Biomedical subjects

R Candau

Publications and source records attributed to R Candau.

At least 19 recordsLinked to original sources

PubMed1998

Energy cost and running mechanics during a treadmill run to voluntary exhaustion in humans.

The aim of the present study was to examine the physiological and mechanical factors which may be concerned in the increase in energy cost during running in a fatigued state. A group of 15 trained triathletes ran on a treadmill at velocities corresponding to their personal records over 3000m [mean 4.53 (SD 0.28) m x s(-1)] until they felt exhausted. The energy cost of running (CR) was quantified from the net O2 uptake and the elevation of blood lactate concentration. Gas exchange was measured over 1 min firstly during the 3rd-4th min and secondly during the last minute of the run. Blood samples were collected before and after the completion of the run. Mechanical changes of the centre of mass were quantified using a kinematic arm. A significant mean increase [6.9 (SD 3.5)%, P < 0.001] in CR from a mean of 4.4 (SD 0.4) J x kg(-1) x m(-1) to a mean of 4.7 (SD 0.4) J x kg(-1) x m(-1) was observed. The increase in the O2 demand of the respiratory muscles estimated from the increase in ventilation accounted for a considerable proportion [mean 25.2 (SD 10.4)%] of the increase in CR. A mean increase [17.0 (SD 26.0)%, P < 0.05] in the mechanical cost (CM) from a mean of 2.36 (SD 0.23) J x kg m(-1) to a mean of 2.74 (SD 0.55) J x kg(-1) x m(-1) was also noted. A significant correlation was found between CR and CM in the non-fatigued state (r=0.68, P < 0.01), but not in the fatigued state (r=0.25, NS). Furthermore, no correlations were found between the changes (from non-fatigued to fatigued state) in CR and the changes in CM suggesting that the increase in CR is not solely dependent on the external work done per unit of distance. Since step frequency decreased slightly in the fatigued state, the internal work would have tended to decrease slightly which would not be compatible with an increase in CR. A stepwise regressions showed that the changes in CR were linked (r=0.77, P < 0.01) to the changes in the variability of step frequency and in the variability of potential cost suggesting that a large proportion of the increase in CR was due to an increase in the step variability. The underlying mechanisms of the relationship between CR and step variability remains unclear.

Adult↗

PubMed1998

External loading does not change energy cost and mechanics of rollerski skating.

The purpose of this study was to examine the effects of external loading on the energy cost and mechanics of roller ski skating. A group of 13 highly skilled male cross-country skiers roller skied at 19.0 ( SD 0.1) km x h(-1) without additional load and with loads of 6% and 12% body mass (mb). Oxygen uptake (VO2), knee and ankle joint kinematics, roller-ski electromyogram (EMG) of the vastus lateralis and gastrocnemius lateralis muscles, and roller ski velocity were recorded during the last 40 s of each 4-min period of roller skiing. One-way repeated measures ANOVA revealed that the VO2 expressed relative to total mass (mtot), joint kinetics, eccentric-to-concentric ratio of the integrated EMG, velocity changes within a cycle, and cycle rate did not change significantly with load. The subsequent analysis of the effect of load on each resistance opposing motion suggested that the power to sustain changes in translational kinetic energy, potential energy, and overcoming rolling resistance increased proportionately with the load. The lack of a significant change in VO2/mtot with external loading was associated with a lack of marked change in external mechanical power relative to mtot. The existence of an EMG signal during the eccentric phase prior to the thrust (concentric phase), as well as the lack of significant delay between the two phases, showed that a stretch-shortening cycle (SSC) occurs in roller ski skating. Taken together, the present results would suggest that external loading up to 12% mb does not increase storage and release of elastic energy of lower limb muscles during SSC in roller ski skating.

Adult↗

PubMed1998

Effect of cycling position on ventilatory and metabolic variables.

Three positions are generally used by cyclists: upright posture (UP), dropped posture (DP) and aero posture (AP). They determine different angles of trunk flexion which could be associated with physiological changes. The purpose of this study was to analyse the effect of these rider positions on ventilatory and metabolic variables. Nine male competitive cyclists (26.3+/-3yrs, mean+/-SD) exercised on a cycle ergometer. Subjects performed three 10 min exercise bouts at 70% VO2max (maximal oxygen uptake, I x min(-1)) in UP, DP and AP, in a randomized order. Each bout was separated by a 5 min active recovery period (50% of VO2max). Ventilatory and gas exchange responses to exercise were averaged every min. Blood lactate concentration ([La]b, mM), blood pH were analysed at the 5th and the 10th min. The ventilation, respiratory exchange ratio, mean inspiratory flow, [La]b and perceived exertion were significantly higher in DP (88.4+/-11.41 x min(-1), 0.96+/-0.05 ml x s(-1), 2.52+/-0.84 Mm and 13.6+/-1.2) than in UP (84.8+/-12.31 x min(-1), 0.94+/-0.05 ml x s(-1), 2.14+/-0.99Mm and 12.1+/-1.5). VO2, tidal volume, carbon dioxide output, respiratory rate, inspiratory duty cycle, heart rate and pH remained unchanged between all riding positions (averaged values for the three positions: 3.09+/-0.0061 x min(-1), 2.34+/-0.0061 x br(-1), 3.01+/-0.041 x min(-1), 37.4+/-0.8 br x min(-1), 0.47+/-0, 162+/-1 beat x min(-1) and 7.38+/-0.015). These results showed that the greater changes in ventilatory and metabolic variables occurred in DP. AP appears to be the more suitable position when the aerodynamic drag becomes predominant.

Adult↗

PubMed1998

Stretch-shortening cycle in roller ski skating: effects of technique.

The purposes of this study were 1) to examine the occurrence of the stretch-shortening cycle (SSC) in the three principal techniques of ski skating and 2) to test the hypothesis that differences in SSC behaviour may explain the choice of a technique when the grade is altered. Ten highly skilled male cross-country skiers roller skied at 5.3+/-0.1 m x s(-1) using the V1 skate (V1), V2-alternate (V2A), and V2 skate (V2) techniques. Knee and ankle joint kinematics, EMG of the vastus lateralis (VL) and gastrocnemius lateralis (GL) muscles, and roller ski velocity were recorded during the last 40 s of each bout of roller skiing. The existence of an EMG signal during the eccentric phase prior to the thrust, as well as the lack of significant delay between the two phases, showed that SSC occurs in the three techniques of roller ski skating with the exception of 50% of the subjects at the weak side knee with V1. The VL stretching velocity over the eccentric phase was higher (p < 0.005) with V2 and V2A than V1, indicating that less elastic energy was stored in the lower limbs extensor muscles with V1. This could partly explain why this technique is not used on flat terrains during races. However, no differences of average EMG activity or IEMG existed between techniques for either GL or VL during the concentric phase, suggesting that the theoretical advantage due to the SSC behaviour is minor in roller ski skating.

Adult↗

PubMed1997

Binding of NF1 to the MMTV promoter in nucleosomes: influence of rotational phasing, translational positioning and histone H1.

To analyse the role of rotational orientation and translational positioning of nucleosomal DNA on transcription factor binding we have generated a series of mutant MMTV promoters containing insertions of various lengths between the hormone-responsive region and the binding site for NF1. These various MMTV promoter fragments were assembled in mononucleosomes and used for structural studies and binding experiments. We show that the insertions change the rotational phase and translational positioning of the NF1 site as predicted if the sequences upstream of the insertion site were the main determinants of nucleosome phasing. In band shift experiments with recombinant NF1 we cannot detect binding of the protein to NF1 sites included within the limits of a nucleosome, independent of their rotational orientation. Moving the NF1 site closer to the nucleosome border also did not permit NF1 binding. This behaviour probably reflects the way NF1 binds DNA, namely it almost completely surrounds the circumference of the double helix establishing a large number of contacts with the bases and the backbone. In contrast to the wild-type and short insertion mutants, NF1 bound readily to nucleosomes containing 30 or 50 bp insertions which placed the NF1 site at the nucleosome edge or within linker DNA. NF1 binding to the linker DNA was unaffected by incorporation of histone H1 into the nucleosome particle. These findings are discussed in relation to chromatin remodelling initiated by steroid hormones during induction of the MMTV promoter.

Animals↗

PubMed1997

Two tandem and independent sub-activation domains in the amino terminus of p53 require the adaptor complex for activity.

The ability of p53 to function as a tumor suppressor is linked to its function as a transcriptional activator, since p53 mutants that do not transactivate are unable to suppress tumor cell growth. Previous studies identified an activation domain in the amino terminal 40 residues of the protein, a region that binds to several general transcription factors and to some oncogene products. For example, mdm-2, a cellular oncoprotein, binds to this region and represses p53 transactivation. Here we describe a new activation domain within the amino terminus of p53 that maps between amino acids 40-83, and whose residues trp-53 and phe-54 are critical for function both in yeast and in mammalian cells. In vivo studies in yeast show that the new activation subdomain, unlike the previously described, is mdm-2 independent. Both p53 activation subdomains (1-40 and 40-83) require the yeast adaptor complex ADA2/ADA3/GCN5 for transcriptional activation. Moreover, since activation by p53 requires GCN5's enzymatic histone acetyltransferase domain, p53 may regulate gene expression by influencing chromatin modification.

DNA-Binding Proteins↗

PubMed1997

Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.

The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8- and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5delta, ada2delta, or ada3delta yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5delta and spt7delta strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. We have named this novel transcription regulatory complex SAGA (Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).

Acetylation↗

PubMed1997

Histone acetyltransferase activity and interaction with ADA2 are critical for GCN5 function in vivo.

Yeast GCN5 is one component of a putative adaptor complex that includes ADA2 and ADA3 and functionally connects DNA-bound transcriptional activators with general transcription factors. GCN5 possesses histone acetyltransferase (HAT) activity, conceptually linking transcriptional activation with enzymatic modification at chromatin. We have identified the minimal catalytic domain within GCN5 necessary to confer HAT activity and have shown that in vivo activity of GCN5 requires this domain. However, complementation of growth and transcriptional activation in gcn5- cells required not only the HAT domain of GCN5, but also interaction with ADA2. The bromodomain in GCN5 was dispensable for HAT activity and for transcriptional activation by strong activators; however, it was required for full complementation in other assays. Fusion of GCN5 to the bacterial lexA DNA binding domain activated transcription in vivo, and required both the HAT domain and the ADA2 interaction domain. These results suggest that both functions of GCN5, HAT activity and interaction with ADA2, are necessary for targeting and acetylation of nucleosomal histones.

Acetyltransferases↗

PubMed1997

Histone acetyltransferase activity is conserved between yeast and human GCN5 and is required for complementation of growth and transcriptional activation.

Yeast and human ADA2 and GCN5 (y- and hADA2 and y- and hGCN5, respectively) have been shown to potentiate transcription in vivo and may function as adaptors to bridge physical interactions between DNA-bound activators and the basal transcriptional machinery. Recently it was shown that yGCN5 is a histone acetyltransferase (HAT), suggesting a link between enzymatic modification of nucleosomes and transcriptional activation. In this report, we demonstrate that hGCN5 is also an HAT and has the same substrate specificity as yGCN5. Since hGCN5 does not complement functional defects caused by deletion of yGCN5, we constructed a series of hGCN5-yGCN5 chimeras to identify human regions capable of activity in yeast. Interestingly, only the putative HAT domain of hGCN5, when fused to the remainder of yGCN5, complemented gcn5- cells for growth and transcriptional activation. Moreover, an amino acid substitution mutation within the HAT domain reduced both HAT activity in vitro and transcription in vivo. These findings directly link enzymatic histone acetylation and transcriptional activation and show evolutionary conservation of this potentially crucial pathway in gene regulation.

Acetylation↗

PubMed1997

[Epileptic syndromes which show continuous spike and wake complexes during slow wave sleep].

INTRODUCTION AND OBJECTIVE: The association of cognitive and/or behavioral disturbances, epileptic seizures and continuous discharges spike-waves during slow sleep in the EEG, has been described in three epileptic syndromes: continuous spike-wave during slow sleep syndrome (CSWS), Landau-Kleffner syndrome (LKS) or acquired epileptic aphasia, and atypical benign partial epilepsy of childhood (ABPE). MATERIAL AND METHODS: We report 13 patients; two of these have been diagnosed of CSWS. four of LKS and seven of ABPE, according to the definition criteria. RESULTS: The main clinical features in each patient are presented here. In all patients, continuous spike-wave discharges were observed for more than > 80% of the total sleep period at some time during the course of the illness. There are many similarities at the age of onset, the occurrence of several types of seizures, the evolution and the electroencephalographic findings in awake state and sleep. CONCLUSIONS: It suggests that the CSWS, LKS and ABPE have probably the same pathophysiology and that they are the severe, moderate and benign forms of a single epileptic syndrome, age-related, with continuous spike waves and various neuropsychological and behavioral disturbances. The neuropsychological manifestations depend probably on the localization of the original epileptic focus.

Age of Onset↗

PubMed1996

Structural and functional analysis of yeast putative adaptors. Evidence for an adaptor complex in vivo.

Putative transcriptional adaptor proteins are found in eukaryotes from yeast to humans and are required for full function of many eukaryotic acidic activators. To study their functional interactions, deletion mutations in the yeast adaptors ADA2, GCN5, and ADA3 were created. We defined a region within the middle of GCN5 required for interaction with ADA2 in vitro. We identified regions of ADA2 required for function in vivo and determined whether these same regions are involved in physical interaction of ADA2 with GCN5 or ADA3 in vitro. Two regions were crucial for ADA2 function in vivo, the amino terminus and a middle region. Immunoprecipitation analysis showed that the amino terminus of ADA2 was required for interaction with GCN5, while a region in the middle of ADA2 was necessary for interaction with ADA3. Deletions of the region that was required for interaction with ADA3 abolished dependence of lexA-ADA2 transcriptional activity on ADA3. Moreover, using coimmunoprecipitation analysis, physical interaction between ADA2, ADA3, and GCN5 was demonstrated in yeast extracts. Taken together, the physical interaction in vivo, along with the correlation observed between regions of ADA2 required for in vitro interaction with GCN5 and ADA3, and regions required for function in vivo, argue for the existence of a physiologically relevant adaptor complex.

Base Sequence↗

PubMed1996

Interaction of steroid hormone receptors with transcription factors involves chromatin remodelling.

The mechanism by which steroid hormones modulate promoter utilization is not clear. Evidence from transfection studies and cell-free assays points to an interaction of the hormone receptors with general transcription factors, as well as with sequence-specific transcription factors. Moreover co-activators or transcription intermediary factors, have been identified which could mediate some of the transcriptional effects of the hormone-receptor complex. However, in addition to this interaction of receptors with proteins directly involved in transcription, a participation of chromatin structure in gene regulation by steroid hormones is becoming increasingly evident. In the case of the MMTV promoter, the nucleosomal organization seems to be responsible for transcriptional repression prior to hormonal stimulation. This effect is due to occlusion by a nucleosome positioned on the MMTV promoter sequences in such a way that essential transcription factors cannot access their recognition sites. Following hormone induction, a remodelling of the nucleosome structure takes place which enables a whole complement of sequence specific transcription factors to assemble on the promoter. Since a complete occupancy of binding sites does not take place when the promoter is present as naked DNA, the nucleosomal organization appears to be required for the proper synergism between transcription factors following hormonal induction. According to this model, the positioning of a nucleosome sets the stage for constitutive repression and hormone induction of the MMTV promoter.

Amino Acid Sequence↗

PubMed1996

The hormone responsive region of mouse mammary tumor virus positions a nucleosome and precludes access of nuclear factor I to the promoter.

The mouse mammary tumor virus (MMTV) promoter is transcriptionally silent prior to hormonal induction, partly because its organization into phased nucleosomes precludes access of transcription factors to their cognate sites. A T47D-derived cell line carrying a single integrated copy of the MMTV promoter exhibited a positioned nucleosome, which prevented binding of nuclear factor I (NFI). To study the molecular mechanisms controlling promoter accessibility we have made use of a strong chimeric transactivator, NFI-VP16, composed of NFI linked to the transactivation function of VP16. T47D cells transiently transfected with an MMTV-CAT reporter show little transcription even after cotransfection of an expression vector for NFI-VP16. However, a truncated MMTV promoter, lacking the hormone regulatory region (HRR) was transactivated by cotransfected NFI-VP16. The repressive effect of the HRR was not due to binding of a sequence-specific transcriptional repressor, and was evident with the DEAE-Dextran transfection procedure but not with the calcium phosphate technique. A similar behavior was observed in Saccharomyces cerevisiae carrying wild type or truncated MMTV-lacZ reporters and expressing NFI-VP16. Reconstitution experiments suggest that the promoter lacking the HHR generates less stable nucleosomes, a fraction of which contain a more accessible NFI site. Recombinant NFI binds to nucleosomes assembled on this truncated promoter but not to nucleosomes encompassing the HRR. These results are compatible with the notion that transiently transfected MMTV promoters behave like their stably integrated counterparts, in that the HRR drives positioning of a nucleosome and mediates transcriptional repression by preventing access of NFI to its cognate site.

Animals↗

PubMed1996

Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Characterization of physical interactions of the putative transcriptional adaptor, ADA2, with acidic activation domains and TATA-binding protein.

RNA polymerase II transcription requires functional interactions between activator proteins bound to upstream DNA sites and general factors bound to the core promoter. Accessory transcription factors, such as adaptors and coactivators, have important, but still unclear, roles in the activation process. We tested physical interactions of the putative adaptor ADA2 with activation domains derived from acidic activator proteins and with certain general transcription factors. ADA2 associated with the herpesvirus VP16 and yeast GCN4 activation domains but not with the activation domain of yeast HAP4, which previously was shown to be independent of ADA2 function in vivo and in vitro. Furthermore, the amino terminus of ADA2 directly interacted with the VP16 activation domain, suggesting that ADA2 provides determinants for interaction between activation domains and the adaptor complex. Both TATA-binding protein (TBP) and TFIIB have previously been shown to interact directly with the VP16 activation domain in vitro (Stringer, K. F., Ingles, C. J., and Greenblatt, J. (1990) Nature 345, 783-786; Lin, Y. S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1991) Nature 353, 569-571). Interestingly, when binding was tested between VP16 and these general factors in yeast nuclear extracts, both factors interacted with VP16, but only the TBP/VP16 association was dependent on ADA2. In addition, ADA2 physically associated with TBP, but not with TFIIB. These results suggest that the role of ADA2 in transcriptional activation is to promote physical interaction between activation domains and TBP.

CCAAT-Binding Factor↗

PubMed1995

Transcriptional control by steroid hormones: the role of chromatin.

The mouse mammary tumour virus (MMTV) promoter contains a complex hormone-responsive unit composed of four hormone-responsive elements, a nuclear factor I (NFI) binding site and two octamer motifs. All these sites are required for optimal hormonal induction. Although synergism has been found between hormone receptors and octamer transcription factor 1 (Oct-1/OTF-1), we were unable to detect a positive interaction between receptors and NFI in vitro. In chromatin, the MMTV hormone-responsive unit is contained in a phased nucleosome. The precise positioning of the DNA double helix on the surface of the histone octamer precludes binding of NFI and Oct-1/OTF-1 to their cognate sequences, while still allowing recognition of two hormone-responsive elements by the hormone receptors. Hormone treatment leads to a characteristic change in chromatin structure that makes the centre of the nucleosome more accessible to digestion by DNase I and facilitates binding of receptors, NFI and Oct-1/OTF-1 to the nucleosomally organized promoter. The MMTV promoter functions in yeast in a hormone receptor-dependent and NFI-dependent fashion. Depletion of nucleosomes activates hormone-independent transcription from the MMTV promoter. These results imply that nucleosome positioning not only represses hormone-independent transcription, but also enables binding of a full complement of transcription factors to the hormone-responsive unit after hormone induction.

Animals↗

PubMed1995

Mechanical step variability during treadmill running.

The present study was designed to study intra-individual step variability measured both on vertical displacement of the body (delta Z) and on step time (delta t) parameters by means of a kinematic arm and during treadmill running. A group of 17 subjects ran successively at 60%, 80%, 100% and 140% of their maximal aerobic velocity (Vamax). The total number of steps analysed was 6116. The absolute delta Z step variability (sigma delta Z) ranged between 5 mm and 21 mm while the absolute delta t variability (sigma delta t) ranged between 6 ms and 40 ms. Step variabilities were due to step asymmetry (from 38.5% to 48.5% of the step variability) and to stride variability. For submaximal velocities (60%, 80%, and 100% Vamax) both sigma delta t and sigma delta Z were independent of velocity or body dimensions whereas differences between subjects were significant (P < 0.01) for sigma delta Z. On the other hand, variabilities were significantly increased when velocity was changed from submaximal to the 140% Vamax level. Furthermore, at submaximal levels sigma delta Z was linked to the subject's energy cost of running (P < 0.05). Therefore, the intra-individual step variability should not be neglected in future studies on mechanical efficiency of running and it is suggested that, to obtain a good accuracy (better than 1%, P < 0.05) on mean value and variability of the mechanical parameters, measurements should be performed on at least 32-64 consecutive steps, which corresponds to about 15 to 20s of running.

Adult↗

PubMed1995

Constitutive repression and nuclear factor I-dependent hormone activation of the mouse mammary tumor virus promoter in Saccharomyces cerevisiae.

To study the influence of various transactivators and the role of nucleosomal structure in gene regulation by steroid hormones, we have introduced mouse mammary tumor virus (MMTV) promoter sequences along with expression vectors for the glucocorticoid receptor (GR) and nuclear factor I (NFI) in Saccharomyces cerevisiae, an organism amenable to genetic manipulation. Both in the context of an episomal multicopy vector and in a centromeric single-copy plasmid, the MMTV promoter was virtually silent in the absence of inducer, even in yeast strains expressing GR and NFI. Induction was optimal with deacylcortivazol and required both GR and NFI. The transactivation function AF1 in the N-terminal half of GR is required for ligand-dependent induction and acts constitutively in truncated GR lacking the ligand binding domain. A piece of the MMTV long terminal repeat extending from -236 to +111 is sufficient to position a nucleosome, B, over the regulatory region of the promoter from -45 to -190 and another nucleosome over the transcription start region. The rotational orientation of the DNA on the surface of nucleosome B is the same as that previously found in animal cells and in reconstitution experiments. This orientation is compatible with binding of GR to two sites, while it should preclude binding of NFI and hence be responsible for constitutive repression. Upon ligand induction, there is no major chromatin rearrangement, but the proximal linker DNA, including the TATA box, becomes hypersensitive to nucleases. The transcriptional behavior of the MMTV promoter was unaffected by deletions of the genes for zuotin or SIN1/SPT2, two proteins which have been claimed to assume some of the functions of linker histones. Thus, despite the lack of histone H1, yeast cells could be a suitable system to study the contribution of nucleosomal organization to the regulated expression of the MMTV promoter.

Animals↗