Silk Gland Factor-2, Involved in Fibroin Gene Transcription, Consists of LIM Homeodomain, LIM-interacting, and Single-stranded DNA-binding Proteins

Background: is Results: SGF-2 is a 1.1 MDa heteromeric complex containing Awh, Ldb, Lcaf and


Isolation and characterization of SGF-2
2 full activation of the fibroin gene in vivo (11).
We have demonstrated the presence of factors that bind specifically to these elements (10,12). Silk gland factor-1 (SGF-1) is a fork head (Fkh) protein binding to the proximal upstream region (12,13). Fibroin-modulatorbinding protein-1 (FMBP-1), which contains a novel DNA-binding domain, binds to both En I and the intronic element En II (12,14). Silk gland factor-2 (SGF-2) binds to the E site with two AT-rich repeat sequences, which resemble the consensus sequence recognized by homeodomain proteins. Of these factors, SGF-2 is specifically detected in PSG (10).
In this study, SGF-2 was purified and its composition was determined. SGF-2 contains at least four components; the silk protein P25/fhx, a LIM -homeodomain (LIM-HD) protein Awh, LIM -domain binding (Ldb) protein and a member of the sequence-specific singlestranded DNA -binding protein (Ssdp) family. By misexpression of Awh in transgenic silkworms, expression of the fibroin gene was induced in the middle silk glands, demonstrating that SGF-2 is a tissue-specific activator complex of the fibroin gene.
Purification of SGF-2 -Commercial silkworm strains (Kin-Shu x Sho-Wa or Shun-Rei x Sho-Getsu from Kanebo Silk Co., Kasugai City, Japan) of B. mori were reared at 27 °C on an artificial diet from Kyodo Shiryo Co. (Yokohama, Japan). SGF-2 was purified from crude nuclear extracts of PSG from V2 instar larvae through six column chromatographic chromatography steps (Fig. 1C), Crude nuclear extract (protein, 80.0 g: volume, 2,040 ml) from 40,000 pairs of PSG from V2 instar larvae was prepared as described previously (6,7,9), and subjected to following purification steps (Fig.  1C). The nuclear extract was diluted 5-fold by adding TEMGTK 0 buffer. TEMGTK buffers contain 20 mM Tris-HCl (pH 7.9), 0.1 mM EDTA, 12.5 mM MgCl 2 , 10 % glycerol, 0.1 % Tween 20, 0.1 % PMSF; the number following TEMGTK denotes the concentration (mM) of KCl. After 30 min stirring at 4 °C, the sample was centrifuged with a JA-10 rotor (Beckman) at 10,000 rpm for 1 hour at 4 °C. The supernatant was filtered through a Y020A047A membrane filter (ADVANTEC, Tokyo), and sequentially applied to a 500 ml of SP Sepharose Fast Flow resin column (Pharmacia GE Healthcare) equilibrated in TEMGTK 20 . The column was washed with 1,000 ml TEMGTK 40 and then eluted with TEMGTK 120 . The eluate (volume: 1,000 ml) was loaded onto a 40 ml Source 30Q resin column (Pharmacia GE Healthcare). The column was washed with 200 ml TEMGTK 140 , and the SGF-2 activity was eluted with TEMGTK 190 . The SGF-2 fraction (protein, 511 mg: volume, 983 ml) was diluted with TEMGTK 0 to adjust the KCl concentration to 100 mM. The diluted eluate underwent DNAaffinity purification using Dynabeads (Dyna-l) on which 4 µg poly(EW) DNA was immobilized per mg beads. Poly(EW) DNA contained tandem repeats of SGF-2 binding sequence derived from the E site of fibroin promoter and was 0.2 to 1.0 kbp length. For one round of DNA-affinity purification, 190 ml diluted eluate was incubated with 10 mg heat-denatured salmon sperm DNA and 10 mg poly(dI-dC) on ice for 10 min, and then mixed with 10 mg of the beads. After the binding reaction at 4 °C for 30 min, the beads were collected using a magnetic stand and the supernatant was removed. The beads were washed 8 times (50 ml, 20 ml, 10 ml, 5 ml, 2 ml, 1 ml, 0.5 ml, 0.2 ml, respectively) with TEMGTK 100 batch-wise, and bound proteins were eluted with 1 ml TEMGTK 1000 twice. The total eluate (0.15 mg, 2.1 ml) was dialyzed against TEMGTK 100 and loaded onto a column of BioSilect 250 (Bio-Rad) equilibrated with TEMGTK 100 . Fractions containing SGF-2 activity (71 µg, 11.8 ml) were pooled and applied to a 0.1 ml Mini S column (Pharmacia GE Healthcare, SMART) equilibrated in TEMGTK 0 . The column was washed with TEMGTK 40 and proteins were eluted with TEMGTK 0 containing 6 M urea, and then with TEMGTK 0 containing 6 M guanidine-HCl. The SGF-2 activity was not detected in the TEMGTK 40 wash fraction. The eluate with 6 M urea was dialyzed against TEMGTK 40 and applied to a 0.1 ml Mini Q column (Pharmacia GE Healthcare, SMART) equilibrated in TEMGTK 40 . The column was washed with TEMGTK 40 and bound proteins were eluted with TEMGTK 0 containing 6 M guanidine-HCl.
Amino acid sequencing of SGF-2 -Purified proteins were resolved byin SDS-PAGE, and the bands of interest were subjected to in gel tryptic Isolation and characterization of SGF-2 3 digestion, as described previously (13). The generated tryptic peptides were fractionated with a reverse-phase column, and the resolved peptide peaks were subjected to automated Edman degradation on an ABI Procise 477A protein sequencer (Applied Biosystems).
Isolation of cDNAs for SGF-2 subunits -cDNA library prepared using poly(A) + RNA from V2 PSG was screened with a random primed probe made from RT-PCR products amplified using primer sets designed on the basis of the results of amino acid sequencing ( Table 1). The positive clones were sequenced. The accession numbers of these cDNA clones of SGF2 subunits, p36 (Awh), p47B (LDBLdb) and p48/p47G/p45 (Lcaf) are AB687553, AB687554 and AB687556, respectively. During the cDNA cloning of SGF2 p47B and p48/p47G/p45, the other clones, named Ldbβ (AB687555) and Lcafβ (AB687557) were also obtained, which is derived probably from alternatively spliced mRNA.
Plasmid construction -Expression plasmids for the yeast two-hybrid assay and yeast onehybrid assay were constructed using pLexA/NLS, which were LexA-fused protein expression vectors carrying the TRP1 gene, and pGAD424 for GAL4-AD-fused protein expression vector with the LEU3 gene (16).
Interaction assay by yeast two-hybrid system -Qualitative interaction assay was performed to measure HIS3 gene expression (16). A pair of fusion gene plasmids was introduced into yeast strain L40 by the standard Lithium acetate transformation procedure. Transformants were plated on an SD agar plate containing 10 mM 3amino-1, 2, 4-triazole (3-AT) without histidine, leucine, tryptophan, lysine and uracil, and incubated overnight at 30 °C.
In vivo transcriptional activity assay by yeast one-hybrid system -The quantitative yeast onehybrid assay was performed to measure the expression of the β-galactosidase gene under the control of four tandem repeated LexA-binding sequences. Equal amounts of logarithmicgrowing yeast transformants expressing each LexA hybrid protein were subjected to βgalactosidase activity assay (17).
Preparation of transgenic silkworms -The Awh ORF was amplified by using primers 5'agtctagaatgaagacggagcaccgcac -3' and 5'agtctagatcagacttcactctgcatgc -3', and inserted into the BlnI site of the pBacUASMCS vector (18), which has a [3xP3-AmCyan] screening marker. The plasmid was injected into w1-pnd embryos to obtain the UAS-Awh strains. The established strains were crossed with the hs-GAL4 strain (19).

RESULTS
AT-rich sequences of E site in En I are essential for SGF-2 binding -Our previous results showed that SGF-2 binds to both C and E sites in the upstream enhancer element En I of the fibroin gene, with a stronger preference for the E site ( Fig. 1A; 10). Further investigation of the sequence important for binding to SGF-2, EMSA was performed using a series of mutant E site (Fig. 1B). Two AT-rich sequences (boxed in Fig. 1B) are critical for SGF-2 binding, overlap with the protected sequences in an in vitro footprint assay using V2 PSG extract, and contain homeodomain protein-binding sequences. A similar AT-rich sequence is found in the C site. The iImportance of these regions for preferential transcription of the fibroin gene in the PSG extracts has been demonstrated repeatedly previously (7)(8)(9).
Purification of SGF-2 -SGF-2 was purified from V2 PSG extract through six chromatographic chromatography steps (Fig.  1C). Figure 1D depicts a silver-stained SDS-PAGE gel containing active fractions from the 3rd step of the purification using DNAimmobilized beads. In this step, we used not only EW oligonucleotide with an intact E site but also with two mutant oligonucleotides, EgcW and EgcM. EgcW contains mutations but maintains SGF-2 binding activity, while mutations in EgcM completely abolish SGF-2 4 binding. The elution profile of SGF-2 activity in the 4th step of purification (size exclusion chromatography using Biosilect 250) correlated with EW oligo-specific polypeptides visualized on silver-stained SDS-PAGE (Fig. 2). The native molecular mass of SGF-2 activity was estimated as about 1.1 MDa by gel filtration chromatography.
cDNA cloning of SGF-2 components -To identify the components of SGF-2, amino acid sequence analysis of the purified polypeptides were performed (Table 1). This analysis revealed that p33, p32, and p30 are derived from the silk protein P25/fhx, which was identified as a fibroin-associated protein (20,21), and other proteins represent novel Bombyx gene products. The peptide sequences from p55, p50B ("B" indicates light-brown protein bands in the silver stained gel) and p47B were mostly identical, and so were those from p48, p47G ("G" indicates gray protein bands) and p45. These results suggest that p55/p50B/p47B and p48/p47G/p45 might represent products from two distinct genes by alternative splicing, respectively.
A 2.2 kb cDNA clone for the 36 kDa protein encodes an LIM-HD protein of 274 amino acids. Since the deduced amino acid sequence is highly homologous to that of the Drosophila Arrowhead protein (22) and orthologues in other species (Fig. 3A), we named the protein as Bombyx Arrowhead (Awh).
Next, we isolated the cDNA clones for p47B. The predicted protein product, which contains 357 amino acid residues, is highly homologous to mouse Ldb1/NLI/CLIM-2, CLIM-1 and Xenopus XLdb1 (23)(24)(25). It possesses a LIM domain-interacting domain (LID), which is identical among all Ldb proteins (Fig. 3B, and 26,27). We designated this protein as Bombyx LIM domain-binding protein (Ldb). Supporting our notion that p47B, p50B and p55 are products derived from the same gene, their peptide sequences are found in the predicted amino acid sequence of Ldb.
Finally, we isolated the cDNA for the SGF-2 components p48, p47G and p45. A 3.0 kb cDNA clone, which encodes a novel protein of 357 amino acid residues containing all peptide sequences from p48, p47G and p45, was isolated and designated as Lcaf (LIM-HD and LDB Ldb complex associated factor). We searched the DNA database for molecules related to Lcaf and identified sequence-specific single-stranded-DNA-binding protein (SSDP) as the closest relative in the vertebrate (Fig. 3C). The amino acid sequence of Lcaf shows high similarity to that of various vertebrate SSDPs, especially in the N-terminal 92 amino-acid sequence. Interestingly, though SSDP was reported originally as a factor binding to the DNase I hypersensitive region of chicken α2(I) collagen gene promoter (28), it was also identified as a factor interacting with Ldb proteins (29,30).
SGF-2 subunits shows restricted or preferential expression in PSG -SGF-2 is detected in the extract of PSG, but not of MSG (10). Northern blot analysis using total RNA derived from the posterior or middle portion of the fifth instar silk glands showed that Awh and P25/fhx transcripts were only detected in PSG (Fig. 3D). On the other hand, Ldb and Lcaf transcripts were found in both regions of the silk gland, but preferentially in the posterior portion.
Lcaf forms a DNA-binding protein complex with Awh and Ldb -To examine whether Lcaf forms a complex with other SGF-2 subunits Awh, Ldb and P25/fhx, all four proteins were co-expressed in Sf9 insect cells by using the baculovirus expression system. We constructed recombinant baculoviruses expressing each of HA-tagged Awh (ha:Awh), FLAG-tagged Ldb (f:Ldb), His-tagged Lcaf (h:Lcaf) and Myctagged P25/fhx (m:P25/fhx). When cells were infected with baculovirus expressing ha:Awh, f:Ldb or m:P25/fhx individually, the recombinant proteins were insoluble and not recovered well. When cells were infected with the h:Lcaf baculovirus, a 45 kDa protein band together with a minor protein band just above it were was detected in the extract affinity purified fraction ( Fig. 4A left panel lane 8). On the other hand, when cells were co-infected with ha:Awh, f:Ldb and h:Lcaf baculiviruses and h:Lcaf protein was purified with Ni affinity chromatography, the 45 kDa protein was copurified with several proteins in an almost stoichiometric manner. Immunoblotting analysis using anti-His6, anti-HA and anti-FLAG antibodies showed that the proteins co-purified with h:Lcaf were ha:Awh and f:Ldb ( Fig. 4A right panel). Co-infection of Sf9 cells with m:fhx/P25 baculovirus and the other three baculoviruses was performed, but we did not detect integration of P25/fhx protein into the Awh/Ldb/Lcaf complex.
To examine the possible DNA-binding activity of the h:Lcaf complex, EMSA was performed. As shown in Fig. 4B, DNA-binding activity to the E site was detected in the 60 and 80 mM imidazole fractions of Ni affinity chromatography. The complex migrated slightly faster than native SGF-2 purified from PSG extract (compare lane 14 with lane 15 in Fig.  4B). This DNA-protein complex is super-shifted by the addition of antibodies against HA, FLAG and His epitopes, but not by anti-Myc antibody (Fig. 4C). Most importantly, similar to SGF-2, this complex was specifically abolished by the addition of anti-SGF-2 antibody (Fig. 4C, compare lanes 1 and 2 with lanes 4 and 9). These results clearly illustrate that SGF-2-like complex with specific DNA-binding activity to the E site can be reconstituted by recombinant proteins encoded by Awh, Ldb and Lcaf cDNA..
We also purified the protein complex from Sf9 cells co-infected with f:Ldb-and ha:Awhexpressing baculoviruses using FLAG-tag, and examined its DNA-binding activity. The f:Ldb complex could bind specifically to the E site in EMSA, but the DNA-protein complex migrated much faster than that of the h:Lcaf complex ( Fig.  4D lanes 1 and 3). The DNA-protein complex was confirmed to contain both f:Ldb and ha:Awh by super-shift migration using anti-HA and anti-FLAG antibodies, but was not affected by anti-SGF-2 antibody. These results demonstrate that the complex of Awh and Ldb is sufficient for the specific binding to the E site, but is not equivalent to the purified SGF-2.
Induction of ectopic expression of the fibroin gene by Awh in MSG -To investigate whether SGF-2 is a tissue-specific transcriptional activator of the fibroin gene, we generated transgenic silkworms that possess a UAS-Awh transgene, in which Bombyx Awh was under the control of an UAS promoter. UAS-Awh silkworms were crossed with hs-GAL4 transgenic silkworms, and hs-GAL4/UAS-Awh offspring were selected. These transgenic worms were kept at 42°C for two hours on day 1 of the fourth instar, and the expression of the fibroin gene was then analyzed. Strikingly, by misexpression of the Awh transgene in transgenic worms, the fibroin gene was induced in MSG (Fig.5), where Ldb and Lcaf genes are expressed (Fig. 3D), indicating that Awh protein is a PSG-specific activator of the fibroin gene.
Self-association of Lcaf -To compare the size of the h:Lcaf complex with native SGF-2, gel filtration chromatography was performed.
The elution profiles of the h:Lcaf complex are shown in the top panel of Fig. 6A, in which the majority was eluted in a peak corresponding to a molecular mass of ~800 kDa. Although previous reports showed that the LIM-HD and LDB Ldb proteins bound to each other to form a heterotetrameric complex in vitro (24,31), it is still possible that Lcaf could oligomerize by itself. When h:Lcaf protein, expressed by the baculovirus system and purified by nickel affinity chromatography, was subjected to gel filtration chromatography, the majority of h:Lcaf was eluted in a peak corresponding to a molecular mass of ~300 kDa (Fig. 6A, bottom panel). This is equivalent to almost six times the predicted molecular mass of a sole h:Lcaf molecule (48 kDa). The self-association ability of the Lcaf protein was confirmed by a yeast two-hybrid system using the GAL4 activation domain (GAL4-AD) and LexA as a DNAbinding portion. We prepared expression plasmids for Lcaf fused to an N-terminal Gal4-AD, called G:Lcaf, along with two Lcaf truncated mutants fused to an N-terminal LexA, which contained the amino-terminal region (1-150 aa) or the carboxyl-terminal region (101-357 aa) of Lcaf, named L:Lcaf∆C151 and L:Lcaf∆N100, respectively. As shown in Fig.  6B, G:Lcaf interacted with L:Lcaf∆C151, but not with L:Lcaf∆N100, in yeast. These findings indicate that Lcaf protein can form a homooligomer through its N-terminal 100 amino acid sequence, which may contribute to the formation of a huge h:Lcaf complex with ha:Awh and f:Ldb.
Lcaf interacts with Ldb -Co-purification of ha:Awh and f:Ldb proteins with h:Lcaf suggested possible direct interactions of Lcaf with Awh and Ldb. The yeast two-hybrid system was used to examine this possibility. We constructed expression plasmids for Ldb as LexA fusion L:Ldb and Awh as GAL4-AD fusion G:Awh (Fig. 7A). Yeast transformants co-expressing L:Ldb and GAL4-AD did not grow on an SD agar plate containing 10 mM 3amino-1,2,4-triazole (3-AT) without histidine. When L:Ldb was co-expressed with G:Awh or G:Lcaf in the reporter yeast strain, both transformants were able to grow under the same conditions (Fig. 7B).
To define the regions on Ldb involved in the interactions with Awh and Lcaf, two LexA hybrid proteins called L:Ldb∆C257 and L:Ldb∆N256 that contain the amino-terminal region (1-256) and the carboxyl-terminal region (257-376) of Ldb, respectively, were examined.

6
Our results showed that G:Lcaf interacts with L:Ldb∆C257 (i.e. the amino-terminal portion of Ldb), while G:Awh binds to L:Ldb∆N256 (i.e. the carboxyl-terminal portion of Ldb) in yeast (Fig. 7B). These observations suggest that Ldb interacts with Awh and Lcaf through distinct binding domains. To determine which portion of Lcaf is necessary for the Lcaf-Ldb interaction, we used two truncation mutants of Lcaf, as described previously. While G:Lcaf∆C151 hybrid protein maintained its interaction with L:Ldb in the reporter yeast strain, G:Lcaf∆N100 lost this ability under the same condition (Fig.  7C), suggesting that the amino-terminal 100 amino acid sequence of Lcaf is necessary for Lcaf-Ldb interaction.
Awh and Lcaf contribute to transcriptional activation -To examine which SGF-2 subunits identified here play a significant role in transcriptional activation, we performed experiments using the yeast one-hybrid system. Yeast transformants expressing L:Awh or L:Lcaf grew well on media containing 10 mM 3-AT without histidine, and were also positive for β-galactosidase activity (Fig. 7D). The βgalactosidase activity of the L:Lcaf expressing transformant was about 8-fold stronger than those expressing L:Awh. On the other hand, yeast transformants expressing L:Ldb or L:P25/fhx did not grow under the same condition and exhibited little or no βgalactosidase activity. These results indicate that both Awh and Lcaf possess intrinsic transcriptional activation ability.

DISCUSSION
SGF-2: A transcriptional activator complex of the fibroin gene -SGF-2 was originally identified by EMSA in extracts of PSG on the basis of the binding activity to the fibroin En I element, and is thought to be a key transactivator for the fibroin gene (6-10). We identified four proteins, Awh, Ldb, Lcaf and P25/fhx, as components of SGF-2. Several lines of evidence support that these proteins constitute SGF-2 and promote transcriptional activation of the fibroin gene. 1. Recombinant Awh, Ldb and Lcaf proteins formed a complex with specific DNA-binding activity to the E site. 2. The protein complex of recombinant Awh, Ldb, and Lcaf was recognized by anti-SGF-2 antibody. 3. Awh, Ldb, Lcaf and P25/fhx are specifically or preferentially expressed in PSG. 4. The purified complex of recombinant Awh, Ldb and Lcaf exhibited almost the same mass as purified SGF-2. 5. Awh and Lcaf showed transcriptional activation activity in yeast onehybrid system. 6. Misexpression of Awh, which is normally restricted to PSG, induced ectopic expression of the fibroin gene in MSG of transgenic silkworms. Another indirect evidence supporting the possible contribution of Awh to tissue-and developmental stage-specific transcriptional activation of the fibroin gene is that in Drosophila transgenic lines carrying the fibroin promoter fused to the β-galactosidase gene, reporter gene expression is restricted to anterior cells of the larval salivary gland, where the Drosophila Awh gene is specifically expressed (22,32).
The silkworm Awh and Ldb are members of the LIM-HD family of transcription factors and the Ldb protein familyDB, respectively. The LIM-HD•/LDB complex appears to be a critical regulator during development and functions as a transcriptional activator (23,24,33,34). Our finding that SGF-2 contains Awh and Ldb is consistent with the notion that LDB Ldb proteins are a requisite component of many transcriptional regulatory complexes involving LIM-HD factors.
P25/fhx is known to be a component of the 2.3 MDa secretory elementary unit of silk fibroin (35). Since P25/fhx is not expressed in MSG and misexpression of Awh can induce fibroin gene expression in MSG, P25/fhx appears to be a non-essential component in the SGF-2 complex. However it will be intriguing to speculate that P25/fhx might play a role in fine-tuning the molecular ratio of fibroin to P25/fhx by regulating the transcription of the fibroin gene through SGF-2. P25/fhx is a glycoprotein (36). The N-linked oligosaccharide chains of P25/fhx are important for maintaining the 2.3 MDa complex of fibroin elementary unit (35). However, recombinant P25/fhx protein in Sf9 cells seemed not to be glycosylated on the mobility on SDS-PAGE. It is possible that glycosilation of P25/fhx is important for its integration into Awh•/Ldb•/Lcaf complex.
Lcaf: Additional component of LIM-HD•/LDBdb complex -The present study identified another protein, Lcaf, a member of the SSDP family, which could transform the Awh/Ldb protein complex into a larger protein complex due to its oligomerization activity. The N-terminal amino acid sequence (~100 amino acids) of Lcaf is almost identical to that of sequence-specific single-stranded-DNA-binding protein (SSDP), which was originally identified as a nuclear protein that binds to the singlestranded pyrimidine-rich element in the chicken α2(I) collagen gene promoter (28). This promoter element is well conserved among different mammalian species and located in a region that is DNase I hypersensitive only when

Isolation and characterization of SGF-2
7 the promoter is active. These observations have led investigators to believe that SSDP might be involved in the transcriptional regulation of the α2(I) collagen gene. The remarkable conservation of the N-terminus between Lcaf and mammalian SSDP (37) suggests that silkworm Lcaf-protein is a functional homologue of vertebrate SSDP. The conserved domain of Lcaf was necessary not only for its self-oligomerization ability but also for Ldb interaction.
Previous studies in flies and vertebrates have revealed the importance of LIM-HD and LDB Ldb proteins in tissue patterning and differentiation (30). However, the molecular mechanisms in which LIM-HD•/LDB protein complex functions as transcriptional regulator are not fully understood. Genetic experiments imply that Chip, a Drosophila homologue of LDB Ldb proteins, may mediate communication between enhancers and promoters (34,38,39). Given that the SSDP/Lcaf protein family is a requisite interaction partner for LDB Ldb proteins, they might play a role in long-range enhancer-promoter communication by cooperating with LDB Ldb proteins. In this scenario, the potential sequence-specific singlestranded DNA -binding activity, interaction activity with LDB Ldb proteins and the selfoligomerization activity of the SSDP/Lcaf protein family could facilitate enhancerpromoter communication by gathering together sequence-and structure-specific cis-elements scattered throughout certain gene loci and by organizing transcriptional regulatory elements on chromatin to form particular higher-order structures that support transcriptional regulation. From this point of view, it is important to stress that besides the En I region of the fibroin gene, a key region in the further upstream enhancer element from -1659 to -1590 detected in vivo and localized near a DNase hypersensitive site also possesses an SGF-2-binding sequence (11,Takiya unpublished results). The mouse LDB Ldb protein was found to occupy numerous DNase I hypersensitive sites on chromatin across a region of ~130 kb in the mouse αglobin locus (40). Long-range genomic interaction via Ldb1 and GATA1 was reported also in mammalian β-globin gene locus (41). It would be interesting to investigate whether the SSDP/Lcaf protein family can co-occupy the same positions as the LDB Ldb proteins, and if so, how it could contribute to the regulation of developmental gene expression, such as the formation of intra-chromosomal loops and histone modification (42). Recently, Brandt and his coworkers (43,44) reported that SSDPs regulate the activity of Ldb-containing complex through stabilization of Ldb proteins by interfering proteasomal degradation. SSDPs may be a multifunctional component in transcriptional regulation.         Oligonucleotides encoding underlined sequences were used for RT-PCR to amplify partial cDNA fragments of the corresponding SGF-2 components. (a) sequence was from undigested p36 protein (a') sequence was included in (a) (b) and (d)-(f) were the same sequence (b) includes (c') sequence (g) sequences were found in every proteins of p48, p47G and p45