Biochemical properties of human full-length aryl hydrocarbon receptor (AhR).

The aryl hydrocarbon receptor (AhR) is a very unstable protein. AhR binds to the molecular chaperone complex (HSP90-p23-XAP2) to maintain a stable structure in the cytoplasm. After binding to ligands, such as dioxin, AhR translocates from the cytoplasm to the nucleus with a molecular chaperone complex. The protein forms a heterodimer with Arnt after nuclear transfer, functions as a transcription factor by binding to a xenobiotic responsive element (XRE), and induces the cytochrome P450 1A1 (CYP1A1). Because of the unstable protein, expression of the full-length AhR in the E. coli expression system is very difficult. Many studies investigated AhR using AhR domains in vitro. We expressed and purified the human full-length AhR in E. coli expression system. Furthermore, specific antibodies were prepared. Purified full-length AhR could bind to ligand. In the presence of ligand, α-helix and random coil of AhR increased and ß-sheet decreased on CD spectrum. Full-length AhR could bind to HSP90, XAP2 and p23 in the presence or absence of ligand. We now show the biochemical properties of full-length AhR.

PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome.

The poly(ADP-ribose) polymerase, PARP1, plays a key role in maintaining genomic integrity by detecting DNA damage and mediating repair. γH2A.X is the primary histone marker for DNA double-strand breaks and PARP1 localizes to H2A.X-enriched chromatin damage sites, but the basis for this association is not clear. We characterize the kinetics of PARP1 binding to a variety of nucleosomes harbouring DNA double-strand breaks, which reveal that PARP1 associates faster with (γ)H2A.X- versus H2A-nucleosomes, resulting in a higher affinity for the former, which is maximal for γH2A.X-nucleosome that is also the activator eliciting the greatest poly-ADP-ribosylation catalytic efficiency. The enhanced activities with γH2A.X-nucleosome coincide with increased accessibility of the DNA termini resulting from the H2A.X-Ser139 phosphorylation. Indeed, H2A- and (γ)H2A.X-nucleosomes have distinct stability characteristics, which are rationalized by mutational analysis and (γ)H2A.X-nucleosome core crystal structures. This suggests that the γH2A.X epigenetic marker directly facilitates DNA repair by stabilizing PARP1 association and promoting catalysis.

Bone morphogenetic protein 4 promotes the survival and preserves the structure of flow-sorted Bhlhb5+ cochlear spiral ganglion neurons in vitro.

SGNs are the primary auditory neurons, and damage or loss of SGNs leads to sensorineural hearing loss. BMP4 is a growth factor that belongs to the TGF-ß superfamily and has been shown to play a key role during development, but little is known about its effect on postnatal cochlear SGNs in mice. In this study, we used the P3 Bhlhb5-cre/tdTomato transgenic mouse model and FACS to isolate a pure population of Bhlhb5+ SGNs. We found that BMP4 significantly promoted SGN survival after 7 days of culture. We observed fewer apoptotic cells and decreased expression of pro-apoptotic marker genes after BMP4 treatment. We also found that BMP4 promoted monopolar neurite outgrowth of isolated SGNs, and high concentrations of BMP4 preserved the number and the length of neurites in the explant culture of the modiolus harboring the SGNs. We showed that high concentration of BMP4 enhanced neurite growth as determined by the higher average number of filopodia and the larger area of the growth cone. Finally, we found that high concentrations of BMP4 significantly elevated the synapse density of SGNs in explant culture. Thus, our findings suggest that BMP4 has the potential to promote the survival and preserve the structure of SGNs.

Genome-wide identification, classification, and functional analysis of the basic helix-loop-helix transcription factors in the cattle, Bos Taurus.

The basic helix-loop-helix (bHLH) transcription factors (TFs) form a huge superfamily and play crucial roles in many essential developmental, genetic, and physiological-biochemical processes of eukaryotes. In total, 109 putative bHLH TFs were identified and categorized successfully in the genomic databases of cattle, Bos Taurus, after removing redundant sequences and merging genetic isoforms. Through phylogenetic analyses, 105 proteins among these bHLH TFs were classified into 44 families with 46, 25, 14, 3, 13, and 4 members in the high-order groups A, B, C, D, E, and F, respectively. The remaining 4 bHLH proteins were sorted out as 'orphans.' Next, these 109 putative bHLH proteins identified were further characterized as significantly enriched in 524 significant Gene Ontology (GO) annotations (corrected P value ≤ 0.05) and 21 significantly enriched pathways (corrected P value ≤ 0.05) that had been mapped by the web server KOBAS 2.0. Furthermore, 95 bHLH proteins were further screened and analyzed together with two uncharacterized proteins in the STRING online database to reconstruct the protein-protein interaction network of cattle bHLH TFs. Ultimately, 89 bHLH proteins were fully mapped in a network with 67 biological process, 13 molecular functions, 5 KEGG pathways, 12 PFAM protein domains, and 25 INTERPRO classified protein domains and features. These results provide much useful information and a good reference for further functional investigations and updated researches on cattle bHLH TFs.

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli.

Genome-wide analysis of bHLH transcription factor and involvement in the infection by yellow leaf curl virus in tomato (Solanum lycopersicum).

BACKGROUND: The basic helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that can bind to specific DNA target sites. They have been well characterized in model plants such as Arabidopsis and rice and have been shown to be important regulatory components in many different biological processes. However, no systemic analysis of the bHLH transcription factor family has yet been reported in tomatoes. Tomato yellow leaf curl virus (TYLCV) threatens tomato production worldwide by causing leaf yellowing, leaf curling, plant stunting and flower abscission. RESULTS: A total of 152 bHLH transcription factors were identified from the entire tomato genome. Phylogenetic analysis of bHLH domain sequences from Arabidopsis and tomato facilitated classification of these genes into 26 subfamilies. The evolutionary and possible functional relationships revealed during this analysis are supported by other criteria, including the chromosomal distribution of these genes, the conservation of motifs and exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Distribution mapping results showed bHLH genes were localized on the 12 tomato chromosomes. Among the 152 bHLH genes from the tomato genome, 96 bHLH genes were detected in the TYLCV-susceptible and resistant tomato breeding line before (0 dpi) and after TYLCV (357 dpi) infection. As anticipated, gene ontology (GO) analysis indicated that most bHLH genes are related to the regulation of macromolecule metabolic processes and gene expression. Only four bHLH genes were differentially expressed between 0 and 357 dpi. Virus-induced gene silencing (VIGS) of one bHLH genes SlybHLH131 in resistant lines can lead to the cell death. CONCLUSION: In the present study, 152 bHLH transcription factor genes were identified. One of which bHLH genes, SlybHLH131, was found to be involved in the TYLCV infection through qRT-PCR expression analysis and VIGS validation. The isolation and identification of these bHLH transcription factors facilitated clarification of the molecular genetic basis for the genetic improvement of tomatoes and the development of functional gene resources for transgenic research. In addition, these findings may aid in uncovering an unexplored mechanism during the TYLCV infection in tomatoes.

Hypoxia-inducible regulation of placental BOK expression.

BOK (BCL-2-related ovarian killer) is a member of the pro-apoptotic BCL-2 family that is highly expressed in the human placenta. BOK excess causes increased trophoblast autophagy and apoptosis in pre-eclampsia, a pathological condition of hypoxia and oxidative stress. In the present study, we identified an HRE (hypoxia-response element) at the junction of exon-1 and intron-1 (+229 to +279) in the human BOK gene, as well as an antisense transcript driven by a promoter located in intron-2. The isolated BOK-HRE bound hypoxia-inducible HIF (hypoxia-inducible factor) proteins in vitro as well as in trophoblastic JEG3 cells and was functional in its natural position as well as in front of a heterologous promoter. Being a reverted repeat, the BOK-HRE functioned in both orientations. This directionless feature of the BOK-HRE facilitates hypoxia regulation via HIF of both BOK and its antisense transcript as demonstrated by RNAi knockdown of the HIF system. Although the antisense transcript was expressed in several human carcinoma cell lines, including choriocarcinoma-derived JEG3 cells, no antisense-regulated mechanism for BOK expression was noted. Taken together, these findings indicate that hypoxia-induced expression of BOK in placental cells is regulated via HIF and is not affected by its antisense transcript.

Expression and roles of As-NUPR1 protein from Artemia sinica during embryo development and in response to salinity stress.

As-NUPR1, a stress-related protein, plays an important role in post-diapause during embryonic development in the brine shrimp Artemia sinica. In the present study, successful expression of As-NUPR1 from the cDNA sequence isolated from A. sinica was demonstrated using a prokaryotic expression system. The recombinant protein consisted of 132 amino acids with a molecular weight of 15 kDa, and a predicted isoelectric point of 7.17. As-NUPR1 polyclonal antibodies were prepared by immunization of Balb/c mice with purified recombinant As-NUPR1 protein as an antigen, and immunological studies were carried out. Expression of As-NUPR1 during different developmental stages of the embryo and in response to salinity stress was analyzed in A. sinica using Western blots. The experimental results showed that the expression of As-NUPR1 is widely distributed at different developmental stages in A. sinica, and there was no tissue or organ specificity. Expression of As-NUPR1 decreased gradually during the diapause termination stage of embryo development, after which there was a general increase in expression after breaking the shell. In addition, As-NUPR1 expression was highly upregulated under conditions of high salinity. These results suggest that the As-NUPR1 protein is a stress-related protein that plays a role in protecting embryos from high salt damage in different embryonic developmental stages, especially during the post-diapause period.

Identification and characterization of a putative basic helix-loop-helix transcription factor involved in the early stage of conidiophore development in Aspergillus oryzae.

The helix-loop-helix (HLH) family of transcriptional factors is a key player in a wide range of developmental processes. HLH proteins form homo- and/or heterodimers with other HLH proteins and bind to E-box motifs. The regulation and functions of these proteins can be complex due to their bifunctional roles as activators and repressors of gene transcription. In this study, we isolated and characterized a novel predicted bHLH protein-encoding gene, AO090023000902, designated ecdR (early conidiophore development regulator), in Aspergillus oryzae. The ecdR gene disruptant produced very few conidia. Conversely, the overexpression of ecdR resulted in the formation of a large number of conidia at an early stage, suggesting that the EcdR protein is required for early asexual development. Additionally, when serially diluted conidia were spread-cultivated onto malt agar medium, we found that conidial number of the control strain depended on the cultivated conidium density, while the ecdR-overexpressing strain showed no significant change in conidiation. These phenotypes of ecdR-disruptant and ecdR-overexpressing strains are partially similar to those of the sclR-overexpressing strain and sclR-disruptant, respectively. Yeast two-hybrid assays and sclR, ecdR-double deletion experiment indicated that EcdR plays a major role in conidiation, and SclR represses this function by competitively interacting with EcdR in A. oryzae.

Affinity purification of microRNA-133a with the cardiac transcription factor, Hand2.

Predictions of microRNA-mRNA interactions typically rely on bioinformatic algorithms, but these algorithms only suggest the possibility of microRNA binding and may miss important interactions as well as falsely predict others. We developed an affinity purification approach to empirically identify microRNAs associated with the 3'UTR of the mRNA encoding Hand2, a transcription factor essential for cardiac development. In addition to miR-1, a known regulator of Hand2 expression, we determined that the Hand2 3'UTR also associated with miR-133a, a microRNA cotranscribed with miR-1 in cardiac and muscle cells. Using a sequential binding assay, we showed that miR-1 and miR-133a could occupy the Hand2 3'UTR concurrently. miR-133a inhibited Hand2 expression in tissue culture models, and miR-133a double knockout mice had elevated levels of Hand2 mRNA and protein. We conclude that Hand2 is regulated by miR-133a in addition to miR-1. The affinity purification assay should be generally applicable for identifying other microRNA-mRNA interactions.

Groucho corepressor functions as a cofactor for the Knirps short-range transcriptional repressor.

Despite the pervasive roles for repressors in transcriptional control, the range of action of these proteins on cis regulatory elements remains poorly understood. Knirps has essential roles in patterning the Drosophila embryo by means of short-range repression, an activity that is essential for proper regulation of complex transcriptional control elements. Short-range repressors function in a local fashion to interfere with the activity of activators or basal promoters within approximately 100 bp. In contrast, long-range repressors such as Hairy act over distances >1 kb. The functional distinction between these two classes of repressors has been suggested to stem from the differential recruitment of the CtBP corepressor to short-range repressors and Groucho to long-range repressors. Contrary to this differential recruitment model, we report that Groucho is a functional part of the Knirps short-range repression complex. The corepressor interaction is mediated via an eh-1 like motif present in the N terminus and a conserved region present in the central portion of Knirps. We also show that this interaction is important for the CtBP-independent repression activity of Knirps and is required for regulation of even-skipped. Our study uncovers a previously uncharacterized interaction between proteins previously thought to function in distinct repression pathways, and indicates that the Groucho corepressor can be differentially harnessed to execute short- and long-range repression.

Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas.

Novel strategies in diabetes therapy would obviously benefit from the use of beta (beta) cell stem/progenitor cells. However, whether or not adult beta cell progenitors exist is one of the most controversial issues in today's diabetes research. Guided by the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, we show that beta cell progenitors can be activated in injured adult mouse pancreas and are located in the ductal lining. Differentiation of the adult progenitors is Ngn3 dependent and gives rise to all islet cell types, including glucose responsive beta cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants. Multipotent progenitor cells thus exist in the pancreas of adult mice and can be activated cell autonomously to increase the functional beta cell mass by differentiation and proliferation rather than by self-duplication of pre-existing beta cells only.

Sohlh2 is a germ cell-specific bHLH transcription factor.

Germ cell-specific transcriptional regulation is essential to understand pathways that confer germ cells their unique biology. Germ cell-specific transcription factors such as Figla and Nobox are critical in oogenesis, while Zfp148 and Taf4b are also critical in spermatogenesis. Identification and characterization of the full complement of germ cell-specific transcription factors is necessary to understand germ cell-specific regulatory networks. Here, we describe a discovery of a novel spermatogenesis- and oogenesis-specific basic helix-loop-helix (bHLH) transcription factor, Sohlh2. Sohlh2 is expressed both in the male and female germline. In females, Sohlh2 transcripts are detectable in the female embryonic gonad but confined to oocytes of small follicles in the immature ovary. In adult ovaries, SOHLH2 protein is present in primordial follicles but not detected in growing oocytes. SOHLH2 expression in testes is confined to spermatogonia. The expression pattern of SOHLH2 suggests that it may be a critical regulator of early germ cell development.

Transgenic analysis of the medaka mesp-b enhancer in somitogenesis.

Somitogenesis is a critical step during the formation of metameric structures in vertebrates. Recent studies in mouse, chick, zebrafish and Xenopus have revealed that several factors, such as T-box genes, Notch/Delta, Wnt, retinoic acid and FGF signaling, are involved in the specification of nascent somites. By interacting with these pathways, the Mesp2-like bHLH transcription factors are transiently expressed in the anterior presomitic mesoderm and play a crucial role in somite formation. The regulatory mechanisms of Mesp2 and its related genes during somitogenesis have been studied in mouse and Xenopus. However, the precise mechanism that regulates the transcriptional activity of Mesp2 has yet to be determined. In our current report, we identify the essential enhancer element of medaka mesp-b, an orthologue of mouse Mesp2, using transgenic techniques and embryo manipulation. Our results demonstrate that a region of approximately 2.8 kb, upstream of the mesp-b gene, is responsible for both the initiation and anterior localization of mesp-b transcription within a somite primordium. Furthermore, putative motifs for both T-box transcription factors and Notch/Delta signaling are present in this enhancer region and are essential for activity.