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J. Biol. Chem., Vol. 275, Issue 35, 27494-27499, September 1, 2000

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The High Mobility Group I/Y Protein Is Hypophosphorylated in Endoreduplicating Maize Endosperm Cells and Is Involved in Alleviating Histone H1-mediated Transcriptional Repression^*

Jing Zhao and Gideon Grafi

From Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel

Received for publication, March 2, 2000, and in revised form, June 22, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

During maize endosperm development, cells shift from a mitotic cycle to endoreduplication, driving the massive synthesis of storage proteins (zeins) and starch. In this developmental context, we studied changes in expression levels of histone H1 and high mobility group I/Y (HMG-I/Y), two chromatin architectural proteins that are known to affect gene transcription. Almost no change was found in the level of histone H1 during endosperm development, despite a dramatic increase in DNA content (endoreduplication); hence, the histone H1/DNA ratio decreased substantially. Concurrently with a reduction in the Cdc2 kinase activity at the shift to endoreduplication, significant changes were found in the level and mobility of the HMG-I/Y protein; the faster migrating forms were, at least partly, hypophosphorylated. Purified maize HMG-I/Y protein was found to be phophorylated in vitro by the Cdc2 kinase and bound efficiently to the -zein promoter AT-rich tract (Z-AT). Using an in vitro transcription assay, we demonstrated the capability of the maize HMG-I/Y protein to relieve the inhibitory effect exerted by histone H1 on templates containing the Z-AT sequence. These data suggest that during maize endosperm development transcription and perhaps replication are controlled, at least partly, by the activity of the Cdc2 kinase and the interplay between histone H1 and HMG-I/Y proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transcription and replication occur in an "open" chromatin configuration that allows transcription/replication factors to approach the DNA. The packaging of DNA into nucleoprotein complexes that make up chromatin is highly conserved among eukaryotes (1, 2). DNA organization within the interphase nucleus provides the means not only for DNA compaction but also for orderly gene expression. In a variety of eukaryotes, histone H1 binds to the internucleosomal AT-rich linker DNA and facilitates the compaction of DNA into transcriptionally inactive chromatin (3). Gene transcription is associated with unfolding of the chromatin fiber and is accompanied by depletion, replacement, or displacement of histone H1 by various proteins (4). The ubiquitous nonhistone HMG¹ chromosomal proteins in vertebrates as well as in plants have been implicated in transcriptional activation (5-7). These small chromosomal proteins comprise a heterogeneous protein family common to eukaryotic organisms. HMG proteins are rich in acidic and basic amino acid residues and are operationally defined as being extractable from chromatin with 0.35 M NaCl and soluble in 2% trichloroacetic acid or in 5% perchloric acid (see Ref. 8, and references therein). On the basis of sequence homology and DNA binding properties, HMG proteins are grouped into three families: HMG-1/2, HMG-14/17, and HMG-I/Y (8). The HMG-I/Y as well as HMG-14/17 proteins are implicated in the initial unfolding of the 30-nm chromatin fiber by displacing histone H1 (7, 9). Besides their architectural function, HMG-I/Y proteins are actively involved in the regulation of gene transcription through their interaction with activator proteins (8, 10, 11). Several HMG-I/Y-related genes were isolated from a variety of plant species (12-18). Sequence analysis showed that plant HMG-I/Y proteins are structurally different from those of animals and display certain unique features, e.g. they lack the COOH-terminal acidic region characteristic of the animal proteins and contain an amino-terminal region that shares homology with the globular domain of histone H1 (13, 18). The role played by plant HMG-I/Y proteins in chromatin structure and gene regulation has not been established yet.

Maize endosperm provides a unique metabolic system in which the mode of the cell cycle is sequentially changed during development. After fertilization, endosperm development begins with rapid, synchronous nuclear divisions (acytokinesis mitosis), followed by a mitotic cycle composed of G₁, S, G₂, and M phases and, finally, changing into endoreduplication (19). The shift into endoreduplication is accompanied by a sharp increase in transcriptional activity and involves a significant reduction in the mitotic CDK activity (20). These changes drive the correct functional maturation of endosperm cells, namely, the massive synthesis of storage proteins and starch. The expression of zein genes, encoding the major storage proteins of maize, is coordinately regulated during endosperm development by multiple trans- and cis-acting elements, both at the transcriptional and posttranscriptional levels (21-23). We investigated the role played by histone H1 and HMG-I/Y proteins in controlling the expression of zein genes during endosperm development. We found that the shift from a mitotic cycle to endoreduplication was accompanied by a decrease in H1/DNA ratio, concomitantly with a significant change in level and mobility of the HMG-I/Y protein. We conclude that during maize endosperm development, activation of gene transcription is controlled, at least partly, by the activity of the Cdc2 kinase and the interplay between histone H1 and HMG-I/Y proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plant Material-- Maize (Pool 33 QPM) plants were grown in the field (Coastal Plain, Israel) during 1998 and 1999. Ears were bagged before silking and self-pollinated. At various days after pollination (DAP), ears were collected and quick-frozen in liquid nitrogen. Kernels were collected and then peeled, and the embryos were removed. Isolated endosperm was ground into flour in liquid nitrogen using mortar and pestle and stored at 80 °C.

Purification of Zeins, HMGs, and Histones from Developing Endosperm-- Zein storage proteins were extracted from developing endosperm with 70% ethanol, essentially as described (24). HMG and histone proteins were extracted with 2% trichloroacetic acid (6). Frozen endosperm flour (50 mg) from different developmental stages was homogenized in 20 volumes of NETN (100 mM NaCl, 1 mM EDTA, 20 mM Tris·HCl, pH 8.0, and 0.5% Nonidet P-40) containing 2% trichloroacetic acid and incubated overnight at 4 °C with constant shaking. Samples were centrifuged at 12,000 × g for 10 min, and supernatants were collected and passed through a 0.2-µm filter. To precipitate acid-soluble proteins, trichloroacetic acid was slowly added to 25% (w/v), followed by incubation at 4 °C for 1 h. Proteins were precipitated by centrifugation at 12,000 × g for 20 min, washed twice with cold acetone, dried, and dissolved in phosphate-buffered saline. Protein concentration was determined with the Bradford reagent (Bio-Rad). Histone H1 and HMG-I/Y proteins were detected by immunoblotting using antiserum to histone H1 (a gift from Dr. S. Spiker, North Carolina University, Raleigh, NC) and a polyclonal antiserum raised against HMG-Y protein of Canavalia gladiata D.C. (a gift from Dr. Minamikawa, Tokyo Metropolitan University, Tokyo, Japan; Ref. 17).

Isolation of the Mitotic CDK and Phosphorylation Assays-- The mitotic CDK (Cdc2/cyclin B kinase) from developing endosperm was purified on glutathione-agarose beads containing GST-Suc1 fusion protein. Glutathione-agarose containing GST-Suc1-associated proteins were tested directly for kinase activity (25) in the presence of [-³²P]ATP using as substrates bovine calf thymus histone H1 (Biochemicals & Laboratory Equipment Ltd.), acid-soluble fractions from developing maize endosperm, or purified GST-ZmHMG-I/Y proteins. To verify phosphorylation of acid-soluble proteins, appropriate samples were re-extracted with 2% trichloroacetic acid and resolved by 15% SDS-PAGE. Gels were either stained with Coomassie Blue or dried and autoradiographed.

Electrophoretic Mobility Shift Assays (EMSAs)-- EMSAs were performed essentially as described by Zhao et al. (7). A 137-base pair fragment of the -zein promoter AT-rich sequence (referred to as Z-AT; accession no. X53514, nucleotides 740-876), containing the homopolymeric (dA·dT)₂₁ tract, was amplified by PCR in the presence of [-³²P]dATP, the sense primer 5'-GGCTCCATATTCACACAACC, and the antisense primer 5'-CTAGTCATATGCCTGTGCATTGC. Binding reactions were performed in EMSA buffer (10 mM Tris·HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, and 5% glycerol) containing 250 ng of DNA ( DNA digested with HindIII and EcoRI), protein extracts from various DAP, and ³²P-labeled Z-AT. Poly(dA-dT) and poly(dI-dC) (Sigma) at various concentrations were used as competitors. Samples were incubated for 20 min at room temperature to allow for binding, and DNA-protein complexes were electrophoresed in 4% non-denaturing acrylamide, 0.25× TBE gels for 2 h (10 V/cm). Gels were vacuum-dried and autoradiographed.

Construction of Plasmids for in Vitro Transcription Assays-- The plasmid Z-AT-Cdc2 was generated by placing the -zein promoter AT-rich region upstream from the T7 promoter. This AT-rich DNA fragment (580-1127; accession no. X53514) was PCR-amplified using as a template pPHI-3630 (kindly provided by B. A. Larkins) and the following primers: a sense primer containing a HindIII site, 5'-GGATCCCGGGAAGCTTCAACGATCGTCTCGTGTC, and an antisense primer flanked with a SmaI site to which the T7 promoter sequence (in lowercase) was attached, 5'-GAGACCCGGGccctatagtgagtcgtattaCGGATACAACGAATTCTTGGAGC. The PCR product was cleaved with HindIII and SmaI restriction enzymes, resolved on 1% agarose gel, and purified using the Agarose Gel DNA Extraction kit (Roche Molecular Biochemicals). As a reporter gene we used the maize Cdc2 cDNA (26) that was amplified by PCR using a sense primer containing a BamHI site, 5'-CGCGAGATCTCGAGGATCCACCATGGAGCAGTACGAGAAGG, and an antisense primer flanked with an EcoRV site, 5'-GCGGGATCCATCGATATCCTGCAGTCACTGTACCACTTCAAGGTC. The PCR product was cleaved with BamHI and EcoRV enzymes and purified from the gel as above. The HindIII-SmaI Z-AT and the EcoRV-BamHI Cdc2 fragments were subcloned into the HindIII-BamHI sites of pUC19 to generate the plasmid Z-AT-Cdc2.

Plasmids K38 and K40 (7) were kindly provided by U. K. Laemmli (University of Geneva, Geneva, Switzerland). pK38 contains 657 base pairs of the histone scaffold attachment region (SAR) upstream from the T7 promoter. pK40 is a control deficient of SAR. Both plasmids were prepared for in vitro transcription assay as described (7).

For T7 transcription assays, pZ-AT-Cdc2 was cleaved with HindIII giving rise to a transcript of 475 nucleotides. Plasmids K38 and K40 were linearized as described (7) giving rise to transcripts of 252 and 150 nucleotides, respectively.

Expression and Purification of GST-HMG-I/Y Fusion Proteins-- For preparation of glutathione S-transferase (GST)-HMG-I/Y fusion proteins, the cDNA clone of maize HMG-I/Y obtained from the ZmDB-Maize Genome Data Base (clone AI770331) was used as a template in PCRs to amplify either the full-length clone or its truncated forms. Because of GC richness of the maize HMG-I/Y cDNA, all PCR reactions were performed in the presence of 10% dimethyl sulfoxide (Me₂SO, Sigma). The PCR primers used to generate the various HMG-I/Y clones are listed: Hmg-S, 5'-GATATCGGATCCGAGATGGCCACCGACGAAGCCACC-3'; Hmg-AS, 5'-GTCGACGAATTCGAGCTCTCAAGCCGCGGCCGTCTCGCTG-3'; Hmg-N-S, 5'-GAGAGGATCCCCCGAGATGATCCTGGCGGCGATCGAG-3'; Hmg-C-AS, 5'-GAGAGAATTCTCAGGACCCGTCGCCAGCAGG-3'.

All PCR fragments were cut with BamHI and EcoRI restriction enzymes and cloned into the same sites of pGEX-2T. To generate the NH₂-terminal 69 amino acids fused to GST (GST-HMG-I/Y 1-69), the pGEX-ZmHMG-I/Y was cleaved with EcoRI and Ecl136II to remove the half COOH-terminal AT-hook region, ends were blunted using Klenow fragment of DNA polymerase I, and the plasmid was religated. Proteins were expressed and purified by glutathione-agarose, eluted essentially as described by the manufacturer's protocol (GST Gene Fusion System, Amersham Pharmacia Biotech) and dialyzed. Protein concentration was determined with either the Bradford reagent or the Lowry method (Sigma 690-A). Protein aliquots were stored at 80 °C until use.

T7 in Vitro Transcription Assays-- T7 transcription assays were performed essentially as described (7). Linearized DNA templates were mixed with purified bovine calf thymus histone H1 (Biochemicals & Laboratory Equipment Ltd.) in a binding buffer (20 mM Hepes, pH 7.4, 60 mM KCl, 1 mM MgCl₂, and 10% glycerol) at room temperature for 15 min in a final volume of 10 µl; in derepression experiments, GST-ZmHMG-I/Y was thereafter added and incubation continued for additional 15 min. Ten µl of T7 transcription mixture (0.5 mM each of ATP, CTP, and GTP, 0.1 mM UTP, 5 mM dithiothreitol, 4 units/µl RNasin, 2 units/µl T7 RNA polymerase, and 0.5 µCi/µl [-³²P]UTP (800 Ci/mmol)) was added, and reactions were incubated at 37 °C for 25 min, then stopped by addition of 180 µl of TE buffer containing 100 mM NaCl and 1% SDS. RNA products were purified with phenol-chloroform, precipitated by ammonium acetate and ethanol, and analyzed by electrophoresis on 7.5% acrylamide, 7 M urea, 1× TBE denaturing gels. Gels were dried, and transcription products were visualized by autoradiography either by exposure to x-ray film or by the Fujifilm imaging apparatus (BAS 2500).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Analysis of Histones and HMG-I/Y Proteins during Maize Endosperm Development-- Among maize storage proteins, -zein is the first to become evident (12 DAP, Fig. 1), concomitantly with the initiation of endoreduplication (20). At this stage, intensive transcriptional activity is initiated, resulting in the synthesis of zein mRNAs as well as mRNAs involved in starch synthesis. A unique feature of the -zein promoter is the presence of short homopolymeric runs of dA·dT base pairs, which act as preferential binding sites for histone H1 and HMG-I/Y proteins (8, 28, 29). At various DAP, we extracted these and other proteins from developing endosperm by acid extraction and analyzed them by SDS-PAGE. The level of histone H1, which appeared in multiple forms (ranging from 35 to 40 kDa), remained almost unchanged (Fig. 2, A and C) despite a significant increase in DNA content resulting from endoreduplication (see Ref. 20); hence, a decrease in histone H1/DNA ratio ensued. In contrast to the H1 linker histones, the levels of the maize acid-soluble core histone proteins (30) were elevated with the advance in endoreduplication cycles (Fig. 2B). Hence, it appears that the first level of DNA organization, namely, the nucleosomal structure, is retained during endoreduplication. Similarly to core histones, the maize HMG-I/Y protein was significantly increased beginning at 12 DAP, accompanied by a slight increase in its mobility (Fig. 2, A and D). The level of acid-soluble proteins migrating faster than HMG-I/Y (16-18 kDa, Fig. 2A), possibly HMG1-like proteins (31), was also increased during endosperm development. We focused on the maize HMG-I/Y protein and examined whether the shift in its mobility could have resulted from dephosphorylation. Because HMG-I/Y proteins have been shown to be phosphorylated by the mitotic CDK (8), we tested the capability of the maize mitotic CDK to phosphorylate the HMG-I/Y protein. We first analyzed the SUC1-associated Cdc2 kinase activity in protein extracts from developing endosperm. Fig. 3A shows a high kinase activity during the mitotic cycle (8 and 10 DAP), followed by a sharp decrease as cells shifted to endoreduplication (12 and 14 DAP). Acid-soluble fractions, enriched with HMG-I/Y protein, from developing endosperm were subjected to phosphorylation by SUC1-associated CDK from 8 DAP endosperm (Fig. 3B). Among the various forms of HMG-I/Y found in 12 and 14 DAP cells (Fig. 3B, Coomassie staining), only the upper, slowly migrating forms are phosphorylated (Fig. 3B, Autoradiogram). This indicates that the HMG-I/Y protein is phosphorylated, at least partly, in dividing endosperm cells (6 and 8 DAP) but is hypophosphorylated as cells shift to endoreduplication, concurrently with the decline in the mitotic kinase activity (12 DAP and onward; Ref. 20).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Synthesis of storage proteins during maize endosperm development. Storage proteins were extracted from developing endosperm at various DAP with 70% ethanol, resolved by 15% SDS-PAGE, and stained with Coomassie Blue. The positions of - and -zeins are indicated. M indicates molecular weight markers. The transition from a mitotic cycle to endoreduplication is shown.

View larger version (64K): [in this window] [in a new window]   Fig. 2.   Composition of acid-soluble proteins in developing endosperm. Flour (50 mg) from developing endosperm at the indicated DAP was extracted with 2% trichloroacetic acid , and soluble proteins were resolved by 15-17% SDS-PAGE. Gels were stained with Coomassie Blue (A and B), or blotted onto membranes and probed with anti-histone H1 (C) or anti-HMG-I/Y (D). Arrows indicate the positions of the various forms of histone H1 and HMG-I/Y proteins. M, molecular weight markers.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   The HMG-I/Y protein is phosphorylated by CDK in dividing endosperm cells and is hypophosphorylated in endoreduplicating cells. A, SUC1-associated CDK activity in developing endosperm during mitotic (8 and 10 DAP) and endoreduplication (12 and 14 DAP) cycles. Histone H1 was used as a substrate for phosphorylation. B, acid-soluble fractions from developing endosperm were subjected to phosphorylation by the 8 DAP SUC1-associated kinase. Proteins were resolved by SDS-PAGE and either stained by Coomassie Blue (right panel) or autoradiographed (left panel). The various forms of HMG-I/Y are indicated on the right by arrows; phosphorylated forms are designated by the letter p. C, in vitro phosphorylation of ZmHMG-I/Y by SUC1-associated Cdc2 kinase. Various ZmHMG-I/Y constructs (numbered 1-5) were generated by PCR, subcloned into a GST gene fusion vector, and overexpressed in bacteria. The schematic diagram of the HMG-I/Y protein is shown to indicate the positions of the NH2-terminal globular domain (GD) and the half COOH-terminal containing the four AT-hooks (I-IV). The positions of potential CDK phosphorylation sites are shown. Uppercase P indicates consensus site and lowercase p minimal sites. Phosphorylation of the various GST-ZmHMG-I/Y fusion proteins by the GST-SUC1-associated Cdc2 kinase is indicated on the right.

We next determined the maize HMG-I/Y protein regions that can be phosphorylated by Cdc2 kinase. The putative amino acid sequence of the protein (18) contains one consensus site (TPGK) and six minimal sites (SP) for CDK phosphorylation; one minimal site is located at the amino-terminal region, and all others are at the half COOH-terminal AT-hook region (Fig. 3C). We generated by PCR various ZmHMG-I/Y constructs (depicted in Fig. 3C) and cloned them into pGEX-2T to produce GST fusion proteins. These bacterially produced proteins were subjected to phosphorylation by the SUC1-associated kinase from 8 DAP endosperm. Fig. 3C summarizes the phosphorylation activity of Cdc2 on the various GST-ZmHMG-I/Y proteins. All truncated HMG-I/Y proteins, except the NH₂-terminal region (amino acids 1-69), were phosphorylated by Cdc2, suggesting that ZmHMG-I/Y CDK-phosphorylation sites are confined to the half COOH-terminal AT-hook region.

AT Binding Activity of Acid-soluble Proteins during Endosperm Development-- Because AT-rich sequences are often implicated in chromatin function through the action of histone H1 and HMG proteins (8, 29), we tested the AT-rich DNA binding activity of acid-soluble fractions from developing maize endosperm. For this analysis we used the AT-rich sequence from the promoter region of the storage -zein gene (referred to as Z-AT). A band-shift assay (Fig. 4A) was carried out with ³²P-labeled Z-AT fragment (137 base pairs) amplified by PCR. Increasing protein levels resulted in multiple complexes of protein-Z-AT identified by a ladder of shifted products. Among the tested acid-soluble fractions, the 14 DAP fraction had the highest affinity to ³²P-Z-AT; formation of protein-DNA complexes required as low as 5 ng of protein from 14 DAP (Fig. 4A, lane 13) but 40 ng of protein from 8 DAP endosperm (Fig. 4A, lane 10). DNA-protein complexes were evident with 20 ng of histone H1 (Fig. 4A, lane 4). Competition experiments indicated that protein binding to Z-AT is AT-specific, inasmuch as Z-AT-protein complexes are efficiently competed out with increasing amounts of poly(dA-dT) but much less efficiently with poly(dI-dC) (data not shown). Thus, the AT-binding affinity is changed during endosperm development, becoming higher at developmental stages corresponding to intensive activities of transcription and replication.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   The maize HMG-I/Y protein binds efficiently to the -zein AT-rich tract. A, acid-soluble proteins from developing endosperm were assayed for binding with the AT-rich fragment from the -zein promoter region (Z-AT). Increasing amounts of 2% trichloroacetic acid -soluble endosperm proteins (1, 5, 20, 40, and 80 ng) from 8 DAP (mitotic cycle, lanes 7-11, respectively) or 14 DAP (endoreduplication cycle, lanes 12-16, respectively) were incubated with 32P-labeled Z-AT DNA fragment. DNA-protein complexes thus formed were resolved on 4% non-denaturing acrylamide gel by low ionic strength electrophoresis. Increasing amounts of a commercially available histone H1 (1, 5, 20, 40, and 80 ng) were used as a reference (lanes 2-6, respectively). B, DNA binding activity of a purified ZmHMG-I/Y protein. Band-shift assays were conducted with purified, bacterially produced ZmHMG-I/Y and its truncated forms fused to GST (constructs 1-5 in Fig. 3C). Reactions were performed with increasing amounts (1, 5, 10, 50, 100, and 500 ng) of GST-ZmHMG-I/Y () fusion protein (lanes 3-8, respectively), or with 100 ng of the indicated GST-ZmHMG-I/Y derivatives (lanes 9-12). GST alone (100 ng) was used as a reference protein. This assay was conducted with two 32P-labeled PCR probes: the Z-AT and a non-AT fragment. DNA-protein complexes were resolved on 4% nondenaturing acrylamide gel, dried and autoradiographed. P (lane 1) indicates the probe mixture only; FP indicates the free probes.

The rise in AT binding activity that accompanies enhanced gene transcription (14 DAP) could be attributed, at least in part, to the increased level and mobility of the HMG-I/Y protein. To assess this possibility, we next conducted band-shift experiments with purified GST-ZmHMG-I/Y proteins described in Fig. 3C. The AT-binding analysis showed that binding to ³²P-Z-AT was a characteristic of the ZmHMG-I/Y but not of the GST portion of the fused protein (Fig. 4B, compare lane 2 to lanes 3-8). In addition, the GST-ZmHMG-I/Y protein selectively bound to the AT-rich DNA sequence (Z-AT), gradually forming two clear complexes as a function of the protein dose. Removal of the 15 NH₂-terminal amino acids (GST-ZmHMG-I/Y (), Fig. 4B, lane 9) or the COOH-terminal AT-hook (GST-ZmHMG-I/Y (), Fig. 4B, lane 11), or both (GST-ZmHMG-I/Y (), Fig. 4B, lane 10), did not affect binding activity; the NH₂-terminal region alone (GST-ZmHMG-I/Y (1-69), Fig. 4B, lane 12) failed to bind to Z-AT. Competition experiments verified that the DNA binding activity of the GST-ZmHMG-I/Y protein is AT-specific (data not shown).

HMG-I/Y Alleviates Histone H1-mediated Transcriptional Repression-- We next investigated the potential of the maize HMG-I/Y protein to induce gene transcription otherwise suppressed by histone H1, by using the previously described T7 RNA polymerase in vitro transcription system (7). In this system, an AT-rich SAR sequence, to which histone H1 and certain HMG proteins can bind, was inserted upstream the T7 promoter. The constructs K38 (AT-rich) and K40 (non-AT) used in these experiments were described previously (7); pZ-AT-Cdc2 is schematically depicted in Fig. 5A. To establish the experimental protocol, we first confirmed previous findings (7) showing that at certain concentrations (up to 30% H1/DNA ratio) histone H1 efficiently repressed transcription by T7 polymerase of AT- but not of non-AT-containing templates (data not shown). At higher concentrations (>35%), transcription of both templates was inhibited (data not shown). Transcription from a template containing the Z-AT sequence upstream the T7 promoter was efficiently inhibited by increasing concentrations of histone H1 (Fig. 5B, lanes 1-5); addition of increasing amounts of GST-ZmHMG-I/Y relieved this transcription inhibitory effect (Fig. 5B, lanes 8-11). Similar transcriptional derepression activity was obtained by the Arabidopsis HMG-I/Y protein (data not shown).

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Alleviation of histone H1-mediated transcriptional inhibition by ZmHMG-I/Y. A, schematic representation of the construct Z-AT-Cdc2 used in these experiments. RI, EcoRI; H, HindIII. B, selective repression of T7 RNA polymerase transcription by histone H1 and derepression by ZmHMG-I/Y protein. Linearized K40 (non-AT; Ref. 7) was mixed with a linearized AT-containing template, Z-AT-Cdc2, and incubated with histone H1 at the indicated H1:DNA weight ratios (%) for 15 min at room temperature. Samples were then either transcribed directly with T7 RNA polymerase (lanes 1-5) or subjected to derepression assays (lanes 6-11) by adding various amounts of GST-ZmHMG-I/Y (2.5, 5, 10, and 20 ng, lanes 8-11, respectively) for additional 15 min. Transcription was performed at 37 °C for 25 min, and transcripts were resolved on 7.5% urea/acrylamide gel. Indicated on the left are the positions of transcripts generated by Z-AT-Cdc2 (Z-AT) and by K40 (non-AT). Results in the right panel were obtained by PhosphorImager.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A unique metabolic feature of maize endosperm is the co-occurrence of replication and transcription leading to orderly development of the storage tissue. Here we showed that the abrupt increase in zein gene expression, concomitantly with the initiation of DNA endoreduplication, is correlated with a decrease in H1/DNA ratio, which in turn may reflect structural modifications in chromatin. Electron microscopy studies showed that H1-DNA complexes appear as thin filaments or as double fibers at a low H1/DNA ratio; increasing the histone H1/DNA ratio lead to a rod- or a cable-like appearance (see Ref. 32, and references therein). The function of H1 in DNA compaction is demonstrated by the abnormally heterochromatinized nuclei in tobacco plants overexpressing the Arabidopsis histone H1 (33), or by the micrococcal nuclease-resistant chromatin in cells enriched with histone H5 (34) or histone H1⁰ (35) variants. Functionally, histone H1 was shown to inhibit replication in Xenopus egg extract by preventing the assembly of pre-replication complexes on added sperm chromatin (36), an effect that may be related to the role of histone H1 in compacting DNA fibers. Hence, a reduction in histone H1/DNA ratio during endosperm development probably contributes to chromatin relaxation leading to enhanced DNA transcription and replication.

The open chromatin configuration suggested by the reduction in H1/DNA ratio is reinforced by an increase in the level and mobility of HMG-I/Y protein during endoreduplication and the enhancement of zein gene expression. The change in HMG-I/Y mobility can be attributed, at least partly, to hypophosphorylated forms of this protein, which in turn enhance its activity. We cannot exclude the possibility that acetylation/deacetylation may also be involved in regulating the activity of the HMG-I/Y protein during endosperm development. Both phosphorylation and acetylation were shown to negatively control HMG-I/Y activities: phosphorylation, by the mitotic CDK, reduces its binding affinity to AT-rich DNA sequences (37-39), and acetylation disrupts the transcriptional enhanceosome complex (40). The pattern of the mitotic CDK activity during maize endosperm development (Fig. 3A and Ref. 20) supports the finding that HMG-I/Y is phosphorylated in cycling cells and hypophosphorylated in cells shifted to endoreduplication.

HMG-I/Y proteins are actively involved in transcription by assembling and stabilizing complexes of transcriptional factors (11). Recent work with the oat HMG-I/Y (PF1) has provided a clue for the involvement of plant HMG-I/Y in trascriptional activation. PF1 has been shown to enhance the recruitment of the transcriptional activator GT-2 to the PHYA gene promoter (41). Is the maize HMG-I/Y protein also involved in enhancing the -zein gene expression? The promoter region of this gene contains short homopolymeric runs of dA·dT base pairs, a preferential binding site for HMG-I/Y proteins. The synthesis of -zein RNA and protein is enhanced significantly in the modified opaque-2 QPM, whose promoter possesses an extended dA·dT sequence (25 pairs). It has been shown previously that proteins have higher binding affinity to this extended dA·dT region than to its wild-type (W64A⁺) counterpart (16 pairs) (42). Possibly, the extended dA·dT motif has higher affinity for the HMG-I/Y protein, which in turn enhances -zein gene transcription.

Plant HMG-I/Y proteins display certain features which are distinct from those of animals. Whereas animal HMG-I/Y proteins contain three copies of the AT-hook motif (RGR, Ref. 43), plant proteins usually have four such copies and lack the COOH-terminal acidic region characteristic of the animal proteins (13, 18). Of particular significance is the finding that plant HMG-I/Y proteins, but not their animal counterparts, contain an amino-terminal region that shares homology with the globular domain of histone H1 (18). Here we demonstrated that histone H1, unlike HMG-I/Y, negatively regulates transcription by the T7 RNA polymerase of AT-containing DNA templates. Conceivably, both H1 and HMG-I/Y proteins compete with each other for binding to the AT-rich sequence upstream the promoter region. This competition-type relationship between H1 and HMG-I/Y is displayed by the reduction in H1/DNA ratio during endosperm development, under which condition the distribution of histone H1 protein is expected to be skewed toward AT-rich sequences (see Fig. 5B and Ref. 7), the preferential binding sites of HMG-I/Y proteins.

Histone H1 may inhibit transcription by its cooperative binding to DNA, thus coating the DNA with H1 molecules that render it inaccessible to RNA polymerases. The occurrence of large H1-DNA complexes (Fig. 4A) incapable of migrating into the gel may suggest cooperative binding; such large complexes could not be detected with the ZmHMG-I/Y protein (Fig. 4B). The mechanism of cooperative binding of H1 to DNA is not fully understood. Clark and Thomas (44) suggested that cooperative binding of H1 is mediated by direct hydrophobic interactions between globular domains, thus contradicting the assumption by Singer and Singer (45) that this domain is not necessary for cooperativity. The fact that the ZmHMG-I/Y, like other plant HMG-I/Y proteins, contains an NH₂-terminal H1-like globular domain may support the view that this domain is not involved in the cooperative binding-mediated transcriptional repression. Considering that HMG-I/Y could relieve the histone H1-mediated transcriptional repression in vitro, we suggest that histone H1 and HMG-I/Y proteins play a role in the activation of gene transcription in maize endosperm, partly by affecting chromatin structure. We currently seek to determine the HMG-I/Y domains that affect chromatin architecture and activate gene transcription.

	ACKNOWLEDGEMENTS

We thank Y. Avivi and T. Trebitsh for critical reading and editing of the manuscript, J. Colasanti and V. Sundaresan for the Cdc2 clone, S. Spiker for providing antibodies to H1, T. Minamikawa for anti-HMG-Y, U. K. Laemmli for providing plasmids K38 and K40, the ZmDB-Maize Genome Data Base (Ames, IA) for providing HMG-I/Y cDNA clone, and the Arabidopsis Biological Resource Center (ABRC) for providing cDNA libraries. We also thank Dr. B. A. Larkins, in whose laboratory this work was initiated, and all the members of our laboratory for helping in the field and on the bench.

	FOOTNOTES

^* This work was supported by BARD (to G. G).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 972-8-934-3505; Fax: 972-8-934-4181; E-mail: gideon.grafi@weizmann.ac.il.

Published, JBC Papers in Press, June 22, 2000, DOI 10.1074/jbc.M001711200

	ABBREVIATIONS

The abbreviations used are: HMG, high mobility group; HMG-I/Y, high mobility group I/Y; DAP, days after pollination; CDK, cyclin-dependent kinase; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; SAR, scaffold attachment region; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TBE, Tris borate/EDTA.

	REFERENCES

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