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The basic helix-loop-helix (bHLH) family is one of the most conserved transcription factor families that plays an important role in regulating cell growth, differentiation and tissue development. Typically, members of this family form homo- or heterodimers to recognize specific motifs and activate transcription. MyoD is a vital transcription factor that regulates muscle cell differentiation. However, it is necessary for MyoD to form a heterodimer with E-proteins to activate transcription. Even though the crystal structure of the MyoD homodimer has been determined, the structure of the MyoD heterodimer in complex with the E-box protein remains unclear. In this study, we determined the crystal structure of the bHLH domain of the MyoD-E47 heterodimer at 2.05 Å. Our structural analysis revealed that MyoD interacts with E47 through a hydrophobic interface. Moreover, we confirmed that heterodimerization could enhance the binding affinity of MyoD to E-box sequences. Our results provide new structural insights into the heterodimer of MyoD and E-box protein, suggesting the molecular mechanism of transcription activation of MyoD upon binding to E-box protein., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH-PAS) domain family of transcription factors mediates cellular responses to a variety of internal and external stimuli. As functional transcription factors, these proteins act as bHLH-PAS heterodimers and can be further sub-classified into sensory/activated subunits and regulatory or ARNT-like proteins. This class of proteins act as master regulators of the bHLH-PAS superfamily of transcription factors that mediate circadian rhythm gene programs, innate and adaptive immune responses, oxygen-sensing mechanisms and compensate for deleterious environmental exposures. Some contribute to the etiology of human pathologies including cancer because of their effects on cell growth and metabolism. We will review the canonical roles of ARNT and ARNT-like proteins with an emphasis on coactivator selectivity and recruitment. We will also discuss recent advances in our understanding of noncanonical DNA-binding independent or off-target roles of ARNT that are uncoupled from its classic heterodimeric bHLH-PAS binding partners. Understanding the DNA binding-independent functions of ARNT may identify novel therapeutic options for the treatment of a large spectrum of disease states.

Zfp423/OAZ, a multi-zinc finger protein, is proposed to participate in neuronal differentiation through interactions with the Olf/EBF (O/E) family of transcription factors and mediate extrinsic BMP signaling pathways. These activities are associated with distinct domains of the Olf/EBF-associated zinc finger (OAZ) protein. Sustained OAZ expression arrests olfactory sensory neurons (OSNs) at an immature state and alters olfactory receptor expression, but the mechanism remains elusive. We show here that constitutive expression of a C-terminal mutant OAZ (OAZΔC) in mice that selectively disrupts OAZ-O/E interaction while retaining other activities, exhibits apparently normal OSN differentiation. Additionally, interfering with potential BMP signaling pathways by inducible Follistatin expression in adult mice does not alter the neuronal lineage or differentiation status. Our results indicate that O/E-mediated processes are essential for the differentiation of OSNs and the establishment of a mature phenotype. BMP signaling pathways, if they are active in normal adult olfactory epithelium, may play a minor role in this tissue.

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nanomolar K(d) values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14, 15, 9, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site.

Efg1p is a key transcriptional regulator in Candida albicans which controls various aspects of morphogenesis and metabolism in this organism. Efg1p contains a central basic helix-loop-helix (bHLH) domain, flanked by sequences highly conserved in fungal APSES proteins, as well as polyglutamine stretches at the N- and C-terminal ends. A systematic deletion approach to specify functional domains of Efg1p revealed that the APSES domain is essential for morphogenesis of the normal yeast and true hyphal cell forms and that bHLH flanking sequences are needed for Efg1p stability. Additional C-terminal sequences were required for hyphal formation on some inducing media, and most Efg1p sequences were needed for chlamydospore morphogenesis. Overexpression of EFG1 led to pseudohypha formation only if a functional APSES domain was present, while a switch from the opaque to the white cell type in addition depended on the presence of certain N- and C-terminal segments. Yeast two-hybrid experiments revealed that binding of Efg1p to its antagonist Czf1p required two regions outside of the APSES domain, which did not coincide with Efg1p sequences needed for its transcriptional repressor activity. Binding of the Flo8 transcription factor to Efg1p did not require the APSES domain but appeared to occur at two or more redundant domains. In contrast, DNA binding of Efg1p to an MluI cell cycle box (MCB) element solely required the APSES domain. Overall, these results suggest that functional domains of Efg1p are spread throughout most of its sequences, including the central APSES domain involved in DNA binding, as well as flanking regions required for various protein interactions and regulatory activities.

The eight twenty-one protein, ETO, is implicated in 12%-15% of acute human leukemias as part of a gene fusion with RUNX1 (also called AML1). Of the four ETO domains related to Drosophila melanogaster Nervy, only two are required to induce spontaneous myeloid leukemia upon transplantation into the mouse. One of these domains is related in sequence to TAF4, a component of TFIID. The structure of this domain, ETO-TAFH, is similar to yeast Rpb4 and to Escherichia coli sigma(70); it is the first TAF-related protein with structural similarity to the multisubunit RNA polymerases. Overlapping surfaces of ETO-TAFH interact with an autonomous repression domain of the nuclear receptor corepressor N-CoR and with a conserved activation domain from the E-box family of transcription factors. Thus, ETO-TAFH acts as a structural platform that can interchange negative and positive coregulatory proteins to control transcription.

The response regulator VicR from the Gram-positive bacterium Enterococcus faecalis forms part of the two-component signal transduction system of the YycFG subfamily. The structure of the DNA-binding domain of VicR, VicR(c), has been solved and belongs to the winged helix-turn-helix family. It is very similar to the DNA-binding domains of Escherichia coli PhoB and OmpR, despite low sequence similarity, but differs in two important loops. The alpha-loop, which links the two helices of the helix-turn-helix motif, is similar to that of PhoB, where it has been implicated in contacting the sigma subunit of RNA polymerase, but differs from that of OmpR. Conversely, the loop following the helix-turn-helix motif is similar to that of OmpR and differs from that of PhoB. YycF/VicR, PhoB and Bacillus subtilis PhoP regulators all recognize almost identical DNA sequences and although there is currently no experimental evidence linking this loop with the DNA, the structure is consistent with possible involvement in selective DNA recognition or binding.

The human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair.

Helix-loop-helix (HLH) proteins are transcriptional regulators that control a wide variety of developmental pathways in both invertebrate and vertebrate organisms. Results obtained in the past decade have shown that HLH proteins also contribute to the development of lymphoid lineages. A subset of HLH proteins, the 'E proteins', seems to be particularly important for proper lymphoid development. Members of the E protein family include E12, E47, E2-2 and HEB. The E proteins contribute to B lineage- and T lineage-specific gene expression programs, regulate lymphocyte survival and cellular proliferation, activate the rearrangement of antigen receptor genes and control progression through critical developmental checkpoints. This review discusses HLH proteins in lymphocyte development and homeostasis.

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM.

The ability of barrier-to-autointegration factor (BAF) to bind and bridge DNA in a sequence-independent manner is crucial for its role in retroviral integration and a variety of cellular processes. To better understand this behavior, we solved the crystal structure of BAF bound to DNA. The structure reveals that BAF bridges DNA using two pairs of helix-hairpin-helix motifs located on opposite surfaces of the BAF dimer without changing its conformation.

Upstream stimulating factors (USF), USF-1 and USF-2, are members of the eucaryotic evolutionary conserved basic-Helix-Loop-Helix-Leucine Zipper transcription factor family. They interact with high affinity to cognate E-box regulatory elements (CANNTG), which are largely represented across the whole genome in eucaryotes. The ubiquitously expressed USF-transcription factors participate in distinct transcriptional processes, mediating recruitment of chromatin remodelling enzymes and interacting with co-activators and members of the transcription pre-initiation complex. Results obtained from both cell lines and knock-out mice indicates that USF factors are key regulators of a wide number of gene regulation networks, including the stress and immune responses, cell cycle and proliferation, lipid and glucid metabolism, and in melanocytes USF-1 has been implicated as a key UV-activated regulator of genes associated with pigmentation. This review will focus on general characteristics of the USF-transcription factors and their place in some regulatory networks.

Circadian rhythms result from feedback loops involving clock genes and their protein products. In mammals, 2 orphan nuclear receptors, REV-ERBalpha and RORalpha, play important roles in the transcription of the clock gene Bmal1. The authors now considerably extend these findings with the demonstration that all members of the REV-ERB (alpha and beta) and ROR (alpha, beta, and gamma) families repress and activate Bmal1 transcription, respectively. The authors further show that transcription of Bmal1 is the result of competition between REV-ERBs and RORs at their specific response elements (RORE). Moreover, they demonstrate that Reverb genes are similarly expressed in the thymus, skeletal muscle, and kidney, whereas Ror genes present distinct expression patterns. Thus, the results indicate that all members of the REV-ERB and ROR families are crucial components of the molecular circadian clock. Furthermore, their strikingly different patterns of expression in nervous and peripheral tissues provide important insights into functional differences between circadian clocks within the organism.

HAND proteins are tissue-restricted members of the basic helix-loop-helix transcription factor family that play critical roles in cell differentiation and organogenesis including placental, cardiovascular, and craniofacial development. Nevertheless, the molecular basis underlying the developmental action of HAND proteins remains undefined. Within the embryo, HAND1 is first detected in the developing heart where it becomes restricted to the atrial and left ventricular compartments, a pattern identical to that of the Nppa gene, which encodes atrial natriuretic factor, the major secretory product of the heart. We hereby report that the cardiac atrial natriuretic factor promoter is directly activated by HAND1, making it the first known HAND1 transcriptional target. The action of HAND1 does not require heterodimerization with class I basic helix-loop-helix factors or DNA binding through E-box elements. Instead, HAND1 is recruited to the promoter via physical interaction with MEF2 proteins. MEF2/HAND1 interaction results in synergistic activation of MEF2-dependent promoters, and MEF2 binding sites are sufficient to mediate this synergy. MEF2 binding to DNA is not enhanced in the presence of HAND1. Instead, cooperativity likely results from corecruitment of co-activators such as CREB-binding protein. The related HAND2 protein can also synergize with MEF2. Thus, HAND proteins act as cell-specific developmental co-activators of the MEF2 family of transcription factors. These findings identify a novel mechanism for HAND action in the heart and provide a general paradigm to understand the mechanism of HAND action in organogenesis.

Interaction with Max via the helix-loop-helix/leucine zipper (HLH-LZ) domain is essential for Myc to function as a transcription factor. Myc is commonly upregulated in tumours, however, its activity can also be potentiated by virally derived mutations. vMyc, derived from the virus, MC29 gag-Myc, differs from its cellular counterpart by five amino acids. The N-terminal mutation stabilizes the protein, however, the significance of the other mutations is not known. We now show that vMyc can sustain longer deletions in the LZ domain than cMyc before complete loss in transforming activity, implicating the viral mutations in contributing to Myc:Max complex formation. We confirmed this both in vitro and in vivo, with loss of Max binding correlating with a loss in the biological activity of Myc. A specific viral mutation, isoleucine383>leucine (I383>L) in helix 2 of the HLH domain, extends the LZ domain from four to five heptad repeats. Significantly, introduction of I383>L into a Myc mutant that is defective for Max binding substantially restored its ability to complex with Max in vitro and in vivo. We therefore propose that this virally derived mutation is functional by significantly contributing to establishing a more hydrophobic interface between the LZs of Myc and Max.

Vertebrate and invertebrate nervous tissue is derived from early embryonic ectoderm, which also gives rise to epidermal derivatives such as skin. Proneural basic helix-loop-helix (bHLH) transcription factors are the key players in the formation of peripheral nervous system (PNS) and central nervous system (CNS) from naïve ectoderm to differentiated postmitotic neurons. The comparative approach and the use of a wide range of animal models have led to increasingly comprehensive investigations of this issue in the last decade. This review will focus on current studies of neural development in vertebrate and invertebrate PNS and on understanding how the bHLH domain structure encodes multiple functions required for neural specification.

Two major families of transcription factors (TFs), basic helix-loop-helix (bHLH) and homeodomain (HD), are known to be involved in cell fate identity. Some recent findings suggest that these TFs are used combinatorially to code for cellular determination in the retina. However, neither the extent nor the efficiency of such a combinatorial coding mechanism has been tested. To look systematically for interactions between these two TF types that would address these questions, we used a matrix analysis. We co-expressed each of six retinally expressed bHLH TFs (XNeuroD; XNgnr-1; Xath3; Xath5; Xash1; Xash3) with each of eight retinally expressed HD TFs (XRx1; XOptx2; XSix3; XPax6; XOtx2; XOtx5b; XBH; XChx10) in retinal progenitors of Xenopus laevis using targeted lipofection. The effects of each of these combinations were assayed on the six major cell types in the retina: Retinal ganglion cells (GCs), Amacrines (ACs), Bipolars (BCs), Horizontals (HCs), Photoreceptors (PRs), and Muller cells (MCs), creating 288 result categories. Multiple-way ANOVA indicated that in 14 categories, there were interactions between the two TFs that produced significantly more or less of a particular cell type than either of the components alone. However, even the most effective combinations were incapable of generating more than 65% of any particular cell type. We therefore used the same techniques to misexpress selected combinations of three TFs in retinal progenitors, but found no further enhancements of particular cell fates, indicating that other factors are probably involved in cell type specification. To test whether particular combinations were essential for horizontal fates, we made VP16 and EnR fusion constructs of some of the factors to provide dominant negative transcriptional activities. Our results confirmed that normal activities of certain combinations were sufficient, and that individually these activities were important for this fate.

The existence of hemangioblasts, which serve as common progenitors for hematopoietic cells and cardioblasts, has suggested a molecular link between cardiogenesis and hematopoiesis in Drosophila. However, the molecular mediators that might link hematopoiesis and cardiogenesis remain unknown. Here, we show that the highly conserved basic helix-loop-helix (bHLH) transcription factor Hand is expressed in cardioblasts, pericardial nephrocytes and hematopoietic progenitors. The homeodomain protein Tinman and the GATA factors Pannier and Serpent directly activate Hand in these cell types through a minimal enhancer, which is necessary and sufficient to drive Hand expression in these different cell types. Hand is activated by Tinman and Pannier in cardioblasts and pericardial nephrocytes, and by Serpent in hematopoietic progenitors in the lymph gland. These findings place Hand at a nexus of the transcriptional networks that govern cardiogenesis and hematopoiesis, and indicate that the transcriptional pathways involved in development of the cardiovascular, excretory and hematopoietic systems may be more closely related than previously appreciated.

Hypoxia inducible factor-1 (HIF-1) is as a key transcriptional mediator of the hypoxic response in eukaryotic cells, regulating the expression of a myriad of genes involved in oxygen transport, glucose uptake and glycolysis and angiogenesis. Deregulation of HIF-1 activity occurs in many human cancers, usually at the level of the HIF-1alpha subunit. HIF-1 is regulated by a variety of mechanisms including transcription, translation post-translational modification, protein-protein interaction and degradation. Our understanding of the key signalling pathways that regulate HIF-1 has significantly progressed in recent years and has highlighted the potential for targeting the HIF-1 pathway as a basis for the development of new cancer therapies.

Cell growth and differentiation require precise coordination of cell cycle and differentiation proteins. This can be achieved by direct interactions between proteins, by indirect interaction in multiprotein complexes, or by modulation of gene expression levels of partner proteins. Contradictory data abound in the literature regarding the binding between some central cell cycle proteins, pRb, and CDK6, with myogenic differentiation promoting, MyoD, and inhibiting, Id-2, factors. We have tested these interactions using pure proteins and in vitro biophysical and biochemical methods, which included mass spectrometry, nuclear magnetic resonance (NMR), the affinity chromatography pull-down assays, and gel filtration chromatography. Using this multimethod approach, we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, CDK6 and p19, and MyoD and DNA. Using the same methods, we could unambiguously show that there is no direct protein-protein interaction in vitro between the small pocket domain of pRb and the bHLH domain of MyoD, the small pocket domain of pRb and Id-2, and CDK6 and a 15-amino-acid peptide from the C-terminal domain of MyoD. Indirect interactions, through additional binding partners in multiprotein complexes or modulation of gene expression levels of these proteins, are therefore their probable mode of action.

Butyrate, a short-chain fatty acid, is the major energy fuel for the colonocytes. We have previously reported that monocarboxylate transporter isoform 1 (MCT1) mediates uptake of butyrate by human colonic Caco-2 cells. To better understand the mechanisms of MCT1 expression and regulation in the human intestine, we examined the activity and regulation of MCT1 promoter in Caco-2 cells. The transcription initiation site in the MCT1 promoter was identified as a guanine nucleotide 281 bp upstream from the translation initiation site and is surrounded by a guanine-cytosine-rich area. The promoter was found to be highly active when transfected into Caco-2 cells, and its activity decreased with deletions at its 5'-end. Gel mobility shift experiments showed binding of the transcription factors upstream stimulatory factor (USF)1 and 2 to the site -114 to -119 of the MCT1 promoter. With the use of site-directed mutagenesis and promoter activity in Caco-2 cells, the USF proteins appeared to have a repressor role on the MCT1 promoter, which was further confirmed by cotransfecting expression vectors encoding USF1 and 2 in Caco-2 cells and determining endogenous MCT1 expression in USF2 overexpressed cells. The two potential SP1 binding sites found in the same region of the promoter were found not to be involved in its regulation.

Nuclear stabilization of beta-catenin and its interaction with TCF/LEF factors are key events in transduction of the Wnt/beta-catenin signal pathway. Our previous study indicated that nuclear beta-catenin accumulation provides an initial signal for trans-differentiation toward the squamoid phenotype of endometrial carcinoma (Em Ca) cells in a TCF4-dependent manner, which makes this a possible factor for a positive prognosis. However, little is known about regulation of TCF4 expression in Em Cas. We show here that beta-catenin can directly induce transcription from the TCF4 promoter, the effect being enhanced by the p300 coactivator. In clinical cases, nuclear beta-catenin accumulation was found to frequently overlap with TCF4 immunoreactivity in morules and surrounding glandular carcinoma lesions, showing a significant positive correlation (r = 0.82, P < 0.0001), in contrast to areas of squamous metaplasia (SqM) within Em Cas. In cases with coexistence of two squamoid features in trans-differentiated areas, loss of nuclear beta-catenin and TCF4 immunoreactivity was closely related to change in the morphology from the morular to the SqM phenotype. The TCF4 promoter contains a single consensus TCF-binding site that is critical for activation by beta-catenin. The p300 coactivator, in particular N-terminal residues 1 to 670, appears sufficient to enhance beta-catenin-dependent transcription, again with TCF4-dependence. These findings indicate that a positive feedback loop of TCF4 expression mediated by beta-catenin/p300 may be important for initial steps during trans-differentiation of Em Ca cells. In addition, its downregulation is associated with induction of a more-differentiated squamoid phenotype.

The jasmonic acid (JA)-responsive gene RERJ1 isolated from suspension-cultured rice cells encodes a transcription factor with a basic helix-loop-helix motif. In this study, we found that RERJ1 is also expressed in rice plants in response to JA, and that its expression in rice leaves is up-regulated by exposure to wounding and drought stress. It is also suggested that JA but not abscisic acid is involved in the up-regulation of RERJ1 expression caused by wounding and drought stress.

Basic-helix-loop-helix-PAS transcription factors play important roles in diverse biological processes including cellular differentiation and specification, oxygen tension regulation and dioxin metabolism. Drosophila tango is orthologous to mammalian Arnt and acts as a common dimerization partner for bHLH-PAS proteins during embryogenesis. A transient transfection assay using Drosophila S2 tissue culture cells and wild-type and mutant Drosophila tango cDNAs was used to localize the activation domain of the Tango protein. An activation domain was identified in the C-terminus of TGO consisting of poly-glutamine and histidine-proline repeats. Transcriptional activation of the fibroblast growth factor receptor (breathless) gene required an intact TGO C-terminus, in vitro. Co-expression assays of trachealess and tgo in the developing eye imaginal disc showed a requirement for the C-terminal transactivation domain of TGO for a cellular response. Genetic analysis of tgo(3) shows that the paired repeat is necessary for tracheal tubule formation in all branches. Lastly, expression of a C-terminal truncated tgo transgene specifically in the CNS midline and trachea resulted in reductions in the number of breathless-expressing cells. These results together identify TGO's transactivation domain and establish its importance for proper target gene regulation and cellular specification.

Prior to puberty the Sertoli cells undergo active cell proliferation, and at the onset of puberty they become a terminally differentiated postmitotic cell population that support spermatogenesis. The molecular mechanisms involved in the postmitotic block of pubertal and adult Sertoli cells are unknown. The four known helix-loop-helix ID proteins (i.e., Id1, Id2, Id3, and Id4) are considered dominant negative regulators of cellular differentiation pathways and act as positive regulators of cellular proliferation. ID proteins are expressed at low levels by postpubertal Sertoli cells and are transiently induced by serum. The hypothesis tested was that ID proteins can induce a terminally differentiated postmitotic Sertoli cell to reenter the cell cycle if they are constitutively expressed. To test this hypothesis, ID1 and ID2 were stably integrated and individually overexpressed in postmitotic rat Sertoli cells. Overexpression of ID1 or ID2 allowed postmitotic Sertoli cells to reenter the cell cycle and undergo mitosis. The cells continued to proliferate even after 300 cell doublings. The functional markers of Sertoli cell differentiation such as transferrin, inhibin alpha, Sert1, and androgen binding protein (ABP) continued to be expressed by the proliferating Sertoli cells, but at lower levels. FSH receptor expression was lost in the proliferating Sertoli cell-Id lines. Some Sertoli cell genes, such as cyclic protein 2 (cathepsin L) and Sry-related HMG box protein-11 (Sox11) increase in expression. At no stage of proliferation did the cells exhibit senescence. The expression profile as determined with a microarray protocol of the Sertoli cell-Id lines suggested an overall increase in cell cycle genes and a decrease in growth inhibitory genes. These results demonstrate that overexpression of ID1 and ID2 genes in a postmitotic, terminally differentiated cell type have the capacity to induce reentry into the cell cycle. The observations are discussed in regards to potential future applications in model systems of terminally differentiated cell types such as neurons or myocytes.

Mxi1 protein is a basic helix-loop-helix, leucine zipper (bHLHZIP) transcriptional factor, which dimerizes with the Max protein. This heterodimer binds a specific DNA sequence (E-box) and suppresses transcription of target genes. On the other hand, c-Myc protein is also a bHLHZIP protein that dimerizes with Max, binds the identical E-box sequence but activates transcription of the target genes. We report that hematopoietic cells have three novel Mxi1 transcripts: Mxi-D lacks exon 3, which encodes the basic region; Mxi-ND lacks the N-terminal mSin3 binding region and the DNA binding region; and Mxi-NF lacks the N-terminal mSin3 binding region. In normal bone marrow and hematological cell lines, the dominantly expressed isoforms are Mxi-D and full-length Mxi1 (Mxi-F). In GST-pull down assays, the Mxi-D protein binds the Max protein and the PHA2 regions of mSin3 proteins. Whereas the Mxi-F/Max heterodimer binds the E-box sequence, Mxi-D/Max heterodimer can not bind this sequence in electrophoretic mobility shift assays. In reporter gene assay, Mxi-D suppresses transcription as strongly as Mxi-F. In colony formation assay using Rat1 fibroblast cells, Mxi-D cannot suppress clonal growth stimulated by c-Myc as strongly as Mxi-F. In summary, Mxi-D may play an important role in the c-Myc family protein network acting as a dominant negative isoform of Mxi-F since: i) Mxi-D can dimerize with Max in vitro; ii) Mxi-D/Max heterodimers cannot bind E-box in vitro, iii) Mxi-D cannot suppress clonal growth stimulated by c-Myc.

Changes in O(2) tension can significantly impact cell survival, yet the mechanisms underlying these effects are not well understood. Here, we report that maintaining sympathetic neurons under low O(2) inhibits apoptosis caused by NGF deprivation. Low O(2) exposure blocked cytochrome c release after NGF withdrawal, in part by suppressing the up-regulation of BIM(EL). Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2). Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells. Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death. These data suggest a new model for how O(2) tension can influence apoptotic events that underlie trophic factor deprivation-induced cell death.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays an important role in tumor growth and metastasis by regulating energy metabolism and inducing angiogenesis. Elevated levels of HIF-1alpha, a subunit of HIF-1, are noted in various malignant tumors, but it is unclear whether this is so in esophageal carcinoma. The purpose of this study was to evaluate the implications of HIF-1alpha expression in esophageal squamous cell carcinoma. In 215 patients with esophageal carcinoma, we examined immunoreactivity for HIF-1alpha protein, vascular endothelial growth factor (VEGF) protein and p53 protein. In 38 patients, we examined the expression of HIF-1alpha messenger ribonucleic acid (mRNA) (using the semiquantitative reverse transcriptase-polymerase chain reaction [RT-PCR]). A positive HIF-1alpha protein expression was recognized in 95% of the patients, and was strongly apparent within both the nuclei and/or cytoplasm of tumor cells. The proportion of patients in the 'high score' group for HIF-1alpha protein expression increased significantly with increasing VEGF protein expression. Immunoreactivity for HIF-1alpha protein was found to have a significant effect on disease-free survival rate in our univariate analysis, but no effect on overall survival rate. In RT-PCR, HIF-1alpha mRNA scores correlated significantly with scores for HIF-1alpha protein expression, but not with any clinicopathologic factor or either of the survival rates. The detection of HIF-1alpha protein and mRNA would appear to offer limited information as to progression and prognosis in esophageal carcinoma.

In the mammalian auditory system, sensory cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. To restore hearing, it is necessary to generate new functional hair cells. One potential way to regenerate hair cells is to induce a phenotypic transdifferentiation of nonsensory cells that remain in the deaf cochlea. Here we report that Atoh1, a gene also known as Math1 encoding a basic helix-loop-helix transcription factor and key regulator of hair cell development, induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells through adenovectors. This is the first demonstration of cellular and functional repair in the organ of Corti of a mature deaf mammal. The data suggest a new therapeutic approach based on expressing crucial developmental genes for cellular and functional restoration in the damaged auditory epithelium and other sensory systems.

Id proteins function as negative regulators for basic helix-loop-helix transcriptional factors that play important roles in cell fate determination. They preferentially associate with ubiquitously expressed E proteins of the basic helix-loop-helix family and prevent them from binding to DNA and activating transcription. Although their small size suggests that Id proteins enter and exit the nucleus by passive diffusion, several studies have indicated that other pathways may regulate their subcellular localization. In this study, we obtained evidence that Id2 has the ability to shuttle between the nucleus and the cytoplasm. When passive diffusion was prevented by fusion with green fluorescent protein (GFP), Id2 was predominantly localized in the cytoplasm. Using GFP fusion constructs, we demonstrated that the C-terminal region is required for cytoplasmic localization. Nuclear accumulation of GFP-Id2 in cells treated with the nuclear export inhibitor leptomycin B suggests that the nuclear export receptor chromosome region maintenance protein 1 mediates the cytoplasmic localization of Id2. Id2 contains two putative leucine-rich nuclear export signals, and the nuclear export signal in the C-terminal region is essential for nuclear export. On the other hand, the helix-loop-helix domain is important for nuclear localization. Finally, experiments using reporter assays revealed an inverse correlation between nuclear export and transcriptional repression via the E-box sequence. Based on all these findings, we propose that nucleo-cytoplasmic shuttling is a novel mechanism for the regulation of Id2 function.

Myelin-forming oligodendrocytes facilitate saltatory nerve conduction and support neuronal functions in the mammalian CNS. Although the processes of oligodendrogliogenesis and differentiation from neural progenitor cells have come to light in recent years, the molecular mechanisms underlying oligodendrocyte myelinogenesis are poorly defined. Herein, we demonstrate the pivotal role of the basic helix-loop-helix transcription factor, Olig1, in oligodendrocyte myelinogenesis in brain development. Mice lacking a functional Olig1 gene develop severe neurological deficits and die in the third postnatal week. In the brains of these mice, expression of myelin-specific genes is abolished, whereas the formation of oligodendrocyte progenitors is not affected. Furthermore, multilamellar wrapping of myelin membranes around axons does not occur, despite recognition and contact of axons by oligodendrocytes, and Olig1-null mice develop widespread progressive axonal degeneration and gliosis. In contrast, myelin sheaths are formed in the spinal cord, although the extent of myelination is severely reduced. At the molecular level, we find that Olig1 regulates transcription of the major myelin-specific genes, Mbp, Plp1, and Mag, and suppresses expression of a major astrocyte-specific gene, Gfap. Together, our data indicate that Olig1 is a central regulator of oligodendrocyte myelinogenesis in brain and that axonal recognition and myelination by oligodendrocytes are separable processes.

Oxygen is the ultimate source of oxidizing power for disulfide bond formation, suggesting that under limiting oxygen proper protein folding might be compromised. We show that secretion of vascular endothelial growth factor (VEGF), a protein with multiple disulfide bonds, was indeed impeded under hypoxia and was partially restored by artificial increase of oxidizing equivalents with diamide. Physiologically, the oxireductase endoplasmic reticulum oxidoreductin-1 (Ero1)-L alpha, but not other proteins in the relay of disulfide formation, was strongly upregulated by hypoxia and independently by hypoglycemia, two known accompaniments of tumors. Further, we provide genetic evidence that induction of Ero1-L alpha by hypoxia and hypoglycemia is mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1) but is independent of p53. In natural human tumors, Ero1-L alpha mRNA was specifically induced in hypoxic microenvironments coinciding with that of upregulated VEGF expression. To establish a physiological relevance to modulations in Ero1-L alpha levels, we showed that even a modest, two- to three-fold reduction in Ero1-L alpha production via siRNA leads to significant inhibition of VEGF secretion, a compromised proliferation capacity and enhanced apoptosis. Together, these findings demonstrate that hypoxic induction of Ero1-L alpha is the key adaptive response in a previously unrecognized HIF-1-mediated pathway that operates to improve protein secretion under hypoxia and might be harnessed for inhibiting tumor growth via inhibiting VEGF-driven angiogenesis.

Wnt glycoproteins regulate homeostasis and development by binding to membrane Frizzled-LRP5/6 receptor complexes. Wnt signaling includes a canonical pathway involving cytosolic beta-catenin stabilization, nuclear translocation and gene regulation, acting as a co-activator of T-cell factor (TCF) proteins, and noncanonical pathways that activate Rho, Rac, JNK and PKC, or modulate Ca(2+) levels. DICKKOPF-1 (DKK-1) encodes a secreted Wnt antagonist that binds to LRP5/6 and induces its endocytosis, leading to inhibition of the canonical pathway. We show that activation of canonical signaling by Wnt1 or ectopic expression of active beta-catenin, TCF4 or LRP6 mutants induces transcription of the human DKK-1 gene. Multiple beta-catenin/TCF4 sites in the DKK-1 gene promoter contribute to this activation. In contrast, Wnt5a, which signals through noncanonical pathways, does not activate DKK-1. Northern and Western blot studies show that activation of the Wnt/beta-catenin pathway by treatment with lithium or Wnt3a-conditioned medium, or by stable expression of either Wnt1 or beta-catenin, increases DKK-1 RNA and protein, thus initiating a negative feedback loop. However, we found that DKK-1 expression decreases in human colon tumors, which suggests that DKK-1 acts as a tumor suppressor gene in this neoplasia. Our data indicate that the Wnt/beta-catenin pathway is downregulated by the induction of DKK-1 expression, a mechanism that is lost in colon cancer.

Influx of excess fatty acids and the resultant accumulation of intracellular triglycerides are linked to impaired insulin secretion and action in the pathogenesis of type 2 diabetes. Sterol regulatory element-binding protein (SREBP)-1c is a transcription factor that controls cellular synthesis of fatty acids and triglycerides. SREBP-1c is highly expressed in high-energy and insulin-resistant states. To investigate effects of this synthetic lipid regulator on insulin secretion, we generated transgenic mice overexpressing nuclear SREBP-1c under the insulin promoter. beta-Cell-specific expression of SREBP-1c caused reduction in islet mass and impaired glucose-stimulated insulin secretion and was associated with accumulation of triglycerides, suppression of pancreas duodenal homeobox-1, and upregulation of uncoupling protein 2 gene expression. The mice presented with impaired glucose tolerance that was exacerbated by a high-energy diet. Taken together with enhanced insulin secretion from SREBP-1-null islets, these data suggest that SREBP-1c and endogenous lipogenesis could be involved in beta-cell dysfunction and diabetes.

Neurogenesis in all animals is triggered by the activity of a group of basic helix-loop-helix transcription factors, the proneural proteins, whose expression endows ectodermal regions with neural potential. The eventual commitment to a neural precursor fate involves the interplay of these proneural transcriptional activators with a number of other transcription factors that fine tune transcriptional responses at target genes. Most prominent among the factors antagonizing proneural protein activity are the HES basic helix-loop-helix proteins. We have previously shown that two HES proteins of Drosophila, E(spl)mgamma and E(spl)m7, interact with the proneural protein Sc and thereby get recruited onto Sc target genes to repress transcription. Using in vivo and in vitro assays we have now discovered an important dual role for the Sc C-terminal domain. On one hand it acts as a transcription activation domain, and on the other it is used to recruit E(spl) proteins. In vivo, the Sc C-terminal domain is required for E(spl) recruitment in an enhancer context-dependent fashion, suggesting that in some enhancers alternative interaction surfaces can be used to recruit E(spl) proteins.

Hyperthermia is an important component of many cancer treatment protocols. In our study the regulation of the multidrug resistance (MDR) transporter P-glycoprotein by hyperthermia was studied in multicellular prostate tumor spheroids. Hyperthermia treatment of small (50-100 microm) tumor spheroids significantly increased P-glycoprotein and mdr-1 mRNA expression with a maximum effect at 42 degrees C, whereas only moderate elevation of P-glycoprotein was found in large (350-450 microm) tumor spheroids. Hyperthermia caused an elevation of intracellular reactive oxygen species (ROS). Inhibition of ROS generation with NADPH-oxidase inhibitors diphenylen iodonium (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) abolished P-glycoprotein expression but did not affect its transcript levels following heat treatment. This indicates that P-glycoprotein levels are controlled by regulating its translation rate or stability. Hyperthermia incubation resulted in a differential activation of p38 mitogen-activated protein kinase (MAPK), extracellular regulated kinase 1,2 (ERK1,2), and c-jun N-terminal kinase (JNK) immediately, 4 hr and 24 hr after treatment. Furthermore, upregulation of hypoxia-inducible factor 1alpha (HIF-1alpha) was observed. Elevation of HIF-1alpha and P-glycoprotein expression following hyperthermia treatment were abolished upon coadministration of the p38 inhibitor SB203580. In contrast the JNK inhibitor SP600125 and the ERK1,2 inhibitor UO126 resulted in increase of HIF-1alpha and P-glycoprotein in the control as well as the hyperthermia-treated samples, indicating negative regulation of intrinsic HIF-1alpha and P-glycoprotein expression by ERK1,2 and JNK signaling cascades. In summary our data demonstrate that hyperthermia-induced upregulation of P-glycoprotein and HIF-1alpha is mediated by activation of p38, whereas ERK1,2 and JNK are involved in repression of P-glycoprotein and HIF-1alpha under control conditions.

Background: Neuroblastoma is derived from cells of neural crest origin and often expresses the transcription factor human achaete-scute homolog 1 (HASH1). The aim of this study was to selectively kill neuroblastoma cells by expressing the suicide gene E. coli purine nucleoside phosphorylase (PNP) under the control of the Mash1 promoter, the murine homolog of HASH1., Procedure: The E. coli PNP gene regulated by the Mash1 promoter was cloned into an expression vector and transfected into neuroblastoma and non-neuroblastoma cell lines. After addition of the prodrug M2-fluoroadenine 9-beta-D-arabinofuranoside (F-araA) the cell-specific toxicity was examined. To optimize the cell specific activity, different sizes of the Mash1 promoter were analyzed in neuroblastoma cell lines and compared with the activity in non-neuroblastoma cells., Results: Estimated as the percentages of CMV enhancer-promoter, the activity was significantly higher in the neuroblastoma cells, ranging from 17 to 58% when the shortest and the most active promoter was measured. The non-neuroblastoma cells yielded only 1-6% of the CMV promoter activity. When the shortest Mash1 promoter was combined with the E. coli PNP gene the cytotoxicity was 65% in the neuroblastoma cells with low cell death in the non-neuroblastoma cell lines, relative to the cytotoxicity where the E.coli PNP gene was regulated by the strong but non-specific CMV enhancer-promoter., Conclusions: We show here that the Mash1 promoter regulating the PNP gene confers a cell-type selective toxicity in neuroblastoma cell lines. These results indicate the feasibility to use the Mash1 promoter for regulating E.coli PNP expression in gene-directed enzyme prodrug therapy (GDEPT) of neuroblastoma., ((c) 2004 Wiley-Liss, Inc.)

NeuroD1, a member of the basic helix-loop-helix (bHLH) protein family, is a transcription factor that plays a pivotal role in terminal differentiation of neural progenitors. The primary objective was to generate an early transcriptional profile triggered by NeuroD1 to guide future studies on mechanisms of neuronal differentiation. The human NeuroD1 coding region was amplified from human fetal brain RNA using specific primers and cloned into a CMV expression vector (CT-GFP-TOPO/pcDNA3.1). Transfection of a fetal glial cell line with this construct resulted in expression of NeuroD1 in 13-15% of the cells. Markers typical of early neuronal development were observed by immunocytochemical staining in a small proportion of transfected cells. To enrich the population of NeuroD1-expressing cells, fluorescence-activated cell sorting (FACS) was used to purify and collect the NeuroD1/GFP+ cells. Total RNA was extracted from the pair of cultures (NeuroD1/GFP vs. control plasmid/GFP) and processed for gene expression studies. A final gene list was composed from those probe sets that were either increased or decreased in the NeuroD1-expressing cells in three independent experiments (p < 0.001). Each gene was investigated further for possible roles in neurogenesis and a subset of 177 genes was chosen based on the following characteristics: a) genes that are potential NeuroD1 dimerization partners, b) genes that modulate other bHLH transcription factors, c) genes related to development, and d) genes associated with neural induction, outgrowth, and terminal differentiation. DNA microarray analysis of NeuroD1 expression in an astroglial cell line produced a "snapshot" transcriptional profile that will be useful in deciphering the complex molecular code that specifies a neuronal fate.

Combinatorial actions of transcription factors in multiprotein complexes dictate gene expression profiles in cardiac development and disease. The Hairy-related transcription factor (HRT) family of basic helix-loop-helix proteins is composed of transcriptional repressors highly expressed in the cardiovascular system. However, it has remained unclear whether HRT proteins modulate gene expression driven by cardiac transcriptional activators. Here, we have shown that HRT proteins inhibit cardiac gene transcription by interfering with GATA transcription factors that are implicated in cardiac development and hypertrophy. HRT proteins inhibited GATA-dependent transcriptional activation of cardiac gene promoters such as the atrial natriuretic factor (ANF) promoter. Adenovirus-mediated expression of Hrt2 suppressed mRNA expression of ANF and other cardiac-specific genes in cultured cardiomyocytes. Among various signaling molecules implicated in cardiomyocyte growth, constitutively active Akt1/protein kinase B alpha relieved Hrt2-mediated inhibition of GATA-dependent transcription. HRT proteins physically interacted with GATA proteins, and the basic domain of HRT was critical for physical association as well as transcriptional inhibition. These results suggest that HRT proteins may regulate specific sets of cardiac genes by modulating the function of GATA proteins and other cardiac transcriptional activators in a signal-dependent manner.

An intricate array of cell-specific multiprotein complexes participate in programs of cell-specific gene expression through combinatorial interaction with different transcription factors and cofactors. The dHAND basic helix-loop-helix (bHLH) transcription factor, which is essential for heart development and extra embryonic structures, is thought to regulate cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted transcription factors such as GATA4 and NKX2.5. Here, we determine that dHAND also interacts with the myocyte enhancer binding factor-2c (MEF2C) protein, which belongs to MADS-box transcription factors and is essential for heart development. dHAND and MEF2C synergistically activated expression of the atrial naturetic peptide gene (ANP) in transfected HeLa cells. GST-pulldown and immunoprecipitation assay demonstrate that full-length MEF2C protein is able to interact with dHAND in vitro and in vivo, just like MEF2A and bHLH transcription factors MyoD in skeletal muscle cells. In addition, electrophoretic mobility shift assays (EMSAs) demonstrate that MEF2C and dHAND do not influence each other's DNA binding activity. Using chromatin immunoprecipitation (ChIP) analysis in H9c2 cells we show that dHAND interact with MEF2C to form protein complex and bind A/T sequence in promoter of ANP. Taken together with previous observations, these results suggest the existence of large multiprotein transcriptional complex with core DNA binding proteins that physically interact with other transcriptional factors to form favorable conformation to potentiate transcription., (Copyright 2004 Wiley-Liss, Inc.)

Tcf family transcription factors are the downstream effectors of the Wnt signal transduction pathway that regulates developmental and oncogenetic processes among species. The alternative splicing of consecutive exons in the 3' part of the Tcf-4 gene and an error-prone A9 repeat in exon 17 have recently been examined in several tumors. To further understand the roles played by differential expression and splicing isoforms of human Tcf-4 in brain tumorigenesis, the expression of the 3' part of the Tcf-4 gene (exons 10-17) was analyzed by RT-PCR, nested RT-PCR and DNA sequencing. The results showed that at least 13 of the Tcf-4 alternative splicing isoforms were found and most of them were overexpressed in various brain tumors. L3 isoform was particularly dominant in metastasis. Several novel splicing isoforms were identified. One that contains different combinations of exon 16 (10-11-12-16-17, S5) was found in normal brain and pituitary adenoma but not in astrocytoma, meningioma or metastasis, whereas the other that contains part of intron 16, designated exon-16', was found in metastasis. Overall 23% of sequencing analysis in brain tumors exhibited frameshift mutations in an A9 repeat region of exon 17. These mutants exhibited 2-base or 1-base deletion (A7, A8) and 1-base insertion (A10). Nonetheless, in vitro functional assay showed that these mutants did not affect the transactivity of Tcf-4 comparing to wild-type Tcf-4. Collectively, our data suggest that a large number of alternative splicing isoforms may together with variable mutations of A9 repeat region maintain balanced pools of Tcf-4 isoforms during brain tumorigenesis.

Sperm are highly specialized cells, and their formation requires the synthesis of a large number of unique mRNAs. However, little is known about the transcriptional mechanisms that direct male germ cell differentiation. Sterol response element binding protein 2gc (SREBP2gc) is a spermatogenic cell-enriched isoform of the ubiquitous transcription factor SREBP2, which in somatic cells is required for homeostatic regulation of cholesterol. SREBP2gc is selectively enriched in spermatocytes and spermatids, and, due to its novel structure, its synthesis is not subject to cholesterol feedback control. This suggested that SREBP2gc has unique cell- and stage-specific functions during spermatogenesis. Here, we demonstrate that this factor activates the promoter for the spermatogenesis-related gene proacrosin in a cell-specific manner. Multiple SREBP2gc response elements were identified within the 5'-flanking and proximal promoter regions of the proacrosin promoter. Mutating these elements greatly diminished in vivo expression of this promoter in spermatogenic cells of transgenic mice. These studies define a totally new function for an SREBP as a transactivator of male germ cell-specific gene expression. We propose that SREBP2gc is part of a cadre of spermatogenic cell-enriched isoforms of ubiquitously expressed transcriptional coregulators that were specifically adapted in concert to direct differentiation of the male germ cell lineage.

Melastatin 1 (MLSN1), originally identified as melanoma metastasis suppressor, represents a TRPM subfamily of transient receptor potential (TRP) proteins which serve diverse biological roles in a wide variety of cell types. Down-regulation of MLSN1 expression in human cutaneous melanoma, as indicated by in situ hybridization, appears to be a prognostic marker for melanoma metastasis. However, the exact physiological function(s) of MLSN1, the mechanism(s) involved in the regulation of its expression and its role in melanoma tumour progression are not yet clear. In this study, we identified a 654 bp upstream sequence of MLSN1, containing four E boxes (E1-E4), including an 11 bp M box (E4), that is sufficient for melanocyte-specific transcription and activation by the melanocyte transcription factor MITF (a bHLH-zip factor). Deletion analysis showed that the two distal E boxes (E3 and E4) in the MLSN1 promoter are required for both its activation by MITF and its constitutive activity in melanoma cells. Western blot analysis using polyclonal rabbit anti-human MLSN1 antibodies identified several polypeptides, presumably generated by both alternative splicing of MLSN1 messenger RNA (mRNA) and proteolytic cleavage, in both melanocytes and metastatic melanoma cells. Thus, multiple mechanisms appear to regulate MLSN1 expression in melanocytes and melanoma cells.

Background & Aims: Our previous work has shown that ethanol induces the fatty acid synthesis pathway by activation of sterol regulatory element-binding protein 1 (SREBP-1). In the present study, we studied the mechanisms of this activation by identifying a new target of ethanol, AMP-activated protein kinase (AMPK)., Methods: The effects of ethanol on AMPK, acetyl-CoA carboxylase (ACC), and SREBP-1 were assessed in rat hepatic cells and in the livers of ethanol-fed mice., Results: In rat hepatoma H4IIEC3 or McA-RH 7777 cell lines, ethanol-induced transcription of an SREBP-regulated promoter was suppressed by the presence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or metformin, 2 known AMPK activators. Consistent with this, over expression of a constitutively active form of AMPK blocked the effect of ethanol, whereas coexpression of a dominant-negative form of AMPK augmented the effect. Moreover, activation of AMPK by metformin or AICAR largely blocked the ability of ethanol to increase levels of mature SREBP-1 protein. These findings suggest that the effect of ethanol on SREBP-regulated promoter activation was partially mediated through AMPK inhibition. We further demonstrated that AMPK was inhibited by ethanol in hepatic cells. In parallel, ethanol increased the activity of ACC and suppressed the rate of palmitic acid oxidation. Finally, feeding mice a low-fat diet with ethanol resulted in significantly reduced hepatic AMPK activity, increased ACC activity, and enhanced malonyl CoA content., Conclusions: Taken together, our findings suggest that AMPK may play a key role in regulating the effects of ethanol on SREBP-1 activation, fatty acid metabolism, and development of alcoholic fatty liver.

The transcriptional enhancer (Emu3') of the IgH locus of the channel catfish, Ictalurus punctatus, differs from enhancers of the mammalian IgH locus in terms of its position, structure, and function. Transcription factors binding to multiple octamer motifs and a single muE5 motif (an E-box site, consensus CANNTG) interact for its function. E-box binding transcription factors of the class I basic helix-loop-helix family were cloned from a catfish B cell cDNA library in this study, and homologs of TF12/HEB were identified as the most highly represented E-proteins. Two alternatively spliced forms of catfish TF12 (termed CFEB1 and -2) were identified and contained regions homologous to the basic helix-loop-helix and activation domains of other vertebrate E-proteins. CFEB message is widely expressed, with CFEB1 message predominating over that of CFEB2. Both CFEB1 and -2 strongly activated transcription from a muE5-dependent artificial promoter. In catfish B cells, CFEB1 and -2 also activated transcription from the core region of the catfish IgH enhancer (Emu3') in a manner dependent on the presence of the muE5 site. Both CFEB1 and -2 bound the muE5 motif, and formed both homo- and heterodimers. CFEB1 and -2 were weakly active or inactive (in a promoter-dependent fashion) in mammalian B-lineage cells. Although E-proteins have been highly conserved in vertebrate evolution, the present results indicate that, at the phylogenetic level of a teleost fish, the TF12/HEB homolog differs from that of mammals in terms of 1) its high level of expression and 2) the presence of isoforms generated by alternative RNA processing.

The basic helix-loop-helix (bHLH) factor Hand1 plays an essential role in cardiac morphogenesis, and yet its precise function remains unknown. Protein-protein interactions involving Hand1 provide a means of determining how Hand1-induced gene expression in the developing heart might be regulated. Hand1 is known to form either heterodimers with near-ubiquitous E-factors and other lineage-restricted class B bHLH proteins or homodimers with itself in vitro. To date, there have been no reported Hand1 protein interactions involving non-bHLH proteins. Heterodimer-versus-homodimer choice is mediated by the phosphorylation status of Hand1; however, little is known about the in vivo function of these dimers or, importantly, how they are regulated. In an effort to understand how Hand1 activity in the heart might be regulated postdimerization, we have investigated tertiary Hand1-protein interactions with non-bHLH factors. We describe a novel interaction of Hand1 with the LIM domain protein FHL2, a known transcriptional coactivator and corepressor expressed in the developing cardiovascular system. FHL2 interacts with Hand1 via the bHLH domain and is able to repress Hand1/E12 heterodimer-induced transcription but has no effect on Hand1/Hand1 homodimer activity. This effect of FHL2 is not mediated either at the level of dimerization or via an effect of Hand1/E12 DNA binding. In summary, our data describe a novel differential regulation of Hand1 heterodimers versus homodimers by association of the cofactor FHL2 and provide insight into the potential for a tertiary level of control of Hand1 activity in the developing heart.

dHAND is a transcription factor belonging to the class B basic helix-loop-helix protein family and is expressed during embryogenesis in the heart, branchial arches, limb buds, and neural crest derivatives. Despite much study, the molecular mechanisms involved in the regulation of dHAND activity are not well understood. We therefore carried out yeast two-hybrid screening using full-length dHAND as bait, which led to identification of several dHAND-binding proteins, including three E-proteins: E2A, ME2, and ALF1. Subsequent analysis revealed that although their heterodimerization and transcriptional activities were similar, dHAND/E-protein heterodimers bind to an E-box element with differing affinities, suggesting they have distinct DNA binding specificities. Moreover, in situ hybridization showed that E-protein genes are expressed fairly ubiquitously among embryonic tissues, including the branchial arches and limb buds. By contrast, little signal was detected in the heart, suggesting that dHAND complexes with partners other than E-proteins in cardiac tissue., (Copyright 2004 Elsevier Inc.)