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.

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.

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.

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.

Both HMG boxes in Hmo1 are essential for DNA binding in vitro and in vivo.

Hmo1, a member of the high mobility group B family proteins in Saccharomyces cerevisiae, associates with the promoters of ribosomal protein genes (RPGs) to direct accurate transcriptional initiation. Here, to identify factors involved in the binding of Hmo1 to its targets and the mechanism of Hmo1-dependent transcriptional initiation, we developed a novel reporter system using the promoter of the RPG RPS5. A genetic screen did not identify any factors that influence Hmo1 binding, but did identify a number of mutations in Hmo1 that impair its DNA binding activity in vivo and in vitro. These results suggest that Hmo1 binds to its target promoters autonomously without any aid of additional factors. Furthermore, characterization of Hmo1 mutants showed that the box A domain plays a pivotal role in DNA binding and may be required for the recognition of structural properties of target promoters that occur in native chromatin.

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.

Highly redundant function of multiple AT-rich sequences as core promoter elements in the TATA-less RPS5 promoter of Saccharomyces cerevisiae.

In eukaryotes, protein-coding genes are transcribed by RNA polymerase II (pol II) together with general transcription factors (GTFs). TFIID, the largest GTF composed of TATA element-binding protein (TBP) and 14 TBP-associated factors (TAFs), plays a critical role in transcription from TATA-less promoters. In metazoans, several core promoter elements other than the TATA element are thought to be recognition sites for TFIID. However, it is unclear whether functionally homologous elements also exist in TATA-less promoters in Saccharomyces cerevisiae. Here, we identify the cis-elements required to support normal levels of transcription and accurate initiation from sites within the TATA-less and TFIID-dependent RPS5 core promoter. Systematic mutational analyses show that multiple AT-rich sequences are required for these activities and appear to function as recognition sites for TFIID. A single copy of these sequences can support accurate initiation from the endogenous promoter, indicating that they carry highly redundant functions. These results show a novel architecture of yeast TATA-less promoters and support a model in which pol II scans DNA downstream from a recruited site, while searching for appropriate initiation site(s).

Hmo1 directs pre-initiation complex assembly to an appropriate site on its target gene promoters by masking a nucleosome-free region.

Saccharomyces cerevisiae Hmo1 binds to the promoters of ∼ 70% of ribosomal protein genes (RPGs) at high occupancy, but is observed at lower occupancy on the remaining RPG promoters. In Δhmo1 cells, the transcription start site (TSS) of the Hmo1-enriched RPS5 promoter shifted upstream, while the TSS of the Hmo1-limited RPL10 promoter did not shift. Analyses of chimeric RPS5/RPL10 promoters revealed a region between the RPS5 upstream activating sequence (UAS) and core promoter, termed the intervening region (IVR), responsible for strong Hmo1 binding and an upstream TSS shift in Δhmo1 cells. Chromatin immunoprecipitation analyses showed that the RPS5-IVR resides within a nucleosome-free region and that pre-initiation complex (PIC) assembly occurs at a site between the IVR and a nucleosome overlapping the TSS (+1 nucleosome). The PIC assembly site was shifted upstream in Δhmo1 cells on this promoter, indicating that Hmo1 normally masks the RPS5-IVR to prevent PIC assembly at inappropriate site(s). This novel mechanism ensures accurate transcriptional initiation by delineating the 5'- and 3'-boundaries of the PIC assembly zone.

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.

Identification and recombinant analysis of botrocetin-2, a snake venom cofactor for von Willebrand factor-induced platelet agglutination.

Botrocetin is a heterodimer snake venom protein that induces von Willebrand factor (VWF)- and platelet glycoprotein Ib (GPIb)-dependent platelet agglutination in vitro. We have cloned cDNAs for a botrocetin-2 from a cDNA library of the venom gland of Bothrops jararaca having a high similarity with botrocetin subunits. Recombinant botrocetin-2, expressed in 293T cells, showed cofactor activity comparable to natural botrocetin. In a single subunit expression experiment, a dimer of the ß subunit was obtained, and it showed reduced, but apparent, platelet agglutination activity. Ala scanning mutagenesis showed that substitutions at Asp62, Asp70, Arg115, or Lys117 in the ß subunit reduced platelet agglutination activity. The 3D homology modeling of botrocetin-2 complexed with the VWF A1 domain and GPIbα indicated that Asp62, Arg115, and Lys117 of the ß subunit are located near Arg218 and Asp222 of GPIbα, respectively, and that Aspß70 is in proximity to Gln1391 of the A1 domain. Our results indicate that these charged amino acid residues in the ß subunit have a preferential role in the activity of botrocetin-2. Since it has been time-consuming and difficult to obtain homogeneous botrocetin from natural venom, recombinant botrocetin-2 has potential benefits for clinical and basic investigations into hemostasis and thrombosis as a standard reagent.

Genome-wide localization analysis of a complete set of Tafs reveals a specific effect of the taf1 mutation on Taf2 occupancy and provides indirect evidence for different TFIID conformations at different promoters.

In Saccharomyces cerevisiae, TFIID and SAGA principally mediate transcription of constitutive housekeeping genes and stress-inducible genes, respectively, by delivering TBP to the core promoter. Both are multi-protein complexes composed of 15 and 20 subunits, respectively, five of which are common and which may constitute a core sub-module in each complex. Although genome-wide gene expression studies have been conducted extensively in several TFIID and/or SAGA mutants, there are only a limited number of studies investigating genome-wide localization of the components of these two complexes. Specifically, there are no previous reports on localization of a complete set of Tafs and the effects of taf mutations on localization. Here, we examine the localization profiles of a complete set of Tafs, Gcn5, Bur6/Ncb2, Sua7, Tfa2, Tfg1, Tfb3 and Rpb1, on chromosomes III, IV and V by chromatin immunoprecipitation (ChIP)-chip analysis in wild-type and taf1-T657K mutant strains. In addition, we conducted conventional and sequential ChIP analysis of several ribosomal protein genes (RPGs) and non-RPGs. Intriguingly, the results revealed a novel relationship between TFIIB and NC2, simultaneous co-localization of SAGA and TFIID on RPG promoters, specific effects of taf1 mutation on Taf2 occupancy, and an indirect evidence for the existence of different TFIID conformations.

Effect of the posterior tibial and peroneal longus on the mechanical properties of the foot arch.

BACKGROUND: The mechanical properties of the foot are controlled by many structures including muscles, tendons, ligaments, tarsal joints and bones. Among them, muscles make the dynamic changes of foot alignment, especially the posterior tibial (PT) and peroneal longus (PL) which contribute to maintaining the foot arch. The purpose of this study was to quantify the effect of PT and PL on the foot mechanical properties. METHODS: The mechanical properties with a longitudinal load to the tibia was measured in eight cadaveric feet. The measurement was carried out with absence of tendon traction (control), the presence of isolated traction of each tendon of the PT or PL, and finally after simultaneous traction of both tendons. RESULTS: The bone displacement significantly decreased with tendon traction. The stiffness significantly increased with PT traction compared to control, and significantly decreased with PL traction and with traction of both tendons. Among the four testing conditions, the energy during loading was least with isolated PT traction. The energy dissipation rate was significantly increased with PL traction and with traction on both tendons, whereas no significant difference existed with PT traction compared to control. CONCLUSIONS: The PT increased the stiffness and reduces the energy stored in the foot. The PT acted to improve the energy efficiency of the load transmission. The PL decreased the stiffness and increased the energy stored. CLINICAL RELEVANCE: PT and PL muscles affect the foot arch. Excessive or insufficient traction may cause some foot disorders.

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.

Saccharomyces cerevisiae Ssd1p promotes CLN2 expression by binding to the 5'-untranslated region of CLN2 mRNA.

In Saccharomyces cerevisiae, TFIID, which is composed of TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs), mediates the transcription of most class II genes. Previous studies have shown that CLN2 expression was significantly reduced by taf1-ts2, but not by taf1-N568Δ, although both mutations display similar temperature-sensitive growth phenotypes and transcriptional defects in other genes. Here, we show that the reduced expression of CLN2 is not because of differences in taf1 alleles in the previous experiments but because of allelic differences at the SSD1 locus in the host strains. Specifically, ssd1-d reduces CLN2 expression when combined with taf1. Ssd1p expressed from SSD1-V, but not from ssd1-d, stabilizes a subpopulation of CLN2 mRNA in wild-type and taf1-N568Δ strains and facilitates the continuous transcription of CLN2 after heat shock in the taf1-N568Δ strain. Reporter assays show that both activities appear to depend on the 5'-untranslated region of CLN2 mRNA and that Ssd1p binds to this region via its amino- and carboxy-terminal domains. Based on these observations, we propose a model for the action of Ssd1p and discuss its biologic role.

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.

Saccharomyces cerevisiae Med9 comprises two functionally distinct domains that play different roles in transcriptional regulation.

Mediator is one of the most important co-activators that function in eukaryotic transcriptional regulation. In Saccharomyces cerevisiae, Mediator is comprised of 25 subunits belonging to four structurally distinct modules: Head, Middle, Tail, and Cyc-C. Although each module plays a critical role in the regulation of a distinct set of genes, the precise molecular mechanisms remain unclear. To gain new insight into the role of the less-characterized Middle module, we analyzed the function of Med9 by constructing a set of mutants and subjecting them to a range of in vivo and in vitro assays. Our results demonstrate that Med9 has two functional domains. The species-specific amino-terminal half (aa 1-63) plays a regulatory role in transcriptional regulation in vivo and in vitro. In contrast, the well-conserved carboxy-terminal half (aa 64-149) has a more fundamental function involved in direct binding to the amino-terminal portions of Med4 and Med7 and the assembly of Med9 into the Middle module. Importantly, activator-dependent recruitment of TBP and Taf11 to the promoter is differentially affected in med9 extracts and in extracts lacking Mediator. Add-back experiments indicate that some unidentified factor(s) in med9 extracts may impact the binding of TFIID to the promoter.

Assembly of regulatory factors on rRNA and ribosomal protein genes in Saccharomyces cerevisiae.

HMO1 is a high-mobility group B protein that plays a role in transcription of genes encoding rRNA and ribosomal proteins (RPGs) in Saccharomyces cerevisiae. This study uses genome-wide chromatin immunoprecipitation to study the roles of HMO1, FHL1, and RAP1 in transcription of these genes as well as other RNA polymerase II-transcribed genes in yeast. The results show that HMO1 associates with the 35S rRNA gene in an RNA polymerase I-dependent manner and that RPG promoters (138 in total) can be classified into several distinct groups based on HMO1 abundance at the promoter and the HMO1 dependence of FHL1 and/or RAP1 binding to the promoter. FHL1, a key regulator of RPGs, binds to most of the HMO1-enriched and transcriptionally HMO1-dependent RPG promoters in an HMO1-dependent manner, whereas it binds to HMO1-limited RPG promoters in an HMO1-independent manner, irrespective of whether they are transcribed in an HMO1-dependent manner. Reporter gene assays indicate that these functional properties are determined by the promoter sequence.

Saccharomyces cerevisiae HMO1 interacts with TFIID and participates in start site selection by RNA polymerase II.

Saccharomyces cerevisiae HMO1, a high mobility group B (HMGB) protein, associates with the rRNA locus and with the promoters of many ribosomal protein genes (RPGs). Here, the Sos recruitment system was used to show that HMO1 interacts with TBP and the N-terminal domain (TAND) of TAF1, which are integral components of TFIID. Biochemical studies revealed that HMO1 copurifies with TFIID and directly interacts with TBP but not with TAND. Deletion of HMO1 (Deltahmo1) causes a severe cold-sensitive growth defect and decreases transcription of some TAND-dependent genes. Deltahmo1 also affects TFIID occupancy at some RPG promoters in a promoter-specific manner. Interestingly, over-expression of HMO1 delays colony formation of taf1 mutants lacking TAND (taf1DeltaTAND), but not of the wild-type strain, indicating a functional link between HMO1 and TAND. Furthermore, Deltahmo1 exhibits synthetic growth defects in some spt15 (TBP) and toa1 (TFIIA) mutants while it rescues growth defects of some sua7 (TFIIB) mutants. Importantly, Deltahmo1 causes an upstream shift in transcriptional start sites of RPS5, RPS16A, RPL23B, RPL27B and RPL32, but not of RPS31, RPL10, TEF2 and ADH1, indicating that HMO1 may participate in start site selection of a subset of class II genes presumably via its interaction with TFIID.

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.