Potent Transactivation Domain Research Articles

An engineered Sox17 induces somatic to neural stem cell fate transitions independently from pluripotency reprogramming.

Advanced strategies to interconvert cell types provide promising avenues to model cellular pathologies and to develop therapies for neurological disorders. Yet, methods to directly transdifferentiate somatic cells into multipotent induced neural stem cells (iNSCs) are slow and inefficient, and it is unclear whether cells pass through a pluripotent state with full epigenetic reset. We report iNSC reprogramming from embryonic and aged mouse fibroblasts as well as from human blood using an engineered Sox17 (eSox17FNV). eSox17FNV efficiently drives iNSC reprogramming while Sox2 or Sox17 fail. eSox17FNV acquires the capacity to bind different protein partners on regulatory DNA to scan the genome more efficiently and has a more potent transactivation domain than Sox2. Lineage tracing and time-resolved transcriptomics show that emerging iNSCs do not transit through a pluripotent state. Our work distinguishes lineage from pluripotency reprogramming with the potential to generate more authentic cell models for aging-associated neurodegenerative diseases.

Identification and functional characterization of transcriptional activators in human cells

Transcription is orchestrated by thousands of transcription factors (TFs) and chromatin-associated proteins, but how these are causally connected to transcriptional activation is poorly understood. Here, we conduct an unbiased proteome-scale screen to systematically uncover human proteins that activate transcription in a natural chromatin context. By combining interaction proteomics and chemical inhibitors, we delineate the preference of these transcriptional activators for specific co-activators, highlighting how even closely related TFs can function via distinct cofactors. We also identify potent transactivation domains among the hits and use AlphaFold2 to predict and experimentally validate interaction interfaces of two activation domains with BRD4. Finally, we show that many novel activators are partners in fusion events in tumors and functionally characterize a myofibroma-associated fusion between SRF and C3orf62, a potent p300-dependent activator. Our work provides a functional catalog of potent transactivators in the human proteome and a platform for discovering transcriptional regulators at genome scale.

Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4

The development of human induced pluripotent stem cells (iPSCs) holds great promise for regenerative medicine. However the iPSC induction efficiency is still very low and with lengthy reprogramming process. We utilized the highly potent transactivation domain (TAD) of MYC protein to engineer the human OCT4 fusion proteins. Applying the MYC-TAD-OCT4 fusion proteins in mouse iPSC generation leads to shorter reprogramming dynamics, with earlier activation of pluripotent markers in reprogrammed cells than wild type OCT4 (wt-OCT4). Dramatic enhancement of iPSC colony induction efficiency and shortened reprogramming dynamics were observed when these MYC-TAD-OCT4 fusion proteins were used to reprogram primary human cells. The OCT4 fusion proteins induced human iPSCs are pluripotent. We further show that the MYC Box I (MBI) is dispensable while both MBII and the linking region between MBI/II are essential for the enhanced reprogramming activity of MYC-TAD-OCT4 fusion protein. Consistent with an enhanced transcription activity, the engineered OCT4 significantly stimulated the expression of genes specifically targeted by OCT4-alone, OCT4/SOX2, and OCT4/SOX2/KLF4 during human iPSC induction, compared with the wt-OCT4. The MYC-TAD-OCT4 fusion proteins we generated will be valuable tools for studying the reprogramming mechanisms and for efficient iPSC generation for humans as well as for other species.

Enhanced MyoD-induced transdifferentiation to a myogenic lineage by fusion to a potent transactivation domain.

Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine.

Nuclear factor of activated T cells (NFAT) in pearl oyster Pinctada fucata: Molecular cloning and functional characterization

Nuclear factor of activated T cells (NFAT) plays an important role in nonimmune cells and also in T cells and many other cells of the immune system, by regulating the expression of a variety of genes involved in the immune response, organ development, developmental apoptosis and angiogenesis. In the present study, the NFAT homology gene, PfNFAT, from the pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfNFAT encodes a putative protein of 1226 amino acids, and contains a highly conserved Rel homology region (RHR) with DNA-binding specificity, and a regulatory domain (NFAT homology region, NHR) containing a potent transactivation domain (TAD). The PfNFAT gene consists of 12 exons and 11 introns, and its promoter contains potential binding sites for transcription factors such as NF-κB (Nuclear factor κB), STATx (signal transducer and activator of transcription), AP-1 (activator protein-1) and Sox-5/9 (SRY type HMG box-5/9), MyoD (Myogenic Differentiation Antigen) and IRF (Interferon regulatory factor). Comparison and phylogenetic analysis revealed that PfNFAT shows high identity with other invertebrate NFAT, and clusters with the NFAT5 subgroup. Furthermore, gene expression analysis revealed that PfNFAT is involved in the immune response to lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus inserting operation. The study of PfNFAT may increase understanding of molluscan innate immunity.

Mouse Y-linked Zfy1 and Zfy2 are expressed during the male-specific interphase between meiosis I and meiosis II and promote the 2nd meiotic division.

Mouse Zfy1 and Zfy2 encode zinc finger transcription factors that map to the short arm of the Y chromosome (Yp). They have previously been shown to promote meiotic quality control during pachytene (Zfy1 and Zfy2) and at the first meiotic metaphase (Zfy2). However, from these previous studies additional roles for genes encoded on Yp during meiotic progression were inferred. In order to identify these genes and investigate their function in later stages of meiosis, we created three models with diminishing Yp and Zfy gene complements (but lacking the Y-long-arm). Since the Y-long-arm mediates pairing and exchange with the X via their pseudoautosomal regions (PARs) we added a minute PAR-bearing X chromosome derivative to enable formation of a sex bivalent, thus avoiding Zfy2-mediated meiotic metaphase I (MI) checkpoint responses to the unpaired (univalent) X chromosome. Using these models we obtained definitive evidence that genetic information on Yp promotes meiosis II, and by transgene addition identified Zfy1 and Zfy2 as the genes responsible. Zfy2 was substantially more effective and proved to have a much more potent transactivation domain than Zfy1. We previously established that only Zfy2 is required for the robust apoptotic elimination of MI spermatocytes in response to a univalent X; the finding that both genes potentiate meiosis II led us to ask whether there was de novo Zfy1 and Zfy2 transcription in the interphase between meiosis I and meiosis II, and this proved to be the case. X-encoded Zfx was also expressed at this stage and Zfx over-expression also potentiated meiosis II. An interphase between the meiotic divisions is male-specific and we previously hypothesised that this allows meiosis II critical X and Y gene reactivation following sex chromosome silencing in meiotic prophase. The interphase transcription and meiosis II function of Zfx, Zfy1 and Zfy2 validate this hypothesis.

PAX3-FOXO1 Induces Up-Regulation of Noxa Sensitizing Alveolar Rhabdomyosarcoma Cells to Apoptosis

Alveolar rhabdomyosarcoma (ARMS) has a much poorer prognosis than the more common embryonal subtype. Most ARMS tumors characteristically possess a specific genomic translocation between the genes of PAX3/7 and FOXO1 (FKHR), which forms fusion proteins possessing the DNA binding domains of PAX3/7 and the more transcriptionally potent transactivation domain of FOXO1. We have shown that the proapoptotic BH3-only family member Noxa is upregulated by the PAX3-FOXO1 fusion transcription factor in a p53-independent manner. The increased expression of Noxa renders PAX3-FOXO1-expressing cells more susceptible to apoptosis induced by a ă-secretase inhibitor (GSI1, Z-LLNle-CHO), the proteasome inhibitor bortezomib, and BH3 mimetic ABT-737. Apoptosis in response to bortezomib can be overcome by shRNA knockdown of Noxa. In vivo treatment with bortezomib reduced the growth of tumors derived from a PAX3-FOXO1-expressing primary myoblast tumor model and RH41 xenografts. We therefore demonstrate that PAX3-FOXO1 up-regulation of Noxa represents an unanticipated aspect of ARMS tumor biology that creates a therapeutic window to allow induction of apoptosis in ARMS cells.

Structure-Based Design of a Potent Artificial Transactivation Domain Based on p53

Malfunctions in transcriptional regulation are associated with a number of critical human diseases. As a result, there is considerable interest in designing artificial transcription activators (ATAs) that specifically control genes linked to human diseases. Like native transcriptional activator proteins, an ATA must minimally contain a DNA-binding domain (DBD) and a transactivation domain (TAD) and, although there are several reliable methods for designing artificial DBDs, designing artificial TADs has proven difficult. In this manuscript, we present a structure-based strategy for designing short peptides containing natural amino acids that function as artificial TADs. Using a segment of the TAD of p53 as the scaffolding, modifications are introduced to increase the helical propensity of the peptides. The most active artificial TAD, termed E-Cap-(LL), is a 13-mer peptide that contains four key residues from p53, an N-capping motif and a dileucine hydrophobic bridge. In vitro analysis demonstrates that E-Cap-(LL) interacts with several known p53 target proteins, while in vivo studies in a yeast model system show that it is a 20-fold more potent transcriptional activator than the native p53-13 peptide. These results demonstrate that structure-based design represents a promising approach for developing artificial TADs that can be combined with artificial DBDs to create potent and specific ATAs.

Unraveling the transcriptional regulatory machinery in chondrogenesis

Since the discovery of SOX9 mutations in the severe human skeletal malformation syndrome campomelic dysplasia in 1994, Sox9 was shown to be both required and sufficient for chondrocyte specification and differentiation. At the same time, its distant relatives Sox5 and Sox6 were shown to act in redundancy with each other to robustly enhance its functions. The Sox trio is currently best known for its ability to activate the genes for cartilage-specific extracellular matrix components. Sox9 and Sox5/6 homodimerize through domains adjacent to their Sry-related high-mobility-group DNA-binding domain to increase the efficiency of their cooperative binding to chondrocyte-specific enhancers. Sox9 possesses a potent transactivation domain and thereby recruits diverse transcriptional co-activators, histone-modifying enzymes, subunits of the mediator complex, and components of the general transcriptional machinery, such as CBP/p300, Med12, Med25, and Wwp2. This information helps us begin to unravel the mechanisms responsible for Sox9-mediated transcription. We review here the discovery of this master chondrogenic trio and its roles in chondrogenesis in vivo and at the molecular level, and we discuss how these pioneering studies open the way for many additional studies that are needed to further increase our understanding of the transcriptional regulatory machinery operating in chondrogenesis.

Identification of Ewing’s sarcoma protein as a G‐quadruplex DNA‐ and RNA‐binding protein

The Ewing's sarcoma (EWS) oncogene contains an N-terminal transcription activation domain and a C-terminal RNA-binding domain. Although the EWS activation domain is a potent transactivation domain that is required for the oncogenic activity of several EWS fusion proteins, the normal role of intact EWS is poorly characterized because little is known about its nucleic acid recognition specificity. Here we show that the Arg-Gly-Gly (RGG) domain of the C-terminal in EWS binds to the G-rich single-stranded DNA and RNA fold in the G-quadruplex structure. Furthermore, inhibition of DNA polymerase on a template containing a human telomere sequence in the presence of RGG occurs in an RGG concentration-dependent manner by the formation of a stabilized G-quadruplex DNA-RGG complex. In addition, mutated RGG containing Lys residues replacing Arg residues at specific Arg-Gly-Gly sites and RGG containing Arg methylated by protein arginine N-methyltransferase 3 decrease the binding ability of EWS to G-quadruplex DNA and RNA. These findings suggest that the RGG of EWS binds to G-quadruplex DNA and RNA via the Arg residues in it.

The Roles of MLL Holocomplex in Embryogenesis, Hematopoiesis and Tumor Suppression.

Abstract 2969Poster Board II-945The roles of MLL holocomplex in embryogenesis, hematopoiesis and tumor suppression MLL is an epigenetic transcriptional regulator that serves critical roles in multiple developmental and homeostatic processes. It is essential for proper maintenance of Hox gene expression during embryogenesis and hematopoiesis, and regulates expression of cyclin-dependent kinase inhibitors (CDKIs) in endocrine tissues such as the pancreas. Conversely, mis-regulation of MLL-dependent transcriptional pathways is associated with various pathologies. Activating mutations of MLL result in constitutive expression of Hox genes leading to acute leukemia, whereas loss of MLL transcriptional function through mutations of menin, an essential MLL-associated cofactor, leads to decreased expression of CDKIs, hyper proliferation of endocrine cells, and development of multiple endocrine neoplasias. Thus, MLL functions in critical growth regulatory transcriptional circuits that are subject to perturbations in various malignancies. MLL is translated as a large precursor protein that subsequently undergoes proteolytic processing into two fragments (MLLN and MLLC) that self-associate through non-covalent interaction to form an intra-molecular complex. MLL is processed by the Taspase I endopeptidase, which specifically cleaves sites that are evolutionally conserved with MLL2 and Drosophila TRX. However the biological requirement for processing remains unknown. MLLN appears to comprise a targeting sub-unit that contains several motifs involved in DNA binding (AT hooks, CXXC domain) and chromatin recognition (PHD fingers, bromo domain). By contrast, MLLC has features of a transcriptional effector sub-unit that possesses a potent transactivation domain and a methyltransferase (SET) domain specific for lysine 4 of histone H3, an epigenetic mark associated with transcriptionally active states. The SET domain also associates with accessory factors (WDR5, RBBP5 and ASH2L) that promote optimal substrate recognition and enzymatic activity. Thus, the MLL intra-molecular complex can be conceptualized as comprised of an MLLC effecter sub-unit tethered to the MLLN targeting sub-unit by non-covalent association. This model has prompted the hypothesis that MLL may serve functionally distinct roles in transcriptional regulation dependent on the conditional association or disassociation of the MLLC subunit. Our biochemical study shows that MLL indeed takes two different forms: an MLLN/MLLC holocomplex and an MLLN complex without MLLC. We examined the roles of the two distinct complexes using a genetic approach employing knock-in mice with targeted mutations of the MLL processing sites. Mice engineered to constitutively express the MLLN subunit without MLLC (thus produce only the MLLN complex) displayed embryonic lethality, hematopoietic defects and transcriptional defects that phenocopy MLL deficiency. By contrast, mutations that disable proteolytic processing, which can assume the MLLN/MLLC holocomplex but not the MLLN complex, had no adverse consequences, demonstrating the lack of an essential role for the MLLN complex. The intra-molecular association of MLLN with MLLC is mediated in part by the PHD1 finger of the MLLN subunit. An MLL mutant lacking PHD1, which mimics deletion mutants associated with human T-cell leukemias, is unable to form an MLLN/MLLC holocomplex. Hence, this oncogenic mutation likely results in loss of function of MLL, suggestive of a potential tumor suppressor role for MLL in the lymphoid lineage similar to that of menin in the endocrine tissues. Our results support the critical roles of the MLLN/MLLC holocomplex in embryogenesis and hematopoiesis and a possible tumor suppressor function in the lymphoid lineage. Disclosures:No relevant conflicts of interest to declare.

Caspase-3-mediated cleavage of p65/RelA results in a carboxy-terminal fragment that inhibits IκBα and enhances HIV-1 replication in human T lymphocytes

BackgroundDegradation of p65/RelA has been involved in both the inhibition of NF-κB-dependent activity and the onset of apoptosis. However, the mechanisms of NF-κB degradation are unclear and can vary depending on the cell type. Cleavage of p65/RelA can produce an amino-terminal fragment that was shown to act as a dominant-negative inhibitor of NF-κB, thereby promoting apoptosis. However, the opposite situation has also been described and the production of a carboxy-terminal fragment that contains two potent transactivation domains has also been related to the onset of apoptosis. In this context, a carboxy-terminal fragment of p65/RelA (ΔNH2p65), detected in non-apoptotic human T lymphocytes upon activation, has been studied. T cells constitute one of the long-lived cellular reservoirs of the human immunodeficiency virus type 1 (HIV-1). Because NF-κB is the most important inducible element involved in initiation of HIV-1 transcription, an adequate control of NF-κB response is of paramount importance for both T cell survival and viral spread. Its major inhibitor IκBα constitutes a master terminator of NF-κB response that is complemented by degradation of p65/RelA.Results and conclusionsIn this study, the function of a caspase-3-mediated carboxy-terminal fragment of p65/RelA, which was detected in activated human peripheral blood lymphocytes (PBLs), was analyzed. Cells producing this truncated p65/RelA did not undergo apoptosis but showed a high viability, in spite of caspase-3 activation. ΔNH2p65 lacked most of DNA-binding domain but retained the dimerization domain, NLS and transactivation domains. Consequently, it could translocate to the nucleus, associate with NF-κB1/p50 and IκBα, but could not bind -κB consensus sites. However, although ΔNH2p65 lacked transcriptional activity by itself, it could increase NF-κB activity in a dose-dependent manner by hijacking IκBα. Thus, its expression resulted in a persistent transactivation activity of wild-type p65/RelA, as well as an improvement of HIV-1 replication in PBLs. Moreover, ΔNH2p65 was increased in the nuclei of PMA-, PHA-, and TNFα-activated T cells, proving this phenomenon was related to cell activation. These data suggest the existence of a novel mechanism for maintaining NF-κB activity in human T cells through the binding of the carboxy-terminal fragment of p65/RelA to IκBα in order to protect wild-type p65/RelA from IκBα inhibition.

Mechanism of Transcriptional Activation by the Proto-oncogene Twist1

Mammalian Twist1, a master regulator in development and a key factor in tumorigenesis, is known to repress transcription by several mechanisms and is therefore considered to mediate its function mainly through inhibition. A role of Twist1 as transactivator has also been reported but, so far, without providing a mechanism for such an activity. Here we show that heterodimeric complexes of Twist1 and E12 mediate E-box-dependent transcriptional activation. We identify a novel Twist1 transactivation domain that coactivates together with the less potent E12 transactivation domain. We found three specific residues in the highly conserved WR domain to be essential for the transactivating function of murine Twist1 and suggest an alpha-helical structure of the transactivation domain.

Transgenic mice expressing PAX3-FKHR have multiple defects in muscle development, including ectopic skeletal myogenesis in the developing neural tube

The t(2;13) chromosomal translocation is found in the majority of human alveolar rhabdomyosarcomas (RMS). The resulting PAX3-FKHR fusion protein contains PAX3 DNA-binding domains fused to the potent transactivation domain of FKHR, suggesting that PAX3-FKHR functions to deregulate PAX3-specific target genes and signaling pathways. We previously developed transgenic mice expressing PAX3-FKHR under the control of mouse Pax3 regulatory sequences to test this hypothesis. We reported that PAX3-FKHR interferes with normal Pax3 developmental functions, with mice exhibiting neural tube and neural crest abnormalities that mimic those found in Pax3-deficient Splotch mice. Here we expanded those studies to show that developmental expression of PAX3-FKHR results in aberrant myogenesis in the developing somites and neural tube, leading to ectopic skeletal muscle formation in the mature spinal cord. Gene expression profiling indicated that PAX3-FKHR expression in the developing neural tube induces a myogenic pattern of gene expression at the expense of the normal neurogenic program. Somite defects in PAX3-FKHR transgenic animals resulted in skeletal malformations that included rib fusions and mis-attachments. As opposed to the neural tube defects, the severity of the rib phenotype was rescued by reducing Pax3 levels through mating with Splotch mice. Embryos from the transgenic line expressing the highest levels of PAX3-FKHR had severe neural tube defects, including exencephaly, and almost half of the embryos died between gestational ages E13.5-E15.5. Nearly all of the embryos that survived to term died after birth due to severe spina bifida, rather than the absence of a muscular diaphragm. These studies reveal a prominent role for PAX3-FKHR in disrupting Pax3 functions and in deregulating skeletal muscle development, suggesting that this fusion protein plays a critical role in the pathogenesis of alveolar RMS by influencing the commitment and differentiation of the myogenic cell lineage.

Identification of a putative transactivation domain in human Nanog

Nanog is a newly identified divergent homeodomain protein that directs the infinite propagation and sustains the pluripotency of embryonic stem cells. It has been reported that murine Nanog has two potent transactivation domains in N-terminal and C-terminal regions. Human Nanog (hNanog) polypeptide shares about 58% and 87% identity to the open reading frame and homeodomain of murine Nanog, respectively. However, the functional domains and molecular mechanisms of hNanog are poorly understood. In this study, for the first time, we presented that only C-terminus of hNanog contains a potent transactivation domain. Based on the amino acid sequences of homeobox domain, we roughly divided hNanog open reading frame into the three regions such as N-terminal, homeodomain and C-terminal regions and constructed either the fusion proteins between hNanog individual and Gal4 DNA binding domain or the context of native hNanog protein. Reporter assays by using reporter plamid containing Gal4 or Nanog binding site revealed that the only C-terminal region exhibited the significant fold induction of transactivation. However, interestingly, there was no significant activation through N-terminal region unlike murine Nanog, suggesting that C-terminal region may have more critical roles in the transcriptional activation of target genes. Taken together, the finding of a putative transactivation domain in hNanog may contribute to the further understanding of molecular mechanism on the regulation of downstream genes involved in self-renewal and pluripotency of human stem cells.

A Potent Transactivation Domain Mimic with Activity in Living Cells

Transcriptional coactivator-binding peptoids were isolated from a large combinatorial library. One of these molecules is shown to function as a potent activation domain surrogate in mammalian cells. Up to a 900-fold increase in expression of a Gal4-responsive reporter gene is observed when a steroid conjugate of the peptoid is incubated with HeLa cells expressing a Gal4 DNA-binding domain (DBD)-glucocorticoid receptor ligand-binding domain (GRLBD) fusion protein.

The Interferon-inducible p202a Protein Modulates NF-κB Activity by Inhibiting the Binding to DNA of p50/p65 Heterodimers and p65 Homodimers While Enhancing the Binding of p50 Homodimers

p202a is a member of the interferon-inducible murine p200 family of proteins. These proteins share 1 or 2 partially conserved 200 amino acid segments of the a or the b type. The known biological activities of p202a include among others the regulation of muscle differentiation, cell proliferation, and apoptosis. These biological activities of p202a can be correlated with the inhibition of the activity of several transcription factors. Thus, the binding of p202a results in the inhibition of the sequence-specific binding to DNA of the c-Fos, c-Jun, E2F1, E2F4, MyoD, myogenin, and c-Myc transcription factors. This study concerns the mechanisms by which p202a inhibits the activity of NF-kappaB, a transcription factor involved among others in host defense, inflammation, immunity, and the apoptotic response. NF-kappaB consists of p50 and p65 subunits. We demonstrate that p202a can inhibit in vitro and in vivo the binding to DNA of p65 homodimers and p50/65 heterodimers, whereas it increases the binding of p50 homodimers. Thus p202a can impair NF-kappaB activity both by inhibiting the binding to DNA of the transcriptionally active p65 homodimers and p50/p65 heterodimers and by boosting the binding of the repressive p50 homodimers. p202a can bind p50 and p65 in vitro and in vivo, and p202a can be part of the p50 homodimer complex bound to DNA. p50 binds in p202a to the a type segment, whereas p65 binds to the b type segment. Transfected ectopic p202a increases the apoptotic effect of tumor necrosis factor (at least in part) by inhibiting NF-kappaB and its antiapoptotic activity.

Host Cell Factor-1 Interacts with and Antagonizes Transactivation by the Cell Cycle Regulatory Factor Miz-1

Human host cell factor-1 (HCF-1) is essential for cell cycle progression and is required, in conjunction with the herpes simplex virus transactivator VP16, for induction of viral immediate-early gene expression. We show here that HCF-1 directly binds to the Myc-interacting protein Miz-1, a transcription factor that induces cell cycle arrest at G(1), in part by directly stimulating expression of the cyclin-dependent kinase inhibitor p15(INK4b). A domain encompassing amino acids 750-836, contained within a subregion of HCF-1 required for cell cycle progression, was sufficient to bind Miz-1. Conversely, HCF-1 interacted with two separate regions in Miz-1: the N-terminal POZ domain and a C-terminal domain (residues 637-803) previously shown to harbor determinants for interaction with c-Myc and the coactivator p300. The latter functioned as a potent transactivation domain when tethered to DNA, indicating that HCF-1 targets a transactivation function in Miz-1. HCF-1 or a Miz-1-binding fragment of HCF-1 repressed transactivation by Gal4-Miz-1 in transfection assays. Moreover, HCF-1 repressed Miz-1-mediated transactivation of a reporter gene linked to the p15(INK4b) promoter. Protein/protein interaction studies and transient transfection assays demonstrated that HCF-1 interferes with recruitment of p300 to Miz-1, similar to what has been reported with c-Myc. Our findings identify Miz-1 as a novel HCF-1-interacting partner and illustrate cross-talk between these two proteins that may be of consequence to their respective functions in gene regulation and their opposing effects on the cell cycle.

MLL-AFX requires the transcriptional effector domains of AFX to transform myeloid progenitors and transdominantly interfere with forkhead protein function.

MLL-AFX is a fusion gene created by t(X;11) chromosomal translocations in a subset of acute leukemias of either myeloid or lymphoid derivation. It codes for a chimeric protein consisting of MLL fused to AFX, a forkhead transcription factor that normally regulates genes involved in apoptosis and cell cycle progression. We demonstrate here that forced expression of MLL-AFX enhances the self-renewal of hematopoietic progenitors in vitro and induces acute myeloid leukemias after long latencies in syngeneic recipient mice. MLL-AFX interacts with the transcriptional coactivator CBP, which is also a fusion partner for MLL in human leukemias. A potent minimal transactivation domain (CR3) at the C terminus of AFX mediates interactions with the KIX domain of CBP and is necessary for transformation of myeloid progenitors by MLL-AFX. However, CR3 alone is not sufficient, suggesting that simple acquisition of a transactivation domain per se does not activate the oncogenic potential of MLL. Rather, two conserved transcriptional effector domains (CR2 and CR3) of AFX are required for full oncogenicity of MLL-AFX and also endow it with the potential to competitively interfere with transcription and apoptosis mediated by wild-type forkhead proteins. Furthermore, a dominant-negative mutant of AFX containing CR2 and CR3 enhances the growth of myeloid progenitors in vitro, although considerably less effectively than does MLL-AFX. Taken together, these data suggest that recruitment of transcriptional cofactors utilized by forkhead proteins is a critical requirement for oncogenic action of MLL-AFX, which may impact both MLL- and forkhead-dependent transcriptional pathways.

Functional analysis of the transcription factor ER71 and its activation of the matrix metalloproteinase-1 promoter.

The ETS transcription factor family is characterized by a conserved ETS DNA-binding domain and its members have been implicated in a plethora of biological processes, including development, cell transformation and metastasis. ER71 is a testis-specific ETS protein that is not homologous to any other protein outside its ETS domain, suggesting that it fulfills a unique physiological role. Here, we report that ER71 is a constitutively nuclear protein whose intracellular localization is dependent on a portion of the ETS domain, namely ER71 amino acids 276-315. Furthermore, the DNA binding activity is intramolecularly regulated, as the N-terminus of ER71 has a negative effect on DNA binding while the C-terminus dramatically enhances this activity. We also demonstrate that ER71 possesses an extremely potent N-terminal transactivation domain comprised of amino acids 1-157. Finally, we show that ER71 is capable of directly activating both an E74 site-driven and the matrix metalloproteinase-1 promoter. Altogether, these data represent the first functional characterization of ER71, which may perform important functions in the developing and adult testis as well as in testicular germ cell tumorigenesis.