Argyris Papantonis

Josef Vanden Broeck

Rena Lecanidou
email: rlecanid@biol.uoa.gr

Architectural factor HMGA induces promoter bending and recruits C/EBP and GATA during silkmoth chorion gene regulation

Keywords: High mobility group protein A, DNA bending, transcriptional regulation, insect choriogenesis, Bombyx mori, follicular epithelium, promoter elements 1

A protein displaying significant similarity to mammalian HMGA proteins, but also bearing unique structural features, was isolated from silkmoth (Bombyx mori) follicular cells. This factor, named BmHMGA, exhibits specific binding preference for chorion gene promoter elements and induces DNA bending thereon. BmHMGA deploys temporal-specific interaction with transcription factors BmC/EBP and BmGATA during follicle maturation. The respective protein complexes can be detected on chorion gene promoters in vivo, with different developmental profiles each time. Analogous interaction takes place on the putative promoter of the BmC/EBP gene, hinting towards a transcriptional circuit responsible for the progress of choriogenesis as a whole. Finally, transient suppression of BmHMGA expression led to down-regulation of chorion genes and the BmC/EBP gene and revealed recruitment of BmC/EBP, BmGATA and TFIID/TBP by BmHMGA. A revised model for chorion gene regulation is discussed in view of these findings.

INTRODUCTION

In eukaryotes, developmental regulation of gene expression in response to environmental or physiological stimuli renders cascades of promoter activation very complex. Our need to thoroughly understand such molecular processes requires a suitable system of study. The developing Bombyx mori ovariole represents an established model for studies on differential gene expression during the execution of long-term developmental programs, all stages of follicular development being organized in a continuous array [START_REF] Kafatos | Chorion genes: an overview of their structure, function and transcriptional regulation[END_REF]. The final process of silkmoth oogenesis is choriogenesis and results in the formation of chorion; this complex protein structure surrounds the oocyte and develops alongside with it. Being expressed solely in the follicular cells surrounding the developing oocyte, chorion genes are regulated through a sequence of gene activation and repression events [START_REF] Kafatos | Chorion genes: an overview of their structure, function and transcriptional regulation[END_REF]. According to their time of expression, chorion genes are characterized as early (5H4, ErA/B, 6F6), middle (A/B), or late (HcA/B) [START_REF] Lecanidou | Evolution of the silk moth chorion gene superfamily: gene families CA and CB[END_REF][START_REF] Hibner | Organization and expression of three genes from the silkmoth early chorion locus[END_REF][START_REF] Lecanidou | Three copies of the early gene 6F6 are interspersed in and around the late chorion gene cluster of Bombyx mori[END_REF][START_REF] Kravariti | Sequence analysis of a small early chorion gene subfamily interspersed within the late gene locus in Bombyx mori[END_REF]. The respective superfamily consists of and genes; and genes of the same developmental specificity are in their vast majority organized in divergently transcribed pairs, sharing a common 5' flanking promoter region [START_REF] Lecanidou | Evolution of the silk moth chorion gene superfamily: gene families CA and CB[END_REF][START_REF] Hibner | Organization and expression of three genes from the silkmoth early chorion locus[END_REF][START_REF] Jones | Structure, organization and evolution of developmentally regulated chorion genes in a silkmoth[END_REF]. These relatively short intergenic sequences (approx. 250 bp) are equipped with all cis-elements required for differential gene regulation [START_REF] Mitsialis | Regulatory elements controlling chorion gene expression are conserved between flies and moths[END_REF]. Transcriptional cascades responsible for tissue and temporal specificity are thought to involve several transcription factors in various combinations during different phases of choriogenesis [START_REF] Skeiky | Synergistic interactions of silkmoth chorion promoter-binding factors[END_REF][START_REF] Sourmeli | A novel role for the Bombyx Slbo homologue, BmC/EBP, in insect choriogenesis[END_REF][START_REF] Papantonis | Chorion gene activation is directly dependent on BmC/EBP expression and binding to cognate cis-elements[END_REF].

In this study we identified yet another protein factor which participates in the formation of multiprotein complexes on chorion gene promoters. We characterized this molecule as a member of the nonhistone High Mobility Group (HMG) protein family. HMG proteins, often termed 'architectural factors', are relatively abundant and ubiquitous; they are classified into three subfamilies: HMGA (HMGI/Y, -C), HMGB (HMG-1, -2) and HMGN (HMG14/17; revised nomenclature according to [START_REF] Bustin | Revised nomenclature for high mobility group (HMG) chromosomal proteins[END_REF]).

Proteins of the HMGA subfamily [START_REF] Reeves | Molecular biology of HMGA proteins: hubs of nuclear function[END_REF], originally characterized by their small molecular size (10-13 kDa), associate with DNA via minor groove interactions by utilizing a set of DNA binding domains (AT hooks) located closer to their N-terminus. The interacting counterparts for efficient binding are the characteristic basic AT hook motif (G/P-R-G-R-P, core consensus) and an approximately 10 bp adeninethymidine rich DNA sequence [START_REF] Huth | The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif[END_REF]. Functionally, HMGA proteins, though not potent activators on their own, play a key role in the transcriptional activation of numerous genes by inducing conformational changes to DNA in vivo and by mediating transcription factor recruitment and interaction [START_REF] Reeves | Molecular biology of HMGA proteins: hubs of nuclear function[END_REF][START_REF] Foti | A nucleoprotein complex containing Sp1, C/EBP beta, and HMGI-Y controls human insulin receptor gene transcription[END_REF][START_REF] Yie | Intra-and intermolecular cooperative binding of high-mobility-group protein I(Y) to the beta-interferon promoter[END_REF]. Contortion in DNA structure is thought to facilitate deployment of additional DNA-protein or proteinprotein contacts, which in the absence of such bending-induced compaction would not be feasible [START_REF] Semsey | A gamut of loops: meandering through DNA[END_REF].

In insects, proteins belonging to the HMG family have been biochemically characterized in the dipterans Drosophila and Chironomus [START_REF] Wisniewski | Insect proteins homologous to mammalian high mobility group protein 1. Characterization and DNA-binding properties[END_REF][START_REF] Claus | Insect proteins homologous to mammalian high mobility group proteins I/Y (HMG I/Y). Characterization and binding to linear and four-way junction DNA[END_REF], whereas analogous lepidopteran factors have only been recently discovered [START_REF] Aleporou-Marinou | High mobility group-like proteins of the insect Plodia interpunctella[END_REF]. The data that follow analyze the functional properties of the novel silkmoth HMGA protein by focusing on how the early-middle/middle A/B.L9 and the middle/middle-late A/B.L1 chorion gene pairs are differentially regulated, their expression profile and promoter architecture displaying only subtle differences. We offer evidence for BmHMGA implication in chorion gene regulation via interaction with BmC/EBP and BmGATA ; a revision of the model for evoking transcriptional activation and imposing repression of chorion gene expression [START_REF] Sourmeli | A novel role for the Bombyx Slbo homologue, BmC/EBP, in insect choriogenesis[END_REF][START_REF] Papantonis | Chorion gene activation is directly dependent on BmC/EBP expression and binding to cognate cis-elements[END_REF] is accordingly discussed.

EXPERIMENTAL Animal strains, follicle staging and preparation of nuclear extracts

One hundred female pupae of the B. mori FengIx54A strain were dissected 8 days after pupation. Isolation of intact developing ovarioles was followed by follicle staging [START_REF] Nadel | Specific protein synthesis in cellular differentiation. IV. The chorion proteins of Bombyx mori and their program of synthesis[END_REF]. Vitellogenic follicles were pooled together (Pre) and choriogenic follicles were divided in six consecutive developmental stages (early, E; early-middle, EM; middle, M; middle-late, ML; late, L; very late, VL). Nuclear extracts from staged follicular cells were prepared as previously described [START_REF] Sourmeli | In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors[END_REF].

Generation of cladograms

Neighbour-joining cladograms were generated using Molecular Evolutionary Genetic Analysis software 3.0 [START_REF] Kumar | MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment[END_REF]. Bootstrap test values were set at 1050 for 'replication' in the Poisson correction analysis applied.

Northern analysis

Total RNA was isolated from pooled late vitellogenic and choriogenic follicles using TriPure (Roche); polyA+ RNA was obtained with the Oligotex purification kit (Qiagen). Following electrophoresis the sample was transferred onto a nylon membrane (Ammersham) using a capillary device. Prehybridization and hybridization was carried out in 0.4M NaCl, 50mM Tris-HCl pH 7.5, 1% SDS, 5mM EDTA, 12x Denhart's solution and 0.5 mg/mL salmon sperm DNA at 67 o C o/n. The full length BmHMGA cDNA probe was labeled by random-priming (Fermentas); the High Range RNA ladder (Fermentas) was used.

Quantitative Real-time-PCR

Transcription levels for BmHMGA and BmC/EBP were assessed using Quantitative Real-time-PCR (Applied Biosystems 7500 series). Reactions were carried out in triplicate on two separately obtained polyA+ RNA template from staged follicles; the set up was repeated twice. For the ( Ct) method chosen calculations were carried out according to manufacturer's instructions (Guide to Performing Relative Quantitation of Gene Expression, ABI). Primers were designed using the Primer Express 3.0 software (qHMGA-F: 5'-caagggcaagggtgaaggta-3', qHMGA-R: 5'-ttcctcagtagaagcagcatctttt-3', qC/EBP-F: 5'-ccccaaccagacctcaaaaa-3', qC/EBP-R: 5'-gtccattgagctcgtcgagtt-3'); actin amplification served as reference.

Expression and purification of His-Tag fusion proteins

Open reading frames for the wild-type and deletion mutants of BmHMGA were generated via PCR (HMGexpF: 5 -ccatatgtctgacgacggttcaacag-3 ; HMGexpR: 5 -gggatccttattggtcttcagatccttcatc-3 ; HMGmutF1: 5'-ccatatggccaatggaacacaacctgagtcg-3'; HMGmutF2: 5'-ccatatggctgccaccagaacaaaag-3'; HMGmutR1:

5'-gggatccttacttgactcccgcttttttcttgga-3'; HMGmutR2: 5'gggatccttacctgttcttcctcagtagaagcagc-3'). Amplified products were cloned into the pET15-b expression vector (Novagen) and ORF integrity was confirmed by DNA sequencing (MWG-Biotech A.G.). Proteins were expressed as 6x His-tagged fusions in the E. coli BL21(DE3) strain (Novagen) and affinity purified from a Ni-NTA resin column (Qiagen) to near homogeneity under native conditions. Samples were dialyzed against 350mM NaCl, 20mM HEPES pH 7.9, 10mM KCl, 1.5mM MgCl 2 , 0.1mM EGTA, 10% Glycerol, 0.5mM DTT and 0.5mM PMSF.

Gel retardation assays

Approximately 2x10 4 cpm (corresponding to an average of 5 ng) of the appropriate probe were incubated with follicular nuclear extracts or bacterially produced proteins and loaded onto 6% non-denaturing polyacrylamide gels as previously described [START_REF] Sourmeli | In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors[END_REF]. The pL1 and pL9 fragments representing the A/B.L1 and A/B.L9 promoter regions located between the and -TATA boxes were used as probes. All promoter fragments were end-labeled by filling in 3' protruding ends with [ -P 32 ]ATP using Klenow fragment (Fermentas). For competition assays double stranded oligonucleotides were added in a 100x molar excess. For supershift assays, the antiserum raised against full length human HMGI(Y) and its corresponding preimmunization serum were used.

DNase I footprinting assays

After radio-labeling each strand of the pL1 or pL9 fragment separately, 10 5 cpm of the appropriate probe was mixed with recombinant BmHMGA and treated with 200-400 ng DNase I; for control reactions the same amount of probe was cleaved with 100-200 ng DNase I. All other procedures were as previously described [START_REF] Sourmeli | In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors[END_REF].

Antisense DNA assays were performed essentially as described in [START_REF] Kendirgi | An ovarian follicular epithelium protein of the silkworm (Bombyx mori) that associates with the vitelline membrane and contributes to the structural integrity of the follicle[END_REF]. Briefly, a 4-day-old pupa was dissected and follicles corresponding to vitellogenic (-10 to -6, Series A) and to early choriogenic stages (+1 to +5, Series B) were collected and allowed to develop ex vivo in appropriate medium (Grace's Insect Medium, 0.5% fetal calf serum, 50 mgr/ml gentamycin) and environment (28 o C, 100% oxygen atmosphere). Different concentrations (0 to 25 µ ) of a PTO-modified oligonucleotide (BmHMGA-AS: 5'-gtcagacatagttcactaaa-3'), that spanned 20 nucleotides around the initiation codon of the BmHMGA transcript, were added to the culture medium, which was renewed every 12 hours. Incubation time was 36 hours in total. For the mock assay (Mock oligo: 5'-tttagtgaactatgtctgac-3') vitellogenic and early choriogenic follicles were pooled together. The interference effect was assessed through RT-PCR using Superscript II (Invitrogen). Primer sets used were: HMGexpF and BmHMGexpR (restriction sites omitted), BmC/EBPF: 5'-gagcgtcctacgcgccagtac-3', BmC/EBPR: 5'-gatcacggagctgtgccggtc-3', Er.A1F: 5'-ctacattcgctgtcttactcctttgc-3', Er.A1R: 5'-gcacctgtcccattacaccgg-3', B.L11F: 5'-gtggcacagccgctttaggc-3', B.L11R:

5'-ccgtagccgcctccgtaac-3', ActinF: 5'-gacgaagaagttgccgcgttgg-3', ActinR: 5'ggcgggcgtgttgaatgtttcg-3'. Assays were performed twice to ensure reproducibility.

RESULTS

BmHMGA structural features and developmental profile

The uncharacterized BmMCBP5 cDNA sequence [START_REF] Kafatos | Chorion genes: an overview of their structure, function and transcriptional regulation[END_REF] was used to design specific primers for PCR on an early-choriogenic follicular cell cDNA library template [START_REF] Sourmeli | A novel role for the Bombyx Slbo homologue, BmC/EBP, in insect choriogenesis[END_REF]. This resulted in the amplification of a single 343 bp fragment. The full length clone, named BmHMGA (GenBank Acc. No. DQ402512), was subsequently isolated via PCR (Herculase II, Stratagene) using 5' or 3' universal primers of the library vector (Clontech). Sequence analysis revealed 1205 bp containing an open reading frame (ORF) coding for a putative polypeptide of 114 amino acids. The latter was found to include characteristic motifs of the High Mobility Group (HMG) protein family: four AT-hook motifs, three of which follow the same amino acid core consensus (G-R-G-R-P), whereas in the second one the first glycine residue was substituted by lysine. In the protein C-terminus a stretch of acidic residues was identified, another common feature of HMG proteins (Figure 1). In order to classify the silkmoth protein, its sequence was aligned against other invertebrate and vertebrate HMG protein sequences. In the neighbour-joining cladogram produced, this novel factor was positioned closer to mammalian HMGA proteins; protein sequences derived from Drosophila and Aedes formed a different outgroup (Figure 2A). The BmHMGA number of AT hook motifs is not encountered in any animal HMGA proteins, but appears to be a characteristic feature of the respective plant factors [START_REF] Grasser | Chromatin-associated HMGA and HMGB proteins: versatile co-regulators of DNA-dependent processes[END_REF]. These proteins are significantly larger (170-210 amino acids long) than their animal counterparts due to the insertion of a N-terminal globular domain. The Triticum aestivum and Oryza sativa-derived proteins (GenBank Acc. Nos. AF502250 and BAC24935, respectively) were also not grouped with BmHMGA, since similarity regarding the AT hook number and amino acid composition did not extend to the rest of the protein sequence. The aforementioned underline a close association of BmHMGA to homologues derived from higher vertebrates and accentuates its differentiation in primary structure and motif composition from any animal HMG factor isolated up to date.

In silico screening of the B. mori genomic database (http://www.nises.affrc.go.jp/sgp) using the cDNA clone as probe revealed the organization of the BmHMGA gene; numerous overlapping contigs were retrieved. Analysis allowed the identification of two intronic sequences (66 bp each, Figure 1) in the open reading frame, positioned 72 and 225 bp from the initiation codon respectively. These introns separate the coding region into three exons, each one containing a single AT hook motif, with the exception of the second and third motif which are situated in the same exon. An additional 574 bp intron was found in the 5 UTR, spanning positions +509 to +1083; a similar feature exists in the human but is absent from the mouse HMGA1 gene [START_REF] Reeves | HMGI/Y proteins: flexible regulators of transcription and chromatin structure[END_REF]. These findings raise the possibility of alternative splicing since mammalian HMGA1a and HMGA1b result from a single transcript [START_REF] Borrmann | HMGA protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity[END_REF]; this was investigated by Northern analysis. Vitellogenic and total choriogenic follicles were probed with the full cDNA sequence and resulted in a single 1.3 kbp band (Figure 2B); it can be thus inferred that BmHMGA is present as a single transcript throughout oogenesis. Screening of the B. mori EST database (http://www.ab.a.utokyo.ac.jp/silkbase/) for BmHMGA transcripts revealed its wide dispersion in almost all available libraries (data not shown) indicative of a ubiquitously expressed factor.

In an effort to correlate the BmHMGA transcription profile to that of BmC/EBP, a molecule of key importance for chorion gene regulation [START_REF] Sourmeli | A novel role for the Bombyx Slbo homologue, BmC/EBP, in insect choriogenesis[END_REF][START_REF] Papantonis | Chorion gene activation is directly dependent on BmC/EBP expression and binding to cognate cis-elements[END_REF], we carried out Quantitative Real-Time PCR for both transcripts using mRNA from staged follicles. Comparative analysis showed that BmC/EBP exhibited higher expression levels during E-EM stages followed by a decline towards late choriogenesis (in aggrement with RNA dot blot [START_REF] Sourmeli | A novel role for the Bombyx Slbo homologue, BmC/EBP, in insect choriogenesis[END_REF]); BmHMGA displayed significantly higher expression levels in three distinct peaks, during vitellogenesis, in mid-choriogenesis and in very late choriogenic stages (Figure 2C).

Specific binding to chorion gene promoters occurs via two AT hooks

Binding of HMG proteins to DNA is mediated by AT hook motifs and preferably occurs on A/T rich sequences of the minor groove [START_REF] Reeves | Molecular biology of HMGA proteins: hubs of nuclear function[END_REF]. We tested bacterially produced BmHMGA for binding to chorion gene promoter fragments. In gel retardation assays, recombinant BmHMGA was allowed to interact with radio-labeled promoter fragments pL1 and pL9 from the A/B.L1 and A/B.L9 chorion gene pairs, respectively (Figure 3A). Binding was evident even when the factor was added in small amounts (100 ng; 1:8 DNA:protein molar ratio), while larger amounts of protein (1:25 and 1:40 molar ratios) resulted in complexes of gradually decreasing mobility. This either suggests multiple HMGA binding sites on these promoters or non-specific interaction. Supershift assays using an anti-HMGA antiserum resulted in lowered complex mobility, thus verifying involvement of this factor in the observed protein-DNA interaction (Figure 3B). Direct binding of BmHMGA was also observed on promoter fragments derived from chorion genes of early and late specificity (data not shown). Substrate preference was assessed using synthetic double-stranded polynucleotides as competitors in gel retardation assays (Figure 3C). Given that HMG family members are prone to binding A/T rich sequences we tested poly(dA-dT) and poly(dG-dC) substrates. BmHMGA-DNA complexes responded differently to each competitor. We observed increased sensitivity to poly(dA-dT): the complex dissociated completely in the presence of 500 ng of the competitor, while treatment with poly(dG-dC) did not produce any obvious effect.

We also investigated the manner by which BmHMGA recognizes and binds chorion gene promoters via Dnase I footprinting assays on the pL1 and pL9 promoter fragments (Figure 4). Footprints revealed involvement of two 10-12 bp long sequences on each promoter (L1-I and -II, L9-I and -II). All four sequences are rich in adenines and thymidines; spacer sequences are vastly composed by pyrimidines. Type I binding elements follow a 5'-ARARWAAYYTYT-3' consensus sequence (W=A/T, R=A/G and Y=C/T) whereas type II elements a 5'-WAKYATTTW-3' consensus. Both BmHMGA recognition sequence pairs are located closer to the -gene and are spaced apart by approximately half and one-and-ahalf turn of the DNA helix, for pL1 and pL9 respectively. Projection of the sites on the double stranded DNA revealed that each pair resides on the same side of the helix. Such phasing of cis-elements has been established as a prerequisite for binding of a single HMGA molecule via two consecutive AT hooks [START_REF] Yie | Intra-and intermolecular cooperative binding of high-mobility-group protein I(Y) to the beta-interferon promoter[END_REF]. Although a single AT hook motif may bind DNA as a monomer (see mut5 in Figure 7), it is possible that footprints I and II represent binding sites for two adjacent AT hooks of BmHMGA. In the case of pL1 the intervening distance between the two sequences may be easily covered by the amino acid stretch connecting adjacent AT hooks of BmHMGA, the shortest being 20 amino acids long. For pL9 the oneand-a-half turn of the helix would require a connecting sequence of approximately 30 amino acids. This would require involvement of either the second and third AT hook pair of BmHMGA (center to center distance is 29 amino acids) or the cooperation of two non-consecutive motifs; an alternate scenario could be binding to a prebent DNA structure [START_REF] Borrmann | HMGA protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity[END_REF] which would decrease the distance to be covered.

In pL1 the type-I and II sites are tightly associated with a late-type C/EBP site, and in pL9 an earlytype C/EBP site [START_REF] Sourmeli | A novel role for the Bombyx Slbo homologue, BmC/EBP, in insect choriogenesis[END_REF] is located 10 bp downstream. These sites are preferred by BmC/EBP for binding in vitro (data not shown). In competition assays (Figure 5A), the BmHMGA-pL9 complex was abolished when the non radio-labeled L9-II fragment was added to the reaction. This finding suggests comparable affinity of BmHMGA binding to L9-I and II elements and is also supported by the fact that both footprints were generated simultaneously upon addition of BmHMGA (Figure 4). We should stress that in both 1:8 and 1:40 DNA-protein molar ratios used for footprinting no additional protected sequences could be identified. We therefore attribute lower migrating complexes seen in gel retardation assays to nonspecific protein-protein contacts, due to protein excess. Using the Actin A3 promoter region (GenBank Acc. no. AF422795) or the C/EBP binding element (oX2) as competitors protein-DNA complex formation was not affected. When pL9 was allowed to interact with EM follicular nuclear extracts, formation of six distinct complexes was observed (Figure 5A). Although complexes X1-5 could be competed out by the C/EBP binding element oX2, the X5 complex was also abolished by the addition of L9-II to the reaction. This suggests BmHMGA-BmC/EBP synergy during complex formation. Enhancement of the G (GATA) complex upon disassociation of X-complexes insinuates an antagonistic binding mode between BmC/EBP and BmGATA (also observed in early chorion genes; [START_REF] Sourmeli | In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors[END_REF]). Both C/EBP and HMGA proteins are characterized by heat-stability [START_REF] Sourmeli | A novel role for the Bombyx Slbo homologue, BmC/EBP, in insect choriogenesis[END_REF][START_REF] Johnson | Eukaryotic transcriptional regulatory proteins[END_REF][START_REF] French | Involvement of a high-mobility-group protein in the transcriptional activity of herpes simplex virus latency-active promoter 2[END_REF]. This was affirmed for BmHMGA by heating at 70 o C for 10 min and proceeding with gel retardation assays, where the factor retained functionality despite thermal shock. Furthermore, pre-heating EM follicular nuclear extracts did not affect X-complex formation (Figure 5A).

Full length BmHMGA induces specific bending on chorion gene promoters

Members of the HMG family have been known to induce bends of 70-130 o to double stranded DNA [START_REF] Ferrari | SRY, like HMG1, recognizes sharp angles in DNA[END_REF][START_REF] Giese | The HMG domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structures[END_REF]. Nevertheless, HMGA proteins were not thought to induce specific bends, but rather to retain proper B-DNA geometry and in cases reverse moderately prebent structures [START_REF] Huth | The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif[END_REF]. In order to examine the bending potential of BmHMGA we prepared constructs of pL1 and pL9 in the pBend2 vector [START_REF] Johnson | Eukaryotic transcriptional regulatory proteins[END_REF]. Circularly permuted restriction fragments of the same length were generated from pBend2 constructs. As has been established, binding of a protein to its cognate cis-element, borne on a DNA fragment, retards mobility of the fragment during gel electrophoresis. If protein binding also results in DNA bending, one should expect alterations in complex migration during electrophoresis, since this is affected by the position and degree of the bend induced [START_REF] Kim | Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector[END_REF]. Gel retardation assays revealed differences in the mobility of pL1 and pL9 permuted fragments. Control electrophoresis of the probes in the absence of BmHMGA or in the presence of a Chd1-like silkmoth protein which also contained AT hook motifs (unpublished data) did not produce differentially migrating bands (data not shown). The above comprise evidence of BmHMGA-induced specific DNA bending of chorion gene promoters.

The altered electrophoretic mobility of each curved fragment may be attributed to the reduction in its end-to-end distance [START_REF] Thompson | Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes[END_REF]: when the bending site is located near the middle of the DNA fragment, mobility is less than when it is closer to either end. The relative mobility of each complex was calculated and plotted against the relative position of the restriction fragment (Figure 6A). In general, if only one major bending locus appears in a DNA fragment, the mobility curve should be symmetrical and roughly sinusoidal with a period equal to the length of the fragment [START_REF] Wu | The locus of sequence-directed and protein-induced DNA bending[END_REF]. Extrapolation of the linear portions of the curves pointed to the bending locus on the construct. For pL1 this is located approximately 67 bp upstream from the 5 -end of the XbaI cloning site and for pL9 78 bp upstream from the same site. This could be translated into a locus of curvature located in both cases between the type I and II cis-elements that BmHMGA recognizes (shown in Figure 4 and Figure 6A). In order to confirm bending point estimates, we subcloned the -and -half of the pL9 promoter region into the pBend2 vector and used permuted restriction fragments (SspI, EcoRV, PvuII, BglII) in bending assays (Figure 6B). Noticeably, complex mobility was different among the L9 restriction fragments, but almost identical for L9 ones, signifying bending within the pL9 -half, as predicted above. Finally, apparent bending angles were calculated according to the µM/ µ =cos / 2 formula [START_REF] Thompson | Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes[END_REF]; they differed for each promoter, probably also due to the difference in BmHMGA binding sequence spacing. In the case of pL9 an angle of 95 o was calculated and for pL1 one of 87 o .

We also proceeded with the expression and purification of five deletion mutants of BmHMGA (Figure 1). In these fusion proteins certain key domains were deleted (one or more AT hook motifs and/or the full length or part of the acidic region) and bending assays were repeated. Mutants bearing at least two AT hook motifs bound DNA fragments with comparable affinity to the full-length protein. BmHMGAmut5, containing a single AT hook, could also bind DNA but with lower affinity (data not shown). Relative complex mobilities were plotted in simple graphs representing the observed 'curve' (Figure 7). Curves generated by the interaction of full-length BmHMGA with the permuted fragments could not be reproduced upon addition of any deletion mutant. Significant differences in DNA contortion and in some cases preclusion of DNA bending were observed. Progressive deletion of functional motifs in the protein mutants did not result in the loss of 'curving' ability in a linear manner. It should be noted that the effect of binding of the same mutant to either promoter resulted in similar DNA contortion. These findings suggest that the full length molecule is required in order to efficiently bend DNA.

BmHMGA develops interaction with other choriogenic transcriptional regulators

The very first evidence for synergy between BmC/EBP and BmHMGA was derived from gel retardation assays using follicular nuclear extracts (Figure 5A). The respective recombinant factors were allowed to interact with the pL9 fragment, either consecutively or simultaneously (Figure 5B). The DNA-protein complexes produced migrated through the gel more slowly than complexes resulting from BmC/EBP (complex C2, corresponding to the X2 complex of Figure 5A) or BmHMGA (complex H). When added to the reaction in a consecutive manner (15 min incubation with BmC/EBP, addition of BmHMGA in equimolar quantity followed by another 15 min of incubation) formation of two distinct complexes (C/H1, C/H2) was observed. However, when the two factors were allowed to interact on ice for 10 min and then added onto the promoter probe, a single complex (C/H3) of further diminished mobility was generated. Since these two proteins do not compete for the same or overlapping binding elements we presume a synergistic mode of action.

We sought to verify their ability to interact in a eukaryotic cell context through Yeast-Two-Hybrid interaction assays. Full length ORFs were cloned into 'prey' and 'bait' vectors and were tested for interaction in alternating pairs. Pairs resulted in comparable amounts of relative interaction, compared to the positive interaction control (Figure 8A). Results were highly reproducible and interaction values were always distinctly higher than those of the negative control, the interaction of BmHMGA with the Cotesia congregata bracovirus protein CcV1 (Acc. No. CAG17443).

Since chorion genes are developmentally regulated, we would expect transcription factors to interact differentially in vivo. This question was pursued by conducting co-immunoprecipitation (IP) assays on protein extracts from staged follicular nuclei (Figure 8B). IPs were carried out using either anti-HMGI/Y, raised against human HMGA [START_REF] Yie | Intra-and intermolecular cooperative binding of high-mobility-group protein I(Y) to the beta-interferon promoter[END_REF] or the C135 antiserum raised against Drosophila C/EBP [START_REF] Spoerel | Negative and positive regulators modulate the activity of a silkmoth chorion gene during choriogenesis[END_REF] or finally the NT2-102 antiserum recognizing BmGATA [START_REF] Papantonis | Chorion gene activation is directly dependent on BmC/EBP expression and binding to cognate cis-elements[END_REF]. For the first two, extracts from developmental stages were evenly combined: Pre, E and EM, M and ML, L and VL, thus representing vitellogenesis, early, middle and late choriogenesis respectively. For the late-activator BmGATA we combined vitellogenic and early choriogenic extracts. We showed that BmHMGA and BmC/EBP interact in the follicular nucleus, regardless of developmental stage, but in an evidently more prominent fashion during early and middle choriogenesis. In the IP between BmGATA and BmHMGA interaction was only observed during late choriogenesis, when the factor is supposed to be implicated in late chorion gene activation [START_REF] Skeiky | Synergistic interactions of silkmoth chorion promoter-binding factors[END_REF]. Control blots using (a) the secondary anti-rabbit IgG (HRP-conjugated) antibody alone, (b) total cytoplasmic protein extracts as the IP input or (c) the non-specific anti-C/EBP antiserum for the IP yielded no detectable signals (Figure 8B). BmHMGA therefore develops a developmentally defined pattern of interaction with these modulators of chorion gene expression.

The amino acid sequence encompassing the third AT hook motif of BmHMGA suffices for interaction with BmC/EBP

In an attempt to map the interaction domain of BmHMGA with BmC/EBP we performed in vitro proteinprotein cross-linking assays; the chemical cross-linker used was BS 3 (Pierce), bearing a spacer arm of 11.4 . Equimolar quantities of the interacting counterparts were allowed to interact and samples were cross-linked by the addition of BS 3 ; formed complexes were identified with the use of a monoclonal antibody raised against the six N-terminal histidine residues of the recombinant proteins (Figure 8C). Using the full length recombinant protein and deletion mutants 2 (lacking the regions encompassing AT hook motif 1 and 4, as well as the acidic C-terminus) and 5 (comprised by the region encompassing the third AT hook) we discovered that an approximately 50 kDa complex representing pairing of BmHMGA-BmC/EBP could be formed. These findings suggest that interaction does not require the involvement of the acidic tail of BmHMGA, nor any of the first, second and fourth AT hooks. In contrast, the 37 amino acid-long sequence encompassing the third AT hook forms a scaffold that suffices for interaction with BmC/EBP. In control reactions, proteins did not form self-aggregates; in the BmHMGA-BmC/EBP (GenBank Acc. No. AY618900) non-specific interaction no complex could be stabilized, thus supporting assay specificity.

BmHMGA, BmC/EBP, BmGATA and TFIID are differentially recruited to chorion gene promoters

Chromatin immunoprecipitation was applied onto nuclei from staged follicles, in order to assess middle chorion gene promoter occupancy by transcription factors during ovarian development. We looked for binding of BmHMGA, BmGATA and the transcription initiation factor TFIID/TBP on A/B.L1 and A/B.L9 gene pair promoters (Figure 8D; lanes concerning BmC/EBP have been recently discussed [START_REF] Papantonis | Chorion gene activation is directly dependent on BmC/EBP expression and binding to cognate cis-elements[END_REF] and are included for correlation purposes).

BmHMGA was found preloaded onto both promoters, already from vitellogenesis, but not uniformly. It bound with noticeably higher affinity to its cognate elements in stages EM to ML; lower signals came from vitellogenic and VL choriogenic stages. On the other hand, BmGATA binding was prominent during M, absent during ML and detectable in L/VL stages. Very faint signals from vitellogenic and E/EM choriogenic stages are in agreement with observations from early chorion gene promoter studies [START_REF] Sourmeli | In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors[END_REF]. As far as TFIID was concerned, its binding profile fitted the transcriptional profile of the studied gene pairs (A/B.L9 displaying shorter and earlier expression than A/B.L1) and indicated orderly recruitment of the general transcriptional machinery. Finally, BmC/EBP could not be positioned on either promoter in vitellogenesis, but occupied sites on both during choriogenesis. Binding to L1 promoter took place at a slightly later time during follicle development than binding to L9. Our results also showed evident rebinding of BmC/EBP during late choriogenesis, when the respective genes were expected to be transcriptionally silenced. The anti-C/EBP (non-specific control) and 'no antibody' control reactions generated no significant background signals.

BmHMGA and BmC/EBP are involved in the regulation of both the BmC/EBP and chorion genes

Taking advantage of the fact that silkmoth ovarioles retain their ability to independently develop outside the living organism [START_REF] Swevers | Early establishment and autonomous implementation of a developmental program controlling silkmoth chorion gene expression[END_REF], the in vivo role of BmHMGA was investigated using antisense DNA interference assays. A short antisense oligo complementing 20 bp around the initiation codon of the BmHMGA transcript was found to transiently block gene expression. We isolated vitellogenic (Series A) and early choriogenic (Series B) follicles and allowed them to develop in a favorable medium. RT-PCR on mRNA isolated from the follicles revealed specific decrease in BmHMGA transcripts which in turn affected chorion gene expression levels (Figure 9A). In more detail, Series A follicles progressed up to early choriogenesis and BmHMGA repression led to an obvious diminishment of ErA.1 gene transcription. Series B follicles progressed until middle choriogenesis. In this case also BmHMGA transcripts were reduced resulting in analogous down-regulation of B.L11 expression. In both series, the actin control displayed uniform levels of expression and PCR reactions seeking to amplify Hc.A/B genes generated no product (data not shown). The latter is indicative of the fact that follicles of neither series progressed into late choriogenesis, when this group of genes is rigorously transcribed. Mock Series C showed prominent Interestingly, diminishment of BmHMGA transcript abundancy affected BmC/EBP transcription; noticeable decrease in BmC/EBP transcripts was observed (especially in Series A). Under the light of this finding, we investigated the in vivo occupancy of the BmC/EBP putative promoter region by transcription factors BmC/EBP, BmHMGA, BmGATA and TFIID/TBP. We used the sequences included in the silkmoth genomic database (http://www.nises.affrc.go.jp/sgp) and identified a candidate promoter region spanning approximately 3 kb upstream from the BmC/EBP transcription initiation site. Specific primers were designed encompassing positions -159 to -639 and -1096 to -1599; these two regions were termed proximal promoter (pp) and distal promoter (dp), respectively. We chose the pp and dp fragments because they contained recognition elements for BmHMGA as well as BmC/EBP and BmGATA . The dp was the sole region containing a typical TATA box sequence, whereas in the -159 to -1 region all three recognition elements were not identified. ChIP assays revealed grave differences between the putative promoter regions. In dp no binding of either factor could be detected, not even TFIID/TBP despite the presence of a TATA box (data not shown). On pp, however, all factors presented specific binding profiles (Figure 9B). BmHMGA was located on the amplified fragment from E to ML/L choriogenic stages, BmC/EBP in E and EM stages, when the gene is rigorously transcribed and BmGATA binding was evident solely during L stages; recruitment of TFIID took place in early choriogenesis. This may be explained under the assumption that TFIID/TBP on this potential TATA-less promoter is only required for initiating transcription but not for maintaining an active state of gene expression.

Lastly, ChIP assays were performed on follicles (Series B) grown to mid-choriogenic stages in the presence of an antisense oligo against BmHMGA. We noticed that binding of HMGA to L9 promoter is a prerequisite for C/EBP recruitment (Figure 9C); recruitment of BmGATA and TFIID/TBP was also evidently impeded. Assays were repeated on chromatin isolated from follicles treated with an antisense oligo targeting BmC/EBP [START_REF] Papantonis | Chorion gene activation is directly dependent on BmC/EBP expression and binding to cognate cis-elements[END_REF] and similar results were obtained. Abolishing BmC/EBP binding to L9 promoter resulted in non-recruitment of TFIID/TBP. Binding of BmHMGA, however, was not hindered and neither was BmGATA binding (Figure 9C). Results were the same for both L1 and pp promoter fragments (data not shown).

DISCUSSION

In general, binding of proteins classified under the HMGA subfamily to DNA induces bending of specific magnitude. Their interaction with promoter/enhancer sequences and participation in multi-protein complexes is of definitive importance for orderly recruitment of transcription factors and of the general transcription machinery [START_REF] Reeves | HMGI/Y proteins: flexible regulators of transcription and chromatin structure[END_REF][START_REF] Agalioti | Ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter[END_REF]. In this study we isolated a gene coding for an HMGA-like protein from the developing follicular epithelium of the silkmoth B. mori. Primary protein structure of this factor contains characteristic HMGA motifs. The protein, accordingly named BmHMGA, is distinguishable from other insect and mammalian orthologues because of the unique presence of a fourth AT hook. In addition, the silkmoth protein does not undergo alternative splicing, at least in follicular cells, unlike mammalian HMGA1 genes. BmHMGA transcripts accumulate in follicular cells throughout oogenesis and distinct peaks of transcription were observed during late vitellogenesis, middle and late-very late choriogenesis.

Silkmoth HMGA recognizes and binds specifically to chorion gene promoters, regardless of their developmental specificity. Binding occurs via the AT hooks of the protein and is of specific nature. Our footprinting data suggest that binding occurs on two adjacent sequences that are phased on the DNA helix. We further propose that a single molecule occupies both sites via two AT hooks. The AT hook pair most likely to engage in DNA binding encompasses the first two consecutive motifs, as in deletion mutants tested this pair (in the presence of the acidic C-terminus) conveyed full functionality. A direct consequence of BmHMGA binding to DNA is induction of bending. Binding of an architectural protein to DNA is known to contribute towards the stabilization of inter-molecular complexes [START_REF] Das | The binding interaction of HMG-1 with the TATA-binding protein/TATA complex[END_REF][START_REF] Melillo | Critical role of the HMGI(Y) proteins in adipocytic cell growth and differentiation[END_REF] and ultimately leads to a single reproducible conformation [START_REF] Semsey | A gamut of loops: meandering through DNA[END_REF]. Our circular permutation assays revealed specific contortion of middle chorion gene promoters of approximately 90 degrees; the bend centers were in both cases located in the spacer sequence between the two binding elements of BmHMGA. The full length protein is required for inducing a specific angle, as use of BmHMGA deletion mutants in bending assays generated less sharp angles. We suggest that the functional purpose of HMGA-mediated DNA bending is deployment of protein-protein interaction for activation of transcription of both -and thegene by a single short promoter of 250 bp. The -half of the studied region seems to cluster most necessary cis-elements for gene activation and this is supported by data from assays where this segment was capable of driving quantitative and qualitative expression of reporter genes [START_REF] Spoerel | Negative and positive regulators modulate the activity of a silkmoth chorion gene during choriogenesis[END_REF].

BmHMGA physically interacts with BmC/EBP during choriogenesis for synergistic binding, until this interaction fades in very late chorion stages when the developmental cascade concludes. BmC/EBP possesses a dual role in chorion gene regulation. Its binding profile on chorion gene promoter modules provides evidence for its implication in the onset of transcription as well as in establishment of repression [START_REF] Papantonis | Chorion gene activation is directly dependent on BmC/EBP expression and binding to cognate cis-elements[END_REF]. We showed that BmC/EBP recruitment to both A/B.L1 and A/B.L9 promoters is dependent on interaction with BmHMGA, their recognition elements being positioned adjacent to one another. Impeding recruitment of BmC/EBP leads to non-recruitment of TFIID/TBP in the promoter vicinity. BmHMGA also interacts with BmGATA , a protein initially proposed to act as a late-specific activator [START_REF] Skeiky | Synergistic interactions of silkmoth chorion promoter-binding factors[END_REF][START_REF] Drevet | Developmental regulation of a silkworm gene encoding multiple GATA-type transcription factors by alternative splicing[END_REF] but later also implicated in early chorion promoter binding [START_REF] Sourmeli | In vitro analysis of Bombyx mori early chorion gene regulation: stage specific expression involves interactions with C/EBP-like and GATA factors[END_REF]. GATA factors in general may undertake the role of a repressor [START_REF] Martin | A novel GATA factor transcriptionally represses yolk protein precursor genes in the mosquito Aedes aegypti via interaction with the CtBP corepressor[END_REF], of an activator [START_REF] John | An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein[END_REF] or even a biphasic role dependent on protein availability [START_REF] Mcnagny | Regulation of eosinophil-specific gene expression by C/EBP-Ets complex and GATA-1[END_REF]. We provided data of its interaction with BmHMGA only during late choriogenesis, presumably for activating late (HcA/B) genes. ChIP assays revealed a peculiar binding profile of BmGATA on middle chorion gene promoters in M and in L/VL stages. Its recruitment to A/B.L1 and A/B.L9 promoters depends on its interaction with BmHMGA, but not BmC/EBP. Alhough we cannot exclude its involvement in A/B.L1 and A/B.L9 activation, we strongly support a scheme according to which BmGATA in an antagonistic relation to BmC/EBP imposes their repression.

In view of these findings, we propose a regulatory role for BmHMGA in terms of intercalation in the BmC/EBP-BmGATA functional equilibrium towards maintaining the developmental program of chorion gene expression (Figure 10). This architectural factor is preloaded on chorion promoters already from vitellogenesis and mediates BmC/EBP binding. Following temporal-specific TFIID/TBP recruitment by BmC/EBP, the formation of higher order complexes on pL1 and pL9 should facilitate assembly of the general transcription machinery. BmC/EBP in turn detaches and interaction is reinstated when repression is to be established. However, a late-specific interaction of BmHMGA with BmGATA may act as the intermediate signal for down-regulation of middle chorion genes. Under this scenario BmHMGA acts a hub where different modes of protein-protein interaction initiate, terminate and establish arrest of chorion gene expression. In our opinion, induction of DNA bending contributes in the direction of facilitating deployment of such interaction and is probably a feature of functional importance for proper follicle development. Furthermore, there seems to be a transcriptional circuit (Figure 10) through which the BmC/EBP gene is self-and heteroregulated and which in turn affects chorion gene expression; self regulation of C/EBP genes is common in eukaryotes [START_REF] Kockar | Analysis of the Xenopus laevis CCAAT-enhancer binding protein alpha gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1[END_REF]. Based on results presented here, we support the scenario that BmHMGA and BmC/EBP co-regulate transcription of the latter in a positive manner, whereas BmGATA antagonizes BmC/EBP for interaction with BmHMGA during late choriogenesis, binds to its cognate elements and potentially signals transcriptional repression. Attenuating BmC/EBP expression gradually results in the down-regulation of chorion genes (at least early and middletype genes). In all the above processes BmHMGA assumes a mediator role and seems to coordinate the deployment of protein-protein interaction towards orchestrated gene expression.

Figure 3 Gel retardation assays using recombinant BmHMGA

(A) BmHMGA binding to middle chorion gene promoter fragments. Increasing amounts of BmHMGA (100 ng, lanes 2, 6; 300 ng, lanes 3, 7; 500 ng lanes 4, 8) were allowed to interact with pL1 (free probe, lane 1) and pL9 (free probe, lane 5) promoter fragments. (B) Supershift using anti-HMGA antiserum. BmHMGA (300 ng, lane 2) was pre-incubated with the antiserum (2µl, lane 3; 5µl lane 4) or the preimmunisation serum (P/S, lane 5) prior to the addition of the pL1 probe (lane 1). (C) Competition assays using synthetic double-stranded polynucleotides. BmHMGA (300ng) was allowed to interact with pL9 (free probe, lanes 1, 6) in the presence of increasing amounts of poly(dA-dT) (0.25 µg, lane 5; 500 ng lane 4; 2 µg, lane 3; 5 µg, lane 2) and poly(dG-dC) (1 µg, lane 9; 3 µg, lane 8; 6 µg, lane 7). (A) Competition assays using oligonucleotides that represent characterized cis-elements. The pL9 promoter fragment (free probe; lanes 1, 7) was allowed to interact with BmHMGA (300 ng; lanes 2-5) and follicular nuclear extracts from EM stages (4 µg; lanes 6, 8-10) in the absence (lanes 5, 6, 9) or presence of non-labeled competitors representing the L9-II binding site (lanes 2, 10), the C/EBP binding site from the 6F6.2 gene promoter (oX2; lanes 3, 8), the actin A3 promoter (lane 4). Preheated extracts were used in lanes 5, 9 (70 o C for 10 min). (B) In vitro BmHMGA-BmC/EBP binding synergy. Proteins were added to the binding reaction in a 1:1 molar ratio (300 and 850 ng respectively) independently (lanes 2, 3), sequentially (lane 4) or simultaneously. In all panels complexes formed are indicated with X-for unknown consistency, C-for C/EBP complexes, H-for HMGA complexes and G-for the GATA attributed complex.

Figure 6 Circular permutation assays

(A) Bending plots for pL1 and pL9. Relative complex mobility was plotted against the distance of each permuted fragment from the left-hand SmaI site. Linear portions of the curve were extrapolated to indicate the bending point on each fragment (arrow below promoter graph). Black boxes represent HMGA binding elements; estimated bending angles are shown. (B) Bending assays on the -and -half of pL9. Permuted fragments of -and L9 (indicative free probes, lanes 1, 6) were allowed to interact with BmHMGA (300 ng, lanes 2-5, 7-10) and displayed different migration patterns.

Figure 7 BmHMGA and deletion mutants bending plots

Bending induced on pL1 and pL9 by wild-type BmHMGA (wt) and deletion mutants (mut1-5). The relative mobility of permuted promoter fragments after binding of each protein is depicted in simple plots. The wt 'curve' was not produced by any of the deletion mutants tested.

Figure 8 BmHMGA interaction and ChIP assays

(A) Interaction of BmHMGA, BmC/EBP and BmGATA in the Yeast-two-hybrid system. The average interaction potential (black line) of each pair is shown in arbitrary units. Boxes include all values obtained from the assays. (B) Co-immunoprecipitation assays using staged follicular nuclear extracts (Pre to VL stages). Protein samples precipitated using anti-C/EBP, anti-HMGA and anti-BmGATA antisera were separated in gradient SDS-PAGE; stage-specific interactions were visualised via Western blots. "Input' lanes represent 20% of the actual input. Cytoplasmic protein extracts (cyt) that underwent the same procedure were used as a control. Additional control assays using the non-specific anti-C/EBP antiserum are grouped below. Protein ladder bands are indicated on the right. (C) Mapping of the BmHMGA interaction domain via chemical cross-linking. BmHMGA (black triangle) was allowed to interact with BmC/EBP (panel 1) and BmC/EBP (open circle; panel 2) and complexes were cross-linked using BS 3 . HMGA deletion mutants 2 and 5 (black triangle; panels 3 and 4, respectively) underwent the same procedure. For western blots an anti-His monoclonal antibody was used; interaction is indicated by the presence of a ~50 kDa complex (circle/triangle). For the non-specific interaction control BmC/EBP and BmHMGA having almost identical mobility in 16% SDS-PAGE appear as a single intense band (panel 1). Indicative bands of the protein ladder are shown. (D) ChIP conducted on chromatin from consecutive developmental stages (Pre to VL) using antibodies against BmHMGA, BmC/EBP, BmGATA and TFIID/TBP and testing for A/B.L9 and A/B.L1 gene promoter amplification; input samples are shown. As controls no antibody and non-specific antibody (anti-C/EBP ) reactions were used.

9

  Biochemical Journal Immediate Publication. Published on 17 Jul 2008 as manuscript BJ20081012 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20081012 S t a g e 2 ( a ) P O S T -P R I N T and uniform expression levels of all amplified gene targets (Figure 9A), confirming specific targeting of the antisense oligo.

Figure 1

 1 Figure 1 Schematic representation of BmHMGA primary structure The BmHMGA open reading frame (open box) contains consecutive AT hook motifs (1-4, consensus above each motif) and a C-terminal acidic region (grey box). 5' and 3' untranslated regions are represented by lines; open triangles denote intron positions. ORFs for BmHMGA deletion mutants (mut1-5) are shown below; numbers next to boxes indicate the number of the first and last amino acid. Putative phosphorylation sites by Casein Kinase II at Ser-44/48 and Ser-97 are indicated by circled 'P'.

Figure 2

 2 Figure 2 BmHMGA features and expression profile (A) Cladogram presenting BmHMGA comparison to High Mobility Group factors from other organisms. Bombyx and Drosophila proteins are boxed and form different outgroups. GenBank accession numbers are listed on the right; numbers next to junctions indicate bootstrap values. (B) Northern blot analysis on total follicular mRNA. The arrow indicates the 1.3 kbp band corresponding to the single BmHMGA transcript. The RNA ladder bands are shown on the right. (C) BmHMGA expression profile. Quantitative real-time PCR revealed BmHMGA transcriptional profile, relative to that of BmC/EBP, during vitellogenic (Pre) and consecutive choriogenic (E to VL) stages.

Figure 4

 4 Figure 4 Dnase I footprinting of the pL1 and pL9 promoter fragments Promoter fragments were treated Dnase I (100 ng, lanes 2 and 7; 200 ng, lanes 3, 4, 8, 9; 300 ng, lanes 5, 10) in the absence (lanes 2, 3, 7, 8) or presence (1 µg, lanes 4, 9; 5 µg, lanes 5, 10) of recombinant BmHMGA. Produced footprints are indicated by black boxes. Grey boxes represent C/EBP recognition elements known to be occupied by BmC/EBP in vitro. Undigested pL9/lower strand and pL1/lower strand probes are shown at the top; G/A ladders in lanes 1 and 6.

Figure 5

 5 Figure 5 Gel retardation assays using BmHMGA, BmC/EBP and follicular nuclear extracts

Figure 9

 9 Figure 9 BmHMGA and BmC/EBP regulate both BmC/EBP and chorion gene expression(A) Antisense DNA interference assays. Electrophoretic analysis of PCR products amplified using the BmHMGA antisense assay cDNA pool as template. Amplification of the BmHMGA, BmC/EBP and ErA.1 transcripts from Series A follicles grown in the absence (0 µM) or presence (5 to 25 µ ) of the antisense oligo; B.L11 transcription was not initiated in this series. In follicles from Series B amplification of BmHMGA, BmC/EBP and B.L11 transcripts grown in the absence (0 µM) or presence[START_REF] Kravariti | Sequence analysis of a small early chorion gene subfamily interspersed within the late gene locus in Bombyx mori[END_REF]

  to 20 µ ) of the antisense oligo is shown; ErA.1 transcription has been terminated as these are more mature follicles. In the mock control setup of the assay the BmHMGA, BmC/EBP and ErA.1 transcripts were amplified. In all three series, lanes C represent no template reactions; actin A3 transcript amplification was used as a control of specific antisense targeting; markers used were 100 bp, 1 kb ladder (Fermentas) and PCR marker (Promega). (B) Chromatin immunoprecipitation assays on the BmC/EBP putative promoter. ChIP conducted on chromatin from consecutive developmental stages (Pre to VL) using antibodies against BmHMGA, BmC/EBP, BmGATA and TFIID/TBP; input samples are shown. As controls a no-antibody and non-specific antibody (anti-C/EBP ) reactions were used. In the graphic representing cis-element content of the BmC/EBP proximal promoter oval shapes denote C/EBP, triangles GATA and boxes HMGA sites, respectively. (C) ChIP assays performed on follicles grown in the absence (0 µM) presence (25 µM) of antisense oligos targeting either BmHMGA or BmC/EBP. Follicles progressed to mid-choriogenesis and the pL9 promoter fragment was amplified. Antibodies and control reactions were the same as in panel B.

Figure 10 A

 10 Figure 10 A model for chorion gene regulationScheme depicting the circuit comprised by BmHMGA, BmC/EBP and BmGATA for regulating chorion and BmC/EBP genes. At the onset of choriogenesis (left panel) HMGA is already bound to chorion gene promoters and to the putative BmC/EBP proximal promoter. Interaction between HMGA and C/EBP recruits TBP (and the general transcription machinery) and leads to gene activation. At a later time (middle panel) chorion and BmC/EBP genes are down-regulated because GATA antagonizes C/EBP for interaction with HMGA and causes repression. Choriogenesis concludes (right panel) with the establishment of chorion gene repression via C/EBP rebinding to multiple cognate elements and the repression of the BmC/EBP gene via the HMGA-GATA interaction.

Biochemical Journal Immediate Publication. Published on 17 Jul 2008 as manuscript BJ20081012 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20081012 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

The authors would like to thank M. Goldsmith for providing the BmMCBP5 clone, S. Van Soest for excellent technical support with the qRT-PCR, S. Adhya for providing the pBend2 vector and S. Tsatsarounos for help with the interaction assays. We are indebted to D. Thanos for the anti-HMGA, P. Rørth for the C135 and K. Iatrou for the NT2-102 antiserum and the BmGATA constructs; also to L.

grant (03E 124) co-funded by the E.U. and the Greek government. A.P. is the recipient of an SSF personal scholarship.