HLH Domain Research Articles

Abstract LB-141: Expression and localization of Id3 in human prostate cancer

Abstract Purpose: The inhibitor of differentiation (Id) proteins are dominant negative basic helix-loop-helix family of transcription factors that lack the DNA-binding basic domain but have intact HLH domain. The four members of the Id protein family (Id1-Id4) are known to play a critical role in the coordinated regulation of gene expression during cell growth, differentiation and tumorigenesis. Although in vitro studies have demonstrated the role of Id1 in regulating prostate cancer (PCa) cell proliferation, apoptosis and androgen independence, its clinical significance in PCa remains controversial. On the other hand there is lack of evidence on the expression of Id2 and Id3 in PCa progression. Studies have demonstrated that silencing of Id3 is significantly more effective in blocking proliferation of PCa cells as compared to Id1 whereas Id2 silencing has been primarily shown to be involved in apoptotic pathway. Hence in this study we re-evaluated the role of Id1 in PCa and extended our study to show that Id3 protein expression has better clinical relevance compared to Id1 and Id2 in PCa. Experimental design: The expression of Id-1, -2 and -3 was determined by real time and Western blotting along with their sub-cellular localization by ICC in PCa cell lines LNCaP, DU145 and PC3. To assess their significance in prostate cancer progression, the protein expression was investigated in clinical prostate tumor samples on high density tissue microarray by IHC. Results: The expression of Id1 and Id3 mRNA was low in LNCaP and DU145 as compared to PC3 whereas the level of Id2 expression was similar between all the PCa cell lines. The protein expression data was consistent with the mRNA levels in the PCa cell lines. All three Id's were primarily cytoplasmic with some nuclear presence. The IHC data showed a significant increase in the expression of Id1 and Id3 in clinical prostate tumor tissue samples compared to normal prostate tissue whereas Id2 showed elevated levels but was not significant as compared to Id1 and Id3. The expression of Id3 protein correlated with the Gleason grading and stage of PCa. Conclusions: This is the first study demonstrating a correlation between increased expression of Id3 and prostate cancer progression. It also reconfirms and establishes the association of Id1with PCa. The results warrant further investigation into isoform specific effects of Id1and Id3 on prostate tumorigenesis. (Research Support: NIH/NCI RO1 CA128914 and NIH/NCRR/RCMI G12RR03062. The first two authors contributed equally to this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-141. doi:10.1158/1538-7445.AM2011-LB-141

The shade avoidance syndrome in Arabidopsis: a fundamental role for atypical basic helix–loop–helix proteins as transcriptional cofactors

The shade avoidance syndrome (SAS) refers to a set of plant responses aimed at anticipating eventual shading by potential competitors. The SAS is initiated after perception of nearby vegetation as a reduction in the red to far-red ratio (R:FR) of the incoming light. Low R:FR light is perceived by the phytochromes, triggering dramatic changes in gene expression that, in seedlings, eventually result in an increased hypocotyl elongation to overgrow competitors. This response is inhibited by genes such as PHYTOCHROME RAPIDLY REGULATED 1 (PAR1), PAR2 and LONG HYPOCOTYL IN FR 1 (HFR1), which are transcriptionally induced by low R:FR. Although PAR1/PAR2 and HFR1 proteins belong to different groups of basic helix-loop-helix (bHLH) transcriptional regulators, they all lack a typical basic domain required for binding to E-box and G-box motifs in the promoter of target genes. By overexpressing derivatives of PAR1 and HFR1 we show that these proteins are actually transcriptional cofactors that do not need to bind DNA to directly regulate transcription. We conclude that protein-protein interactions involving the HLH domain of PAR1 and HFR1 are a fundamental aspect of the mechanism by which these proteins regulate gene expression, most likely through interaction with true transcription factors that do bind to the target genes and eventually unleash the observed SAS responses.

The Transcriptional Repressor ID2 Can Interact with the Canonical Clock Components CLOCK and BMAL1 and Mediate Inhibitory Effects on mPer1 Expression

ID2 is a rhythmically expressed HLH transcriptional repressor. Deletion of Id2 in mice results in circadian phenotypes, highlighted by disrupted locomotor activity rhythms and an enhanced photoentrainment response. ID2 can suppress the transactivation potential of the positive elements of the clock, CLOCK-BMAL1, on mPer1 and clock-controlled gene (CCG) activity. Misregulation of CCGs is observed in Id2(-/-) liver, and mutant mice exhibit associated alterations in lipid homeostasis. These data suggest that ID2 contributes to both input and output components of the clock and that this may be via interaction with the bHLH clock proteins CLOCK and BMAL1. The aim of the present study was to explore this potential interaction. Coimmunoprecipitation analysis revealed the capability of ID2 to complex with both CLOCK and BMAL1, and mammalian two-hybrid analysis revealed direct interactions of ID2, ID1 and ID3 with CLOCK and BMAL1. Deletion of the ID2 HLH domain rendered ID2 ineffective at inhibiting CLOCK-BMAL1 transactivation, suggesting that interaction between the proteins is via the HLH region. Immunofluorescence analysis revealed overlapping localization of ID2 with CLOCK and BMAL1 in the cytoplasm. Overexpression of CLOCK and BMAL1 in the presence of ID2 resulted in a significant reduction in their nuclear localization, revealing that ID2 can sequester CLOCK and BMAL1 to the cytoplasm. Serum stimulation of Id2(-/-) mouse embryonic fibroblasts resulted in an enhanced induction of mPer1 expression. These data provide the basis for a molecular mechanism through which ID2 could regulate aspects of both clock input and output through a time-of-day specific interaction with CLOCK and BMAL1.

Poky/chuk/ikk1 is required for differentiation of the zebrafish embryonic epidermis

An epidermis surrounds all vertebrates, forming a water barrier between the external environment and the internal space of the organism. In the zebrafish, the embryonic epidermis consists of an outer enveloping layer (EVL) and an inner basal layer that have distinct embryonic origins. Differentiation of the EVL requires the maternal effect gene poky/ikk1 in EVL cells prior to establishment of the basal layer. This requirement is transient and maternal Ikk1 is sufficient to allow establishment of the EVL and formation of normal skin in adults. Similar to the requirement for Ikk1 in mouse epidermis, EVL cells in poky mutants fail to exit the cell cycle or express specific markers of differentiation. In spite of the similarity in phenotype, the molecular requirement for Ikk1 is different between mouse and zebrafish. Unlike the mouse, EVL differentiation requires functioning Poky/Ikk1 kinase activity but does not require the HLH domain. Previous work suggested that the EVL was a transient embryonic structure, and that maturation of the epidermis required replacement of the EVL with cells from the basal layer. We show here that the EVL is not lost during embryogenesis but persists to larval stages. Our results show that while the requirement for poky/ikk1 is conserved, the differences in molecular activity indicate that diversification of an epithelial differentiation program has allowed at least two developmental modes of establishing a multilayered epidermis in vertebrates.

Inhibition of endothelial cell activation by bHLH protein E2-2 and its impairment of angiogenesis

E2-2 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. E2-2 associates with inhibitor of DNA binding (Id) 1, which is involved in angiogenesis. In this paper, we demonstrate that E2-2 interacts with Id1 and provide evidence that this interaction potentiates angiogenesis. Mutational analysis revealed that the HLH domain of E2-2 is required for the interaction with Id1 and vice versa. In addition, Id1 interfered with E2-2-mediated effects on luciferase reporter activities. Interestingly, injection of E2-2-expressing adenoviruses into Matrigel plugs implanted under the skin blocked in vivo angiogenesis. In contrast, the injection of Id1-expressing adenoviruses rescued E2-2-mediated inhibition of in vivo angiogenic reaction. Consistent with the results of the Matrigel plug assay, E2-2 could inhibit endothelial cell (EC) migration, network formation, and proliferation. On the other hand, knockdown of E2-2 in ECs increased EC migration. The blockade of EC migration by E2-2 was relieved by exogenous expression of Id1. We also demonstrated that E2-2 can perturb VEGFR2 expression via inhibition of VEGFR2 promoter activity. This study suggests that E2-2 can maintain EC quiescence and that Id1 can counter this effect.

The Central Region of TEL (ETV6) Is Dispensable for the TEL/AML1 (ETV6/RUNX1) Leukemogenesis.

Abstract 1601Poster Board I-627TEL/AML1 (ETV6/RUNX1) fusion gene, the most frequent genetic abnormality of childhood ALL, usually results from genomic breakpoint in TEL intron 5 and AML1 introns 1 or 2 (“classical” TEL/AML1). At the protein level the helix-loop-helix domain and exon 5 coded corepressor domain of TEL are typically fused to almost entire AML1 including transactivation and DNA binding domains. TEL/AML1 fusion as a single event does not lead to leukemia development but induces a preleukemic state characterized by expansion of B-cell precursors with enhanced self-renewal and impaired differentiation to more mature B-cell stages. It was demonstrated that TEL/AML1 preleukemic activity requires three functional domains: HLH domain, corepressor domain and DNA binding domain. One of the proposed mechanisms of TEL/AML1 function is that TEL/AML1 acts as an aberrant transcription factor and deregulates AML1 targets. TEL/AML1 induced transcriptional repression is mediated through interaction with corepressors and histone deacetylases. Both HLH and corepressor domains are able to interact with corepressors and are essential for the TEL-induced transcriptional repression. Parallel screening of TEL/AML1 fusion in 305 consecutive patients treated on BFM protocol by routine FISH and RT-PCR revealed TEL/AML1-positivity in 69 patients by both methods and RT-PCR false negativity in 2 out of 71 FISH-positive cases. In these two patients, we performed further transcriptomic and also genomic fusion site analysis of both TEL/AML1 and reciprocal AML1/TEL fusions and found an unusual genomic breakpoint within the TEL intron 4 resulting in a “variant” TEL/AML1 fusion lacking TEL exon 5-coded corepressor domain in both patients. Previous studies on animal models showed that TEL exon 5-coded region modulates the disease phenotype induced by TEL/ABL1 and TEL/TRKC fusion genes. We aimed to investigate the possible impact of the exon 5 loss in the “variant” TEL/AML1 fusion on disease behavior and TEL/AML1 function. We compared clinical features (age, white blood cell count, proportion of blasts in bone marrow and peripheral blood, detailed immunophenotype including TEL/AML1-specific combination of CD27 and CD44 antigens) and treatment outcome of all the 71 TEL/AML-positive patients from the same treatment protocol and we did not find any difference between the 2 “variant” and 69 “classical” cases. Moreover, we performed genome-wide gene expression profiling (GEP) of the two “variant” TEL/AML1 cases and the results show that the “variant” patients cluster within the “classical” TEL/AML1 cases and thus they are characterized by identical pattern of gene expression. We cloned the “variant” TEL/AML1 fusion into expression vector and analyzed its potential to repress transcription from AML1 target (granzyme B) promoter using luciferase assay. While the wild type AML1 substantially activates the promoter (350% of basal activity), the “variant” and the “classical” TEL/AML1 exhibited identical level of transcriptional repression (63% and 66%, respectively). Moreover, the repressive effect of both “variant” and “classical” TEL/AML1 is dependent on the presence of the AML1 consensus binding sites within the promoter region - mutation of both binding motifs results in elimination of the repressive effect (99% and 112% of the basal activity, respectively). Taken together, our data show that loss of the corepressor region of TEL does not significantly influence the TEL/AML1 function (or at least its ability to repress transcription of AML1 targets) and that the “variant” TEL/AML1 fusion is associated with leukemias that are undistinguishable from the “classical” TEL/AML1 cases. In contrast to recent studies, performed on artificial mouse or in-vitro models, we conclude - based on the data acquired from the genuine, naturally occurring childhood leukemias - that the corepressor region of TEL is dispensable for the TEL/AML1 leukemogenesis. This work was supported by grants MSM0021620813 and MZO00064203. DisclosuresNo relevant conflicts of interest to declare.

FHL2 interacts with and acts as a functional repressor of Id2 in human neuroblastoma cells

ObjectiveId2 is a natural inhibitor of the basic helix-loop-helix(bHLH) transcription factors. Although it is well known that active Id2 prevents differentiation and promotes cell cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated.MethodsYeast two-hybrid, mammalian two-hybrid, GST-pulldown and immunoprecipitation (CoIP) assays were used to screen and identify novel Id2 interactors. Luciferase assays were used to detect E47-mediated transcription activity. Colony formation and BrdU incorporation assays were used to determine cellular proliferation abilities. Northorn blot, western blot and quantitative PCR methods were used to measure gene expression levels. Electrophoretic mobility shift assays (EMSAs) were performed to investigate protein/DNA binding.ResultsThe LIM-only protein FHL2 (four-and-a-half-LIM-only protein 2) was identified to be a novel Id2 interactor. The HLH domain within Id2 is not required for its interaction with FHL2. FHL2 antagonizes the inhibitory effect of Id2 on the basic helix-loop-helix protein E47-mediated transcription. FHL2 prevents the formation of Id2-E47 heterdimer, thus releasing E47 to its target DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 is opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2.ConclusionThese results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.

Loss of Id4 Accelerates CLL Progression in TCL1 Mice

The inhibitor of DNA binding protein 4 (ID4) is a member of dominant negative basic helix loop helix (bHLH) transcription factor family that has a HLH domain but lacking DNA-binding domain. Methylation of ID4 in B-cell lineage malignancies including CLL and germinal center non-Hodgkin lymphomas (GC-NHLs) has been described. To date, the impact of Id4 loss in CLL has not been demonstrated. We utilized the TCL1 transgenic mouse model of human CLL to study the importance of loss of ID4 in disease progression. We demonstrated that ID4 was silenced by methylation only at the time mice had symptomatic leukemia. We therefore sought to investigate if earlier loss of Id4 would accelerate CLL progression. We generated mice with haploid loss of Id4 in TCL1 transgenic background by breeding homozygous TCL1 transgenic mice and mice with heterozygous Id4 mutant gene. By comparing to the control littermates with heterozygous TCL1 oncogene, mice with the loss of Id4 occur to have significantly accelerated CLL disease progression with shorter overall survival (12 versus 16 months, p-value<0.0001). The pathological analysis on Id4 mutant mice demonstrated that the CLL symptoms such as the elevated white blood cell counts, enlarged spleen and lymphoid tissues. We then performed the microarray studies on 1 month old TCL1 mice with control or mutant Id4 to identify ID4-dependent transcriptional changes in primary B-cells. Among the identified targets, genes involved in cell proliferation and anti-apoptosis were then verified. Supportively, B-cells from Id4 mutant TCL1 mice have significantly (p-value<0.001) diminished apoptosis in response to dexamethasone treatment. Additionally, both significant in vitro (p-value<0.001) and in vivo (p-value=0.025) increased proliferation of Id4 mutant TCL1 B-cells after the stimulation by CpG685NO168 was demonstrated. These data provide support that Id4 acts as tumor suppressor in transformed B-lymphocytes and is silenced through the process of methylation. The Effort to identify binding partners of Id4 in CLL cells and targeting its re-expression is warranted.

TAL1/SCL Relieves the E2-2-Mediated Repression of VEGFR2 Promoter Activity

The basic helix-loop-helix (bHLH) protein TAL1/SCL is essential for embryonic-vascular development. TAL1/SCL regulates the activation of endothelial cells by binding directly or indirectly to DNA sequences in critical target genes. We recently demonstrated that E-box protein E2-2 blocks endothelial cell activation via perturbation of VEGFR2 promoter activity. Herein, we report that TAL1/SCL interacts with E2-2 and inhibits E2-2-mediated effects on reporter activity. Mutational analysis revealed that the HLH domain of TAL1/SCL, but not its basic region, is required for interaction with E2-2. Importantly, TAL1/SCL relieves the E2-2-mediated repression of VEGFR2 reporter activity in endothelial cells. Our data elaborate on the bHLH protein interactions that regulate endothelial cell activation.

Integration of CREB and bHLH transcriptional signaling pathways through direct heterodimerization of the proteins: Role in muscle and testis development

The cAMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors is hormone responsive and critical for nearly all mammalian cell types. The basic helix-loop-helix (bHLH) family of transcription factors is important during the development and differentiation of a wide variety of cell types. Independent studies of the role of the bHLH protein scleraxis in testicular Sertoli cells and paraxis in muscle development using yeast-2-hybrid screens provided the novel observation that bHLH proteins can directly interact with ATF/CREB family members. Analysis of the interactions demonstrated the helix-loop-helix domain of bHLH proteins directly interacts with the leucine zipper (ZIP) region of CREB2/ATF4 to form heterodimers. The direct bHLH-CREB2 binding interactions were supported using co-immunoprecipitation of recombinant proteins. Structural analysis of bHLH and ATF4 heterodimer using previous crystal structures demonstrated the heterodimer likely involves the HLH and Zip domains and has the potential capacity to bind DNA. Transfection assays demonstrated CREB2/ATF4 over-expression blocked stimulatory actions of scleraxis or paraxis. CREB1 inhibited MyoD induced myogenic conversion of C3H10T1/2 cells. CREB2/ATF4 and scleraxis are expressed throughout embryonic and postnatal testis development, with scleraxis specifically expressed in Sertoli cells. ATF4 and scleraxis null mutant mice both had similar adult testis phenotypes of reduced spermatogenic capacity. In summary, bHLH and CREB family members were found to directly heterodimerize and inhibit the actions of bHLH dimers on Sertoli cells and myogenic precursor cells. The observations suggest a mechanism for direct cross-talk between cAMP induced and bHLH controlled cellular differentiation.

Identification of SCL and LMO Oncogenes as Transcriptional Repressors of HEB/E2A in the Thymus through Functional Genomic/Genetic Approaches.

The SCL and LMO oncogenes are frequently activated in childhood T cell acute leukemia (T-ALL). SCL is a bHLH transcription factor that forms heterodimers with other members, specifically HEB and E2A. SCL can either activate or repress transcription but its mechanism of action as an oncogene remains to be clarified. Ectopic expression of SCL and LMO in transgenic mice causes thymocyte differentiation arrest during the preleukemic phase with aberrant differentiation at the DN3–DN4 stage, prior to the acquisition of CD4 and CD8. We therefore took several approaches to define the mechanism underlying this differentiation arrest. Using a CD3e-deficient background to eliminate preTCR signaling effects, an event that is not essential for leukemogenesis, we first analyzed global gene expression of pre-leukemic SCLtg/LMOtg DN3 thymocytes against their non-transgenic littermates via the Affymetrix platform. In this context, SCL/LMO globally affects the expression of both positive and negative targets of E2A/HEB. These results were confirmed by real-time quantitative PCR, showing that SCL/LMO repress E2A/HEB activity. Second, bioinformatic analysis of genes significantly affected by SCL/LMO allowed us to identify phylogenetically conserved HEB/E2A binding sites within the promoter regions of half of these genes. E2A/HEB binding was directly assessed through chromatin immunoprecipitation of primary thymocytes, confirming known target genes and revealing novel ones. This assay also showed that SCL associates with HEB and/or E2A on DNA. Moreover, structure function analysis indicate that transcription inhibition by SCL depends on its HLH domain but not on LMO interaction. We therefore conclude that SCL inhibits thymocyte differentiation by inhibiting E2A/HEB targets that include genes required for thymocyte differentiation, cell signalling, DNA repair and replication. Finally, using a genetic approach we show that SCL/LMO collaborates with HEB haploinsufficiency in inducing leukemia. Our observations therefore reveal that the repression of HEB/E2A by SCL/LMO is a crucial step in T cell transformation.Support from CIHR and FRSQ.

Transcriptional repression by the basic helix-loop-helix protein Dec2: Multiple mechanisms through E-box elements

Dec2, a member of the basic helix-loop-helix (bHLH) superfamily, has been shown to function as a transcriptional repressor and is implicated in cell proliferation and differentiation. In addition, Dec2 transcripts exhibit a striking circadian oscillation in the suprachiasmatic nucleus. To identify the molecular mechanisms by which Dec2 regulates gene expression, we carried out structure-function analyses. Gel retardation and luciferase assays showed that Dec2, as well as its related protein Dec1, preferentially binds to class B E-box elements (CACGTG) as a homodimer and represses the transcription of target genes in a histone deacetylase (HDAC)-dependent manner. Functional studies with the GAL4-DNA binding domain fusion proteins identified the domain responsible for the repression activity of Dec2 in its C-terminal region, which is also necessary to recruit HDAC1. In addition, the basic and HLH domains of Dec2 were required for DNA binding and homodimerization, respectively. In contrast, Dec proteins repressed a MyoD-activated promoter activity of muscle creatine kinase gene through class A E-box in an HDAC1-independent manner. Dec2 formed a heterodimer with MyoD through the basic and HLH domains. Consistent with this, both the basic and HLH domains were required for the ability of Dec2 to inhibit the transcriptional activity of MyoD. These findings indicate that Dec2 employs multiple mechanisms, including DNA-binding and protein-protein interactions, to achieve E-box-dependent transcriptional repressions.

Molecular cloning and characterization of the Xenopus hypoxia‐inducible factor 1α (xHIF1α)

AbstractWe report the molecular cloning and the characterization of the Xenopus homolog of mammalian hypoxia‐inducible factor 1α (HIF1α), a member of the bHLH/PAS transcription factor family. Searches in Xenopus genome sequences and phylogenetic analysis reveal the existence of HIF1α and HIF2α paralogs in the Xenopus laevis species. Sequence data analyses indicate that the organization of protein domains in Xenopus HIF1α (xHIF1α) is strongly conserved. We also show that xHIF1α heterodimerizes with the Xenopus Arnt1 protein (xArnt1) with the proteic complex being mediated by the HLH and PAS domains. Subcellular analysis in a Xenopus XTC cell line using chimeric GFP constructs show that over‐expression of xHIF1α and xArnt1 allows us to detect the xHIF1α/xArnt1 complex in the nucleus, but only in the presence of both partners. Further analyses in XTC cell line show that over‐producing xHIF1α and xArnt1 mediates trans‐activation of the hypoxia response element (HRE) reporter. The trans‐activation level can be increased in hypoxia conditions. Interestingly such trans‐activation properties can be also observed when human Arnt1 is used together with the xHIF1α. J. Cell. Biochem. 102: 1542–1552, 2007. © 2007 Wiley‐Liss, Inc.

Molecular Cloning and Expression of Grass Carp MyoD in Yeast Pichia pastoris

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84(th) amino acids) and HLH domain (98-142(th) amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZalphaA and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

VMD2 Promoter Requires Two Proximal E-box Sites for Its Activity in Vivo and Is Regulated by the MITF-TFE Family

The retinal pigment epithelium (RPE) is crucial for the function and survival of retinal photoreceptors. VMD2 encodes bestrophin, an oligomeric chloride channel that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Previously, we defined the VMD2 upstream region from -253 to +38 bp as being sufficient to direct RPE-specific expression in the eye, and we suggested microphthalmia-associated transcription factor (MITF) as a possible positive regulator. Here we show that in transgenic mice the -154 to +38 bp region is sufficient for RPE expression, and mutation of two E-boxes, 1 and 2, within this region leads to loss of promoter activity. A yeast one-hybrid screen using bait containing E-box 1 identified clones encoding MITF, TFE3, and TFEB, and chromatin immunoprecipitation with antibodies against these proteins enriched the VMD2 proximal promoter. Analysis using in vivo electroporation with constructs containing mutation of each E-box indicated that expression in native RPE requires both E-boxes, yet in vitro DNA binding studies suggested that MITF binds well to E-box 1 but only minimally to E-box 2. MITF knockdown by small interfering RNA (siRNA) in cell culture revealed a strong correlation between MITF and VMD2 mRNA levels. Sequential transfection of a luciferase construct with expression vectors following MITF siRNA revealed that TFE3 and TFEB can also transactivate the VMD2 promoter. Taken together, we suggest that VMD2 is regulated by the MITF-TFE family through two E-boxes, with E-box 1 required for a direct interaction of MITF-TFE factors and E-box 2 for binding of the as yet unidentified factor(s).

TEL/ETV6 Binds to Corepressor KAP1 via the HLH Domain

TEL/ETV6 Binds to Corepressor KAP1 via the HLH Domain

Neuronal PAS Domain Protein 1 Is a Transcriptional Repressor and Requires Arylhydrocarbon Nuclear Translocator for Its Nuclear Localization

Neuronal PAS domain protein 1 (NPAS1), a basic helix-loop-helix-PAS transcription factor expressed in the central nervous system, has been suggested to be involved in neuronal differentiation. However, relatively little is known about the molecular mechanism underlying the role of NPAS1 during development. In this study we set out to characterize the different domains within NPAS1. We showed that the nuclear localization of NPAS1 is dependent on the presence of ARNT. In addition, the transcriptional potential of ARNT is not required for this localization. In the absence of ARNT, NPAS1 is excluded from the nucleus, and this exclusion is due to the presence of a nuclear export signal within the N terminus of NPAS1. The interaction between NPAS1 and ARNT is via their N termini. We found no transactivation domain within NPAS1; instead, we mapped out at least three repression domains within NPAS1, suggesting that NPAS1 acts as a repressor. Furthermore, our experiments showed that NPAS1 is able to repress the transactivation functions of ARNT and ARNT2. We suggest that NPAS1 is guided into the nucleus by ARNT via the ARNT nuclear localization signal, and NPAS1 can override the activation function of adjacent transcription factors, providing a mechanism by which NPAS1 may inhibit transcription.

Regulation of IκB Kinase (IKK) Complex by IKKγ-dependent Phosphorylation of the T-loop and C Terminus of IKKβ

Regulation of IκB Kinase (IKK) Complex by IKKγ-dependent Phosphorylation of the T-loop and C Terminus of IKKβ

KIDARI, Encoding a Non-DNA Binding bHLH Protein, Represses Light Signal Transduction in Arabidopsis thaliana

Through activation tagging mutagenesis, we isolated a kidari-D (kdr-D) mutant, which exhibited a defect in blue and far-red light mediated photomorphogenesis. Under continuous blue light, the kdr-D mutant showed long hypocotyl phenotype, whereas it showed normal cotyledon opening and expansion. In addition, the kdr-D showed slightly longer hypocotyl under continuous far-red light, suggesting that KDR functions in a branch of cry signaling and mediates a cross-talk between cry and phyA. In the kdr-D mutant, a gene encoding a putative basic/Helix-Loop-Helix (bHLH) protein was overexpressed by the insertion of 35S enhancer into 10 kb upstream of the gene. Consistently, overexpression of this gene recapitulated the phenotype of kdr-D. KDR is composed of 94 amino acids with non-DNA binding HLH domain, a structure found in human Inhibitor of DNA binding 1 (Id-1) which functions as a negative regulator of bHLH proteins through heterodimerization with them. The KDR specifically interacted with HFR1, a bHLH protein regulating photomorphogenesis, in yeast two hybrid assay and the kdr-D was epistatic to 35S::HFR1 in the hypocotyl phenotype. Thus, it shows that KDR functions as a negative regulator of HFR1, similar to Id-1 in human. The KDR exhibited circadian expression pattern with an increase during the day. Taken together, our results suggest that KDR attenuates light mediated responses in day light condition through inhibition of the activity of bHLH proteins involved in light signaling.

Synthesis and conformational analysis of Id2 protein fragments: impact of chain length and point mutations on the structural HLH motif

The Id proteins are negative regulators of several basic-helix-loop-helix (HLH) transcription factors, including the ubiquitous E factors and the tissue-specific myogenin-regulating factors. Id1 through Id4 contain highly identical HLH domains but different N- and C-terminal extensions. Beside the heterodimerization with the parent HLH factors, Id2 was shown to additionally interact with the retinoblastoma protein and to be overexpressed in neuroblastoma. Thus, Id2 represents an interesting target for cancer therapy based on the inhibition of protein-protein interactions. Here we present the synthesis and circular dichroism (CD) analysis of peptides derived from point mutations and N-/C-terminal truncations of Id2. The helix character of the HLH domain (residues 36-76) was reduced upon substitution of Met39/-62 and Cys42 with Nle and Ser, respectively, suggesting a structural role of these side chains. The largest sequence that could be obtained by stepwise solid-phase peptide synthesis (SPPS) with Fmoc strategy spanned the entire HLH motif (with Cys42 replaced by Ser) and part of the C-terminus (residues 77-110). This 75-residue long fragment was less helical than the isolated HLH domain and had propensity to aggregate, which was correlated with the presence of the flanking residues C-terminal to helix-2. By CD analysis of an equimolar mixture of the sequence 36-110 with the N-terminus 1-35, noncovalent interactions between the two peptides were detected, which, however, changed upon aging. In contrast, the mixture of the HLH sequence 36-76 with the N-terminus was characterized by a stabilized helix structure that was maintained also upon aging. Presumably, the N-terminal region interacted with the folded HLH motif in a specific manner, whereas only unspecific, weak contacts occurred with the partly unfolded HLH domain and/or the immediate flanking residues 77-110.