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R Mantovani

Publications and source records attributed to R Mantovani.

At least 37 records · Page 2Linked to original sources
PubMed1999

NF-Y histone fold alpha1 helices help impart CCAAT specificity.

NF-Y is a conserved trimeric transcriptional activator with an extremely high specificity for CCAAT boxes. The NF-YB and NF-YC subunits have histone fold motifs with a high degree of homology to NC2alpha/beta, a TBP-binding repressor. The histone fold is composed of three alpha helices, alpha1, alpha2, alpha3, separated by short loops. Structural data on core histones showed that alpha1 are involved in DNA-binding. To understand the molecular basis of NF-Y sequence-specificity, we constructed deletion and swapping mutants, in which the alpha1 of NC2 and archeal HMfB, a bona fide histonic protein, was placed in NF-YB and NF-YC. Our analysis indicates that (i) subunit interactions are normal; (ii) NF-YB-NF-YC and NC2alpha/beta do not form heterodimers and NC2 cannot associate NF-YA. (iii) None of the NF-Y swaps can complex with TBP on a TATA box. (iv) Specific residues, R47 and K49 in NF-YC and N61 in NF-YB, are crucial for CCAAT-binding. We conclude that specificity of the NF-Y trimer is not due to NF-YA only, but stems in part from the contribution of the histone fold alpha1, particularly that of NF-YB.

Amino Acid Sequence↗
PubMed1999

NF-Y binding to twin CCAAT boxes: role of Q-rich domains and histone fold helices.

NF-Y (CBF) is a CCAAT-binding trimer that activates 25 % of eukaryotic promoters. It contains putative histone fold motifs (HFMs) and distorts DNA. By using electrophoretic mobility shift assays with the twin CCAAT boxes of the human gamma-globin promoter and several combinations of subunit mutants, we dissected some of the structural features of CCAAT-box binding. NF-YA and NF-YC Q-rich domains significantly influence bending angles quantitatively, but not qualitatively, since they do not modify DNA orientation. They are both required for co-operative interactions among NF-Y molecules: for this, a precise alignement of two CCAAT boxes, 32 bp, three turns of the helix, is essential. Unlike the wild-type (wt) protein, steric hindrance does not impede simultaneous binding of the mutant composed of the short homology domains to CCAAT boxes closer than 22 bp: the addition of 11 amino acid residues to NF-YB and 13 to NF-YC flanking the HFM, restores wt behaviour. These stretches are predicted to form H2B-like alphaC and H2A-like alphaN fourth helices. A further support to this hypothesis comes from off-rates analysis of mutant combinations: the half-life of NF-Y, which is dependent on the type of NF-YB used, is extremely shortened, when the putative alphaC is present, nearly as much as in the wt NF-YB. These data (i) provide further evidence that NF-YB-NF-YC belong to the H2B-H2A subclasses, (ii) uncover new features of Q-rich domains, and (iii) define rules for NF-Y synergy that are potentially important for the regulation of many eukaryotic promoters.

Amino Acid Sequence↗
PubMed1999

Interactions of the CCAAT-binding trimer NF-Y with nucleosomes.

NF-Y is a sequence-specific evolutionary conserved activator binding to CCAAT boxes with high affinity and specificity. It is a trimer formed by NF-YA and two putative histone-like subunits, NF-YB and NF-YC, showing similarity to histones H2B and H2A, respectively. We investigated the relationships between NF-Y and chromatin using an Artemia franciscana chromatin assembly system with plasmids containing the Major HistoCompatibility complex class II Ea promoter. The NF-Y trimer, but not single subunits, protects the Y box in the presence of reconstituted chromatin, and it can bind the target sequence during and after assembly. Using reconstitution assays with purified chicken histones, we show that NF-Y associates with preformed nucleosomes. Translational analysis of various Ea fragments of identical length in which the CCAAT box is at different positions indicated that the lateral fragment was slightly more prone to NF-Y binding. In competition experiments, NF-Y is able to prevent formation of nucleosomes significantly. These data support the idea that NF-Y is a gene-specific activator with a built-in capacity to interface with chromatin structures.

Animals↗
PubMed1999

The activity of the CCAAT-box binding factor NF-Y is modulated through the regulated expression of its A subunit during monocyte to macrophage differentiation: regulation of tissue-specific genes through a ubiquitous transcription factor.

In this study, we analyzed the regulation of NF-Y expression during human monocyte to macrophage maturation. NF-Y is a ubiquitous and evolutionarily conserved transcription factor that binds specifically to the CCAAT motif present in the 5' promoter region of a wide variety of genes. We show here that in circulating monocytes, NF-Y binding activity is not detected on the CCAAT motif present in the promoters of genes such as major histocompatibility complex (MHC) class II, gp91-phox, mig, and fibronectin, whereas during macrophage differentiation, a progressive increase in NF-Y binding activity is observed on these promoters. Analysis of NF-Y subunit expression indicates that the absence of NF-Y activity in circulating monocytes is caused by a lack of the A subunit. Furthermore, addition of the recombinant NF-YA subunit restores NF-Y binding. We show that the lack of NF-YA protein is due to posttranscriptional regulation and not to a specific proteolytic activity. In fact, NF-YA mRNA is present at the same level at all days of monocyte cultivation, whereas the protein is absent in freshly isolated monocytes but is progressively synthesized during the maturation process. We thus conclude that the NF-YA subunit plays a relevant role in activating transcription of genes highly expressed in mature monocytes. In line with this conclusion, we show that the cut/CDP protein, a transcriptional repressor that inhibits gpc91-phox gene expression by preventing NF-Y binding to the CAAT box, is absent in monocytes.

CCAAT-Enhancer-Binding Proteins↗
PubMed1999

GH/IGF-I axis in azoospermia in primary and secondary hypogonadism: a study before and during replacement therapy.

The growth hormone/insulin-like growth factor-I (GH/IGF-I) axis was studied in 15 azoospermic patients and in 10 control men. Eight patients were affected by hypergonadotrophic hypogonadism and 7 by hypogonadotrophic hypogonadism. All were studied before and during replacement therapy with testosterone and gonadotrophin, respectively, using the alpha 2 adrenergic agonist, clonidine (clonidine test). The data demonstrate no differences in basal levels for IGF-I and for the GH response to clonidine in azoospermic patients, affected by primary and secondary hypogonadism, before and during replacement therapy when compared with control fertile men. In contrast to some studies which describe a reduced GH response in azoospermia and oligozoospermia, we conclude that basal serum levels of IGF-I and the GH response to clonidine are not impaired in azoospermic patients affected by primary hypogonadism before and after the restoration of normal androgenization, and in azoospermic patients affected by secondary hypogonadism, both before and after restoration of spermatogenesis.

Adolescent↗
PubMed1999

NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms.

NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.

Animals↗
PubMed1999

Effect of ecteinascidin-743 on the interaction between DNA binding proteins and DNA.

Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, a tunicate growing in mangrove roots in Caribbean. It has been shown to bind in the minor groove of DNA forming covalent adducts by reaction of the N2 of guanine with the carbinolamine moiety. We investigated ET-743 ability to inhibit the binding of different transcription factors to their consensus sequences by using gel shift assays. We have selected three types of factors: (i) oncogene products such as MYC, c-MYB and Maf; (ii) transcriptional activators regulated during the cell cycle as E2F and SRF; and (iii) general transcription factors such as TATA binding protein (TBP), Sp1 and NF-Y. We observed no inhibition of the binding of Sp1, Maf, MYB and MYC. Inhibition of DNA binding was observed for TBP, E2F, SRF at ET-743 concentrations ranging from 50 to 300 microM. The inhibition of binding of NF-Y occurs at even lower concentrations (i.e. 10-30 microM) when the recombinant subunits of NF-Y are preincubated with the drug, indicating that the inhibition of NF-Y binding does not require previous ET-743 DNA binding. Since NF-Y is a trimer containing two subunits with high resemblance to histones H2B and H2A, we have investigated the effect of ET-743 on nucleosome reconstitution. ET-743 caused a decrease of the nucleosomal band at 100 nM, with the complete disappearance of the band at 3-10 microM. These data suggest that the mode of action of this novel anticancer drug is related to its ability to modify the interaction between some DNA binding proteins and DNA.

Animals↗
PubMed1998

Conservation and divergence of NF-Y transcriptional activation function.

The CCAAT-binding protein NF-Y is involved in the regulation of a variety of eukaryotic genes and is formed in higher eukaryotes by three subunits NF-YA/B/C. We have characterized NF-Y of the trematode parasite Schistosoma mansoni and studied the structure and the function of the SMNF-YA subunit. In this work, we present the cloning and sequence analysis of the B subunit of the parasite factor. SMNF-YB contains the conserved HAP-3 homology domain but the remaining part of the protein was found to be highly divergent from all other species. We demonstrated by transfections of GAL4 fusion constructs, that mouse NF-YB does not contain activation domains while the C-terminal part of SMNF-YB has transcriptional activation potential. On the other hand, the N-terminal parts of SMNF-YA and mouse NF-YA were shown to mediate transactivation; the integrity of a large 160 amino acid glutamine-rich domain of NF-YA was required for this function and an adjacent serine- and threonine-rich domain was necessary for full activity in HepG2, but redundant in other cell types. Transactivation domains identified in SMNF-YB are also rich in serine and threonine residues. Our results indicate that serine/threonine-richsequences from helminth parasites potentiate trans-cription and that such structures have diverged during evolution within the same transcription factor.

3T3 Cells↗
PubMed1998

DNA binding of NF-Y: the effect of HMGI proteins depends upon the CCAAT box.

NF-Y is a conserved sequence-specific transcription factor binding to CCAAT boxes. The chromatin-associated HMGI proteins influence promoter activities through positive and negative effects on binding of transcription factors. It was previously shown that HMGI(Y) synergizes the binding of NF-Y to the alpha2-collagen CCAAT box [Currie, R.A. (1997) J. Biol Chem. 272, 30880-30888]. Using recombinant proteins, we confirm that at low concentrations of NF-Y, HMGI(Y) acts synergistically on the alpha2-collagen CCAAT and we extend this observation to HMGI and HMGI-C. However, enhancement of DNA binding to gamma-globin, alpha-globin and MHC class II Ea CCAAT boxes was not observed. At high concentrations, HMGI proteins inhibit binding to alpha2-collagen and to gamma-globin, but not to high affinity Ea or a-globin CCAAT. In none of our experiments did we see a ternary complex between NF-Y, HMGI(Y) and DNA. In protein competition experiments, NF-Y affinity was at least two orders of magnitude higher, even in the context of the suboptimal gamma-globin CCAAT. Our data prove that HMGI proteins have complex positive and negative effects on NF binding to some, but not to all CCAAT boxes, suggesting that this phenomenon is dictated by the sequences flanking the pentanucleotide rather than direct protein-protein interactions.

Animals↗
PubMed1998

NF-Y organizes the gamma-globin CCAAT boxes region.

The CCAAT-binding activator NF-Y is formed by three evolutionary conserved subunits, two of which contain putative histone-like domains. We investigated NF-Y binding to all CCAAT boxes of globin promoters in direct binding, competition, and supershift electrophoretic mobility shift assay; we found that the alpha, zeta, and proximal gamma CCAAT boxes of human and the prosimian Galago bind avidly, and distal gamma CCAAT boxes have intermediate affinity, whereas the epsilon and beta sequences bind NF-Y very poorly. We developed an efficient in vitro transcription system from erythroid K562 cells and established that both the distal and the proximal CCAAT boxes are important for optimal gamma-globin promoter activity. Surprisingly, NF-Y binding to a mutated distal CCAAT box (a C to T at position -114) is remarkably increased upon occupancy of the high affinity proximal element, located 27 base pairs away. Shortening the distance between the two CCAAT boxes progressively prevents simultaneous CCAAT binding, indicating that NF-Y interacts in a mutually exclusive way with CCAAT boxes closer than 24 base pairs apart. A combination of circular permutation and phasing analysis proved that (i) NF-Y-induced angles of the two gamma-globin CCAAT boxes have similar amplitudes; (ii) occupancy of the two CCAAT boxes leads to compensatory distortions; (iii) the two NF-Y bends are spatially oriented with combined twisting angles of about 100 degrees. Interestingly, such distortions are reminiscent of core histone-DNA interactions. We conclude that NF-Y binding imposes a high level of functionally important coordinate organization to the gamma-globin promoter.

Base Sequence↗
PubMed1998

A survey of 178 NF-Y binding CCAAT boxes.

The CCAAT box is one of the most common elements in eukaryotic promoters, found in the forward or reverse orientation. Among the various DNA binding proteins that interact with this sequence, only NF-Y (CBF, HAP2/3/4/5) has been shown to absolutely require all 5 nt. Analysis of a database with 178 bona fide NF-Y binding sites in 96 unrelated promoters confirms this need and points to specific additional flanking nucleotides (C, Pu, Pu on the 5'-side and C/G, A/G, G,A/C, G on the 3'-side) required for efficient binding. The frequency of CCAAT boxes appears to be relatively higher in TATA-less promoters, particularly in the reverse ATTGG orientation. In TATA-containing promoters the CCAAT box is preferentially located in the -80/-100 region (mean position -89) and is not found nearer to the Start site than -50. In TATA-less promoters it is usually closer to the +1 signal (at -66 on average) and is sometimes present in proximity to the Cap site. The consensus and location of NF-Y binding sites parallel almost perfectly a previous general statistical study on CCAAT boxes in 502 unrelated promoters. This is an indication that NF-Y is the major, if not the sole, CCAAT box recognizing protein and that it might serve different roles in TATA-containing and TATA-less promoters.

Animals↗
PubMed1998

Material properties assignment to finite element models of bone structures: a new method.

Finite element analysis (FEA) is widely adopted to investigate the mechanical behaviour of bone structures. Computed tomography (CT) data are frequently used to generate FE models of bone. If properly calibrated, CT images are capable of providing accurate information about the bone morphology and tissue density. The aim of this work was to develop a special program able to read a CT data set as well as the FEA mesh generated from it, and to assign to each element of the mesh the material properties derived from the bone tissue density at the element location. The program was tested on phantom data sets and was adopted to evaluate the effects of the discrete description of the bone material properties. A three-dimensional FE model was generated automatically from a 16 bit CT data set of a distal femur acquired in vivo. The strain energy density (SED) was evaluated for each model element for increasing model complexity (number of different material cards assigned to the model). The computed SED were strongly dependent on the material mapping strategy.

Algorithms↗
PubMed1998

Synergistic activation of transcription by nuclear factor Y and sterol regulatory element binding protein.

The current studies define the role of three distinct cis-elements in the proximal promoter of the rat farnesyl diphosphate (FPP) synthase gene. The three cis-elements, a sterol regulatory element 3 (SRE-3) flanked by an ATTGG motif (inverted CCAAT box), and a CCAAT box, form a sterol regulatory unit that is necessary and sufficient for sterol-regulated expression of FPP synthase promoter-reporter genes. FPP synthase promoter-reporter genes, that contain promoters with either wild-type nucleotide sequences or mutations in one or more of the three cis-elements, were transiently transfected into CV-1 cells. The activity of the wild-type promoter-reporter gene increased when the cells were incubated in sterol-depleted media or when the cells were co-transfected with a plasmid encoding the mature form of SRE binding protein (SREBP-1a). The results with the mutant promoter-reporter genes demonstrated that all three cis-elements were necessary for normal expression/regulation of the reporter gene by either sterols or by co-expressed SREBP-1a. Gel mobility shift assays demonstrated that the synergistic binding of SREBP-1a to SRE-3 was dependent on the binding of recombinant nuclear factor Y (NF-Y) to the DNA, consistent with the in vivo regulation studies.

Alkyl and Aryl Transferases↗
PubMed1997

Cloning and expression of human NF-YC.

The CCAAT box is an important element in eukaryotic promoters and NF-Y (CBF) is a conserved heterotrimeric protein binding to it. Two subunits, NF-YB and NF-YC, contain a histone-like motif. We cloned the complete cDNA coding for the human NF-YC gene. The ORF codes for a 335 aa protein that shows virtual identity to the rat sequence, confirming the stunning invariance of NF-Y genes across species. We expressed and purified the yeast homology domain of NF-YC in bacteria and performed EMSA together with the corresponding conserved domains of NF-YA and NF-YB, obtaining a CCAAT-binding mini-NF-Y. We evaluated the expression of NF-YC and found that mRNA levels are similar in different human tissues except in testis.

Amino Acid Sequence↗
PubMed1997

CCAAT binding NF-Y-TBP interactions: NF-YB and NF-YC require short domains adjacent to their histone fold motifs for association with TBP basic residues.

Both the TATA and CCAAT boxes are widespread promoter elements and their binding proteins, TBP and NF-Y, are extremely conserved in evolution. NF-Y is composed of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding. NF-YB and NF-YC contain a putative histone-like motif, a domain also present in TBP-associated factors (TAFIIs) and in the subunits of the transcriptional repressor NC2. Immunopurification of holo-TFIID with anti-TBP and anti-TAFII100 antibodies indicates that a fraction of NF-YB associates with TFIID in the absence of NF-YA. Sedimentation velocity centrifugation experiments confirm that two pools of NF-YB, and most likely NF-YC, exist: one associated with NF-YA and binding to the CCAAT box; another involved in high molecular weight complexes. We started to dissect NF-Y-TFIID interactions by showing that: (i) NF-YB and NF-YC interact with TBP in solution, both separately and once bound to each other; (ii) short stretches of both NF-YB and NF-YC located within the evolutionary conserved domains, adjacent to the putative histone fold motifs, are necessary for TBP binding; (iii) TBP single amino acid mutants in the HS2 helix, previously shown to be defective in NC2 binding, are also unable to bind NF-YB and NF-YC.

Amino Acid Sequence↗
PubMed1997

The effect of immunization of heifers against alpha 1-26 inhibin conjugate on the superovulatory response to pFSH.

A total of 121 heifers was blocked by time and diet and then randomly assigned, within block, to an inhibin-immunized (I) or a control (C) group. Immunized heifers (n = 61) received a primary immunization (Day 0) with 0.33 mg of an alpha 1-26 bovine inhibin fragment-human serum albumin (HSA) conjugate injected with non-ulcerative Freund's and DEAE-dextran adjuvants. Booster injections were given on Days 28 and 56. Control heifers (n = 60) received HSA and adjuvants. On Days 56 and 83 the ovaries of heifers were examined by ultrasound to determine the ovulation rate, and blood samples were collected for antibody titer determination. On Day 84, 61 heifers (C, n = 30; I, n = 31) received a total of 24 mg of porcine follicle stimulating hormone (pFSH), while 60 heifers (C, n = 30; I, n = 30) received 12 mg im pFSH, which was administered twice daily for 4 d in decreasing doses during the mid-luteal phase of the estrous cycle. Luteolysis was induced with prostaglandin F(2alpha) analog. The heifers were artificially inseminated and were slaughtered 7 d after estrus. Embryos were recovered and morphologically graded on a scale of 1 to 5 (1 = excellent; 5 = degenerated). Antibody titers (percentage binding at 1:125 serum dilution) differed (P < 0.01) between Group C and I heifers at Days 56 (0.1 vs 30%) and 83 (0.2 vs 37%), and 26% of Group I and 1% of Group C heifers (P < 0.01) had twin ovulations on Day 83. The mean number of embryos recovered was reduced (P = 0.02) in Group I heifers (8.9 +/- 1.2) compared with C heifers (12.1 +/- 1.1); however, the mean number of freezable embryos (Grades 1 and 2) was not affected (P = 0.61) by immunization, and there was no interaction with pFSH (P = 0.36). Ovulation rate as well as embryo yield and quality were not different (P > 0.10) between Group C and I heifers when 12 mg pFSH were administered; however, immunization decreased the superovulatory response to 24 mg of pFSH.

Journal Article↗
PubMed1996

Motifs resembling hepatocyte nuclear factor 1 and activator protein 3 mediate the tissue specificity of the human plasminogen gene.

Plasminogen is one of the key elements in the fibrinolytic process. Like most of the gene products that participate in such reactions and which interact with plasminogen, the site of its synthesis is mainly confined to the hepatocyte. Plasminogen RNA has additionally been detected in kidney and very low amounts also in testes. Deletional analysis has indicated that two 5' sequences located within 2.5 kb of the first ATG are responsible for the transcriptional activation and the tissue specificity of the expression of the gene. By DNase protection and gel mobility shift assays with HepG2 nuclear extracts, the two sequences were localized and found to be the recognition sites for the widely known hepatocyte nuclear factor 1 (HNF-1) a trans-acting factor, and a nuclear factor like activator protein 3 (AP-3). The first one lies in a rather unusual position, i.e. within the 5'-untranslated region. The latter is located further upstream in a region between --2200 and --2100 from the plasminogen mRNA cap site. Moreover, site-directed mutagenesis coupled by functional experiments in HepG2 cells has demonstrated a synergism between these two positively acting elements in controlling the transcription of the human plasminogen gene.

Base Sequence↗