Association of polymorphism within LTF gene promoter with lactoferrin concentration in milk of Holstein cows

This study analyzed the association between single nucleotide polymorphism (A/C) in position -28 located in the TATA box of LTF gene and the lactoferrin concentration in bovine milk secreted by healthy and infected udders. Out of 241, 69 cows were selected into the experimental group and were divided into 3 groups according to mean value of somatic cell count (SCC): I <180 000 cells/mL, II: 180 000 - 350 000 cells/mL and III > 350 000 cells/mL. In each SCC group, three LTF genotypes: AA, AC and CC were identified by PCR-SSCP method. A total of 604 milk samples were collected monthly and lactoferrin concentration was measured by ELISA. The 1-way ANOVA within SCC groups was performed to estimate association of -28 A/C genotypes with mean lactoferrin concentration per lactation. In the group of healthy cows (<180 000 cells/mL) LTF concentration in milk cows with the AA genotype (107.58 ± 17.92 μg/mL) was significantly higher than in homozygotes CC (52.09 ± 19.01 μg/mL). Unexpectedly, in cows with elevated SCC (>350 000 cells/mL) we observed a significant opposite relationship (207.21 ± 28.50 in CC vs 115.0 ± 28.6 μg/mL in AA). We hypothesized that a promoter with allele C, which cannot be recognized as a TATA sequence is becoming more accessible for other transcription factors, which may induce alternative LTF gene expression. We assume that our results demonstrate a very interesting effect of differential gene expression depending on polymorphism in a key regulatory motif (TATA box) and also on the health status of mammary tissues.

Noncanonical TATA Sequence in the UL44 Late Promoter of Human Cytomegalovirus Is Required for the Accumulation of Late Viral Transcripts

ABSTRACT During productive infection, human cytomegalovirus (HCMV) UL44 transcription initiates at three distinct start sites that are differentially regulated. Two of the start sites, the distal and the proximal, are active at early times, whereas the middle start site is active only at late times after infection. The UL44 early viral gene product is essential for viral DNA synthesis. The UL44 gene product from the late viral promoter affects primarily viral gene expression at late times after infection rather than viral DNA synthesis (H. Isomura, M. F. Stinski, A. Kudoh, S. Nakayama, S. Iwahori, Y. Sato, and T. Tsurumi, J. Virol. 81:6197, 2007). The UL44 early viral promoters have a canonical TATA sequence, “TATAA.” In contrast, the UL44 late viral promoter has a noncanonical TATA sequence. Using recombinant viruses, we found that the noncanonical TATA sequence is required for the accumulation of late viral transcripts. The GC boxes that surround the middle TATA element did not affect the kinetics or the start site of UL44 late transcription. Replacement of the distal TATA element with a noncanonical TATA sequence did not affect the kinetics of transcription or the transcription start site, but it did induce an alternative transcript at late times after infection. The data indicate that a noncanonical TATA box is used at late times after HCMV infection.

Distribution of UGT1A1 (TA) polymorphisms in Caucasian and Asian subjects

2063 Background: The hepatic isoform 1A1 of uridine diphosphate glucuronosyltransferase (UGT) is responsible for glucuronidation and detoxification of SN-38, the active metabolite of irinotecan. The presence of an additional TA repeat in the TATA sequence of the UGT1A1 gene is a common polymorphism, leading to a significant decrease in SN-38 glucuronidation. Patients with the UGT1A1 (TA)7 allele (either [TA]6/7 or [TA]7/7 ) are more likely to experience severe neutropenia and diarrhea following irinotecan chemotherapy. We assessed the distribution of the UGT1A1 (TA) polymorphism in Caucasian and Asian subjects. Methods: We used a fluorescent PCR-based assay to detect UGT1A1 (TA) polymorphisms in 129 healthy subjects (52 Caucasian, 34 Chinese, 36 Filipino, and 7 Japanese). The chi-square test was used to assess between-group differences in the distribution of UGT1A1 (TA) genotypes. Results: UGT1A1 (TA) genotype distribution differed significantly between Caucasian and Asian subjects (P = 0.003). The UGT1A1 (TA)6/7 and (TA)7/7 genotypes were more common in Caucasians than Asians. Genotype distributions did not differ significantly between men and women in either group ( Table ). Conclusions: The frequency of the deleterious UGT1A1 (TA)7 polymorphism was greater in Caucasians than in Asians; genotype frequencies were consistent with previous reports. In both groups, UGT1A1 (TA) genotype distributions were similar in men and women. [Table: see text] No significant financial relationships to disclose.

The Yeast FACT Complex Has a Role in Transcriptional Initiation

ABSTRACT A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation.

A TATA Binding Protein Mutant with Increased Affinity for DNA Directs Transcription from a Reversed TATA Sequence In Vivo

ABSTRACT The TATA-binding protein (TBP) nucleates the assembly and determines the position of the preinitiation complex at RNA polymerase II-transcribed genes. We investigated the importance of two conserved residues on the DNA binding surface of Saccharomyces cerevisiae TBP to DNA binding and sequence discrimination. Because they define a significant break in the twofold symmetry of the TBP-TATA interface, Ala100 and Pro191 have been proposed to be key determinants of TBP binding orientation and transcription directionality. In contrast to previous predictions, we found that substitution of an alanine for Pro191 did not allow recognition of a reversed TATA box in vivo; however, the reciprocal change, Ala100 to proline, resulted in efficient utilization of this and other variant TATA sequences. In vitro assays demonstrated that TBP mutants with the A100P and P191A substitutions have increased and decreased affinity for DNA, respectively. The TATA binding defect of TBP with the P191A mutation could be intragenically suppressed by the A100P substitution. Our results suggest that Ala100 and Pro191 are important for DNA binding and sequence recognition by TBP, that the naturally occurring asymmetry of Ala100 and Pro191 is not essential for function, and that a single amino acid change in TBP can lead to elevated DNA binding affinity and recognition of a reversed TATA sequence.

Binding of TATA Binding Protein to a Naturally Positioned Nucleosome Is Facilitated by Histone Acetylation

ABSTRACT The TATA sequence of the human, estrogen-responsive pS2 promoter is complexed in vivo with a rotationally and translationally positioned nucleosome (NUC T). Using a chromatin immunoprecipitation assay, we demonstrate that TATA binding protein (TBP) does not detectably interact with this genomic binding site in MCF-7 cells in the absence of transcriptional stimuli. Estrogen stimulation of these cells results in hyperacetylation of both histones H3 and H4 within the pS2 chromatin encompassing NUC T and the TATA sequence. Concurrently, TBP becomes associated with the pS2 promoter region. The relationship between histone hyperacetylation and the binding of TBP was assayed in vitro using an in vivo-assembled nucleosomal array over the pS2 promoter. With chromatin in its basal state, the binding of TBP to the pS2 TATA sequence at the edge of NUC T was severely restricted, consistent with our in vivo data. Acetylation of the core histones facilitated the binding of TBP to this nucleosomal TATA sequence. Therefore, we demonstrate that one specific, functional consequence of induced histone acetylation at a native promoter is the alleviation of nucleosome-mediated repression of the binding of TBP. Our data support a fundamental role for histone acetylation at genomic promoters in transcriptional activation by nuclear receptors and provide a general mechanism for rapid and reversible transcriptional activation from a chromatin template.

Structure of African Swine Fever Virus Late Promoters: Requirement of a TATA Sequence at the Initiation Region

ABSTRACT A number of mutations, including deletions, linker scan substitutions, and point mutations, were performed in the promoter of the late African swine fever virus (ASFV) gene coding for the capsid protein p72. The consequences of the mutations in terms of promoter activity were analyzed by luciferase assays using plasmids transfected into infected cells. The results showed that the promoter function is contained between nucleotides −36 and +5 relative to the transcription initiation site. Moreover, two major essential regions for promoter activity, centered at positions −13 and +3, were located along the 41-bp sequence, the latter mapping in the transcription start site. Sequence alignment with other ASFV late promoters showed homology in the region of transcriptional initiation, where the presence of the sequence TATA was observed in most of the promoters. Substitution of these four residues in three other late viral promoters strongly reduced their respective activities. These results show thatcis-acting control elements of ASFV p72 gene transcription are restricted to a short sequence of about 40 bp and suggest that transcription of late genes is initiated around a TATA sequence that would function as an initiator element.

Functional Interaction of CCR4-NOT Proteins With TATAA-Binding Protein (TBP) and Its Associated Factors in Yeast

The CCR4-NOT transcriptional regulatory complex affects expression of a number of genes both positively and negatively. We report here that components of the CCR4-NOT complex functionally and physically interact with TBP and TBP-associated factors. First, mutations in CCR4-NOT components suppressed the his4-912δ insertion in a manner similar to that observed for the defective TBP allele spt15-122. Second, using modified HIS3 promoter derivatives containing specific mutations within the TATA sequence, we found that the NOT proteins were general repressors that disrupt TBP function irrespective of the DNA sequence. Third, increasing the dosage of NOT1 specifically inhibited the ability of spt15-122 to suppress the his4-912δ insertion but did not affect the Spt− phenotype of spt3 or spt10 at this locus. Fourth, spt3, spt8, and spt15-21 alleles (all involved in affecting interaction of SPT3 with TBP) suppressed ccr4 and caf1 defects. Finally, we show that NOT2 and NOT5 can be immunoprecipitated by TBP. NOT5 was subsequently shown to associate with TBP and TAFs and this association was dependent on the integrity of TFIID. These genetic and physical interactions indicate that one role of the CCR4-NOT proteins is to inhibit functional TBP-DNA interactions, perhaps by interacting with and modulating the function of TFIID.

An n-Alkane-Responsive Promoter Element Found in the Gene Encoding the Peroxisomal Protein of Candida tropicalis Does Not Contain a C6 Zinc Cluster DNA-Binding Motif

ABSTRACT When an asporogenic diploid yeast, Candida tropicalis, is cultivated on n-alkane, the expression of the genes encoding enzymes of the peroxisomal β-oxidation pathway is highly induced. An upstream activation sequence (UAS) which can induce transcription in response to n-alkane (UASALK) was identified on the promoter region of the peroxisomal 3-ketoacyl coenzyme A (CoA) thiolase gene of C. tropicalis(CT-T3A). The 29-bp region (from −289 to −261) present upstream of the TATA sequence was sufficient to inducen-alkane-dependent expression of a reporter gene. Besidesn-alkane, UASALK-dependent gene expression also occurred in the cells grown on oleic acid. Several kinds of mutant UASALK were constructed and tested for their UAS activity. It was clarified that the important nucleotides for UASALKactivity were located within 10-bp region from −273 to −264 (5′-TCCTGCACAC-3′). This region did not contain a CGG triplet and therefore differed from the sequence of the oleate-response element (ORE), which is a UAS found on the promoter region of 3-ketoacyl-CoA thiolase gene of Saccharomyces cerevisiae. Similar sequences to UASALK were also found on several peroxisomal enzyme-encoding genes of C. tropicalis.

The Mating-Type Proteins of Fission Yeast Induce Meiosis by Directly Activating mei3 Transcription

ABSTRACT Cell type control of meiotic gene regulation in the budding yeastSaccharomyces cerevisiae is mediated by a cascade of transcriptional repressors, a1-α2 and Rme1. Here, we investigate the analogous regulatory pathway in the fission yeastSchizosaccharomyces pombe by analyzing the promoter ofmei3, the single gene whose expression is sufficient to trigger meiosis. The mei3 promoter does not appear to contain a negative regulatory element that represses transcription in haploid cells. Instead, correct regulation of mei3transcription depends on a complex promoter that contains at least five positive elements upstream of the TATA sequence. These elements synergistically activate mei3 transcription, thereby constituting an on-off switch for the meiosis pathway. Element C is a large region containing multiple sequences that resemble binding sites for Mc, an HMG domain protein encoded by the mating-type locus. The function of element C is extremely sensitive to spacing changes but not to linker-scanning mutations, suggesting the possibility that Mc functions as an architectural transcription factor. Altered-specificity experiments indicate that element D interacts with Pm, a homeodomain protein encoded by the mating-type locus. This indicates that Pm functions as a direct activator of the meiosis pathway, whereas the homologous mating-type protein in S. cerevisiae (α2) functions as a repressor. Thus, despite the strong similarities between the mating-type loci of S. cerevisiae and S. pombe, the regulatory logic that governs the tight control of the key meiosis-inducing genes in these organisms is completely different.