Long noncoding RNA SNHG1 alleviates high glucose-induced vascular smooth muscle cells calcification/senescence by post-transcriptionally regulating Bhlhe40 and autophagy via Atg10

Long noncoding RNAs (lncRNAs) are emerging regulators of vascular diseases, yet their role in diabetic vascular calcification/aging remains poorly understood. In this study, we identified a down-expressed lncRNA SNHG1 in high glucose (HG)-induced vascular smooth muscle cells (HA-VSMCs), which induced excessive autophagy and promoted HA-VSMCs calcification/senescence. Overexpression of SNHG1 alleviated HG-induced HA-VSMCs calcification/senescence. The molecular mechanisms of SNHG1 in HA-VSMCs calcification/senescence were explored by RNA pull-down, RNA immunoprecipitation, RNA stability assay, luciferase reporter assay, immunoprecipitation and Western blot assays. In one mechanism, SNHG1 directly interacted with Bhlhe40 mRNA 3′-untranslated region and increased Bhlhe40 mRNA stability and expression. In another mechanism, SNHG1 enhanced Bhlhe40 protein SUMOylation by serving as a scaffold to facilitate the binding of SUMO E3 ligase PIAS3 and Bhlhe40 protein, resulting in increased nuclear translocation of Bhlhe40 protein. Moreover, Bhlhe40 suppressed the expression of Atg10, which is involved in the process of autophagosome formation. Collectively, the protective effect of SNHG1 on HG-induced HA-VSMCs calcification/senescence is accomplished by stabilizing Bhlhe40 mRNA and promoting the nuclear translocation of Bhlhe40 protein. Our study could provide a novel approach for diabetic vascular calcification/aging. (AU)

The α7 nicotinic acetylcholine receptor agonist PNU-282987 ameliorates sepsis-induced acute kidney injury through CD4+CD25+ regulatory T cells in rats.

The ameliorative effects of α7 nicotinic acetylcholine receptor (α7nAChR) agonists have been demonstrated in acute kidney injury (AKI) caused by multiple stimulations. However, the ameliorative effect of α7nAChR on sepsis-induced acute kidney injury (SAKI) in the cecal ligation and puncture (CLP) model is unclear. Previous studies have demonstrated that α7nAChR is highly expressed on the surface of CD4+CD25+ regulatory T cells (Tregs). However, the role of Tregs in SAKI is unclear. We hypothesized that Tregs might play a role in the ameliorative effect of α7nAChR on SAKI. Hence, in this study, we determined the effects of PNU-282987 (a selective α7nAchR agonist) on SAKI and evaluated whether PNU-282987 would attenuate SAKI via regulating Tregs. Our study showed that immediate administration of PNU-282987 after CLP surgery in rats improved renal function, reduced levels of systemic inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), etc.), inflammatory cell infiltration and tubular apoptosis in renal tissues, and increased forkhead/winged helix transcription factor p3 (Foxp3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression indicating activated Tregs. Moreover, in in vitro experiments, isolated Tregs co-cultured with PNU-282987 also displayed enhanced expression of CTLA-4 and Foxp3. Furthermore, Tregs were co-cultured with PNU-282987 for 24 hours and then reinfused into rats through the tail vein immediately after CLP surgery, and a significant renal protective effect was observed 24 hours postoperatively. These results demonstrate that PNU-282987 exerts its renal protective effects on SAKI through activation of Tregs.

Structural basis of ubiquitin recognition by the winged-helix domain of Cockayne syndrome group B protein.

Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.

Functions of the TFIIE-Related Tandem Winged-Helix Domain of Rpc34 in RNA Polymerase III Initiation and Elongation.

Rpc34 is a subunit of the Rpc82/34/31 subcomplex residing on the DNA-binding cleft of RNA polymerase (Pol) III. Rpc34 contains a structurally flexible N-terminal tandem winged-helix (tWH) domain related to the TFIIE transcription factor. While the second WH (WH2) fold of the tWH domain is known to function in DNA melting activity during transcription initiation, the functional role of the WH1 fold is unknown. In this study, we generated a series of new Rpc34 tWH mutants conferring a cold-sensitive growth phenotype. We found that the tWH mutations severely compromised in vitro transcription activity due to destabilization of the preinitiation complex (PIC). Site-specific protein photo-cross-linking analysis indicated that the tWH domain persistently interacts with protein subunits of the Pol III cleft in the PIC and the ternary elongation complex (TEC). Furthermore, purified Pol III proteins with tWH mutations also showed reduced efficiency in RNA elongation. Our study results suggest that the tWH domain is an important protein module above the Pol III cleft that integrates protein and nucleic acid interactions for initiation and elongation.

[Association of ulcerative colitis with fork head/winged helix transcription factor-3 gene polymorphisms in Chinese patients].

Objective: To investigate the association of ulcerative colitis (UC) with fork head/winged helix transcription factor-3 (Foxp3) polymorphisms in Han population in Zhejiang province, China. Methods: A total of 381 UC patients and 490 healthy controls were enrolled in this study.The four single nucleotide polymorphisms (SNPs) of Foxp3 (rs3761547, rs2232365, rs2294021, rs3761548) were examined by SNaPshot.The analyses of linkage disequilibrium (LD) and haplotype were also performed in all study subjects. Results: When male and female UC patients were compared with their corresponding controls respectively, the alleles and genotypes of the four SNPs were not statistically different (all P>0.05). According to severity and location of the disease, the UC patients were divided into different subgroups. The alleles (C, G, A) of (rs2232365, rs2294021, rs3761548) were more frequent in male patients with severe UC than in the male controls (69.6% vs 34.3%, P=0.001; 69.6% vs 34.3%, P=0.001; 39.1% vs 14.4%, P=0.002, respectively). As compared with the female controls, the alleles (C, G, A) and genotypes (TC+ CC, AG+ GG, CA+ AA) of (rs2232365, rs2294021, rs3761548) were significantly increased in the female patients with severe UC (51.9% vs 38.0%, 63.5% vs 39.2%, 53.8% vs 21.4%, 80.8% vs 57.7%, 84.6% vs 58.4%, 76.9% vs 34.7%, all P<0.05). The four SNPs above were shown to be in a strong LD both in male and in female subjects.When male and female UC patients were compared with their corresponding controls respectively, nevertheless, each haplotype frequency was not statistically different (all P>0.05). Conclusions:Foxp3 (rs2232365, rs2294021, rs3761548) variations might engender the increased risk of severe UC in Chinese Han patients.

Association of the winged helix motif of the TFIIEα subunit of TFIIE with either the TFIIEß subunit or TFIIB distinguishes its functions in transcription.

In eukaryotes, the general transcription factor TFIIE consists of two subunits, α and ß, and plays essential roles in transcription. Structure-function studies indicate that TFIIE has three-winged helix (WH) motifs, with one in TFIIEα and two in TFIIEß. Recent studies suggested that, by binding to the clamp region of RNA polymerase II, TFIIEα-WH promotes the conformational change that transforms the promoter-bound inactive preinitiation complex to the active complex. Here, to elucidate its roles in transcription, functional analyses of point-mutated human TFIIEα-WH proteins were carried out. In vitro transcription analyses identified two classes of mutants. One class was defective in transcription initiation, and the other was defective in the transition from initiation to elongation. Analyses of the binding of this motif to other general transcription factors showed that the former class was defective in binding to the basic helix-loop-helix motif of TFIIEß and the latter class was defective in binding to the N-terminal cyclin homology region of TFIIB. Furthermore, TFIIEα-WH bound to the TFIIH XPB subunit at a third distinct region. Therefore, these results provide further insights into the mechanisms underlying RNA polymerase II activation at the initial stages of transcription.

Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid-virus pSSVx: a novel peculiar member of the winged helix-turn-helix transcription factor family.

The hybrid plasmid-virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix-turn-helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76-DNA complex has been built using as template a structurally related homolog.

The C-terminal domain of the transcriptional regulator BldD from Streptomyces coelicolor A3(2) constitutes a novel fold of winged-helix domains.

BldD regulates transcription of key developmental genes in Streptomyces coelicolor. While the N-terminal domain is responsible for both dimerization and DNA binding, the structural and functional roles of the C-terminal domain (CTD) remain largely unexplored. Here, the solution structure of the BldD-CTD shows a novel winged-helix domain fold not compatible with DNA binding, due to the negatively charged surface and presence of an additional helix. Meanwhile, a small elongated groove with conserved hydrophobic patches surrounded by charged residues suggests that the BldD-CTD could be involved in protein-protein interactions that provide transcriptional regulation.

The wing of a winged helix-turn-helix transcription factor organizes the active site of BirA, a bifunctional repressor/ligase.

The BirA biotin protein ligase of Escherichia coli belongs to the winged helix-turn-helix (wHTH) family of transcriptional regulators. The N-terminal BirA domain is required for both transcriptional regulation of biotin synthesis and biotin protein ligase activity. We addressed the structural and functional role of the wing of the wHTH motif in both BirA functions. A panel of N-terminal deletion mutant proteins including a discrete deletion of the wing motif were unable to bind DNA. However, all the N-terminal deletion mutants weakly complemented growth of a ΔbirA strain at low biotin concentrations, indicating compromised ligase activity. A wing domain chimera was constructed by replacing the BirA wing with the nearly isosteric wing of the E. coli OmpR transcription factor. Although this chimera BirA was defective in operator binding, it was much more efficient in complementation of a ΔbirA strain than was the wing-less protein. The enzymatic activities of the wing deletion and chimera proteins in the in vitro synthesis of biotinoyl-5'-AMP differed greatly. The wing deletion BirA accumulated an off pathway compound, ADP, whereas the chimera protein did not. Finally, we report that a single residue alteration in the wing bypasses the deleterious effects caused by mutations in the biotin-binding loop of the ligase active site. We believe that the role of the wing in the BirA enzymatic reaction is to orient the active site and thereby protect biotinoyl-5'-AMP from attack by solvent. This is the first evidence that the wing domain of a wHTH protein can play an important role in enzymatic activity.

From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins.

The winged helix domain (WHD) is a widespread nucleic-acid-binding protein structural element found in all kingdoms of life. Although the overall structure of the WHD is conserved, its functional properties and interaction profiles are extremely versatile. WHD-containing proteins can exploit nearly the full spectrum of nucleic acid structural features for recognition and even covalent modification or noncovalent rearrangement of target molecules. WHD functions range from sequence-recognizing keys in transcription factors and bulldozer-like strand-separating wedges in helicases to mediators of protein-protein interactions (PPIs). Further investigations are needed to understand the contribution of WHD structural dynamics to nucleic-acid-modifying enzymatic functions.

Structure of a novel winged-helix like domain from human NFRKB protein.

The human nuclear factor related to kappa-B-binding protein (NFRKB) is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370-495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.

Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation.

The ubiquitin-proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein-protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

Crystal structure of the N-terminal region of human Ash2L shows a winged-helix motif involved in DNA binding.

Ash2L is a core component of the MLL family histone methyltransferases and has an important role in regulating the methylation of histone H3 on lysine 4. Here, we report the crystal structure of the N-terminal domain of Ash2L and reveal a new function of Ash2L. The structure shows that Ash2L contains an atypical PHD finger that does not have histone tail-binding activity. Unexpectedly, the structure shows a previously unrecognized winged-helix motif that directly binds to DNA. The DNA-binding-deficient mutants of Ash2L reduced Ash2L localization to the HOX locus. Strikingly, a single mutation in Ash2L(WH) (K131A) breaks the chromatin domain boundary, suggesting that Ash2L also has a role in chromosome demarcation.

Solution structure of the human HSPC280 protein.

The human HSPC280 protein belongs to a new family of low molecular weight proteins, which is only present in eukaryotes, and is absent in fungi. The solution structure of HSPC280 was determined using multidimensional NMR spectroscopy. The overall structure consists of three α-helices and four antiparallel ß-strands and has a winged helix-like fold. However, HEPC280 is not a typical DNA-binding winged helix protein in that it lacks DNA-binding activity. Unlike most winged-helix proteins, HSPC280 has an unusually long 13-residue (P62-V74) wing 1 loop connecting the ß3 and ß4 strands of the protein. Molecules of HSPC280 have a positively charged surface on one side and a negatively charged surface on the other side of the protein structure. Comparisons with the C-terminal 80-residue domain of proteins in the Abra family reveal a conserved hydrophobic groove in the HSPC280 family, which may allow HSPC280 to interact with other proteins.

Structure of the LexA-DNA complex and implications for SOS box measurement.

The eubacterial SOS system is a paradigm of cellular DNA damage and repair, and its activation can contribute to antibiotic resistance. Under normal conditions, LexA represses the transcription of many DNA repair proteins by binding to SOS 'boxes' in their operators. Under genotoxic stress, accumulating complexes of RecA, ATP and single-stranded DNA (ssDNA) activate LexA for autocleavage. To address how LexA recognizes its binding sites, we determined three crystal structures of Escherichia coli LexA in complex with SOS boxes. Here we report the structure of these LexA-DNA complexes. The DNA-binding domains of the LexA dimer interact with the DNA in the classical fashion of a winged helix-turn-helix motif. However, the wings of these two DNA-binding domains bind to the same minor groove of the DNA. These wing-wing contacts may explain why the spacing between the two half-sites of E. coli SOS boxes is invariant.

Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development.

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

Functional mode of FoxD1/CBF2 for the establishment of temporal retinal specificity in the developing chick retina.

Two winged-helix transcription factors, FoxG1 (previously called chick brain factor1, CBF1) and FoxD1 (chick brain factor2, CBF2), are expressed specifically in the nasal and temporal regions of the developing chick retina, respectively. We previously demonstrated that FoxG1 controls the expression of topographic molecules including FoxD1, and determines the regional specificity of the nasal retina. FoxD1 is known to prescribe temporal specificity, however, molecular mechanisms and downstream targets have not been elucidated. Here we addressed the genetic mechanisms for establishing temporal specificity in the developing retina using an in ovo electroporation technique. Fibroblast growth factor (Fgf) and Wnt first play pivotal roles in inducing the region-specific expression of FoxG1 and FoxD1 in the optic vesicle. Misexpression of FoxD1 represses the expression of FoxG1, GH6, SOHo1, and ephrin-A5, and induces that of EphA3 in the retina. GH6 and SOHo1 repress the expression of FoxD1. In contrast to the inhibitory effect of FoxG1 on bone morphogenic protein (BMP) signaling, FoxD1 does not alter the expression of BMP4 or BMP2. Studies with chimeric mutants of FoxD1 showed that FoxD1 acts as a transcription repressor in controlling its downstream targets in the retina. Taken together with previous findings, our data suggest that FoxG1 and FoxD1 are located at the top of the gene cascade for regional specification along the nasotemporal (anteroposterior) axis in the retina, and FoxD1 determines temporal specificity.

The copper-responsive repressor CopR of Lactococcus lactis is a 'winged helix' protein.

CopR of Lactococcus lactis is a copper-responsive repressor involved in copper homoeostasis. It controls the expression of a total of 11 genes, the CopR regulon, in a copper-dependent manner. In the absence of copper, CopR binds to the promoters of the CopR regulon. Copper releases CopR from the promoters, allowing transcription of the downstream genes to proceed. CopR binds through its N-terminal domain to a 'cop box' of consensus TACANNTGTA, which is conserved in Firmicutes. We have solved the NMR solution structure of the N-terminal DNA-binding domain of CopR. The protein fold has a winged helix structure resembling that of the BlaI repressor which regulates antibiotic resistance in Bacillus licheniformis. CopR differs from other copper-responsive repressors, and the present structure represents a novel family of copper regulators, which we propose to call the CopY family.