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.

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.

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.

Son of Notch, a winged-helix gene involved in boundary formation in the Drosophila wing.

A P element enhancer trap screen was conducted to identify genes involved in dorsal-ventral boundary formation in Drosophila. The son of Notch (son) gene was identified by the son(2205) enhancer trap insertion, which is a partial loss-of-function mutation. Based on son(2205) mutant phenotypes and genetic interactions with Notch and wingless mutations, we conclude that son participates in wing development, and functions in the Notch signaling pathway at the dorsal-ventral boundary in the wing. Notch signaling pathway components activate son enhancer trap expression in wing cells. son enhancer trap expression is regulated positively by wingless, and negatively by cut in boundary cells. Ectopic Son protein induces wingless and cut expression in wing discs. We hypothesize that there is positive feedback regulation of son by wingless, and negative regulation by cut at the dorsal-ventral boundary during wing development.

The carboxyl-terminal nucleoplasmic region of MAN1 exhibits a DNA binding winged helix domain.

MAN1 is an integral protein of the inner nuclear membrane that interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2, and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1-R-Smads interaction, thus perturbing transforming growth factor beta superfamily signaling pathway. As a first step to understanding the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences.