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D E Ayer

Publications and source records attributed to D E Ayer.

At least 19 recordsLinked to original sources
PubMed2006

The Mlx network: evidence for a parallel Max-like transcriptional network that regulates energy metabolism.

Recent experiments suggest the existence of a transcriptional network that functions in parallel to the canonical Myc/Max/Mad transcriptional network. Unlike the Myc/Max/Mad network, our understanding of this network is still in its infancy. At the center of this network is a Max-like protein called Mlx; hence we have called this network the Mlx network. Like Max, Mix interacts with transcriptional repressors and transcriptional activators, namely the Mad family and the Mondo family, respectively. Similar to Max-containing heterodimers, Mlx-containing heterodimers recognize CACGTG E-box elements, suggesting that the transcriptional targets of these two networks may overlap. Supporting this hypothesis, we have observed genetic interactions between the Drosophila melanogaster orthologs of Myc and Mondo. In higher eukaryotes, two proteins, MondoA and MondoB/CHREBP/WBSCR14, constitute the Mondo family. At present little is known about the transcriptional targets of MondoA; however, pyruvate kinase is a putative target of MondoB/CHREBP/WBSCR14, suggesting a function for the Mondo family in glucose and/or lipid metabolism. Finally, unlike the predominant nuclear localization of Myc family proteins, both Mondo family members localize to the cytoplasm. Therefore, while the Myc and Mondo families may share some biological functions, it is likely each family is under distinct regulatory control.

Amino Acid Sequence↗
PubMed2001

Pf1, a novel PHD zinc finger protein that links the TLE corepressor to the mSin3A-histone deacetylase complex.

The mSin3A-histone deacetylase corepressor is a multiprotein complex that is recruited by DNA binding transcriptional repressors. Sin3 has four paired amphipathic alpha helices (PAH1 to -4) that are protein-protein interaction motifs and is the scaffold upon which the complex assembles. We identified a novel mSin3A-interacting protein that has two plant homeodomain (PHD) zinc fingers we term Pf1, for PHD factor one. Pf1 associates with mSin3A in vivo and recruits the mSin3A complex to repress transcription when fused to the DNA binding domain of Gal4. Pf1 interacts with Sin3 through two independent Sin3 interaction domains (SIDs), Pf1SID1 and Pf1SID2. Pf1SID1 binds PAH2, while Pf1SID2 binds PAH1. Pf1SID1 has sequence and structural similarity to the well-characterized 13-amino-acid SID of the Mad bHLHZip repressor. Pf1SID2 does not have sequence similarity with either Mad SID or Pf1SID1 and therefore represents a novel Sin3 binding domain. Mutations in a minimal fragment of Pf1 that encompasses Pf1SID1 inhibited mSin3A binding yet only slightly impaired repression when targeted to DNA, implying that Pf1 might interact with other corepressors. We show that Pf1 interacts with a mammalian homolog of the Drosophila Groucho corepressor, transducin-like enhancer (TLE). Pf1 binds TLE in an mSin3A-independent manner and recruits functional TLE complexes to repress transcription. These findings suggest that Pf1 may serve to bridge two global transcription networks, mSin3A and TLE.

Amino Acid Sequence↗
PubMed2000

Solution structure of the interacting domains of the Mad-Sin3 complex: implications for recruitment of a chromatin-modifying complex.

Gene-specific targeting of the Sin3 corepressor complex by DNA-bound repressors is an important mechanism of gene silencing in eukaryotes. The Sin3 corepressor specifically associates with a diverse group of transcriptional repressors, including members of the Mad family, that play crucial roles in development. The NMR structure of the complex formed by the PAH2 domain of mammalian Sin3A with the transrepression domain (SID) of human Mad1 reveals that both domains undergo mutual folding transitions upon complex formation generating an unusual left-handed four-helix bundle structure and an amphipathic alpha helix, respectively. The SID helix is wedged within a deep hydrophobic pocket defined by two PAH2 helices. Structure-function analyses of the Mad-Sin3 complex provide a basis for understanding the underlying mechanism(s) that lead to gene silencing.

Amino Acid Sequence↗
PubMed2000

Beta-catenin-histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator.

Recent evidence suggests that certain LEF/TCF family members act as repressors in the absence of Wnt signaling. We show here that repression by LEF1 requires histone deacetylase (HDAC) activity. Further, LEF1 associates in vivo with HDAC1, and transcription of a model LEF1-dependent target gene is modulated by the ratio of HDAC1 to beta-catenin, implying that repression by LEF1 is mediated by promoter-targeted HDAC. Consistent with this hypothesis, under repression conditions the promoter region of a LEF1 target gene is hypoacetylated. By contrast, when the reporter is activated, its promoter becomes hyperacetylated. Coexpression of beta-catenin with LEF1 and HDAC1 results in the formation of a beta-catenin/HDAC1 complex. Surprisingly, the enzymatic activity of HDAC1 associated with beta-catenin is attenuated. Together, these findings imply that activation of LEF1-dependent genes by beta-catenin involves a two-step mechanism. First, HDAC1 is dissociated from LEF1 and its enzymatic activity is attenuated. This first step yields a promoter that is inactive but poised for activation. Second, once HDAC1-dependent repression has been overridden, beta-catenin binds LEF1 and the beta-catenin-LEF1 complex is competent to activate the expression of downstream target genes.

Cell Line↗
PubMed2000

MondoA, a novel basic helix-loop-helix-leucine zipper transcriptional activator that constitutes a positive branch of a max-like network.

Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix-leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

Amino Acid Sequence↗
PubMed1999

Mlx, a novel Max-like BHLHZip protein that interacts with the Max network of transcription factors.

Mad:Max heterodimers oppose the growth-promoting action of Myc:Max heterodimers by recruiting the mSin3-histone deacetylase (mSin3. HDAC) complex to DNA and functioning as potent transcriptional repressors. There are four known members of the Mad family that are indistinguishable in their abilities to interact with Max, bind DNA, repress transcription, and block Myc + Ras co-transformation. To investigate functional differences between Mad family proteins, we have identified additional proteins that interact with this family. Here we present the identification and characterization of the novel basic-helix-loop-helix zipper protein Mlx (Max-like protein x), which is structurally and functionally related to Max. The similarities between Mlx and Max include 1) broad expression in many tissues, 2) long protein half-life, and 3) formation of heterodimers with Mad family proteins that are capable of specific CACGTG binding. We show that transcriptional repression by Mad1:Mlx heterodimers is dependent on dimerization, DNA binding, and recruitment of the mSin3A.HDAC corepressor complex. In contrast with Max, Mlx interacts only with Mad1 and Mad4. Together, these findings suggest that Mlx may act to diversify Mad family function by its restricted association with a subset of the Mad family of transcriptional repressors.

3T3 Cells↗
PubMed1999

A 13-amino acid amphipathic alpha-helix is required for the functional interaction between the transcriptional repressor Mad1 and mSin3A.

Members of the Mad family of bHLHZip proteins heterodimerize with Max and function to repress the transcriptional and transforming activities of the Myc proto-oncogene. Mad:Max heterodimers repress transcription by recruiting a large multi-protein complex containing the histone deacetylases, HDAC1 and HDAC2, to DNA. The interaction between Mad proteins and HDAC1/2 is mediated by the corepressor mSin3A and requires sequences at the amino terminus of the Mad proteins, termed the SID, for Sin3 interaction domain, and the second of four paired amphipathic alpha-helices (PAH2) in mSin3A. To better understand the requirements for the interaction between the SID and PAH2, we have performed mutagenesis and structural studies on the SID. These studies show that amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction. Further, this minimal 13-residue SID peptide forms an amphipathic alpha-helix in solution, and residues on the hydrophobic face of the SID helix are required for interaction with PAH2. Finally, the minimal SID can function as an autonomous and portable repression domain, demonstrating that it is sufficient to target a functional mSin3A/HDAC corepressor complex.

Amino Acid Sequence↗
PubMed1999

Histone deacetylases: transcriptional repression with SINers and NuRDs.

The DNA in eukaryotic cells is packaged into chromatin, which functions as a boundary to the transcriptional activation process. The nucleosome is the basic repeating unit of chromatin. The purification and characterization of several chromatin-remodelling complexes and the demonstration that histone acetyltransferases and histone deacetylases are regulatory components of coactivator and corepressor complexes, respectively, demonstrates that the nucleosome is not simply a static architectural feature of chromatin but, rather, plays a dynamic and integral role in the regulation of gene expression. This review focuses primarily on histone deacetylases and deacetylase-containing complexes and their role in mediating transcriptional repression.

Cell Nucleus↗
PubMed1998

A role for histone deacetylase activity in HDAC1-mediated transcriptional repression.

Treatment of mammalian cells with small molecule histone deacetylase (HDAC) inhibitors induces changes in the transcription of specific genes. These changes correlate directly with an increase in the acetylation levels of all four core histones in vivo. Antibodies directed against endogenous HDAC1, HDAC2, or HDAC3 immunoprecipitate histone deacetylase activity that is inhibited in vitro by the small molecule trapoxin (TPX), and all three HDACs are retained by a TPX-affinity matrix. HDAC1 and HDAC2 are associated in HeLa cells in a complex that is predominantly separate from an HDAC3 immune complex. Both Jurkat HDAC1 and HeLa HDAC1/2 immune complexes deacetylate all four core histones and recombinant HDAC1 deacetylates free and nucleosomal histones in vitro. Purified recombinant HDAC1 deacetylates core histones in the absence of protein cofactors. Site-directed mutagenesis was used to identify residues required for the enzymatic and structural integrity of HDAC1. Mutation of any one of four conserved residues causes deleterious effects on deacetylase activity and a reduced ability to bind a TPX-affinity matrix. A subset of these mutations also cause a decreased interaction with the HDAC1-associated proteins RbAp48 and mSin3A. Disruption of histone deacetylase activity either by TPX or by direct mutation of a histidine presumed to be in the active site abrogates HDAC1-mediated transcriptional repression of a targeted reporter gene in vivo.

Amino Acid Sequence↗
PubMed1998

SAP30, a component of the mSin3 corepressor complex involved in N-CoR-mediated repression by specific transcription factors.

The transcriptional corepressor mSin3 is found in a large multiprotein complex containing the histone deacetylases HDAC1 and HDAC2, in addition to at least five tightly associated polypeptides. We have cloned and characterized a novel component of the mSin3 complex, SAP30, SAP30 binds to mSin3 and is capable of mediating transcriptional repression via histone deacetylases. SAP30 also binds the N-CoR corepressor and is required for N-CoR-mediated repression by antagonist-bound estrogen receptor and the homeodomain protein Rpx, as well as N-CoR suppression of transactivation by the POU domain protein Pit-1. However, SAP30 is not required for N-CoR-mediated repression by unliganded retinoic acid receptor or thyroid hormone receptor, suggesting that SAP30 is involved in the functional recruitment of the mSin3-histone deacetylase complex to a specific subset of N-CoR corepressor complexes.

Amino Acid Sequence↗
PubMed1997

Histone deacetylase activity is required for full transcriptional repression by mSin3A.

Members of the Mad family of bHLH-Zip proteins heterodimerize with Max to repress transcription in a sequence-specific manner. Transcriptional repression by Mad:Max heterodimers is mediated by ternary complex formation with either of the corepressors mSin3A or mSin3B. We report here that mSin3A is an in vivo component of large, heterogeneous multiprotein complexes and is tightly and specifically associated with at least seven polypeptides. Two of the mSin3A-associated proteins, p50 and p55, are highly related to the histone deacetylase HDAC1. The mSin3A immunocomplexes possess histone deacetylase activity that is sensitive to the specific deacetylase inhibitor trapoxin. mSin3A-targeted repression of a reporter gene is reduced by trapoxin treatment, suggesting that histone deacetylation mediates transcriptional repression through Mad-Max-mSin3A multimeric complexes.

Acetylation↗
PubMed1997

Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase.

The transcriptional corepressors SMRT and N-CoR function as silencing mediators for retinoid and thyroid hormone receptors. Here we show that SMRT and N-CoR directly interact with mSin3A, a corepressor for the Mad-Max heterodimer and a homolog of the yeast global-transcriptional repressor Sin3p. In addition, we demonstrate that the recently characterized histone deacetylase 1 (HDAC1) interacts with Sin3A and SMRT to form a multisubunit repressor complex. Consistent with this model, we find that HDAC inhibitors synergize with retinoic acid to stimulate hormone-responsive genes and differentiation of myeloid leukemia (HL-60) cells. This work establishes a convergence of repression pathways for bHLH-Zip proteins and nuclear receptors and suggests this type of regulation may be more widely conserved than previously suspected.

Antineoplastic Agents↗
PubMed1996

Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism.

Amino Acid Sequence↗
PubMed1996

Neuroprotective efficacy of microvascularly-localized versus brain-penetrating antioxidants.

The 21-aminosteroid (lazaroid) tirilazad mesylate has been demonstrated to be a potent inhibitor of lipid peroxidation and to reduce traumatic and ischemic damage in a number of experimental models. Currently, tirilazad is being actively investigated in phase III clinical trials in head and spinal cord injury, ischemic stroke and subarachnoid hemorrhage. This compound acts in large part to protect the microvascular endothelium and consequently to maintain normal blood-brain barrier (BBB) permeability and cerebral blood flow autoregulatory mechanisms. However, due to its limited penetration into brain parenchyma, tirilazad has generally failed to affect delayed neuronal damage to the selectively vulnerable hippocampal CA1 and striatal regions. Recently, we have discovered a new group of antioxidant compounds, the pyrrolopyrimidines, which possess significantly improved ability to penetrate the BBB and gain direct access to neural tissue. Several compounds in the series, such as U-101033E, have demonstrated greater ability to protect the CA1 region in the gerbil transient forebrain ischemia model with a post-ischemic therapeutic window of at least four hours. In addition, U-101033E has been found to reduce infarct size in the mouse permanent middle cerebral artery occlusion model in contrast to tirilazad which is minimally effective. These results suggest that antioxidant compounds with improved brain parenchymal penetration are better able to limit certain types of ischemic brain damage compared to those which are localized in the cerebral microvasculature. On the other hand, microvascularly-localized agents like tirilazad appear to have better ability to limit BBB damage.

Animals↗
PubMed1996

Mad proteins contain a dominant transcription repression domain.

Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 were also capable of repression. We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators.

Amino Acid Sequence↗
PubMed1996

Inhibition of cell proliferation by the Mad1 transcriptional repressor.

Mad1 is a basic helix-loop-helix-leucine zipper protein that is induced upon differentiation of a number of distinct cell types. Mad1 dimerizes with Max and recognizes the same DNA sequences as do Myc:Max dimers. However, Mad1 and Myc appear to have opposing functions. Myc:Max heterodimers activate transcription while Mad:Max heterodimers repress transcription from the same promoter. In addition Mad1 has been shown to block the oncogenic activity of Myc. Here we show that ectopic expression of Mad1 inhibits the proliferative response of 3T3 cells to signaling through the colony-stimulating factor-1 (CSF-1) receptor. The ability of over-expressed Myc and cyclin D1 to complement the mutant CSF-1 receptor Y809F (containing a Y-to-F mutation at position 809) is also inhibited by Mad1. Cell cycle analysis of proliferating 3T3 cells transfected with Mad1 demonstrates a significant decrease in the fraction of cells in the S and G2/M phases and a concomitant increase in the fraction of G1 phase cells, indicating that Mad1 negatively influences cell cycle progression from the G1 to the S phase. Mutations in Mad1 which inhibit its activity as a transcription repressor also result in loss of Mad1 cell cycle inhibitory activity. Thus, the ability of Mad1 to inhibit cell cycle progression is tightly coupled to its function as a transcriptional repressor.

3T3 Cells↗