors to the nuclear matrix at distinct developmental stages. These results are consistent with a role for the nuclear matrix in regulating the expression of bone-tissue specific genes during development of the mature osteocytic phenotype (Lindenmuth, 1997).

The Pebpb2/Cbfb gene encodes the non-DNA binding beta subunit of the heterodimeric transcription factor, PEBP2/CBF, and has been implicated in a subtype of human acute myeloid leukemia, as well as being indispensable for the development of definitive hematopoiesis in the murine fetal liver. By examining a subcellular localization of the PEBP2beta/CBFbeta protein in tissue culture cells, an additional aspect of the protein has been revealed, other than to be a subunit of a transcription factor. Immunoblot and immunocytochemical staining showed that PEBP2beta/CBFbeta is mostly present in the cytoplasm. This PEBP2beta/CBFbeta is free from its DNA-binding partner, the alpha subunit of PEBP2/CBF, as judged by the electrophoretic mobility shift assays. A significant amount of PEBP2beta/CBFbeta is retained in the cytoskeleton preparation after detergent extraction of the cells and is found by double immunofluorescence to colocalize with the F-actin on stress fibers and the vinculin in membrane processes. Thus, the present study extends the character of the nuclear protein PEBP2beta/CBFbeta to be a cytoskeleton-affinitive as well. (Tanaka, 1997).

A protein module called the WW domain recognizes and binds to a short oligopeptide called the PY motif, PPxY, to mediate protein-protein interactions. The PY motif is present in the transcription activation domains of a wide range of transcription factors including c-Jun, AP-2, NF-E2, C/EBPalpha and PEBP2/CBF, suggesting that it plays an important role in transcriptional activation. Mutation of the PY motif in the subregion of the activation domain of the DNA-binding subunit of PEBP2, PEBP2alpha, abolishes the PY motif's transactivation function. Using yeast two-hybrid screening, it has been demonstrated that Yes-associated protein (YAP) binds to the PY motif of PEBP2alpha through its WW domain. The C-terminal region of YAP fused to the DNA-binding domain of GAL4 shows transactivation as strong as that of GAL4-VP16. Exogenously expressed YAP confers transcription-stimulating activity on the PY motif fused to the GAL4 DNA-binding domain as well as to native PEBP2alpha. The osteocalcin promoter is stimulated by exogenous PEBP2alphaA and a dominant negative form of YAP strongly inhibits this activity, suggesting YAP involvement in this promoter activity in vivo. These results indicate that the PY motif is a novel transcription activation domain that functions by recruiting YAP as a strong transcription activator to target genes (Yagi, 1999).

The RUNX family genes are the mammalian homologs of the Drosophila genes runt and lozenge, and members of this family function as master regulators of definitive hematopoiesis and osteogenesis. The RUNX genes encode the alpha subunit of the transcription factor PEBP2/CBF. The ß subunit consists of the non-RUNX protein PEBP2ß. RUNX1/AML1, which is essential for hematopoiesis, is continuously subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. When PEBP2ß is present, however, the ubiquitylation of RUNX1 is abrogated and this causes a dramatic inhibition of RUNX1 proteolysis. Heterodimerization between PEBP2ß and RUNX1 thus appears to be an essential step in the generation of transcriptionally competent RUNX1. Consistent with this notion, RUNX1 is barely detected in PEBP2ß-/- mouse. CBF(PEBP2)ß-SMMHC, the chimeric protein associated with chromosome rearrangement inversion 16 acute myeloid leukemia, is found to protect RUNX1 from proteolytic degradation more efficiently than PEBP2ß. These results reveal a hitherto unknown and major role of PEBP2ß, namely that it regulates RUNX1 by controlling its turnover. This has stimulated new insights into the mechanism of leukemogenesis by CBFß-SMMHC (Huang, 2001).

PEBP2ß is known to regulate RUNX in several ways. PEBP2ß increases the affinity of RUNX proteins for DNA by heterodimerizing with RUNX without interacting with the DNA by itself. Molecular analysis of this heterodimerization has facilitated the understanding of why the affinity of RUNX for DNA is increased by PEBP2ß binding. The ability of the Runt domain to bind to DNA is blocked intramolecularly by additional N- and C-terminal regions. These regions are termed the negative regulatory domains for DNA binding and are denoted as NRDBn and NRDBc. RUNX1 alone binds very poorly to its cognate DNA site because of masking of the DNA-binding surface of the Runt domain by NRDBn and NRDBc. The N- and C-terminal regions of the RUNX proteins each contain a negative regulatory domain for heterodimerization, denoted NRHn and NRHc, respectively. When the C-terminal 80 amino acids of RUNX1 corresponding to NRHc are cleaved off, leaving NRDBc intact for dimerization with PEBP2ß, the resulting dimer binds strongly to DNA. It is thought that the dimerization induces structural changes in RUNX1 in such a way that the DNA-binding surface is unmasked. The unmasking of the DNA-binding surface is thus an important role of PEBP2ß. Heterodimerization also increases the affinity of the minimum DNA-binding domain (not having NRHs and NRDBs) for DNA. This must also be considered another role of PEBP2ß. Finally, as shown by structural analysis, PEBP2ß maintains the SH form of the cysteine residue at position 81 in the Runt domain of RUNX1, which may contribute to better DNA binding by the Runt domain (Huang, 2001).

The core binding factor (CBF) heterodimeric transcription factors comprised of AML/CBFA/PEBP2alpha/Runx and CBFß/PEBP2ß subunits are essential for differentiation of hematopoietic and bone cells, and their mutation is intimately related to the development of acute leukemias and cleidocranial dysplasia. The crystal structures of the AML1/Runx-1/CBFalpha(Runt domain)-CBFß(core domain)-C/EBPß(bZip)-DNA, AML1/Runx-1/CBFalpha(Runt domain)-C/EBPß(bZip)-DNA, and AML1/Runx-1/CBFalpha(Runt domain)-DNA complexes are presented. The hydrogen bonding network formed among CBFalpha(Runt domain) and CBFß, and CBFalpha(Runt domain) and DNA reveals the allosteric regulation mechanism of CBFalpha(Runt domain)-DNA binding by CBFß (Tahirov, 2001).

CBFalpha belongs to a group of transcriptional regulatory proteins that possess an immunoglobulin fold in their DNA binding domains—these include NFAT, STAT1, STAT3ß, T-domain, p53, and NF-kappaB. These proteins all bind to DNA mainly using loops and a C-terminal linker located on the same end of a ß barrel, though their three-dimensional orientations vary widely, leading to differences of their respective target DNA sequences. Since there is now a better understanding of the mechanism by which CBFß regulates CBFalpha-DNA binding, it becomes of great interest to compare that mechanism with those regulating the binding to DNA of other proteins containing immunoglobulin folds. Within the NFAT-Fos-Jun-DNA complex, the minor groove-interacting loop (E'F loop) of NFAT is stabilized by Fos-Jun heterodimer. This mode of interaction has a distant resemblance to the CBFß-mediated stabilization of L9 of CBFalpha via L5. Furthermore, when NFAT within the NFAT-Fos-Jun-DNA complex and CBFalpha within the CBFalpha-ß-C/EBPß-DNA complex are superimposed using their conserved ß strand regions for fitting, Fos-Jun partially overlaps CBFß. Within NF-kappaB (p50, p52, and p65)-DNA complexes, the 'mobile' insert region, which may interact with other DNA binding proteins, could stabilize the interaction between the protein and the DNA minor groove. When the NF-kappaB N-terminal domain within the NF-kappaB (p50)-DNA complex and CBFalpha within the CBFalpha-ß-C/EBPß-DNA complex are superimposed, the insert region of p50, which is also contained in p52 and p65, overlaps CBFß, suggesting the regulatory role of the insert region is similar to that of CBFß. Within the p53-DNA complex, the minor groove-interacting loop (L3), which sandwiches the DNA sugar-phosphate ridge with the major groove-interacting ß strand (S10), is stabilized by zinc coordination such that the zinc atom can modulate the interaction between p53 and the DNA. Interestingly, Arg248 in L3, Arg273 in S10 and some point mutations destabilizing the L3 conformation are very frequently mutated in human tumors. Thus, a number of transcriptional regulatory factors possessing immunoglobulin folds in their DNA binding domains also seem to possess related mechanisms -- mediated by other transcription factors or by chemical modifications -- that regulate DNA binding by affecting areas of interaction sandwiched between the protein and the DNA, although the detailed mechanisms differ. Analogous to CBFs, the Ets domain transcription factor GABP also consists of a subunit that binds to the DNA (GABPalpha) and a regulatory subunit that does not (GABPß). In this case, the minor groove-interacting helix of GABPalpha is stabilized by the ankyrin-like repeats of GABPß. From the structural evidences of the protein-DNA complexes explained above, it can be found that stabilization of the protein backbone amide-DNA phosphate interaction at the protein-DNA minor groove interface appears to be a common feature of mechanisms in which a regulatory protein, having or not having the DNA binding activity, affects its partner protein-DNA interaction (Tahirov, 2001 and references therein).

Key components of DNA replication and the basal transcriptional machinery as well as several tissue-specific transcription factors are compartmentalized in specialized nuclear domains. In the present study, determinants of subnuclear targeting of the bone-related Runx2/Cbfa1 protein are shown to reside in the C-terminus. With a panel of C-terminal mutations, it is further demonstrated that targeting of Runx2 to discrete subnuclear foci is mediated by a 38 amino acid sequence (aa 397-434). This nuclear matrix-targeting signal (NMTS) directs the heterologous Gal4 protein to nuclear-matrix-associated Runx2 foci and enhances transactivation of a luciferase gene controlled by Gal4 binding sites. Importantly, it is shown that targeting of Runx2 to the NM-associated foci contributes to transactivation of the osteoblast-specific osteocalcin gene in osseous cells. Taken together, these findings identify a critical component of the mechanisms mediating Runx2 targeting to subnuclear foci and provide functional linkage between subnuclear organization of Runx2 and bone-specific transcriptional control (Zaidi, 2001).

Runx2 is essential for bone development in mice, and mutations in RUNX2 are found in 65%-80% of individuals with cleidocranial dysplasia. Although all Runx family members can interact with Cbfbeta, a role for Cbfbeta in bone development has not been demonstrated owing to lethality in Cbfb(-/-) mouse embryos at 12.5 days of development from hemorrhages and lack of definitive hematopoiesis. Using a 'knock-in' strategy, mouse embryonic stem cells were generated that express Cbfb fused in-frame to a cDNA encoding green fluorescent protein (GFP). Cbfb(+/GFP) mice had normal life spans and appeared normal, but Cbfb(GFP/GFP) pups died within the first day after birth. The Cbfb(GFP/GFP) mice exhibited a delay in endochondral and intramembranous ossification as well as in chondrocyte differentiation, similar to but less severe than delays observed in Runx2(-/-) mice. Cbfbeta is expressed in developing bone and forms a functional interaction with Runx2, and Cbfb(GFP) is a hypomorphic allele. The fusion allele maintains sufficient function in hematopoietic cells to bypass the early embryonic lethality, and identifies a new role for Cbfb in bone development. These findings raise the possibility that mutations in CBFB may be responsible for some cases of cleidocranial dysplasia that are not linked to mutations in RUNX2 (Kundu, 2002).

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. 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. It is proposed 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 (Harrington, 2002).

CBF interaction with Ets proteins on T-cell receptor (TCR) and other enhancers

An examination was made of the molecular basis for the synergistic regulation of the minimal TCR alpha enhancer by multiple proteins was examined. Reconstitution of TCR alpha enhancer function in nonlymphoid cells requires expression of the lymphoid-specific proteins LEF-1, Ets-1 and PEBP2 alpha (CBF alpha), and a specific arrangement of their binding sites in the enhancer. Ets-1 cooperates with PEBP2 alpha to bind adjacent sites at one end of the enhancer, forming a ternary complex that is unstable by itself. Stable occupancy of the Ets-1- and PEBP2 alpha-binding sites in a DNase I protection assay was found to depend on both a specific helical phasing relationship with a nonadjacent ATF/CREB-binding site at the other end of the enhancer and on LEF-1. The HMG domain of LEF-1 bends the DNA helix in the center of the TCR alpha enhancer. The HMG domain of the distantly related SRY protein, which also bends DNA, can partially replace LEF-1 in stimulating enhancer function in transfection assays. Taken together with the observation that Ets-1 and members of the ATF/CREB family have the potential to associate in vitro, these data suggest that LEF-1 can coordinate the assembly of a specific higher-order enhancer complex by facilitating interactions between proteins bound at nonadjacent sites (Giese, 1995).

Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bind individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. CBF and Ets-1 bind together to beta E2 and beta E3: Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increases the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes are not observed when either the Ets or core site is mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appears to be little constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA-binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant. Ets and core sites are found in several T-cell-specific enhancers, suggesting that this interaction is of general importance in T-cell-specific transcription (Wotton, 1994).

LEF-1 (see Drosophila Pangolin) is a transcription factor that participates in the regulation of the T-cell receptor alpha (TCRalpha) enhancer by facilitating the assembly of multiple proteins into a higher order nucleoprotein complex. The function of LEF-1 is dependent, in part, on the HMG domain that induces a sharp bend in the DNA helix, and on an activation domain that stimulates transcription only in a specific context of other enhancer-binding proteins. ALY, a novel LEF-1-interacting protein was cloned in order to gain insight into the function of context-dependent activation domains. ALY is a ubiquitously expressed, nuclear protein that specifically associates with the activation domains of LEF-1 and AML-1, which is another protein component of the TCRalpha enhancer complex. In addition, ALY can increase DNA binding by both LEF-1 and AML proteins. Overexpression of ALY stimulates the activity of the TCRalpha enhancer complex reconstituted in transfected nonlymphoid HeLa cells, whereas down-regulation of ALY by anti-sense oligonucleotides virtually eliminates TCRalpha enhancer activity in T cells. Similar to LEF-1, ALY can stimulate transcription in the context of the TCRalpha enhancer but apparently not when tethered to DNA through an heterologous DNA-binding domain. It is proposed that ALY mediates context-dependent transcriptional activation by facilitating the functional collaboration of multiple proteins in the TCRalpha enhancer complex (Bruhn, 1997).

Control elements of many genes are regulated by multiple activators working in concert to confer the maximal level of expression, but the mechanism of such synergy is not completely understood. The promoter of the human macrophage colony-stimulating factor (M-CSF) receptor presents an excellent model with which synergistic, tissue-specific activation can be studied. Myeloid-specific expression of the M-CSF receptor is regulated transcriptionally by three factors that are crucial for normal hematopoiesis: PU.1 (an ETS domain transcription factor, see Pointed), AML1 (the mammalian homolog of Drosophila Runt), and C/EBPalpha. These proteins interact in such a way as to demonstrate at least two examples of synergistic activation. AML1 and C/EBPalpha are shown to activate the M-CSF receptor promoter in a synergistic manner. AML1 also synergizes, and interacts physically, with PU.1. Detailed analysis of the physical and functional interaction of AML1 with PU.1 and C/EBPalpha has revealed that the proteins contact one another through their DNA-binding domains and that AML1 exhibits cooperative DNA binding with C/EBPalpha, but not with PU.1. This difference in DNA-binding abilities may explain, in part, the differences observed in synergistic activation. Furthermore, the activation domains of all three factors are required for synergistic activation, and the region of AML1 required for synergy with PU.1 is distinct from that required for synergy with C/EBPalpha. These observations present the possibility that synergistic activation is mediated by secondary proteins contacted through the activation domains of AML1, C/EBPalpha, and PU.1 (Petrovick, 1998).

To understand the molecular basis for the dramatic functional synergy between transcription factors that bind to the minimal T-cell receptor alpha enhancer (Ealpha), enhancer occupancy was examined in thymocytes of transgenic mice in vivo by genomic footprinting. Formation of a multiprotein complex on this enhancer in vivo results from the occupancy of previously identified sites for CREB/ATF, TCF/LEF, CBF/PEBP2, and Ets factors as well as from the occupancy of two new sites 5' to the CRE site, GC-I (which binds Sp1 in vitro) and GC-II. Significantly, although all sites are occupied on a wild-type Ealpha, all sites are unoccupied on versions of Ealpha with mutations in the TCF/LEF or Ets sites. Previous in vitro experiments have demonstrated hierarchical enhancer occupancy with independent binding of LEF-1 and CREB. The data presented here indicate that the formation of a multiprotein complex on the enhancer in vivo is highly cooperative and that no single Ealpha binding factor can access chromatin in vivo to play a unique initiating role in its assembly. Rather, the simultaneous availability of multiple enhancer binding proteins is required for chromatin disruption and stable binding site occupancy as well as the activation of transcription and V(D)J recombination (Hernandez-Munain, 1998).

The transcription factors Ets-1 and AML1 (the alphaBl subunit of PEBP2/CBF) play critical roles in hematopoiesis and leukemogenesis, and cooperate in the transactivation of the T cell receptor (TCR) beta chain enhancer. The DNA binding capacity of both factors is blocked intramolecularly but can be activated by the removal of negative regulatory domains. These include the exon VII domain for Ets-1 and the negative regulatory domain for DNA binding (NRDB) for alphaB1. The direct interaction between the two factors leads to a reciprocal stimulation of their DNA binding activity and activation of their transactivation function. Detailed mapping reveals two independent contact points involving the exon VII and NRDB regions as well as the two DNA binding domains. Using deletion variants and dominant interfering mutants, it has been demonstrated that the interaction between exon VII and NRDB is necessary and sufficient for cooperative DNA binding. The exon VII and NRDB motifs are highly conserved in evolution yet deleted in natural variants, suggesting that the mechanism described is of biological relevance. The mutual activation of DNA binding of Ets and AML1 through the intermolecular interaction of autoinhibitory domains may represent a novel principle for the regulation of transcription factor function (Kim, 1999).

Different isoforms of a new Ets transcription factor family member, NERF/ELF-2, NERF-2, NERF-1a, and NERF-1b, have been isolated. In contrast to the inhibitory isoforms NERF-1a and NERF-1b, NERF-2 acts as a transactivator of the B cell-specific blk promoter. NERF-2 and NERF-1 physically interact with AML1 (RUNX1), a frequent target for chromosomal translocations in leukemia. NERF-2 binds to AML1 via an interaction site located in a basic region upstream of the Ets domain. This is in contrast to most other Ets factors such as Ets-1 that bind to AML1 via the Ets domain, suggesting that different Ets factors utilize different domains for interaction with AML1. The interaction between AML1 and NERF-2 leads to cooperative transactivation of the blk promoter, whereas the interaction between AML1 and NERF-1a leads to repression of AML1-mediated transactivation. To delineate the differences in function of the different NERF isoforms, it was determined that the transactivation domain of NERF-2 is encoded by the N-terminal 100 amino acids, which have been replaced in NERF-1a by a 19-amino acid transcriptionally inactive sequence. Furthermore, acidic domains A and B, which are conserved in NERF-2 and the related proteins ELF-1 and MEF/ELF-4, but not in NERF-1a, are largely responsible for NERF-2-mediated transactivation. Because translocation of the Ets factor Tel to AML1 is a frequent event in childhood pre-B leukemia, understanding the interaction of Ets factors with AML1 in the context of a B cell-specific promoter might help to determine the function of Ets factors and AML1 in leukemia (Cho, 2004).

Groucho proteins are AML corepressors

The mammalian AML/CBFalpha runt domain (RD) transcription factors regulate hematopoiesis and osteoblast differentiation. Like their Drosophila counterparts, most mammalian RD proteins terminate in a common pentapeptide, VWRPY, which serves to recruit the corepressor Groucho (Gro). Using a yeast two-hybrid assay, in vitro association and pull-down experiments, it has been demonstrated that Gro and its mammalian homolog TLE1 specifically interact with AML1 and AML2. In addition to the VWRPY motif, other C-terminal sequences are required for these interactions with Gro/TLE1. TLE1 inhibits AML1-dependent transactivation of the T cell receptor (TCR) enhancers alpha and beta, which, in transfected Jurkat T cells, contain functional AML binding sites. LEF-1 is an additional transcription factor that mediates transactivation of TCR enhancers. LEF-1 and its Drosophila homolog Pangolin (Pan) are involved in the Wnt/Wg signaling pathway through interactions with the coactivator beta-catenin and its highly conserved fly homolog Armadillo (Arm). TLE/Gro interacts with LEF-1 and Pan, and inhibits LEF-1:beta-catenin-dependent transcription. These data indicate that, in addition to their activity as transcriptional activators, AML1 and LEF-1 can act, through recruitment of the corepressor TLE1, as transcriptional repressors in TCR regulation and Wnt/Wg signaling (Levanon, 1998).

The AML1 gene encodes DNA-binding proteins that contain the Runt domain. The gene is found at the breakpoints of some translocations associated with leukemias. It has been reported that AML1 plays pivotal roles in myeloid differentiation, probably through the transcriptional regulation of various hematopoietic genes. This study demonstrates the physical and functional interaction between AML1 and TLE1 (transducin-like Enhancer of split), the human homolog of Groucho that is known to be a corepressor of Hairy-related proteins. TLE1 binds to AML1 through the Runt domain and the C terminus of AML1, which includes the VWRPY motif. The interaction is mainly mediated by the SP domain of TLE1. Moreover, TLE1 inhibits AML1-induced transactivation of the target promoters through the C terminus of AML1. These results suggest that TLE1 acts as a repressor of AML1 and provide important insights into the mechanism of the negative regulation of the AML1 functions in hematopoiesis and leukemogenesis (Imai, 1998).

Regulation of gene expression by tissue-specific transcription factors involves both turning on and turning off transcription of target genes. Runx3, a runt-domain transcription factor, regulates cell-intrinsic functions by activating and repressing gene expression in sensory neurons, dendritic cells (DC), and T cells. To investigate the mechanism of Runx3-mediated repression in an in vivo context, mice were generated expressing a mutant Runx3 lacking the C-terminal VWRPY, a motif required for Runx3 interaction with the corepressor Groucho/transducin-like Enhancer-of-split (TLE). In contrast with Runx3–/– mice, which displayed ataxia due to the death of dorsal root ganglia TrkC neurons, Runx3VWRPY–/– mice are not ataxic and have intact dorsal root ganglia neurons, indicating that ability of Runx3 to tether Groucho/TLE is not essential for neurogenesis. In the DC compartment, the mutant protein Runx3VWRPY– promoted normally developed skin Langerhans cells but failed to restrain DC spontaneous maturation, indicating that this latter process involves Runx3-mediated repression through recruitment of Groucho/TLE. Moreover, in CD8+ thymocytes, Runx3VWRPY– up-regulates alphaE/CD103-like WT Runx3, whereas unlike wild type, it fails to repress alphaE/CD103 in CD8+ splenocytes. Thus, in CD8-lineage T cells, Runx3 regulates alphaE/CD103 in opposing regulatory modes and recruits Groucho/TLE to facilitate the transition from activation to repression. Runx3VWRPY– also failed to mediate the epigenetic silencing of CD4 gene in CD8+ T cells, but normally regulated other pan-CD8+ T cell genes. These data provide evidence for the requirement of Groucho/TLE for Runx3-mediated epigenetic silencing of CD4 and pertain to the mechanism through which other Runx3-regulated genes are epigenetically silenced (Yarmus, 2006).

Hairy-related proteins interact with Runt-related proteins

Drosophila Runt is the founding member of a family of related transcription factors involved in the regulation of a variety of cell-differentiation events in invertebrates and vertebrates. Runt-related proteins act as both transactivators and transcriptional repressors, suggesting that context-dependent mechanisms modulate their transcriptional properties. The aim of this study was to elucidate the molecular mechanisms that contribute to the regulation of the functions of the mammalian Runt-related protein, Cbfa1. Cbfa1 (as well as the related protein, Cbfa2/AML1) physically interacts with the basic helix loop helix transcription factor, HES-1, a mammalian counterpart of the Drosophila Hairy and Enhancer of split proteins. This interaction is mediated by the carboxyl-terminal domains of Cbfa1 and HES-1, but does not require their respective tetrapeptide motifs, WRPY and WRPW. These studies also show that HES-1 can antagonize the binding of Cbfa1 to mammalian transcriptional corepressors of the Groucho family. Moreover, HES-1 can potentiate Cbfa1-mediated transactivation in transfected cells. Taken together, these findings implicate HES-1 in the transcriptional functions of Cbfa1 and suggest that the concerted activities of Groucho and HES proteins modulate the functions of mammalian Runt-related proteins (McLarren, 2000).

The finding that HES and Cbfa proteins can physically interact with one another is consistent with a number of previous results. (1) Expression studies show that, in both invertebrates and vertebrates, HES and Runt-related proteins are coexpressed in a variety of cell types. (2) Both of these proteins interact with Groucho/TLE family members, suggesting that HES- and Runt-related proteins may come in contact with each other at least during mechanisms involving Groucho/TLEs. (3) Genetic studies in Drosophila show that runt and HES genes participate in common developmental mechanisms involved in the control of sex determination and segmentation. For instance, both Runt and the HES family member, Deadpan, can bind to the promoter of the Sex-lethal gene and regulate its expression. (4) Cbfa1 and HES-1 contribute to the regulation of mammalian osteoblast-specific genes; for instance, they provide antagonistic inputs to the control of the expression of the osteopontin gene. This first demonstration of a direct link between mammalian HES and Cbfa proteins will now facilitate the study of how these factors interact with each other and with TLE proteins. Moreover, it will be important to determine whether invertebrate members of these protein families also interact with each other in similar ways (McLarren, 2000).

These studies have also shown that the carboxyl-terminal domains of Cbfa1 and HES-1 are involved in these proteins' interactions . Specifically, the last 60 amino acids of Cbfa1 can interact with the last 88 residues of HES-1. Importantly, the same carboxyl-terminal region of Cbfa1 involved in HES-1 binding also contains binding sites for TLE proteins, raising the possibility that HES-1 and TLE proteins may compete with each other for Cbfa1 binding (McLarren, 2000).

The observation that the carboxyl-terminal region of Cbfa1 is involved in TLE binding is consistent with the identification of a transcriptional repressor function within this domain and suggests that this repressor activity is due to the recruitment of the TLE corepressors. Interestingly, the region of Cbfa1 containing residues 443-516 is ~70% identical to amino acids 366 through 438 of mouse Cbfa2. Since this domain of Cbfa2 also harbors a transcription repression function, it is possible that TLE-binding sites are present within this carboxyl-terminal region of Cbfa2 (McLarren, 2000).

Binding of TLE proteins to Cbfa1 is not dependent on the presence of a carboxyl-terminal WRPY motif. This result is in agreement with the fact that the binding of TLE1 to AML1 occurs even in the absence of the WRPY motif. Moreover, these findings are consistent with transcription studies in transfected mammalian cells showing that TLE overexpression reduces transactivation by both Cbfa1 and a truncated Cbfa1 form lacking the WRPY motif (albeit not as effectively in the latter case). These combined results differ from the previous report that binding of Drosophila Groucho to Runt requires the carboxyl-terminal WRPY motif of the latter. However, those same studies show a weak Groucho/Runt interaction when a truncated form of Runt lacking solely the WRPY motif is used. Only when additional sequences are deleted together with the WRPY motif does Runt fail to bind to Groucho, suggesting that other elements in addition to the WRPY tetrapeptide may mediate this interaction. It is also possible that the difference between the investigations in Drosophila and mammals may reflect differences between Drosophila Runt and its mammalian counterparts or may derive from the use of different experimental protocols (McLarren, 2000).

Cbfa1 can interact with two separate TLE domains located within either the amino-terminal Q region or the carboxyl-terminal WDR domain, both of which are highly conserved among all Groucho/TLE family members. The identification of the WDR domain of Groucho/TLEs as a protein-protein interaction element is not surprising given the demonstrated involvement of WD40 repeats in molecular interactions and the demonstration that the WDR domain of Drosophila Groucho is involved in the interaction with the HES protein, Hairy. The amino-terminal Q domain of TLE proteins has also been shown to mediate protein-protein interactions, including those with the PRDI-BF1/Blimp-1 and UTY proteins. Moreover, in agreement with these results, Cbfa1 has recently been shown to interact with the product of the Grg5 gene, which encodes a roughly 200-amino acid protein homologous to the amino-terminal Q domain of Groucho/TLEs but lacking the carboxyl-terminal SP and WDR regions. Thus, it appears that TLE proteins utilize both of their recognized protein-protein interaction domains to interact with Cbfa family members. Although the specific contributions of these separate TLE domains to the interaction with Cbfa proteins remain to be determined, it is worth mentioning that TLEs also utilize both the amino-terminal Q domain and the carboxyl-terminal WDR domain to associate with specific members of the family of winged-helix DNA-binding proteins. This suggests that the use of separate protein-protein interaction domains may be a feature underlying the association of Groucho/TLE proteins with distinct DNA-binding factors (McLarren, 2000).

The present demonstration that both Cbfa1 and AML1 can interact with HES-1 suggests that members of these two protein families can regulate each other's transcriptional functions. In agreement with this possibility, HES-1 can potentiate Cbfa1-mediated transactivation in transfected cells. A number of observations suggest that HES-1 may perform this function by binding directly to Cbfa1 and inhibiting the interaction between Cbfa1 and endogenous TLE proteins, thereby reducing/inhibiting the repressive effect that TLEs can exert on the transactivating function of Cbfa1. (1) Cbfa1 and HES-1 can directly bind to each other. (2) Binding sites for both HES-1 and TLE proteins are present within the same carboxyl-terminal domain of Cbfa1. (3) HES-1 can interfere with the Cbfa1/TLE interaction in in vitro binding assays. (4) Cbfa1-mediated transactivation can be potentiated by a truncated form of HES-1 that does not interact with TLEs due to HES-1's loss of the carboxyl-terminal WRPW motif but is still competent to bind to Cbfa proteins. Together, these observations suggest that the positive effect of HES-1 on the transcriptional activity of Cbfa1 may involve an active competition with TLEs for direct binding to Cbfa1, rather than a situation in which HES-1 simply titrates away TLEs from Cbfa1 but does not associate with the latter (McLarren, 2000).

Alternative mechanisms can also be proposed. In particular, HES factors may mediate transcriptional activation, instead of repression, when they are associated with Cbfa proteins rather than with TLEs. Although a number of previous studies have shown that invertebrate and vertebrate HES proteins generally act as transcriptional repressors, recent investigations in Xenopus have implicated certain HES family members in both negative and positive feedback loop mechanisms that either repress or maintain the expression of genes of the Notch signaling pathway during embryonic somitogenesis. Together with the present observations, this finding suggests that, perhaps under appropriate conditions in which they escape interactions with Groucho/TLE proteins, HES factors may contribute to the transcriptional activity of other transcription factors (McLarren, 2000).

The possibility that HES-1 may interfere with the Cbfa1/TLE interaction and, vice versa, that Cbfa1 may interfere with the HES-1/TLE interaction may help to explain the finding that HES-1 can repress the expression of the osteopontin gene in osteoblasts, whereas Cbfa1 can activate osteopontin expression. It is possible that HES-1·TLE complexes keep the osteopontin promoter silent and that, by becoming recruited to the promoter, Cbfa1 may contribute to gene activation both directly -- by providing a transactivating function -- and indirectly, by interfering with the TLE/HES-1 interaction. These combined functions may mediate a shift from transcriptional repression mediated by DNA-bound HES-1·TLE complexes to transcriptional activation mediated by Cbfa1. In this model, the direct interaction between Cbfa1 and HES-1 may provide a way to prevent the interaction of Cbfa1 with TLEs. Specifically, by interacting with HES-1, Cbfa1 may become unavailable to TLE proteins and thus protect its transactivation ability from the repressive effect of the TLEs. This situation may provide a molecular explanation for the ability of Cbfa1 to promote transactivation of osteopontin and other osteoblast-specific genes even in the presence of TLE proteins. This would likely not be possible if Cbfa1 were simply titrating away TLEs from HES-1, because the resulting Cbfa1·TLE complexes would probably not be able to promote transactivation (McLarren, 2000).

This model is also consistent with the involvement of Drosophila Runt and Deadpan in the regulation of the Sex-lethal gene. Deadpan mediates repression of Sex-lethal and Groucho is required for this function. Conversely, Runt can bind to the Sex-lethal promoter and stimulate its activation. It is possible that in males, where Runt dosage is one-half of that in females, Deadpan binds to the Sex-lethal promoter and, together with Groucho, mediates transcriptional repression. In females, Runt may be able to antagonize the Deadpan/Groucho-mediated repression by interacting with Deadpan and disrupting the repressive complexes of Deadpan and Groucho. The ensuing Runt-Deadpan complexes may then be able to promote transcription. This model would thus provide a way to regulate the Runt/Groucho interaction through the formation of Runt-Deadpan complexes, a situation that might help to explain the apparent paradox that Runt can activate Sex-lethal expression while at the same time mediating repression of other target genes in the same cells (McLarren, 2000).

Oct-1 counteracts autoinhibition of Runx2 DNA binding to form a novel Runx2/Oct-1 complex on the promoter of the mammary gland-specific gene ß-casein

The transcription factor Runx2 is essential for the expression of a number of bone-specific genes and is primarily considered a master regulator of bone development. Runx2 is also expressed in mammary epithelial cells, but its role in the mammary gland has not been established. Runx2 is shown to form a novel complex with the ubiquitous transcription factor Oct-1 to regulate the expression of the mammary gland-specific gene ß-casein. The Runx2/Oct-1 complex forms on a Runx/octamer element that is highly conserved in casein promoters. The Runt domain is a DNA-binding domain that specifically recognizes a consensus binding site (TGT/cGGT) found in the promoters of several cell type-specific genes. Oct1 regulates transcription from a consensus site ATGC(A/T)AAT. Chromatin immunoprecipitation, RNA interference, promoter mutagenesis, and transient expression analyses were used to demonstrate that the Runx2/Oct-1 complex contributes to the transcriptional regulation of the ß-casein gene. Analysis of the complex revealed autoinhibitory domains for DNA binding in both the N-terminal and the C-terminal regions of Runx2. Oct-1 stimulates the recruitment of Runx2 to the ß-casein promoter by interacting with the C-terminal region of Runx2, suggesting that Oct-1 stimulates Runx2 recruitment by relieving the autoinhibition of Runx2 DNA binding. The regulatory element is actually a composite element consisting of a consensus Runx-binding site adjacent to an octamer sequence. These findings demonstrate that Runx2 collaborates with Oct-1 and contributes to the expression of a mammary gland-specific gene (Inman, 2005).

CBP/p300 is a coactivator of Runx1 and Runx2

The AML1 transcription factor and the transcriptional coactivators p300 and CBP are the targets of chromosome translocations associated with acute myeloid leukemia and myelodysplastic syndrome. In the t(8;21) translocation, the AML1 (CBFA2/PEBP2alphaB) gene becomes fused to the MTG8 (ETO) gene. The terminal differentiation step leading to mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF) is inhibited by the ectopic expression of the AML1-MTG8 fusion protein in L-G murine myeloid progenitor cells. Overexpression of normal AML1 proteins reverses this inhibition and restores the competence to differentiate. Immunoprecipitation analysis shows that p300 and CREB-binding protein (CBP) interact with AML1. The C-terminal region of AML1 is responsible for the induction of cell differentiation and for the interaction with p300. Overexpression of p300 stimulates AML1-dependent transcription and the induction of cell differentiation. These results suggest that p300 plays critical roles in AML1-dependent transcription during the differentiation of myeloid cells. Thus, AML1 and its associated factors p300 and CBFbeta, all of which are targets of chromosomal rearrangements in human leukemia, function cooperatively in the differentiation of myeloid cells (Kitabayashi, 1998).

The complex between AML1 (Runx1) and CBFß is the most frequent target of specific chromosome translocations in human leukemia. The MOZ gene, which encodes a histone acetyltransferase (HAT), is also involved in some leukemia-associated translocations. MOZ is part of the AML1 complex and strongly stimulates AML1-mediated transcription. The stimulation of AML1-mediated transcription is independent of the inherent HAT activity of MOZ. Rather, a potent transactivation domain within MOZ appears to be essential for stimulation of AML1-mediated transcription. MOZ, as well as CBP and MOZ-CBP, can acetylate AML1 in vitro. The amount of AML1-MOZ complex increases during the differentiation of M1 myeloid cells into monocytes/macrophages, suggesting that the AML1-MOZ complex might play a role in cell differentiation. However, the MOZ-CBP fusion protein, which is created by the t(8;16) translocation associated with acute monocytic leukemia, inhibits AML1-mediated transcription and differentiation of M1 cells. These results suggest that MOZ-CBP might induce leukemia by antagonizing the function of the AML1 complex (Kitabayashi, 2001).

p300 is a multifunctional transcriptional coactivator that serves as an adapter for several transcription factors including nuclear steroid hormone receptors. p300 possesses an intrinsic histone acetyltransferase (HAT) activity that may be critical for promoting steroid-dependent transcriptional activation. The vitamin D receptor (VDR) is a member of the steroid and nuclear hormone receptor superfamily of eukaryotic transcription factors and binds target DNA, or response elements, as a homodimer or heterodimer with the 9-cis retinoid X receptor (RXR). In osteoblastic cells, transcription of the bone-specific osteocalcin (OC) gene is principally regulated by the Runx2/Cbfa1 transcription factor and is stimulated in response to vitamin D3 via the vitamin D3 receptor complex. Therefore, p300 control of basal and vitamin D3-enhanced activity of the OC promoter was addressed. Transient overexpression of p300 was found to result in a significant dose-dependent increase of both basal and vitamin D3-stimulated OC gene activity. This stimulatory effect requires intact Runx2/Cbfa1 binding sites and the vitamin D-responsive element. In addition, by coimmunoprecipitation, it has been shown that the endogenous Runx2/Cbfa1 and p300 proteins are components of the same complexes within osteoblastic cells under physiological concentrations. It has also been demonstrated, by chromatin immunoprecipitation assays, that p300, Runx2/Cbfa1, and 1alpha,25-dihydroxyvitamin D3 receptor interact with the OC promoter in intact osteoblastic cells expressing this gene. The effect of p300 on the OC promoter is independent of its intrinsic HAT activity, since a HAT-deficient p300 mutant protein up-regulates expression and cooperates with P/CAF to the same extent as the wild-type p300. On the basis of these results, it is proposed that p300 interacts with key transcriptional regulators of the OC gene and bridges distal and proximal OC promoter sequences to facilitate responsiveness to vitamin D3 (Sierra, 2003).

CCAAT/enhancer-binding proteins (C/EBP) are critical determinants for cellular differentiation and cell type-specific gene expression. Their functional roles in osteoblast development have not been determined. A key component of the mechanisms by which C/EBP factors regulate transcription of a tissue-specific gene during osteoblast differentiation was addressed. Expression of both C/EBPbeta and C/EBPdelta increases from the growth to maturation developmental stages and, like the bone-specific osteocalcin (OC) gene, is also stimulated 3-6-fold by vitamin D3, a regulator of osteoblast differentiation. A C/EBP enhancer element was characterized in the proximal promoter of the rat osteocalcin gene, which resides in close proximity to a Runx2 (Cbfa1) element, essential for tissue-specific activation. C/EBP and Runx2 factors interact together in a synergistic manner to enhance OC transcription (35-40-fold) in cell culture systems. It has been shown by mutational analysis that this synergism is mediated through the C/EBP-responsive element in the OC promoter and by a direct interaction between Runx2 and C/EBPbeta. Furthermore, a domain in Runx2 has been mapped that is necessary for this interaction by immunoprecipitation. A Runx2 mutant lacking this interaction domain does not exhibit functional synergism. It is concluded that, in addition to Runx2 DNA binding functions, Runx2 can also form a protein complex at C/EBP sites to regulate transcription. Taken together, these findings indicate that C/EBP is a principal transactivator of the OC gene and the synergism with Runx2 suggests that a combinatorial interaction of these factors is a principal mechanism for regulating tissue-specific expression during osteoblast differentiation (Gutierrez, 2002).

Transcriptional regulation of Runt homologs

The Runt family transcription factor CBFalpha2 (AML1, PEBP2alphaB, or Runx1) is required by hematopoietic stem cells and expressed at high levels in T-lineage cells. In human T cells CBFalpha2 is usually transcribed from a different promoter (distal promoter) than in myeloid cells (proximal promoter), but the developmental and functional significance of this promoter switch has not been known. Both coding and noncoding sequences of the distal 5' end are highly conserved between the human and the murine genes, and the distal promoter is responsible for the overwhelming majority of CBFalpha2 expression in murine hematopoietic stem cells as well as in T cells. Distal promoter activity is maintained throughout T cell development and at lower levels in B cell development, but downregulated in natural killer cell development (Telfer, 2001).

The distal 5' ends of both human and murine CBFalpha2 include a translational start site, such that CBFalpha2 proteins translated from these mRNAs have an N-terminal sequence different from those encoded by mRNAs with proximal 5' ends. The murine distal 5' end codes for 19 amino acids, rather than the 32 amino acids coded for by the human distal 5' end of AML1B, AML1c, or AML1g. The additional amino acids in the human distal N-terminus are encoded by a separate, intermediate exon, located only ~6 kb upstream of the proximal promoter but ~150 kb downstream of the distal promoter. This intermediate exon is evidently spliced into the human distal mRNA but omitted from the murine mRNA. However, both murine and human distal 5' ends are then spliced into exon 3 to lead into the same sequence, replacing 5 amino acids at the proximal N-terminus. The first 19 amino acids of the distal N-terminus are identical between mouse and human and 63% identical between mouse CBFalpha2, the 'distal' N-terminus of human CBFalpha3, and the only Runt family member isolated from chicken. The distal N-terminus of CBFalpha2 is even 37% identical to the 'distal' N-terminus of its fellow Runt family member Osf2, an osteoblast form of CBFalpha1, or til-1, the same gene inappropriately expressed in thymus via retroviral insertion. This high degree of conservation at the protein and nucleotide levels suggests that both the structure and the regulation of the distal N-terminus are functionally significant (Telfer, 2001).

The distal N-terminal isoform binds to functionally important regulatory sites from known target genes with two- to threefold higher affinity than the proximal N-terminal isoform. Neither full-length isoform alters growth of a myeloid cell line under nondifferentiating conditions, but the proximal isoform selectively delays mitotic arrest of the cell line under differentiating conditions, resulting in the generation of greater numbers of neutrophils (Telfer, 2001).

Runx and sensory neurons

Subpopulations of sensory neurons in the dorsal root ganglion (DRG) can be characterized on the basis of sensory modalities that convey distinct peripheral stimuli, but the molecular mechanisms that underlie sensory neuronal diversification remain unclear. This study used genetic manipulations in the mouse embryo to examine how Runx transcription factor signaling controls the acquisition of distinct DRG neuronal subtype identities. Runx3 acts to diversify an Ngn1-independent neuronal cohort by promoting the differentiation of proprioceptive sensory neurons through erosion of TrkB expression in prospective TrkC+ sensory neurons. In contrast, Runx1 controls neuronal diversification within Ngn1-dependent TrkA+ neurons by repression of neuropeptide CGRP expression and controlling the fine pattern of laminar termination in the dorsal spinal cord. Together, these findings suggest that Runx transcription factor signaling plays a key role in sensory neuron diversification (Kramer, 2006).

Different functional classes of dorsal root ganglion sensory neurons project their axons to distinct target zones within the developing spinal cord. To explore the mechanisms that link sensory neuron subtype identity and axonal projection pattern, the roles of Runx and ETS transcription factors were analyzed in the laminar targeting of sensory afferents. Gain- and loss-of-function studies in chick embryos reveal that the status of Runx3 expression is a major determinant of the dorso-ventral position of termination of proprioceptive and cutaneous sensory axons. In addition, the level of expression and/or activity of Runx3 in individual proprioceptive sensory neurons appears to specify whether their axons terminate in intermediate or ventral regions. These findings suggest that the selectivity of Runx3 expression, and its level of activity, control sensory afferent targeting in the developing spinal cord (Chen, 2006a).

In mammals, the perception of pain is initiated by the transduction of noxious stimuli through specialized ion channels and receptors expressed by nociceptive sensory neurons. The molecular mechanisms responsible for the specification of distinct sensory modality are, however, largely unknown. Runx1, a Runt domain transcription factor, is expressed in most nociceptors during embryonic development but in adult mice, becomes restricted to nociceptors marked by expression of the neurotrophin receptor Ret. In these neurons, Runx1 regulates the expression of many ion channels and receptors, including TRP class thermal receptors, Na+-gated, ATP-gated, and H+-gated channels, the opioid receptor MOR, and Mrgpr class G protein coupled receptors. Runx1 also controls the lamina-specific innervation pattern of nociceptive afferents in the spinal cord. Moreover, mice lacking Runx1 exhibit specific defects in thermal and neuropathic pain. Thus, Runx1 coordinates the phenotype of a large cohort of nociceptors, a finding with implications for pain therapy (Chen, 2006b).

Runt homologs and cancer

Runx3/Pebp2alpha C null mouse gastric mucosa exhibits hyperplasias due to stimulated proliferation and suppressed apoptosis in epithelial cells, and the cells are resistant to growth-inhibitory and apoptosis-inducing action of TGF-ß, indicating that Runx3 is a major growth regulator of gastric epithelial cells. Between 45% and 60% of human gastric cancer cells do not significantly express RUNX3 due to hemizygous deletion and hypermethylation of the RUNX3 promoter region. Tumorigenicity of human gastric cancer cell lines in nude mice is inversely related to their level of RUNX3 expression, and a mutation (R122C) occurring within the conserved Runt domain abolished the tumor-suppressive effect of RUNX3, suggesting that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer (Li, 2002).

Pontin is a critical regulator for AML1-ETO-induced leukemia

The oncogenic fusion protein AML1-ETO, also known as RUNX1-RUNX1T1 is generated by the t(8;21)(q22;q22) translocation, one of the most frequent chromosomal rearrangements in acute myeloid leukemia (AML). Identifying the genes that cooperate with or are required for the oncogenic activity of this chimeric transcription factor remains a major challenge. Previous studies showed that Drosophila provides a genuine model to study how AML1-ETO promotes leukemia. Using an in vivo RNAi screen for suppressors of AML1-ETO activity, pontin/RUVBL1 was identified as a gene required for AML1-ETO-induced lethality and blood cell proliferation in Drosophila. Pontin inhibition strongly impairs the growth of human t(8;21)+ or AML1-ETO-expressing leukemic blood cells. Interestingly, AML1-ETO promotes the transcription of PONTIN. Moreover transcriptome analysis in Kasumi-1 cells revealed a strong correlation between PONTIN and AML1-ETO gene signatures and demonstrated that PONTIN chiefly regulated the expression of genes implicated in cell cycle progression. Concordantly, PONTIN depletion inhibited leukemic self-renewal and caused cell cycle arrest. All together these data suggest that the up-regulation of PONTIN by AML1-ETO participate in the oncogenic growth of t(8;21) cells (Breig, 2013).

The Hematopoietic transcription factors RUNX1 and ERG prevent AML1-ETO oncogene overexpression and onset of the apoptosis program in t(8;21) AMLs

The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. This study investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the bHLH hematopoietic transcription factor LYL1 and LMO2 (see Drosophila Beadex). In contrast, ETS transcription factor ERG, ETS transcription factor FLI1, TAL1, and RUNX1 (see Drosophila Runt) bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. Expression of wild-type transcription factors RUNX1 and ERG in AML was found to be required to prevent this oncogene overexpression. Together these results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells (Mandoli , 2016).

back to Runt Evolutionary homologs part 1/2

runt : Biological Overview | Regulation | Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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