Transcription initiation in vivo without classical transactivators: DNA kinks ̄anking the core promoter of the housekeeping yeast adenylate kinase gene, AKY2, position nucleosomes and constitutively activate transcription

The housekeeping gene of the major adenylate kinase in Saccharomyces cerevisiae (AKY2, ADK1) is constitutively transcribed at a moderate level. The promoter has been dissected in order to de®ne elements that effect constitutive transcription. Initiation of mRNA synthesis at the AKY2 promoter is shown to be mediated by a non-canonic core promoter, (TA)6. Nucleotide sequences 5¢ of this element only marginally affect transcription suggesting that promoter activation can dispense with transactivators and essentially involves basal transcription. We show that the core promoter of AKY2 is constitutively kept free of nucleosomes. Analyses of permutated AKY2 promoter DNA revealed the presence of bent DNA. DNA structure analysis by computer and by mutation identi®ed two kinks ̄anking an interstitial stretch of 65 bp of moderately bent core promoter DNA. Kinked DNA is likely incompatible with packaging into nucleosomes and responsible for positioning nucleosomes at the ̄anks allowing unimpeded access of the basal transcription machinery to the core promoter. The data show that in yeast, constitutive gene expression can dispense with classical transcriptional activator proteins, if two prerequisites are met: (i) the core promoter is kept free of nucleosomes; this can be due to structural properties of the DNA as an alternative to chromatin remodeling factors; and (ii) the core promoter is pre-bent to allow a high rate of basal transcription initiation. INTRODUCTION Transcription activation of regulated genes is generally assumed to rely on interactions of speci®c transcriptional activator proteins with one or more constituents of the polymerase II holoenzyme complex. The prime role of transactivators is either to effect remodeling or removal of nucleosomes repressing the promoter in its inactive state or to enhance the rate of transcription initiation by recruiting, directly or indirectly, the basal transcription machinery to the core promoter (1±7). Some factors may exert both activities. The ®rst type of transcription factors will induce recruitment either of additional transactivators or of the basal transcription machinery to their target sequences without exerting a signi®cant activation potential of their own (8,9). Underlining the repressive role of nucleosomes, histone deprivation and concomitant depletion of nucleosomes have been shown to lead toÐat least partialÐactivation of inducible genes even under conditions of genetic repression. This implies that at some promoters, speci®c activator proteins may be dispensable, once accessibility for the transcription machinery has been established (7,10). The PHO5 promoter is one example in which positioned nucleosomes exclude transcription factors and the polymerase II holoenzyme complex from their binding sites. Transcription activation of PHO5 requires the binding of the active transactivator, Pho4p, to a low af®nity motif which is permanently accessible in a gap on promoter DNA between two exactly positioned nucleosomes. Upon induction by phosphate exhaustion, binding to the low af®nity site leads to removal or excessive remodeling of four accurately positioned nucleosomes which, in the repressed state, occlude the high af®nity binding sites of transcription factors that are essential for gene activation (7,11,12). If, however, nucleosome depletion is achieved experimentally in vivo by switching off expression of histone H4, transcription of PHO5 is *To whom correspondence should be addressed. Tel: +49 89 2180 6176; Fax: +49 89 2180 6160; Email: [email protected] Present addresses: Ulrich Oechsner, Lichtenstein Pharmaceutica GmbH und Co., Industriestrasse 10, D-82256 FuÈrstenfeldbruck, Germany Gary P. Schroth, Genelabs Technologies Inc., 505 Penobscot Drive, Redwood City, CA 94063, USA ã 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30 No. 19 4199±4207 Downloaded from https://academic.oup.com/nar/article-abstract/30/19/4199/2376091/Transcription-initiation-in-vivo-without-classical by guest on 16 September 2017 maximally induced even under repressing conditions and can dispense with additional transcription activators (7,10). In constitutive promoters, on the other hand, a static situation is presumed to keep the promoter permanently in an activated state to allow transcription (13). These promoters likely are constitutively kept free of nucleosomes. In the case of the relatively strong promoter of the ACT1 gene encoding yeast actin it has been speculated that two binding sites for the abundant general regulatory factor Reb1p [possibly in combination or synergistically with a poly(dA ́dT) element] are involved in positioning nucleosomes and in keeping the core promoter accessible (13). In the PFY1 promoter, encoding the G-actin-sequestering pro®lin, one binding site for the abundant regulatory factor Reb1p has been found to be necessary and suf®cient to keep nucleosomes off the DNA region spanning the core promoter and the transcription initiation sites (M.Angermayr, unpublished results). However, the general regulatory factor Reb1p does not have a signi®cant transcriptional activation potential of its own. Rather, its main role in promoters appears to rely on its property to keep a stretch of DNA in the ̄anks of its binding site free of nucleosomes and to position them at a distance (14,15). The lack of binding sites for classical transactivators implies that at these promoters transcription may ensue spontaneously, if the core promoter is readily accessible to polymerase II holoenzyme. We have analyzed the promoter of the yeast major adenylate kinase (ADK1 or AKY2, called AKY2 hereafter) which is constitutively expressed at a moderate level (16,17). The protein is unusually slowly turned over, and translation rates are low (18). Adenylate kinases are ubiquitous, abundant enzymes that ful®ll an essential housekeeping function. They provide the ADP required for oxidative and substrate chain phosphorylations and, because of the reversibility of the catalytic reaction, contribute to the maintenance of the homeostasis of high energy adenine nucleoside phosphate pools in the cell. We have identi®ed the minimal promoter of the gene which mainly consists of a non-consensus TATA sequence and enables expression rates of reporter constructs that are only slightly lower than the complete HTA1±AKY2intergenic region. Since this suggested that promoter activation of AKY2 dispenses with classical transcription factors, we have examined the possibility that structural peculiarities of the promoter DNA create a constitutively open chromatin conformation. We have found that two kinks in the DNA structure closely ̄anking the TATA-like element suf®ce to position nucleosomes at a distance and to allow moderately high levels of basal transcription without the necessity of transactivators. MATERIALS AND METHODS Strains and plasmids pBluescript M13 KS(6)-based vectors (Stratagene, Heidelberg, Germany) were used as templates for promoter truncations by polymerase chain reactions (PCR), or for sitedirected mutagenesis, base deletions or insertions in vitro using SOE-PCR (19). Plasmids were maintained and propagated in Escherichia coli strain XL1-Blue or SURE (both from Stratagene). The AKY2-upstream region together with the nine N-terminal coding triplets was ampli®ed by PCR as EcoRI±BamHI fragment and ligated to the respective restriction sites of pBluescript. All constructs were then fused in the proper orientation to the bacterial lacZ gene of the yeast low copy plasmid pYLZ7. pYLZ7 was constructed from pYLZ6 (20) by inversion of the bacterial selective marker gene bla, because with very short test promoter constructs interference with the bacterial bla gene sequence has been observed. Analyses of expression from the AKY2 promoter were performed in the genetic background of yeast strain W3031A (21) which had been transformed with the respective reporter constructs. Yeast were grown on standard media (22). PCR primers and in vitro mutagenesis Promoter truncations were produced by PCR using upstream primers with an EcoRI restriction site and a reverse primer with a BamHI site for fusion to the lacZ reporter (restriction sites not shown). The following forward primers were used: HN, 5¢-ACGGTAACATATGT-3¢; H5B, 5¢-CTTGAACATGATTGAGTAGC-3¢; H5C, 5¢-TTCACTTTGATAGTGTGACG-3¢; H4, 5¢-GCTCACGATTGCGCGATCC-3¢; H2, 5¢CTGTCCGCAGCAGCCCGCGGC-3¢; H1, 5¢-ATTCGCCCATTTTTTTTTGATTTTCGAC-3¢; H0, 5¢-TTCACTCTGGCTAGTTTTATTAC-3¢; H6, 5¢-GTATATATATATACGCATAAATTTCTC-3¢; H7, 5¢-CGCATAAATT-TCTGAAATGG-3¢. TIsh served as the reverse primer in most constructs, 5¢-ATTAGGACCATTCTAATGGATTCTG-3¢. Promoter mutations or internal deletions were obtained by site-directed mutagenesis using the kit and prescriptions from Stratagene. Permutation analysis was performed with DNA fragment H2/TIsh. Tandem cloning and permutation analysis The cloned PCR-ampli®ed 243 bp DNA fragment H2/TIsh was consecutively restricted with BamHI (and blunted by digestion of protruding 5¢ ends) and then with EcoRI (blunted by a ®ll-in reaction). Tandem ligation generated an EcoRI restriction site. The tandem DNA construct was digested in separate incubations using the set of restriction endonucleases indicated in Figure 2 to yield a set of DNA fragments of equal lengths permutated with respect to the fragment ends (23). After PAGE (8% gel, acrylamide:bisacrylamide = 38:2, 4°C), DNA was visualized by ethidium bromide staining. Analyses of chromatin structure Crude nuclei were prepared as described (24). DNase I or micrococcal nuclease digestions were performed as described by Thoma (25). Chromatin or naked DNA was digested (5 min, 37°C) by different concentrations of DNase I (Roche, Mannheim, Germany) (chromatin at 10.0, 15.0, 20.0 or 30.0 U/ml; naked DNA at 0.01, 0.05 or 0.1 U/ml) or micrococcal nuclease (MBI Fermentas, St Leon-Rot, Germany) (chromatin at 30, 60 or 120 U/ml; naked DNA at 1.5 or 3.0 U/ml). Reactions were stopped by the addition of 0.5% SDS, 4 mM EDTA, 50 mM Tris±HCl pH 8.0 and 200 mg of Proteinase K (Merck, Darmstadt, Germany) and incubated at 37°C for 30 min. DNA was extracted twice with phenol/ chloroform and precipitated by ethanol. The pellet was dissolved in TE buffer (10 mM Tris±HCl, pH 7.5, 1 mM EDTA) and RNA digested with 400 mg of RNase A (Boehringer, Mannheim, Germany) at 37°C for 60 min. 4200 Nucleic Acids Research, 2002, Vol. 30 No. 19 Downloaded from https://academic.oup.com/nar/article-abstract/30/19/4199/2376091/Transcription-initiation-in-vivo-without-classical by guest on 16 September 2017 After extraction once with phenol/chloroform and once with chloroform, DNA was ethanol-precipitated and digested with EcoRV. Gel electrophoresis was at 100 V in 1.5% agarose gels. After Southern transfer to nylon membranes (Biodyne A, Pall, Dreieich, Germany) DNA was detected by a randomly primed, radiolabeled 527 bp HindIII±EcoRV DNA fragment which hybridized to the 3¢ region of the AKY2 gene. DNA structure analysis DNA structure was calculated using the program BioCad which was developed in VMS-Fortran working on a DEC Microvax II GPX-station (26). The algorithm is essentially based on the wedge angles between nearest neighbor base pairs listed by Bolshoy et al. (27) and the assumption of 10.6 bp for a helical turn. Miscellaneous procedures The yeast expression vector pYLZ7 (above) was used for determining b-galactosidase activities of the respective promoter/lacZ fusions as described (28). Values give the mean expression activity of at least four independent clones. Generally, individual values varied in the range of 5±15%. Protein concentrations were determined according to the method described by Bradford (29). Yeast transformations were performed using the procedure described by Gietz et al. (30). Other molecular operations were performed according to standard procedures (31) or as recommended by the manufacturer.