Plant Molecular Biology 19: 985-1000, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

985

AT-rich promoter elements of soybean heat shock gene Gmhspl7.5E bind two distinct sets of nuclear proteins in vitro Eva Czarnecka, John C. Ingersoll and William B. Gurley

Department of Microbiology and Cell Science, 3103 McCarty Hall, University of Florida, Gainesville, FL 32611-0100, USA Received 23 September 1991; accepted in revised form 13 April 1992

Key words: high mobility group proteins, HMG, small molecular weight hsp, gel retardation, UV crosslinking Abstract

A 33 bp double-stranded oligonucleotide homologous to two AT-rich sequences located upstream ( -907 to -889 and -843 to -826) to the start of transcription of heat shock gene Gmhsp17.5E of soybean stimulated transcription when placed 5' to a truncated (-140) maize Adhl promoter. The chimeric promoter was assayed in vivo utilizing anaerobically stressed sunflower tumors transformed by a pTibased vector of Agrobacterium tumefaciens. Nuclear proteins extracted from soybean plumules were shown to bind double-stranded oligonucleotides homologous to AT-rich sequences in the 5' flanking regions of soybean/3-conglycinin, lectin, leghemoglobin and heat shock genes. These proteins were also shown to bind AT-rich probes homologous to homeobox protein binding sites from the Antennapedia and engrailed/fushi tarazu genes of Drosophila. Binding activity specific for AT-rich sequences showed a wide distribution among various plant organs and species. Preliminary characterization indicated that two sets of nuclear proteins from soybean bind AT-rich DNA sequences: a diverse high-molecular-weight (ca. 46-69 kDa) group, and a low-molecular-weight (23 and 32 kDa) group of proteins. The latter meets the operational criteria for high-mobility group proteins (HMGs).

Introduction

The soybean heat shock gene Gmhspl7.5E encodes a heat shock protein (hsp) related to the low-molecular-weight class first characterized in Drosophila [44]. This class of hsps contains from 20 to 30 members in soybean and is the predominant type expressed during the heat shock (HS) response in many plants [32]. Heat shock gene Gmhspl 7.5E is an actively transcribed member of this family with a promoter composed of multiple upstream elements in addition to the TATA motif (Fig. 1A). Transcriptional regulatory sequences

include heat shock consensus elements (HSE) positioned TATA proximal and AT-rich regions centered at -140 (AT proximal 1) and -220 (TATA dyad) [10]. The HSEs are primarily responsible for the thermoinducibility of the gene with the AT-rich elements acting in concert to amplify the magnitude of induction. Other ATrich sequences are located far upstream in the region from -907 to -830 and have been shown to bind nuclear proteins, but do not appear to enhance transcription of Gmhsp17.5E when assayed in transgenic sunflower tumors utilizing a pTi-based vector [ 10].

986 A. Gmhspl7.5E Promoter AT distal IV III II I

i~al -907

AT proximal 1

TATA-dyad

-830

HSE Site

-153

1

TATA

-120-103-87-72 -40 | • GT m o t i f

CCAAT

o

I

'.8

.oor

A Tcorn

HSE Site

i

-226 -214

B. Probes:

2

I

e .os.9

5'-tcgacAAAAATAATA.TTAATATTATATTGAA .Ag-3' -843 i

-826 I

I

.ls9

-12o

A Tprox 1

5'-tcgacGAAGTGGAAGAAAAATAAATATAATGATGTGTAGTAAACAg.3'

TA TAdyad

5'-tcoacGTTATAGGTATAAAGAATTTCTATATGATGATGAg.3'

1~3B

5'-tcgacCTCATTAATAAAAAAAAAAAAAATCATTTGTg*3'

LegHb

5'-tcgaGATATATTAA TATTTTATTTTATAag-3'

Locl

5'-tcgacTTTTTGAA TTTAATTAATTAAAATATATATg.3'

Lec2

5'-tcgacAAAATATATATGCTAACAACATTAAATTTTAAATTTACGTCT

-234

-2.01

-2SO

-2.so

-246

-223

-189

-160

- 170

AATTATAT.Ago3' .e.19

-120

-7..

B.conl

5'.tcgacATTTAATACGTATTATTTATTAAAAAAATATg-3 '

B.con2

.r.. .7.ss 5'-tcgacAAAAAAATATGTAATAATATATTTATATTTTAATATiI-3'

Anna

5'-tcgagATAATATAATAATAAAAATAATAATAATAATAATAATATAATg-3'

NP2

5'-tcgagTCAATTAAATgaTCAATTAAATgag-3'

HSEI

5'.tcgacGTAGGATTTTTCTGGAACATACAAG-3'

.Ls

-47

Fig. 1. A. Schematic diagram of the Gmhspl7.5E promoter. Sites 1 and 2 represent high (9 out of 10 bp match) and low (7 out of 10 bp match) homology to the Drosophila HSE. A CCAAT-like motif is centered at -84 and a GT-rich sequence with homology to the SV40 enhancer core [34] at -96. The remaining open boxes represent AT-rich sequences that bind nuclear proteins

in vitro. B. Synthetic double-stranded oligonucleotide probes used in gel mobility retardation and UV crosslinking assays. The numbers above the sequences refer to transcription coordinates within their respective genes. Lower-case bases correspond to either Sal I or Xho I linkers or, as in the case of NP2, sequences not present in the consensus. The AT composite (A Tcom), AT proximal 1 (ATproxl), TATA dyad (TATAdyad) and HSE1 oligomer sequences are from the Gmhsp17.5E promoter [8, 10], whereas 17.3B is from soybean HS gene Gmhspl 7.3B [3]. The A Tcom oligomer is homologous to regions I, IV and 0 of the Gmhspl7.5E promoter shown by brackets. In the genornic D N A there is a G in position -692 within the 0 region. The LegHb oligonucleofide (oligo I) is derived from binding site 1 of the soybean leghemoglobin lbc3 gene [28]. Lectin 1 and 2 are protein binding sequences from soybean lectin [29] and fl-conglycinin 1 and 2 oligomers are homologous to upstream sequences of ~' -type fl-conglycinin [ 14]. Antpa and NP2 oligomers are homologous to homeodomain protein binding sites from Drosophila Antennapedia and fushi tarazu/ engrailed genes, respectively [38].

Numerous plant genes contain AT-rich sequences upstream of the start of transcription. Developmentally regulated genes in this category include lectin, Kunitz inhibitor [29], leghemoglobin [27,28], nodulin [27], helianthinin [31],

French bean fl-phaseolin [7], and potato class I patatin [41]. Light-regulated genes such as rbcS and cabE, the stress-regulated chalcone synthase gene, and HS genes Gmhspl7.5E and Gmhsp17.3B also contain AT-rich elements [3,

987 10, 11, 24, 25]. Two lines of evidence suggest that many of these AT elements act as sites of transcriptional regulation: analyses of mutations and chimeric promoter constructions in transgenic plants or electroporated protoplasts, and the finding that AT-rich elements often bind nuclear proteins in vitro. Initial studies of AT elements within soybean lectin [29] and leghemoglobin [28] genes suggested that nuclear proteins that bind these sequences in vitro show a strong developmental or tissue-specific bias in their distribution in the plant which correlates with the pattern of expression of the particular gene in which the AT element is located. However, in the case of leghemoglobin (lbc3), which is only expressed in nodules, recent studies indicate that AT element binding activity is also found in roots and leaves in addition to nodules [27]. At present the role that AT elements may play in determining the developmental specificity of plant gene expression is unclear. In this study we show that a 33 bp doublestranded oligonucleotide homologous to AT-rich sequences located upstream of soybean HS gene Gmhsp17.5E can increase anaerobic stress-induced transcription of the maize Adhl promoter. Consistent with its activity as a promoter element is the finding that nuclear proteins from soybean plumules bind this AT element in vitro. We also characterize the in vitro binding of soybean nuclear proteins to a variety of AT-rich DNA sequences from the upstream region of several plant and animal genes including Gmhsp17.5E of soybean. Gel mobility retardation assays, UV crosslinking, and protein renaturation studies indicate that proteins showing affinity for AT-rich sequences are either HMGs, or belong to a diverse set of proteins of higher molecular weight which have some HMG-like properties. Materials and methods

Construction of Adh l /A T element chimeric promoter mutants The 33 bp A Tcom synthetic oligomer was inserted upstream of the maize Adhl promoter [ 12] (see

Fig. 1B and2C) at positions -140 and -410 (Sal I linker sites) and assayed in vivo using sunflower tumors. These Adhl/A Tcom promoter constructs (test genes) were fused to promoterless (position -1 to + 1.2 kb) constructs of the 780 gene of T-DNA [4] to facilitate cloning manipulations and S1 nuclease mapping of the transcripts. Use of the 780 gene as a reporter also eliminated cross-hybridization with the endogenous Adhl gene. The test genes were cloned into the shuttle vector p251 [26] which contained a T-left fragment to target integration of the shuttie vector into the T-DNA of pTi 15955 by homologous recombination in Agrobacterium tumefaciens. An additional copy of the Adhl promoter (Adh1/780 chimeric)was incorporated into the shuttle vector to serve as an internal standard (reference gene) for transcript quantification. The reference gene consisted of a full-length Adhl promoter (wild type) which included 5' flanking sequences to position -1094 (Bam HI site). In order to distinguish between test and reference gene transcripts, the test gene differed from the reference gene by 22 extra bp in the 5' untranslated leader sequence. The test gene was constructed by the ligation of a Sal I linker at position + 78 of the Adhl leader to an Xho I linker at -1 of the 780 gene. The Sal I/Xho I linker-derived sequences contributed 8 bp to the leader of the chimeric gene. In the reference gene, the Adhl leader at position + 65 was directly ligated to position + 1 of the 780 gene leader [26]. Double gene shuttle vectors (reference plus test genes) were transferred into A. tumefaciens strain Ag5260 (str r) (derived from strain 15955 [4]) via tripartite matings as previously described [4, 18].

Tumor formation and anaerobic stress The test and reference gene constructs were integrated into sunflower (Helianthus annuus cv. Large Grey) genomic DNA by inoculation of sixday-old seedlings with A. tumefaciens harboring shuttle vector:pTi co-integrate plasmids. Crown gall tumors were harvested two weeks later and either frozen immediately in liquid N2 (uninduced

988 control), or subjected to 3 to 9 h incubations under anaerobic stress using a buffered solution [22] saturated with argon gas.

Assay of Adhl /A T element chimeric promoters Transcripts derived from the test and reference genes were analyzed by S 1 nuclease hybrid protection mapping [ 16]. The end-labeled hybridization probe consisted of a D N A fragment from the Adhl gene which included sequences from -132 to + 78 fused via an 8 bp tinker to the promoterless 780 gene from -1 to + 59. Total RNA (100 #g) was heat-denatured (85 °C, 30 rain) in hybridization solution containing 80~o formamide, 40 m M PIPES, pH 6.4, 400 m M NaC1 and 1 m M EDTA [16], and annealed overnight at 45 ° C. Analyses of S 1 nuclease-resistant hybrids (test 146 bp, reference 59 bp) and determination of relative transcription levels (RTLs) were conducted as previously described [ 10]. RTLs were determined by averaging the results from at least three separate mapping experiments. The range of variation in RTLs for each construction was no more than + 10~ of the wild-type level due to the pooling of 200 to 300 tumors for each RNA extraction.

Isolation of nuclear extracts Soybean (Glycine max), sunflower and pea (Pisum sativum cv: Laxton's var. Progress number 9) seedlings were grown in a mixture of vermiculite and pearlite in the dark for seven days at 28 °C and the plumules harvested for use in the preparation of crude nuclear extracts. Extracts from the roots of soybean and maize (Zea mays), and maize coleoptiles were prepared from seedlings grown in the dark at 28 °C for three days on moist layers of cheese cloth-covered absorbant paper. For heat-shocked extracts, plumules, coleoptiles and primary roots were harvested and then incubated at 40 °C for 2 h. Non-heat-shock nuclear extracts were prepared from cauliflower florets and also from HeLa cell suspensions

(1 x 10 9 cells) kindly provided by J.B. Flanegan, University of Florida. The preparation of nuclear pellets and extracts was according to the method of Wu [50]. Nuclear proteins were solubilized from the crude nuclear pellets by high salt (0.4 M KCI), clarified by centrifugation at 100000xg for 1 h (Beckman/ Dupont SW55 rotor) at 4 °C and then frozen with liquid N2. From 50 to 100 g of tissue was used in a typical preparation and protein concentrations varied from 0.5 to 2.0 #g/#l.

Purification of nuclear proteins Crude nuclear extract from control seven-day-old soybean plumules was ammonium sulfate-precipitated at 40~o saturation. The precipitated proteins were recovered and resolved on a phosphocellulose column over a 0.1-2.0 M NaCI gradient in Wu's solution III [50]. Fractions 19 through 45 containing the peak of activity of low-mobility complex (L-complex) formation were collected and subjected to heparin Sepharose chromatography (Econo-pac cartridge, BioRad). Proteins were eluted over a 0.05-2.0 M NaC1 gradient in solution III. Fractions 16 to 29 from the heparin Sepharose column were collected and loaded onto a DNA-affinity column (ATcom-Sepharose) at 0.1 M NaCI and activity eluted using a linear gradient of 0.1-2.0 M NaC1 in solution III. Lcomplex activity eluted at 1.4-2.0 M NaC1. After desalting the fractions containing activity, the sample was reapplied to the DNA-affinity column. Combined fractions 20 to 25 from the second column were desalted, concentrated, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12~o polyacrylamide gels [33].

Synthetic oligomerprobes Equal amounts of complementary single strands of various oligomers were denatured for 3 min at 100 °C and then annealed by placing sample tubes in 30 ml of water at 65 °C that was allowed

989 to cool to room temperature over a period of 20-30 min. The annealed double-stranded DNA oligomers (Fig. 1B) homologous to protein-binding sites were employed as binding probes in gel mobility retardation assays. The oligomers were 3' end-labeled with [oc-32p] dCTP and dATP by filling in with Escheriehia coli DNA polymerase large fragment [37]. The AT composite oligomer probe was very AT-rich and showed a tendency to disassociate into single strands (bottom band of free probe) under our electrophoresis conditions. Only the double-stranded form of the A Tcom probe (top band of free probe) was bound by nuclear proteins.

Gel retardation assays

DNA binding activity was monitored by gel mobility retardation assays according to Singh et al. [43] as described previously [8]. Aliquots (510 #g unless otherwise stated) of nuclear protein were generally incubated with 11 #g of yeast t R N A and 500 ng of Hae III restriction fragments of ~X174 bacteriophage replicative form DNA and 50 x 103 cpm of the end-labeled oligomer probe in 20/~1 of a solution containing HEPES (pH 7.9), 0.1 mM EDTA, 0.5 mM DTT, 70100 mM NaC1, 0.4 mM MgC12, and 2.5~o glycerol solution. Protein-DNA complexes were resolved at room temperature by 4% polyacrylamide gel electrophoresis at 110 V for 2 h.

Photoaffinity crosslinking

The double-stranded AT composite oligomer (ATcom) probe was prepared by the incorporation of 5-bromo-2'-deoxyuridine-5'-triphosphate into the bottom strand during a primer-extension radiolabeling reaction with Klenow large fragment [37]. Typically 0.8 ng of the probe (1 × 106 cpm) was present in the 10 #1 standard binding reaction supplemented with 0.1 #g//~l of bovine serum albumin. Samples were crosslinked at room temperature (RT) for 20 min at a distance of 4.5 cm from a UV light bank (Fotodyne transilluminator

model UV 440, short-wave UV radiation at 254 nm). DNase I digestion buffer and DNase I (10 ng) were added and incubation was continued for 10 rain at 30 oC. Reactions were terminated by adjusting EDTA (pH 8.0) to 25 mM. Crosslinked proteins were analyzed by SDSPAGE with either 12~o or 20~o acrylamide gels that were subsequently dried under vacuum and exposed to X-ray film.

Recovery of DNA binding activity from denatured nuclear proteins

Crude nuclear extracts prepared from seven-dayold soybean plumules were denatured by guanidine-HC1 (6 M) treatment for 30 min at RT and subsequently passed through a BioRad Spin-6 column to exchange protein dilution buffer [23] for the denaturant. The renaturation was conducted at RT for various times up to 12 h. Sampies were frozen with liquid N2 and later tested by gel retardation assays,

Results

A T composite oligomer acts as a positive promoter element with the maize Adhl promoter

The maize Adhl gene is transcribed in sunflower tumors in an anaerobically inducible manner similar to that seen in maize roots (unpublished data). Sequences between - 140 and -92, harboring the anaerobic response element (ARE) [49], are responsible for this inducibility, though the promoter region 5' to -140 (-140 to -410) also contributes to overall transcriptional activity [26]. We tested the ability of AT-rich sequences present upstream of the soybean Gmhsp17.5E gene to enhance transcriptional expression of the maize Adhl promoter using the sunflower tumor system. Representative gels showing the results of S 1 nuclease mapping of sunflower transcripts derived from the reference and chimeric Adh1:780 test genes are shown in Fig. 2A. The 33 bp A Tcom oligomer acted as a positive upstream element,

990

B

+1

,

,

.¢/

/(

DNA probe

,

test RNA ref. RNA S1 nuclease

~, test 146 bp

protected hybrids

59 bp

rst.

Fig. 2A/B. Stimulation of the maizeAdhl promoter by insertion oftheA Tcom oligomer. A. Transcripts homologous to the Adhl /780 chimeric gene were mapped by S1 nuclease hybrid protection. Test gene constructs are as follows: wild-type promoter with 1094 bp of upstream sequences (lane 1); 5' deletion to position -140 (lanes 2 and 3); A Tcom oligomer inserted at -140 (lanes 4 through 10); 5' deletion to -410 (lanes 11 and 12); ATcom oligomer inserted at position -410 (lanes 13 through 18). RNAs isolated from uninduced tumors are designated as U. All other lanes contain RNA from anaerobically stressed (3.5 h to 9 h) tumors. Anaerobic stress was 3 h 30 min for the sample in lane 9, and 9 h for the sample in lane 10. Legend: P, full-length probe; Test, hybrids formed with test gene RNA (146 bp); Ref., hybrids formed with reference gene RNA (59 bp). Number ofA Tcorn oligomers inserted is indicated above the lanes and ranges from 0 to 5 copies. B. Diagram shows the DNA probe (test gene), transcripts and S 1 nuclease digested hybrids. The open box indicates the position of the extra 22 bp in the test gene that provide a site for S 1 nuclease cleavage of hybrids formed between the reference RNA and the test DNA probe. i n d e p e n d e n t o f orientation, when placed 5' to the A d h l p r o m o t e r truncated to positions - 1 4 0 and - 4 1 0 . Anaerobically inducible expression o f the - 1 4 0 a n d - 4 1 0 5' deletions were assigned a fold induction value o f one. N o transcripts were detectable in unstressed tumors. Addition of multiple copies o f the A T c o m oligomer at - 1 4 0 increased gene expression in a linear m a n n e r , roughly 5-fold per c o p y o f inserted oligomer (Fig. 2C). The e n h a n c e m e n t per inserted oligomer was m u c h reduced ( < 1-fold) w h e n positioned

further u p s t r e a m at - 4 1 0 which suggests a strong position d e p e n d e n c e for the A Tcom element. ( N o t e that the e n h a n c e m e n t effect o f the A Tcom oligomer was similar at 31/2 and 9 h o f anaerobic treatment; Fig. 2A, lanes 9-10.)

A common set o f proteins binds A T-rich probes T o determine the binding specificity o f nuclear proteins for AT-rich sequences, a n u m b e r o f syn-

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C maize Adhl promoter -410

.140

I

+1

I.

I

I

II

ARE

AT~

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~r

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780

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Fig. 2C. Plasmid constructions and summary of results displayed in panel A. The number and orientation of arrows indicate the copy and orientation of the soybean A Tcom oligomer introduced upstream of the maize Adhl promoter at positions -140 or -410. Fold induction refers to the relative amount of transcription observed after anaerobic induction. A value of one was assigned to the -140 and -410 5' deletion constructs containing no inserted ATcom oligomers.

thetic double-stranded oligonucleotides were used as competitors and probes in gel mobility retardation assays. The AT-rich sequences selected represent a sampling of sites previously characterized as either sites of in vitro protein-DNA interaction [8] or, in the case of HS gene Gmhsp17.3B, are located within a block of upstream sequences required for full expression of a soybean HS promoter [3]. Fig. 1B lists D N A sequences that were either used as probes or competitors for in vitro protein binding assays. These oligonucleotides were homologous to D N A sequences in the promoters of Grnhsp17.5E, Gmhspl 7.3B, leghemoglobin (lbc3), lectin, and flconglycinin genes of soybean. Two Drosophila sequences were also used as probes: a TAA-repeat sequence that binds a synthetic homeodomain peptide derived from the Antennapedia gene product [38], and the NP2 sequence 5'-TCAATTAAAT-3' that binds the proteins encoded

by engrailed (en) and fushi tarazu (ftz) genes [ 13]. The competition between the A Tcorn probe and other AT-rich probes for D N A binding is presented in Fig. 3. The nuclear extract was prepared from heat-shocked tissues since the A Tcom probe is from Gmhspl 7.5E which is active in soybean only during HS. The probe was incubated with nuclear proteins either in the absence (lane 2) or presence (lanes 3 to 20) of an excess of competitor oligomers. The resulting protein-DNA complexes were resolved from unbound D N A by PAGE. In order to suppress nonspecific binding, tRNA and ~X174 replicative form (RF) D N A were added to each binding reaction. All of the AT-rich oligomers showed effective competition for protein binding to the labeled A Tcom probe at either 100- or 1000-fold molar excess with the exception of Lec2 and ATproxl. At 1000-fold molar excess, the A Tproxl oligomer was able to compete approximately 80~o of the binding ac-

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Fig. 3. Gel mobility retardation assays using soybean nuclear extract. Crude nuclear extracts from heat-shocked (40.°C, 2 h) soybean plumules were incubated with the ATcom oligomer probe. Protein-DNA complexes (L and H) were fractionated from

unbound DNA (F) by electrophoresis on 4% nondenaturing polyacrylamide gels. Lane 1 represents nonspecific binding without carrier nucleic acids. Lane 2 shows specific binding without unlabeled competitor probe. In vitro binding in lanes 3 through 20 was competed with 100-fold (odd-numbered lanes), or 1000-fold (even-numbered lanes') molar excess of unlabeled oligonucleotides as indicated above lanes.

tivity (lane 20), whereas under the same conditions Lec2 competed only 20~o of,the binding (lane 14). In the reciprocal experiments, radioactively labeled oligomers corresponding to the ATrich sequences from leghemoglobin, lectin and flconglycinin promoters were all capable ofproteinD N A complex formation as monitored by the mobility shift assay (data not shown). The Antennapedia probe readily formed protein: D N A complexes in band shift assays and southwestern blots, and was the most efficient competitor (data not shown). The specificity of in vitro binding to AT-rich probes was demonstrated when the HSE1 oligomer showed no visible interference with ATcom binding at 1000-fold molar excess (lane 18). The competition ofATcom binding by each of the AT-rich oligomers indicated that a common group of nuclear proteins was able to bind with varying affinities to all of the AT-rich sequences tested. The large difference in affinity between Lecl and Lec2 probes indicated that these binding events may occur at specific AT-rich blocks, and are not merely the result of general binding to random clusters of A and T residues. The distribution of proteins able to bind ATrich sequences was surveyed in nuclear extracts from various sources. Nuclear extracts from

monocotyledonous and dicotyledonous plants, heat- shocked or non-heat-shocked plant organs, as well as HeLa cells, were analyzed by gel retardation assays using the A Tcom probe (Fig. 4). Complexes were formed in all samples tested, indicating that activity was neither tissue- nor species-specific. It is interesting that a similar binding activity was also present in human cells (HeLa). The low-mobility complexes (L, Fig. 4) all had roughly the same electrophoretic mobility. In most cases, L-complexes formed a broad band raising the possibility that more than one size class of proteins may bind the probe, or multiple proteins were bound to single-probe molecules. In addition to the low-mobility complexes, fastermigrating complexes (H, Fig. 4) were also formed with the sunflower, soybean and pea extracts.

Partial characterization of proteins that form highand low-mobility complexes The crude nuclear extract was partially purified in an attempt to resolve the two size classes of binding activity evident in the gel retardation analyses. Extracts were sequentially fractionated by ammonium sulfate precipitation at 4 0 ~ saturation, phosphocellulose column chromato-

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Fig. 4. Gel mobility retardation assays using the A Tcom probe and nuclear extracts from a variety of plant organs and species, and HeLa cells. Crude heat-shocked (40 °C, 2h; lanes 1-6) and control (28 °C, 2 h; lanes 7 and 8) nuclear extracts were obtained from soybean plumules (lane 1) or roots (lane 4), sunflower plumules (lane 2), pea plumules (lane 3), maize coleoptiles (lane 5) and roots (lane 6), cauliflower florets (lane 7) and HeLa cells (lane 8). Protein-DNA complexes were fractionated by electrophoresis on 4 ~ polyacrylamide gels to resolve bound and free ATcom probe.

graphy, heparin agarose (BioRad Econo-pac) chromatography and two passes through an ATcom-Sepharose DNA affinity column. The phosphocellulose fractions were screened in duplicate by gel retardation assays using the ATcom probe with (Fig. 5A) and without (Fig. 5B) carrier nucleic acids. Resolution of proteins involved in formation of the L- and H-complexes was achieved using a linear NaCI gradient. Proteins capable of forming L-complexes eluted at salt concentrations ranging from 0.64 M to 1.26 M in fractions 20 through 44. Proteins forming the Hcomplexes eluted from approximately 1.29to 1.64 M NaCI (fractions 42-54; Fig. 5B) The formation of H-complexes was preferentially suppressed by the presence of heterologous carrier nucleic acids (compare Figs. 5A and B). The differential elution properties and competition by heterologous carrier DNA suggest that proteins forming the H- and L-complexes were not the same.

Further evidence for differences between proteins forming the L- and H-complexes was obtained by analysis of protein denaturation and renaturation properties as shown in Fig. 6. Equal amounts of crude nuclear proteins were dena-

Fig. 5. Phosophocellulose and DNA-al~ity chromatography. A and B. Phosphocellulose fractions (Fr) were tested for protein-DNA complex formation with double-stranded A Tcorn monomer probe in the presence (A) or absence (B) of carrier nucleic acids (1.6 gg of yeast tRNA, 83 ng of ¢X174/ Hae III RF DNA). C. Protein-DNA complexes after a single pass (lane 2; 4/~1) or double pass (lanes 3-6; 4, 8, 16 and 32 #1 respectively) through an ATcom-Sepharose affinity column. Fractions were tested in band shift assays without carrier nucleic acids. The interaction of the ATcom probe with 1/~1 of crude nuclear extract from soybean plumules is shown in lane 1.

tured by treatment with increasing concentrations of guanidine-HCl (Fig. 6, lanes 3-6) and subsequently renatured for 12 h. The nuclear proteins that formed L-complexes were very sensitive to denaturation in 6 M guanidine-HCl and unable to regain DNA binding activity even after long pe-

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Fig. 6. Guanidine-HC1 denaturation of the L- and H- complexes. The aliquots of crude nuclear extracts were treated for 15 min at RT by increasing concentrations of guanidine-HCl: 0 M (lane 3), 1 M (lane 4), 3 M (lane 5), 6 M (lane 6) which was removed prior to assembly of the binding reaction by gel filtration. Equal amounts of protein of each renatured sample were tested by the band shift assay. The extent of binding of 0.5 #1 aliquots of crude nuclear protein in the presence (lane 1) or absence (lane 2) of carrier nucleic acids can be compared roughly with the level of protein-DNA complex formation after guanidine-HC1 treatment. No carrier nucleic acids were present in the binding reactions (lanes 3-6) in order to detect the H-complex.

riods of renaturation (lane 6). The proteins forming H-complexes, on the other hand, were easily renatured and retained activity in DNA binding assays. The decrease in L-complexes did not result in an increase of H-complex formation suggesting that the two mobility classes comprise different sets of proteins. The L-complex was, therefore, not generated by multiple bindings of proteins that form the H-complex. DNA-affinity chromatography utilizingA TcomSepharose was employed to characterize proteins of the L-complex (Fig. 5C). Proteins forming the H-complex were excluded in two passes over the affinity matrix by pooling fractions containing the L-complex. Increasing the amount of affinitypurified protein per binding assay did not generate the H-complex, again demonstrating that the L-complex was not formed by multiple binding of H-complex proteins. The DNA-affinity-purified proteins were resolved by SDS-PAGE and either analyzed by photoaffinity crosslinking, or silverstained (not shown). Three to four major bands corresponding to proteins ranging from 46 to

69 kDa were crosslinked to the A Tcom probe. No proteins were detected by silver staining unless samples were concentrated 28-fold. The silverstained gel of concentrated sample revealed numerous (8-10) low-abundance proteins ranging from ca. 40 kDa to > 200 kDa. Although these proteins appeared to be retained on the DNAaffinity column, we were not able to demonstrate their specific binding to the probe since many did not correspond to proteins detected in the UV crosslinking assay. Some of the bands detected by silver staining may represent degradation products of larger proteins. Degradation is consistent with the marked increase in lability of Lcomplex proteins after DNA-affinity chromatography evidenced by the drastic loss in DNA binding activity following protein concentration or dialysis of affinity-pure preparations. Harrison etal. [25] reported a similar instability after DNA-affinity purification for Phaseolus vulgaris SBF-1 factor that interacts with AT-rich sequences of bean chalcone synthase gene. The affinity purification approach did not lead to the purification of a homogeneous species of protein, but showed that proteins forming the L-complex represent a diverse set larger than ca. 40 kDa.

UV crosslinking of proteins recovered from L- and H-complexes UV crosslinked DNA-protein complexes were eluted from band shift gels in regions corresponding to L- and H-complexes and subsequently fractionated by SDS-PAGE (Fig. 7). The Lcomplex comprised multiple proteins ranging from ca. 46 to 69 kDa. The H-complex was predominantly formed by two proteins of 32 kDa and 23 kDa. The involvement of the 23 and 32 kDa proteins in formation of the H-complex was confirmed by the correlation in the disappearance of these proteins in the UV cross-linking assay (Fig. 7, lane 2; Fig. 8B, lanes 1 and 2) and competition for the H-complex in band shift experiments (Figs. 3, 5A and B, and 8A) by the addition of carrier nucleic acids. There is some indication that high-mobility group proteins

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Fig. 7. L- and H-complexes are formed by two different sets of proteins. UV crosslinked A Tcom DNA-protein complexes formed with crude nuclear proteins (lanes 1, 2 and 4) or with ATcom-Sepharose affinity purified proteins (lane 3) were resolved on a 4% polyacrylamide gel using band shift conditions and extracted from the gel in regions corresponding to L- and H-complexes. Recovered proteins were fractionated by SDSP A G E on 12% polyacrylamide resolving gels. Heterologous carrier nucleic acids were only used during the binding assay shown in lane 2. Lanes 1-3 show proteins extracted from Lcomplexes and lane 4 from the H-complex. M = protein molecular weight markers.

(HMGs; see below) may also be present in the L-complex (Fig. 7, lane 1). These most likely result from binding both L-complex proteins and HMGs to the same probe molecule.

The HMG-like properties of nuclear proteins forming L- and H-complexes H M G proteins are defined on an operational basis as those extractable from chromatin by 0.35 M NaC1 and soluble in 2~o trichloroacetic acid (TCA) [20]. We examined the possibility that nuclear proteins forming the L- and Hcomplexes were related to HMGs. Soybean HMGs were isolated from nuclei according to Spiker [45]. Recovered HMGs (TCA supernatant) and non-HMGs (TCA precipitant) were tested for their ability to bind the A Tcom oligomer in gel retardation assays and UV crosslinking (Fig. 8). In the absence of heterologous carrier nucleic acids, isolated HMGs formed the Hcomplex (Fig. 8A, lane 5) while the non-HMG fraction generated the L-complex (Fig. 8A,

Fig. 8. HMG-like properties of proteins forming H- and Lcomplexes. Crude nuclear extracts (0.4 M NaCI) were fractionated into H M G (TCA-soluble) and non-HMG (TCAinsoluble) proteins according to Spiker etal. [45]. Each fraction was assessed by gel mobility retardation assays (panel A) and UV crosslinking (panel B) using the A Tcom oligomer as probe. In panel A, lanes show the effect of competition with carrier nucleic acids on formation ofprotein-DNA complexes. Binding activities present in crude nuclear extracts (2.6/~g of protein) are shown in lanes 1-4, activity in the H M G fraction in lanes 5-8, and activity in the non-HMG fraction in lanes 9 and 10. Increasing amounts of carrier nucleic acids were included as competitors in lanes 2-4, 6-8 and 9. The carrier nucleic acids mixture was comprised of a 23:1 ratio of yeast tRNA to ¢X174 RF DNA. No carrier nucleic acids were added in lanes 1, 5 and 10. Lanes 2 and 6 each contained 0.96/zg of tRNA and 0.042 #g of ~X174 DNA. Amount of carrier was increased 2-fold in lanes 3 and 7, and 4-fold in lanes 4, 8, and 10. Panel B shows results of crosslinking of H M G and non-HMG fractions. Lanes: 1-2, crude nuclear extract; 3, H M G fraction; 4, non-HMG fraction. No cartier nucleic acids were included except in lane 2 which contained 22.5#g of tRNA and 1.0#g of ¢X174 DNA. M =protein molecular weight markers.

lanes9 and 10). Competition with increasing amounts of ~X174 DNA and yeast tRNA dramatically inhibited H-complex formation in crude

996 nuclear extracts (Fig. 8A, lanes 1 through 4) and with isolated HMGs (Fig. 8A, lanes 5 through 8), but had no effect on L-complex formation by the non-HMG fraction as expected (Fig. 8A, lanes 9 and 10). The 23 kDa and 32 kDa proteins isolated from the H-complex (Fig. 7, lane 4) were clearly detected by UV crosslinking in the H M G fraction shown in Fig. 8B, lane 3. The 46-69 kDa proteins isolated from the L-complex (Fig. 7, lanes 13) were primarily in the non-HMG fraction when assayed by UV crosslinking (Fig. 8B, lane 4), but were also present in the H M G fraction as seen in Fig. 8B, lane 3. The presence of the 46 to 69 kDa proteins in the H M G fraction appears to contradict the absence of an L-complex in the band shift assay shown in Fig. 8A, lane 5. In this case, the lack of an L-complex may be due to the difficulty in renaturation of these proteins after acetone precipitation. This is plausible in view of the poor recovery of L-complex activity after guanidineHC1 treatment shown in Fig. 6. The slight amount of the 23 and 32 kDa proteins of the H-complex in the crosslinked non-HMG fraction (Fig. 8A, lane 9; 8B, lane 4) probably represents TCA pellet contamination. In summary, guanidine-HC1 renaturation studies, competition of DNA binding by carrier nucleic acids, H M G extraction and UV crosslinking assays indicate that the H-complex was formed in vitro by HMGs of 23-32 kDa. The Lcomplex was predominantly composed of proteins from 46 to 69kDa. Proteins of the Lcomplex have both H M G and non-HMG properties since they showed only partial solubility in 2 ~o TCA.

Discussion

AT-rich elements boost transcriptional activity in vivo

The A Tcom oligomer was shown to boost transcription of the maize Adhl promoter in sunflower tumors. Activity of the AT-element was strongly affected by its position within the 5' flanking re-

gion and was inversely related to its distance from the start of transcription. This strong dependence on distance may explain its relatively small contribution to Gmhsp17.5E promoter activity (ca. 15~o) in its native configuration where it is located between positions -907 and -830 [10]. When located upstream from promoter elements that mediate inducible transcription, AT elements appear to increase the amplitude of induction but do not override the primary on/off switch mechanism. This conclusion is based on the failure of the A Tcom sequence to activate basal expression of the Adhl promoter and on the lack of basal expression of Gmhsp17.5E in its natural configuration in soybean [10]. Although AT elements do not seem to be the primary determinants of induction specificity, they still may confer a developmental bias upon inducible expression. When a 55 bp AT element from the fl-phaseolin gene ( -682 bp to -628 bp) was placed immediately upstream of a minimal CaMV 35S promoter ( - 9 0 bp 5' deletion) fused to a GU S reporter gene, expression was enhanced in transgenic tobacco seedlings with the greatest expression seen in roots and in the hypocotyl apex of the embryonic axis [7]. The enhancement of 35 S promoter activity suggests that AT elements, in the proper context, may be able to function in the absence of developmental or environmental response elements. In the case of soybean HS genes, ancillary elements of this type may provide a mechanism for the integration of environmental and developmental control ofgene expression. In addition to higher plants, AT-rich sequences have also been shown to stimulate transcriptional activity in yeast where they have been estimated to occur upstream from the start of transcription in 25 ~ of the genes [46]. Stretches of poly(dAdT) in yeast genes show similarities with the ATrich elements of plants such as a strong distance dependence and a low to moderate level of activity [36]. The yeast, poly(dA-dT) tracts (T elements) act synergistically with other weak upstream activator sequences (UASs) such as those comprising ABF1- or GRFl-binding sites [6].

997 Proteins that interact with A T-rich sequences

There are at least three distinct types of ATelement-binding proteins (NAT1, NAT2 and LAT1) in soybean that interact with sequences upstream of the nodulin N23 gene [27]. The binding activity designated NAT1 is present in roots and nodules, NAT2 is localized to nodules, and LAT1 is found only in leaves. LAT1 proteinDNA complexes (leaf extract-specific) appear to be due to the binding of two peptides (21 and 23 kDa) which interact with equal affinity with AT elements of leghemoglobin genes [27]. Both NAT1 and LAT1 form high-mobility complexes in gel retardation assays similar in mobility to the H-complexes formed by plumule proteins. NAT 1 and LAT1 seem to be H M G proteins based on their extraction by 0.35 M NaCI and solubility in 2 ~ TCA, while NAT2 was reported to be insoluble under these conditions [27]. A similar pattern of in vitro complex formation was obtained from nodule and root nuclear extracts from French bean [ 17]. Nodule extracts formed a lowmobility complex designated PNF-1 when incubated with oligomers homologous to AT-rich repeats present upstream of the glutamine synthase gene (gln-7). A complex of slightly higher mobility, PRF-1, was observed with root extracts. Both binding activities were also capable of specifically interacting with the AT-rich site 2 [29] of the lectin gene. Phaseolin PNF-1 seems to be closely related to soybean NAT2 based on a similar low mobility of the in vitro complex, its relatively high specificity in binding and its relative insolubility in TCA [17]. Pederson et al. [40] demonstrated in vitro binding of highly purified plant H M G proteins to AT-rich sequences located upstream of the pea ferredoxin 1 gene (Fed-l) and a member of the wheat E M family. The similarity in mobility of H M G - D N A complexes ofPederson et al. [40] and the H-complexes reported here support the conclusion that H M G proteins in crude nuclear extracts are responsible for high-mobility complexes seen in soybean and other plants. In addition to the low-molecular-weight HMGlike proteins, other proteins of higher molecular weight have been shown to specifically interact

with AT-rich elements in vitro. In addition to the 46 to 69 kDa proteins of the L-complex identified in this study, Jofuku et al. [29] showed that a 60 kDa protein of soybean was able to bind ATrich elements of the lectin gene. Its presence in nuclear extracts was correlated with the developmental pattern of lectin expression in the embryo. Proteins that form low-mobility complexes in gel retardation assays are designated AT-binding proteins (ATBPs) to distinguish them from the more typical HMG-like proteins that contribute to the high-mobility complexes. By this criteria, NAT2 of nodules, the 60 kDa protein binding the AT-elements of the lectin gene, and the 46 to 69 kDa proteins from plumules are examples of soybean ATBPs. The PNF-1 complex of French bean, AT-1 of pea [ 11 ], and complex II proteins from sunflower [31] are examples of analogous complexes in other plant species. The oligomer binding and competition studies with the plumule ATBPs and those reported for French bean PNF-1 suggest that many plant ATBPs may exhibit some degree of cross-binding to AT elements present in a variety of plant genes. ATBPs show similarities in DNA recognition to both H M G proteins and to some members of the OCT superfamily, such as Drosophila homeodomain proteins of Antennapedia, engrailed and fushi tarazu. Homeodomain DNA recognition involves AT-rich consensus elements that show considerable latitude in binding specificity [19, 42, 47]. The degree of promiscuity in D N A recognition observed for the ATBPs is consistent with properties of both H M G proteins and homeodomains. The direct involvement of homeodomain proteins in transcriptional regulation of animal genes compares with the demonstration that AT-rich elements of plants can stimulate in vivo promoter activity. However, we have no evidence ofhomeodomain involvement in formation of L-complexes in vitro since addition of chicken polyclonal antibodies prepared against a synthetic peptide homologous to the highly conserved helix-3 region of the Antennapedia homeodomain failed to inhibit in vitro binding (data not shown). The partial TCA solubility of plumule proteins of the L-complex (ATBPs) suggests that these

998 proteins may share some similarity in amino acid composition with HMGs. Although the relatively high molecular weights of ATBPs (46 to 69 kDa) are outside the normal range for H M G proteins characterized in animals (10 to 29 kDa), there are examples of high-molecular-weight H M G proteins in animals that show a preference for double-stranded AT-rich DNA. These include the P1 protein found in mammalian cells [39] and D1 of Drosophila [1]. P1 proteins are from 48 to 53 kDa and the D1 protein is estimated to be 50 kDa in D. melanogaster and much larger in D. virilis [1, 39]. Both of these proteins are phosphorylated by casein kinase-2 and bind to long AT-rich tracts. D1 shows a preference for the AT-rich satellite DNA of D. melanogaster which comprises tandem arrays of two repeats: AATAT and AATATAT [5, 15]. Both tandem arrays of these repeats form the motif TAAT, which is also a component of most of the AT-rich oligomers shown to bind plumule HMG-like proteins in this study (Fig. 1). In plants, proteins which meet the operational definition of HMGs have been identified in wheat [45], maize and barley [48] and pea [40]. The transcription factor 3AF1 binds to an ATrich element (5'-AAATAGATAAATAAAAACATT-3'; Box VI) present in the rbcS-3A promoter of pea and has been cloned from a tobacco leaf library [35]. Although the TCA solubility of this protein has not been reported, the large number of charged amino acids deduced from the partial cDNA clone suggests that a portion of it may resemble an H M G protein. A repeated domain containing a putative zinc finger motif is preceded by the amino acid sequence glycinearginine-proline which is conserved in HMG-I positioned near a cluster of basic amino acids [2]. The presence of four serines in the loop of the zinc finger provides a hypothetical mechanism for the inhibition of DNA binding by phosphorylation seen with AT- 1 of pea [ 11 ] and plumule proteins from soybean [ 21 ]. The element to which 3ATFI binds seems to act only when adjacent to other cis-elements, consistent with the hypothetical role of other AT elements in plants thought to boost the amplitude of inducible gene expres-

sion. Although 3AF1 seems to possess many of the characteristics predicted of proteins forming the L-complex, at present is not clear whether any proteins within this relatively large group are related to 3AF1.

Interaction of nuclear proteins with A T-elements in vivo We view the specificity of ATBP enhancement of transcription to be dependent on parameters outlined in the 'jigsaw puzzle hypothesis' of Johnson and McKnight [30] which suggests that in vivo interactions are a function of relative factor binding affinities for the cis-elements, the relative nuclear concentration of these factors, and the context of binding with regard to protein-protein interactions. These parameters must operate in the competition between HMGs and ATBFs for occupancy of AT-rich elements. The degree of regulatory specificity that can be attributed to ATBPs is still unknown. The wide distribution of AT-elements within plant 5'-flanking sequences suggests that they may be intermediate between low abundance/high affinity factors that recognize a tight consensus for DNA binding, and more abundant DNA-binding proteins that play a structural role in maintaining appropriate chromatin structure.

Acknowledgements We thank the Department of Microbiology and Cell Science and the Interdepartmental Center for Biotechnology Research (University of Florida) for synthesis of oligomer probes. The LegHb oligonucleotide (oligo 1) was a kind gift of Dr Erik O. Jensen (University of Aarhus, Denmark). We thank Drs Dulce Barros, Paul Fox and Donald Baldwin for critical reading of the manuscript. This work was supported in part by National Institutes of Health grant GM39732. Florida Agricultural Experiment Station journal series number R-02445.

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