Morphological features of the non-affected side of the hindfoot in patients with unilateral varus ankle osteoarthritis.

BACKGROUND: The innate shape characteristics of the hindfoot bones alter the loading conditions of the foot and thus may be associated with an increased risk of developing varus ankle osteoarthritis (OA). This study aimed to clarify the innate morphological patterns of the hindfoot bones that may be associated with ankle OA by analyzing the differences between the bone morphology of the non-affected side of patients with unilateral varus ankle OA and that of healthy participants. METHODS: In this case-control study, computed tomography images were used to develop three-dimensional models of three hindfoot bones (distal tibia with fibula, talus, and calcaneus) from 23 non-affected sides of patients with radiography-diagnosed unilateral ankle OA and 22 healthy control participants. Anatomical and sliding landmarks were placed on the surface of each bone, and the principal components (PCs) of shape variation among specimens were independently calculated for each bone, preserving homology between individuals. The PC modes representing 5% or more of the overall variation were statistically compared between the ankle OA and control groups. RESULTS: Significant differences were identified between the OA and control groups in the fifth PC mode for the tibia with fibula (proportion of variance, 5.1%; p =.025), fifth PC mode for the talus (6.7%, p =.031), and third PC mode for the calcaneus (7.4%, p =.001). The hindfoot bones of the participants who developed ankle OA had the following innate morphological characteristics: the lateral malleolar articular surface of the fibula was shifted superiorly, tibial plafond was enlarged posteroinferiorly, posterior width of the talar trochlea was narrower, talonavicular articular surface of the talus was oriented more frontally, anterior-middle talocalcaneal articular surfaces of the talus were more medially shifted and those of the calcaneus were flatter, calcaneal sustentaculum tali was less protruding, and lateral plantar process of the calcaneus was more superiorly positioned. CONCLUSIONS: These distinctive morphological alterations may increase the incidence and progression of varus ankle OA through aberrant anterior translation, internal rotation, and varus tilting of the talus.

Taf1 N-terminal domain 2 (TAND2) of TFIID promotes formation of stable and mobile unstable TBP-TATA complexes.

In eukaryotes, TATA-binding protein (TBP) occupancy of the core promoter globally correlates with transcriptional activity of class II genes. Elucidating how TBP is delivered to the TATA box or TATA-like element is crucial to understand the mechanisms of transcriptional regulation. A previous study demonstrated that the inhibitory DNA binding (IDB) surface of human TBP plays an indispensable role during the two-step formation of the TBP-TATA complex, first assuming an unstable and unbent intermediate conformation, and subsequently converting slowly to a stable and bent conformation. The DNA binding property of TBP is altered by physical contact of this surface with TBP regulators. In the present study, we examined whether the interaction between Taf1 N-terminal domain 2 (TAND2) and the IDB surface affected DNA binding property of yeast TBP by exploiting TAND2-fused TBP derivatives. TAND2 promoted formation of two distinct types of TBP-TATA complexes, which we arbitrarily designated as complex I and II. While complex I was stable and similar to the well-characterized original TBP-TATA complex, complex II was unstable and moved along DNA. Removal of TAND2 from TBP after complex formation revealed that continuous contact of TAND2 with the IDB surface was required for formation of complex II but not complex I. Further, TFIIA could be incorporated into the complex of TAND2-fused TBP and the TATA box, which was dependent on the amino-terminal non-conserved region of TBP, implying that this region could facilitate the exchange between TAND2 and TFIIA on the IDB surface. Collectively, these findings provide novel insights into the mechanism by which TBP is relieved from the interaction with TAND to bind the TATA box or TATA-like element within promoter-bound TFIID.

TFIID dependency of steady-state mRNA transcription altered epigenetically by simultaneous functional loss of Taf1 and Spt3 is Hsp104-dependent.

In Saccharomyces cerevisiae, class II gene promoters have been divided into two subclasses, TFIID- and SAGA-dominated promoters or TFIID-dependent and coactivator-redundant promoters, depending on the experimental methods used to measure mRNA levels. A prior study demonstrated that Spt3, a TBP-delivering subunit of SAGA, functionally regulates the PGK1 promoter via two mechanisms: by stimulating TATA box-dependent transcriptional activity and conferring Taf1/TFIID independence. However, only the former could be restored by plasmid-borne SPT3. In the present study, we sought to determine why ectopically expressed SPT3 is unable to restore Taf1/TFIID independence to the PGK1 promoter, identifying that this function was dependent on the construction protocol for the SPT3 taf1 strain. Specifically, simultaneous functional loss of Spt3 and Taf1 during strain construction was a prerequisite to render the PGK1 promoter Taf1/TFIID-dependent in this strain. Intriguingly, genetic approaches revealed that an as-yet unidentified trans-acting factor reprogrammed the transcriptional mode of the PGK1 promoter from the Taf1/TFIID-independent state to the Taf1/TFIID-dependent state. This factor was generated in the haploid SPT3 taf1 strain in an Hsp104-dependent manner and inherited meiotically in a non-Mendelian fashion. Furthermore, RNA-seq analyses demonstrated that this factor likely affects the transcription mode of not only the PGK1 promoter, but also of many other class II gene promoters. Collectively, these findings suggest that a prion or biomolecular condensate is generated in a Hsp104-dependent manner upon simultaneous functional loss of TFIID and SAGA, and could alter the roles of these transcription complexes on a wide variety of class II gene promoters without altering their primary sequences. Therefore, these findings could provide the first evidence that TFIID dependence of class II gene transcription can be altered epigenetically, at least in Saccharomyces cerevisiae.

Anatomical Tenodesis Reconstruction Using Free Split Peroneal Brevis Tendon for Severe Chronic Lateral Ankle Instability.

Many operative procedures have been reported for the management of chronic lateral ankle instability, and anatomical reconstructions are an excellent option. However, if the remnants of the ligaments are considerably damaged, anatomical reconstructions using such remnants can be difficult. In cases such as these, tenodesis stabilization may be required. However, tenodesis stabilization often restricts the range of ankle movement. The purpose of this study was to determine the effectiveness of a new procedure that we developed to mitigate the problems associated with tenodesis stabilization procedures. We installed grafts in the original anatomical position by devising a system for positioning the drill holes in the bones so that our procedure did not restrict the range of ankle movement. A retrospective review of 37 patients (13 men, 24 women) with a mean age of 30.2 (range, 16-66) years was performed at an average of 69 (range, 47-77) months after the surgery. The average American Orthopaedic Foot and Ankle Society ankle-hindfoot score improved significantly from 65.6 (range, 47-77) points preoperatively to 98.0 (range, 87-100) points postoperatively (P < 0.001). With the number of subjects available, no significant differences were detected between the postoperative mean ranges of movement of the ankle and subtalar joints and those of the preoperative ankle. Patients who underwent anatomical tenodesis reconstructions with a free split peroneal brevis tendon showed good outcomes after a 69-month follow-up period.

Visualization and quantification of the degenerative pattern of the distal tibia and fibula in unilateral varus ankle osteoarthritis.

The present study aimed to quantify and visualize the degenerative patterns of the distal tibia and fibula due to ankle osteoarthritis (OA). We analyzed differences in tibial and fibular surface deviation between sides of patients with unilateral varus ankle OA (medial talar tilt > 4°) by registering each surface model to the mirror image of corresponding bone. Computed tomography images of both feet of 33 patients (OA: 22, control: 11) were examined. Statistically significant surface depression of approximately 2.5 mm on the anterior articular surface of the medial malleolus, and surface elevation of approximately 1 mm on the anterodistal edge of the tibiofibular joint and the lateral malleolus were observed in OA patients. These bone degenerations were found to be correlated with those on the other side of the ankle joint, the medial margin of the talar trochlea and the lateral articular surface of the talus, respectively. In contrast, the amount of bone depression on the plafond was smaller than previously anticipated. Such quantitative information about stereotypical patterns of bone degeneration in ankle OA would contribute to better understanding of the development of ankle OA and possible therapeutic interventions.

The function of Spt3, a subunit of the SAGA complex, in PGK1 transcription is restored only partially when reintroduced by plasmid into taf1 spt3 double mutant yeast strains.

In Saccharomyces cerevisiae, class II gene promoters contain two classes of TATA elements: the TATA box and the TATA-like element. Functional loss of TFIID and SAGA transcription complexes selectively impacts steady-state mRNA levels expressed from TATA-like element-containing (i.e., TATA-less) and TATA box-containing promoters, respectively. While nascent RNA analysis has revealed that TFIID and SAGA are required for both types of promoters, the division of their roles remains unclear. We show here that transcription from the PGK1 promoter decreased in some cases by more than half after disruption of the TATA box or SPT3 (spt3Δ), whereas spt3Δ did not affect transcription from the TATA-less promoter, consistent with the prevailing view that Spt3 functions specifically in a TATA box-dependent manner. Transcription from this promoter was abolished in the spt3Δ taf1-N568Δ strain but unaffected in the taf1-N568Δ strain, regardless of TATA box presence, suggesting that TFIID was functionally dispensable for PGK1 transcription at least in the SPT3 strain. Furthermore, transcription from the TATA box-containing PGK1 promoter was slightly reduced in the taf1 strain lacking TAND (taf1-ΔTAND) upon temperature shift from 25 to 37 ℃, with restoration to normal levels within 2 h, in an Spt3-dependent manner. Interestingly, when SPT3 was reintroduced into the spt3Δ TAF1, spt3Δ taf1-N568Δ or spt3Δ taf1-ΔTAND strain, TATA box-dependent transcription from this promoter was largely restored, but TFIID independence in transcription was not restored, especially from the TATA-less promoter, and transient TAND/Spt3-dependent fluctuations of transcription after the temperature shift were also not recapitulated. Collectively, these observations suggest that Spt3 has multiple functions in PGK1 transcription, some of which may be intimately connected to Taf1 function and may therefore become unrestorable when the TFIID and SAGA functions are simultaneously compromised.

Fpr1, a primary target of rapamycin, functions as a transcription factor for ribosomal protein genes cooperatively with Hmo1 in Saccharomyces cerevisiae.

Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1Δ (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1Δfpr1Δ cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.

Author Correction: Visualization and quantification of the degenerative pattern of the talus in unilateral varus ankle osteoarthritis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Three-Dimensional Analysis of the First Metatarsal Bone in Minimally Invasive Distal Linear Metatarsal Osteotomy for Hallux Valgus.

BACKGROUND: Modified Bösch osteotomy (distal linear metatarsal osteotomy [DLMO]) is one of the minimally invasive correctional surgeries for hallux valgus. The 3-dimensional correctional angles and distances of the first metatarsal bone in DLMO have not been clarified. The purpose of this study was to analyze the 3-dimensional postoperative morphological changes of the first metatarsal bone in DLMO. METHODS: Twenty patients (30 feet) who underwent DLMO were enrolled. Preoperative plain radiographs and computed tomography (CT) scans of the feet were examined. Postoperative radiographs and CT scans were also obtained after bone union. The surface data of the pre- and postoperative first metatarsals were reconstructed from the CT data. The positions of the distal ends of the first metatarsals described with respect to the proximal ends were calculated using CT surface-matching technique. RESULTS: The distal end of the first metatarsal after DLMO was significantly supinated (10.2 ± 6.0 degrees, P < .001), adducted (6.0 ± 11.8 degrees, P = .004), dorsiflexed (11.1 ± 10.9, P < .001), shortened (7.4 ± 2.5 mm, P < .001), elevated (2.3 ± 3.1 mm, P = .001), and laterally shifted (8.2 ± 3.0 mm, P < .001) compared to the preoperative metatarsal distal end. Supination correction demonstrated a significant correlation with adduction correction (r = 0.659, P < .001) on correlation analyses between these parameters. CONCLUSION: The 3-dimensional corrections of the first metatarsal bone after DLMO were evaluated. Pronation and abduction were successfully corrected. Furthermore, adduction correction might be an important factor affecting correction of pronation. LEVEL OF EVIDENCE: Level IV, retrospective case series.

Visualization and quantification of the degenerative pattern of the talus in unilateral varus ankle osteoarthritis.

The aim of this study was to quantify and visualize the degenerative patterns of the talus in ankle osteoarthritis (OA). The differences in talar morphology between sides of patients with unilateral varus ankle OA (medial talar tilt > 4°) were compared. Computed tomography images of both feet of 35 patients (OA: 22 patients, control: 13 patients) were analyzed. Each surface model of the right and left tali was registered to the opposite talus via a mirror-image technique and an iterative closest point algorithm. The surface deviation between the two models was quantified and visualized by deviation color maps. The results quantitatively demonstrated that osteophytes are generated in the area under the antero-medial margin of the trochlea in OA tali. In severe OA tali, bone resorption of more than 2 mm in the medial portion of the trochlea, as well as a similar degree of osteophyte formation on the lateral surface, was also seen. Stereotypical patterns of degeneration occurring in OA tali were successfully visualized and quantified by left-right comparison of patients with unilateral ankle OA. Such information would contribute to better understanding of the development of ankle OA and preoperative planning of total ankle arthroplasty and arthrodesis.

Multiple direct interactions of TBP with the MYC oncoprotein.

Transcription factor c-MYC is a potent oncoprotein; however, the mechanism of transcriptional regulation via MYC-protein interactions remains poorly understood. The TATA-binding protein (TBP) is an essential component of the transcription initiation complex TFIID and is required for gene expression. We identify two discrete regions mediating MYC-TBP interactions using structural, biochemical and cellular approaches. A 2.4 -Å resolution crystal structure reveals that human MYC amino acids 98-111 interact with TBP in the presence of the amino-terminal domain 1 of TBP-associated factor 1 (TAF1TAND1). Using biochemical approaches, we have shown that MYC amino acids 115-124 also interact with TBP independently of TAF1TAND1. Modeling reveals that this region of MYC resembles a TBP anchor motif found in factors that regulate TBP promoter loading. Site-specific MYC mutants that abrogate MYC-TBP interaction compromise MYC activity. We propose that MYC-TBP interactions propagate transcription by modulating the energetic landscape of transcription initiation complex assembly.

Transcriptional activation is weakened when Taf1p N-terminal domain 1 is substituted with its Drosophila counterpart in yeast TFIID.

Transcription factor II D (TFIID), a multiprotein complex consisting of TATA-binding protein (TBP) and 13-14 TBP-associated factors (Tafs), plays a central role in transcription and regulates nearly all class II genes. The N-terminal domain of Taf1p (TAND) can be divided into two subdomains, TAND1 and TAND2, which bind to the concave and convex surfaces of TBP, respectively. The interaction between TAND and TBP is thought to be regulated by TFIIA, activators and/or DNA during transcriptional activation, as the TAND1-bound form of TBP cannot bind to the TATA box. We previously demonstrated that Drosophila TAND1 binds to TBP with a much stronger affinity than yeast TAND1 and that the expression levels of full-length chimeric Taf1p, whose TAND1 is replaced with the Drosophila counterpart, can be varied in vivo by substituting several methionine residues downstream of TAND2 with alanine residues in various combinations. In this study, we examined the transcriptional activation of the GAL1-lacZ reporter or endogenous genes such as RNR3 or GAL1 in yeast cells expressing various levels of full-length chimeric Taf1p. The results showed that the substitution of TAND1 with the Drosophila counterpart in yeast TFIID weakened the transcriptional activation of GAL1-lacZ and RNR3 but not that of GAL1. These findings strongly support a model in which TBP must be released efficiently from TAND1 within TFIID upon transcriptional activation.

Minimally invasive distal linear metatarsal osteotomy combined with selective release of lateral soft tissue for severe hallux valgus.

BACKGROUND: Minimally invasive techniques for hallux valgus have been widely used to treat mild to moderate hallux valgus deformities. The purpose of this study was to evaluate the clinical and radiographic outcomes of distal linear metatarsal osteotomy (DLMO), which is one of the minimally invasive techniques, for severe hallux valgus. METHODS: 95 patients (141 feet) with severe hallux valgus underwent DLMOs. Lateral soft tissue release (LSTR) was performed at the same time for the cases selected by an original manual test. The satisfaction level, the Japanese Society of Surgery of the Foot (JSSF) hallux scale score, and weight-bearing radiographs of the foot were assessed preoperatively and after more than 24 months. In addition, the clinical and radiographic outcomes were compared among three groups divided by the kind of LSTR: no LSTR; manual correction; and open release through skin incision. RESULTS: Although the first metatarsal bone was significantly shortened, dorsiflexed, and elevated on postoperative radiographs, the rate of satisfaction was 87.2% (123/141), and the mean JSSF hallux scale score improved significantly from 60.4 (44-73) to 90.4 (65-100). The mean hallux valgus and intermetatarsal angles also improved significantly from 45.5° (40.0-60.0°) to 10.3° (-28.0-40.9°) and from 19.9° (14.0-28.7°) to 8.3° (-1.6-18.5°), respectively. Delayed union (18 feet), metatarsalgia (16 feet), recurrence (22 feet), and hallux varus (22 feet) were observed, and they were more obvious in DLMO combined with open release through a skin incision. CONCLUSIONS: DLMO combined selectively with LSTR is an effective procedure for correcting severe hallux valgus. However, the indication for open release with DLMO should be considered carefully.

SAGA mediates transcription from the TATA-like element independently of Taf1p/TFIID but dependent on core promoter structures in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, core promoters of class II genes contain a TATA element, either a TATA box (TATA[A/T]A[A/T][A/G]) or TATA-like element (1 or 2 bp mismatched version of the TATA box). The TATA element directs the assembly of the preinitiation complex (PIC) to ensure accurate transcriptional initiation. It has been proposed the PIC is assembled by two distinct pathways in which TBP is delivered by TFIID or SAGA, leading to the widely accepted model that these complexes mediate transcription mainly from TATA-like element- or TATA box-containing promoters, respectively. Although both complexes are involved in transcription of nearly all class II genes, it remains unclear how efficiently SAGA mediates transcription from TATA-like element-containing promoters independently of TFIID. We found that transcription from the TATA box-containing AGP1 promoter was greatly stimulated in a Spt3p-dependent manner after inactivation of Taf1p/TFIID. Thus, this promoter provides a novel experimental system in which to evaluate SAGA-mediated transcription from TATA-like element(s). We quantitatively measured transcription from various TATA-like elements in the Taf1p-dependent CYC1 promoter and Taf1p-independent AGP1 promoter. The results revealed that SAGA could mediate transcription from at least some TATA-like elements independently of Taf1p/TFIID, and that Taf1p-dependence or -independence is highly robust with respect to variation of the TATA sequence. Furthermore, chimeric promoter mapping revealed that Taf1p-dependence or independence was conferred by the upstream activating sequence (UAS), whereas Spt3p-dependent transcriptional stimulation after inactivation of Taf1p/TFIID was specific to the AGP1 promoter and dependent on core promoter regions other than the TATA box. These results suggest that TFIID and/or SAGA are regulated in two steps: the UAS first specifies TFIID or SAGA as the predominant factor on a given promoter, and then the core promoter structure guides the pertinent factor to conduct transcription in an appropriate manner.

Etiological factors in hallux valgus, a three-dimensional analysis of the first metatarsal.

BACKGROUND: It has been reported that hallux valgus (HV) is associated with axial rotation of the first metatarsal (1MT). However, the association between HV and torsion of the 1MT head with respect to the base has not been previously investigated. The present study examined whether there was a significant difference in 1MT torsion between HV and control groups. METHODS: Three-dimensional (3D) computed tomography (CT) scans of 39 ft were obtained, and 3D surface models of the 1MT were generated to quantify the torsion of the head with respect to the base. The HV group consisted of 27 ft from 27 women (69.5 ± 7.5 years old). Only the feet of HV patients with an HV angle >20° on weight-bearing radiography were selected for analysis. The control group consisted of 12 ft from 12 women (67.7 ± 7.2 years old). In a virtual 3D space, two unit vectors, which describe the orientation of the 1MT head and base, were calculated. The angle formed by these two unit vectors representing 1MT torsion was compared between the control and hallux valgus groups. RESULTS: The mean (± standard deviation) of the torsional angle of the 1MT was 17.6 (± 7.7)° and 4.7 (± 4.0)° in the HV and control groups, respectively, and the difference was significant (p < 0.01). CONCLUSIONS: This is the first study, to the best of our knowledge, to investigate 1MT torsion in HV patients using CT-based 3D analysis. The 1MT showed significant eversion in hallux valgus patients compared to control group patients.

Radiographic Shape of Foot With Second Metatarsophalangeal Joint Dislocation Associated With Hallux Valgus.

BACKGROUND: Second metatarsophalangeal (MTP) joint dislocation is associated with hallux valgus, and the treatment of complete dislocation can be difficult. The purpose of this study was to radiographically clarify the characteristic foot shape in the presence of second MTP joint dislocation. METHODS: Weight-bearing foot radiographs of the 268 patients (358 feet) with hallux valgus were examined. They were divided into 2 groups: those with second MTP joint dislocation (study group = 179 feet) and those without dislocation (control group = 179 feet). Parameters measured included the hallux valgus angle (HVA), first-second intermetatarsal angle (IMA), second MTP joint angle, hallux interphalangeal angle (IPA), second metatarsal protrusion distance (MPD), metatarsus adductus angle (MAA), and the second metatarsal declination angle (2MDA). Furthermore, the dislocation group was divided into 3 subgroups according to second toe deviation direction: group M (medial type), group N (neutral type), and group L (lateral type). RESULTS: The IPA and the 2MDA were significantly greater in the study group than in the control group. By multiple comparison analysis, the IMA was greatest in group M and smallest in group L. The IPA was smaller and 2MDA greater in group N than in group L. The HVA and MAA in group L were greatest, and MPD in group L was smallest. CONCLUSIONS: The patients with second MTP joint dislocation associated with hallux valgus had greater hallux interphalangeal joint varus and a second metatarsal more inclined than with hallux valgus alone. The second toe deviated in a different direction according to the foot shape. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Age- and sex-associated morphological variations of metatarsal torsional patterns in humans.

It has been demonstrated that the torsional patterns of the metatarsal heads are associated with the presence or absence of the medial longitudinal arch in hominoid feet. The relatively untwisted second metatarsal is unique in humans, but that of the African apes is much more inverted, suggesting that the torsion of the second metatarsal might represent the overall shape and flatness of the foot. Some clinical studies have recently argued that the onset of foot pathologies such as hallux valgus might be related to the torsional pattern of the metatarsals. However, to date, no studies have systematically investigated the morphological variations of the torsional patterns of human metatarsals. In this study, therefore, the aim was to clarify the age- and sex-associated variations in the torsional patterns of human metatarsals using three-dimensional computed tomography. The torsion angles of the five metatarsals were calculated by defining the dorsopalmar vector of the metatarsal base and the vector corresponding to the rotational axis of the metatarsal head. The present result demonstrated that the second metatarsals of females were significantly more inverted with increasing age. Flat foot is known to be most common in elderly women. Whether there is a cause-effect relationship between second metatarsal torsion and flattening of the medial longitudinal arch has yet to be answered, but this study suggested that torsion of the second metatarsal might possibly be used as an indicator for the early diagnosis of flat foot and associated foot pathologies. Clin. Anat. 30:1058-1063, 2017. © 2017 Wiley Periodicals, Inc.

Correction: A Novel Quantitative Hemolytic Assay Coupled with Restriction Fragment Length Polymorphisms Analysis Enabled Early Diagnosis of Atypical Hemolytic Uremic Syndrome and Identified Unique Predisposing Mutations in Japan.

[This corrects the article DOI: 10.1371/journal.pone.0124655.].

Oligomerization of Hmo1 mediated by box A is essential for DNA binding in vitro and in vivo.

Hmo1, a member of HMGB family proteins in Saccharomyces cerevisiae, binds to and regulates the transcription of genes encoding ribosomal RNA and ribosomal proteins. The functional motifs of Hmo1 include two HMG-like motifs, box A and box B, and a C-terminal tail. To elucidate the molecular roles of the HMG-like boxes in DNA binding in vivo, we analyzed the DNA-binding activity of various Hmo1 mutants using ChIP or reporter assays that enabled us to conveniently detect Hmo1 binding to the promoter of RPS5, a major target gene of Hmo1. Our mutational analyses showed that box B is a bona fide DNA-binding motif and that it also plays other important roles in cell growth. However, box A, especially its first α-helix, contributes to DNA binding of Hmo1 by inducing self-assembly of Hmo1. Intriguingly, box A mediated formation of oligomers of more than two proteins on DNA in vivo. Furthermore, duplication of the box B partially alleviates the requirement for box A. These findings suggest that the principal role of box A is to assemble multiple box B in the appropriate orientation, thereby stabilizing the binding of Hmo1 to DNA and nucleating specific chromosomal architecture on its target genes.

A Random Screen Using a Novel Reporter Assay System Reveals a Set of Sequences That Are Preferred as the TATA or TATA-Like Elements in the CYC1 Promoter of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the core promoters of class II genes contain either TATA or TATA-like elements to direct accurate transcriptional initiation. Genome-wide analyses show that the consensus sequence of the TATA element is TATAWAWR (8 bp), whereas TATA-like elements carry one or two mismatches to this consensus. The fact that several functionally distinct TATA sequences have been identified indicates that these elements may function, at least to some extent, in a gene-specific manner. The purpose of the present study was to identify functional TATA sequences enriched in one particular core promoter and compare them with the TATA or TATA-like elements that serve as the pre-initiation complex (PIC) assembly sites on the yeast genome. For this purpose, we conducted a randomized screen of the TATA element in the CYC1 promoter by using a novel reporter assay system and identified several hundreds of unique sequences that were tentatively classified into nine groups. The results indicated that the 7 bp TATA element (i.e., TATAWAD) and several sets of TATA-like sequences are preferred specifically by this promoter. Furthermore, we find that the most frequently isolated TATA-like sequence, i.e., TATTTAAA, is actually utilized as a functional core promoter element for the endogenous genes, e.g., ADE5,7 and ADE6. Collectively, these results indicate that the sequence requirements for the functional TATA or TATA-like elements in one particular core promoter are not as stringent. However, the variation of these sequences differs significantly from that of the PIC assembly sites on the genome, presumably depending on promoter structures and reflecting the gene-specific function of these sequences.