Domain-specific Interactions of Human HP1-type Chromodomain Proteins and Inner Nuclear Membrane Protein LBR*

(Received for publication, March 17, 1997, and in revised form, April 1, 1997)

Qian Ye Dagger §, Isabelle Callebaut , Arash Pezhman Dagger , Jean-Claude Courvalin par ** and Howard J. Worman Dagger Dagger Dagger

From the Dagger  Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, the  Systèmes Moléculaires and Biologie Structurale, Laboratoire de Mineralogie-Cristallographie Paris, (LMCP), CNRS URA09, Université Paris 6 and 7, 4 Place Jussieu, 75252 Paris Cedex 05, France, and the par  Departement de Biologie Cellulaire, Institut Jacques Monod, CNRS, Université Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

HP1-type chromodomain proteins self-associate as well as interact with the inner nuclear membrane protein LBR (lamin B receptor) and transcriptional coactivators TIF1alpha and TIF1beta . The domains of these proteins that mediate their various interactions have not been entirely defined. HP1-type proteins are predicted by hydrophobic cluster analysis to consist of two homologous but distinct globular domains, corresponding to the chromodomain and chromo shadow domain, separated by a hinge region. We show here that the chromo shadow domain mediates the self-associations of HP1-type proteins and is also necessary for binding to LBR both in vitro and in the yeast two-hybrid assay. Hydrophobic cluster analysis also predicts that the nucleoplasmic amino-terminal portion of LBR contains two globular domains separated by a hinge region. The interactions of the LBR domains with an HP1-type protein were also analyzed by the yeast two-hybrid and in vitro binding assays, which showed that a portion of the second globular domain is necessary for binding. The modular domain organization of HP1-type proteins and LBR can explain some of the diverse protein-protein interactions at the chromatin-lamina-membrane interface of the nuclear envelope.


INTRODUCTION

HP1 is a heterochromatin protein originally identified in Drosophila melanogaster, where it functions as a suppressor of position effect variegation (1, 2). HP1 and homologous proteins in other species are included in the chromo superfamily of proteins (3, 4). The identifying feature of this superfamily is the chromodomain, which was first shown to be common to HP1 and Polycomb, a Drosophila protein involved in the down-regulation of homeotic selector genes during development (5). HP1-type proteins, which are included in Class A of the chromo superfamily, contain a second domain, homologous to but distinct from the chromodomain, which has been termed the chromo shadow domain (3).

In contrast to Drosophila, the functions of the highly conserved mammalian HP1-type proteins remain mostly unknown. In humans, three HP1-type proteins that arise from different genes, termed HP1Hsalpha , HP1Hsbeta , and HP1Hsgamma , have been described (6-8). In higher eukaryotic cells, a portion of the transcriptionally inactive heterochromatin is associated with the nuclear envelope (9, 10), and we have demonstrated that human HP1-type proteins bind to the inner nuclear membrane protein LBR (8). LBR (lamin B receptor), which also binds to B-type lamins, has a nucleoplasmic amino-terminal domain of ~210 amino acids and a hydrophobic domain with eight putative transmembrane segments (11-14). Besides possibly playing a role in the attachment of the nuclear lamina and the heterochromatin to the nuclear envelope in interphase, LBR may also function in targeting inner nuclear membrane vesicles to chromatin at the end of mitosis (15, 16). Mouse HP1-type polypeptides have also been shown to bind to the transcriptional coactivators TIF1alpha and TIF1beta (17). TIF transcriptional coactivators interact with the ligand-binding domains of retinoic acid and other nuclear receptors and may mediate their activation functions (18). In the yeast two-hybrid assay, mouse HP1-type proteins have also been shown to self-associate (17).

The structure of HP1-type proteins suggests that different domains may mediate their different interactions. In this study, we have used hydrophobic cluster analysis (HCA)1 (19-21) to predict the secondary structures of human HP1-type proteins and LBR. We then used the structural information obtained by using HCA to identify the domains of these proteins that mediate some of their protein-protein interactions.


EXPERIMENTAL PROCEDURES

Protein Sequence Analysis Using HCA

Guidelines for the use of this method have been published previously (19-21). HCA allows comparisons not only of amino acid sequences, but also the protein secondary structures statistically centered on hydrophobic clusters and their distributions (20). The effectiveness of HCA in predicting protein secondary structure and identifying low levels of sequence homology has been widely demonstrated (for some examples, see Refs. 22-27).

For sequence alignments, the accuracy was assessed by computing identity, similarity, and HCA scores as well as the corresponding Z-scores (27). Z-scores represent the differences between the alignment score under consideration and the mean score of a distribution computed from the alignment of sequence 1 versus a large number of randomly shuffled versions of sequence 2. Differences are then expressed relative to the standard deviation of the random distribution. Scores that are 3.0 standard deviations or greater above the scrambled mean suggest authentic relationships (27).

Plasmid Construction

Complementary DNAs were generated by the polymerase chain reaction (28) using the Gene Amp kit (Hoffmann-La Roche) and a PCR System 2400 thermocycler (Perkin-Elmer Corp.). To amplify DNA sequences, custom oligonucleotide primers (DNAgency, Malvern, PA) designed with restriction endonuclease sites at their 5'-ends were used with clones for human LBR (13) and HP1Hsalpha (8) as templates. Amplified cDNAs were purified and cloned into the plasmids of choice by standard methods.

As our previously characterized cDNA clone for HP1Hsgamma was lacking the first 17 putative amino acids based on sequence homology to other HP1-type proteins (8), we generated a full-length clone by performing the polymerase chain reaction with HeLa cell cDNA from a plasmid library (CLONTECH) as template. Oligonucleotide primers (DNAgency) based on the 3'-sequence of the partial HP1Hsgamma cDNA clone (GenBankTM accession number U26312[GenBank]) and a putative 5'-sequence in GenBankTM (accession number Z15820[GenBank]) were used in the reaction. The amplified cDNA was cloned into plasmid pACT2 (CLONTECH) and sequenced (the entry with GenBankTM accession number U26313[GenBank] has been updated to reflect the complete cDNA sequence of HP1Hsgamma ). This full-length HP1Hsgamma cDNA clone was used as a template for the polymerase chain reaction to generate HP1Hsgamma cDNAs for cloning as described above.

Binding Assays Using in Vitro Translated Proteins

For in vitro transcription-translation, cDNAs were cloned into pBFT4 (supplied by J. Licht, Mount Sinai School of Medicine, New York). Proteins expressed using this plasmid contained a FLAG epitope at their amino termini. In vitro translation was performed using the TNT T7 coupled reticulocyte lysate system (Promega) with [35S]methionine (Amersham Corp). SDS-polyacrylamide slab gel electrophoresis (PAGE) (29) followed by autoradiography was performed to confirm the synthesis of proteins. In binding experiments, 20 µl of in vitro translation lysate were added to 200 µl of binding buffer (150 mM NaCl, 20 mM NaHepes (pH 7.4), 10% glycerol, 0.5% bovine serum albumin, and 0.05% Nonidet P-40) with 20 µl of glutathione-Sepharose (Pharmacia Biotech Inc.) coupled to 3-5 µg of a glutathione S-transferase (GST) fusion protein. Plasmids that expressed GST fusion proteins were constructed by cloning the desired cDNA into pGEX-2T (Pharmacia Biotech Inc.). Fusion proteins were expressed and purified as described (30). Suspensions containing lysates and GST fusion proteins were incubated at 4 °C with rotation for ~2 h. After incubation, glutathione-Sepharose was washed five times with binding buffer, and bound proteins were then eluted with 4% SDS, separated by SDS-PAGE, and detected by autoradiography.

Yeast Two-hybrid Assay

To generate plasmids for use in the yeast two-hybrid assay, cDNAs were cloned into pACT2, which expresses fusion proteins with the GAL4 transcription activation domain, or pGBT9 (provided by S. Fields, University of Washington, Seattle), which expresses fusion proteins with the GAL4 DNA-binding domain. The desired plasmids were cotransformed into Saccharomyces cerevisiae strain Y190 (provided by S. J. Elledge, Baylor College of Medicine, Houston, TX). The two-hybrid assay and measurement of beta -galactosidase activity were performed as described previously (31, 32).


RESULTS

HCA of Human HP1-type Protein Sequences

We have used HCA to analyze the sequences of human HP1Hsalpha , HP1Hsbeta , and HP1Hsgamma . This method is highly effective in detecting three-dimensional similarities between proteins with limited sequence identities (typically 10-30%), primarily by its ability to detect related secondary structure elements statistically centered on hydrophobic clusters. Similar HCA plots for two sequences are therefore indicative of putative three-dimensional relationships. HCA of human HP1Hsalpha revealed two globular domains separated by a hinge region of ~70 amino acids (Fig. 1). Similar results were obtained for HP1Hsbeta and HP1Hsgamma (data not shown). The first of the two globular domains in the HP1 polypeptides was originally identified by Paro and Hogness (5) as the chromodomain common to the Drosophila proteins Polycomb and HP1. Analysis of the second globular domain of HP1Hsalpha suggested that it contained a three-dimensional fold similar to the chromodomain, probably as a result of an internal duplication (Fig. 1). The validity of this prediction was assessed by calculation of Z-scores from the comparison of the alignment of the tandemly duplicated domains with 10,000 alignments performed after randomization of one of the compared sequences. The Z-scores allow for evaluation of how the signal emerges from the background, with values exceeding 3.0 expected to represent an authentic relationship. The Z-identity value of 4.75, the Z-similarity value of 6.30, and the Z-HCA score of 4.72 confirm an authentic relationship between the chromodomain and the second globular domain in human HP1Hsalpha . Similar values were obtained for HP1Hsbeta and HP1Hsgamma . While this work was in progress, Koonin et al. (4) and Aasland and Stewart (3), who termed the second globular domain the chromo shadow domain, published similar results obtained using different sequence analysis methods.


Fig. 1. General organization of HP1Hsalpha as predicted from the analysis of its HCA plot. HCA readily detects two globular domains (boxed) of similar structure separated by a hinge region largely made up of hydrophilic amino acids. The first globular domain is the chromodomain (labeled A), and the second is the chromo shadow domain (labeled B). Protein sequences are shown on a duplicated alpha -helical net with amino acid positions indicated above. The contours of the hydrophobic residues are automatically drawn to form clusters that mainly correspond to the internal faces of regular secondary structures (20). Similarities between the distribution of clusters and their features (light shading) as well as sequence identities (dark shading) are readily observed between the chromodomain and chromo shadow domain, suggesting that they would adopt a similar fold. The symbols used in the plot are as follows: open square, threonine; square with dot inside, serine; diamond, glycine; and star, proline.
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Self-associations of HP1 Proteins

Mouse HP1-type proteins, which are 98-100% identical to their human homologues, self-associate via their chromo shadow domains in the yeast two-hybrid assay (17). We further examined the interactions of HP1Hsalpha and HP1Hsgamma in an in vitro solution binding assay (Fig. 2). 35S-Labeled HP1Hsalpha produced by in vitro translation in reticulocyte lysates was precipitated by the GST-HP1Hsalpha and GST-HP1Hsgamma fusion proteins, but not by GST (Fig. 2, lanes 1-4). Similarly, 35S-labeled HP1Hsgamma was precipitated by the GST-HP1 fusion proteins, but not by GST (Fig. 2, lanes 5-8). These results show that HP1Hsalpha and HP1Hsgamma can self-associate and associate with each other in vitro.


Fig. 2. HP1Hsalpha and HP1Hsgamma self-associate and bind to each other. 35S-Labeled HP1Hsalpha (lanes 1-4) and HP1Hsgamma (lanes 5-8) were synthesized in reticulocyte lysates by in vitro translation and incubated with GST (lanes 2 and 6), GST-HP1Hsalpha (lanes 3 and 7), or GST-HP1Hsgamma (lanes 4 and 8) coupled to glutathione-Sepharose. Ten percent of the amount of incubated 35S-labeled protein used in each binding reaction is also shown (lanes 1 and 5). After washing, the proteins that remained bound to glutathione-Sepharose were eluted with SDS, separated by SDS-PAGE, and detected by autoradiography. Migration of molecular mass standards (in kilodaltons) is indicated on the left.
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To identify the domain of HP1-type proteins responsible for these associations, we expressed various portions of 35S-labeled HP1Hsalpha by in vitro translation (Fig. 3A). GST-HP1Hsalpha and GST-HP1Hsgamma fusion proteins precipitated full-length HP1Hsalpha (Fig. 3B, lanes 1 and 5) from reticulocyte lysates, but not the portion of HP1Hsalpha that contained the chromodomain plus the hinge region (Fig. 3B, lanes 2 and 6). The GST fusion proteins also bound to the hinge region plus the chromo shadow domain of HP1Hsalpha (Fig. 3B, lanes 3 and 7) and to the chromo shadow domain alone (Fig. 3B, lanes 4 and 8). Hence, the chromo shadow domain was necessary and sufficient for the self-association of HP1Hsalpha and its binding to HP1Hsgamma . These results are consistent with those previously reported using the yeast two-hybrid assay, which showed that the chromo shadow domain of the mouse homologue of HP1Hsalpha interacted with mouse HP1Hsalpha and a portion of MOD1, the mouse homologue of HP1Hsgamma (17).


Fig. 3. The chromo shadow domain of HP1Hsalpha mediates its binding to itself and to HP1Hsgamma . A, shown is a schematic diagram of HP1Hsalpha indicating the chromodomain and chromo shadow domain and the positions of amino acids 1, 69, 109, and 191. cDNAs for different domains of HP1Hsalpha were cloned into pBFT4 to express the polypeptides represented by bars 1-4. The corresponding in vitro translated products of each are shown in the autoradiogram. Migration of molecular mass standards (in kilodaltons) is indicated on the left. B, 35S-labeled portions of HP1Hsalpha shown in A were synthesized in reticulocyte lysates by in vitro translation and incubated with GST-HP1Hsalpha (lanes 1-4) or GST-HP1Hsgamma (lanes 5-8) coupled to glutathione-Sepharose. After washing, the proteins that remained bound to glutathione-Sepharose were eluted with SDS, separated by SDS-PAGE, and detected by autoradiography. Approximately 10-15% of the input material bound specifically to the proteins, which generated positive results. Migration of molecular mass standards (in kilodaltons) is indicated on the left.
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The Chromo Shadow Domain of HP1Hsalpha Is Necessary for Binding to LBR

We used the yeast two-hybrid assay to identify the domain of HP1Hsalpha that binds to the nucleoplasmic amino-terminal domain of LBR (Fig. 4). Full-length HP1Hsalpha and the hinge region plus the chromo shadow domain interacted with LBR in the two-hybrid assay. The chromo shadow domain alone also gave a positive result. Neither the portion of HP1Hsalpha that contained the chromodomain plus the hinge region nor the hinge region alone interacted with the amino-terminal domain of LBR. The chromo shadow domain was therefore necessary and sufficient for the interaction of HP1Hsalpha with LBR in the yeast two-hybrid assay.


Fig. 4. The chromo shadow domain of HP1Hsalpha is necessary and sufficient for binding to the amino-terminal domain of LBR in the yeast two-hybrid assay. Schematic diagrams of the various HP1Hsalpha polypeptides (shaded rectangles) fused to the transcription activation domain of GAL4 (white rectangles) used in the two-hybrid assay are shown. The names of the plasmids that encoded each construct are given to the right of each schematic diagram. Plasmid pACT2 expresses the GAL4 transcription activation domain alone and was used as a negative control. These plasmids were cotransformed with pGBT9-LBRAT, which expresses the amino-terminal domain of LBR fused to the DNA-binding domain of GAL4. The panel on the right shows the results of filter assays for beta -galactosidase (beta -Gal) activity, in which the dark rectangles indicate detectable beta -galactosidase activity. The results of a liquid culture assay to measure beta -galactosidase activities are also shown, with the values given as means ± S.E. in arbitrary units (n = 3).
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We also examined the binding of various domains of HP1Hsalpha , synthesized by in vitro translation, to a GST fusion protein of the amino-terminal domain of LBR (Fig. 5). 35S-Labeled full-length HP1Hsalpha and polypeptides containing the chromodomain plus the hinge region, the chromo shadow domain plus the hinge region, and the chromo shadow domain alone were synthesized by in vitro translation in reticulocyte lysates. Full-length HP1Hsalpha was precipitated by the GST-LBR fusion protein (Fig. 5, lane 1), as was the protein that contained the chromo shadow domain plus the hinge region (lane 3). Deletion of the chromo shadow domain from HP1Hsalpha abolished its binding to LBR (Fig. 5, lane 2). The chromo shadow domain alone, however, did not associate with LBR in this assay (Fig. 5, lane 4). Hence, the chromo shadow domain was necessary for the binding of HP1Hsalpha to LBR. In contrast to the results obtained in the yeast two-hybrid assay, it was not sufficient and required additional amino acids of the hinge region at its amino-terminal side. These additional amino acids plus the chromodomain did not bind to LBR, suggesting that they alone did not mediate the interaction.


Fig. 5. The chromo shadow domain of HP1Hsalpha is necessary for its binding to the amino-terminal domain of LBR. 35S-Labeled portions of full-length HP1Hsalpha (lane 1), HP1Hsalpha from amino acids 2 to 109 (lane 2), HP1Hsalpha from amino acids 69 to 191 (lane 3), and HP1Hsalpha from amino acids 104 to 191 (lane 4) were incubated with a GST fusion protein containing the amino-terminal domain of LBR. After washing, the proteins that remained bound to glutathione-Sepharose were eluted with SDS, separated by SDS-PAGE, and detected by autoradiography. Approximately 10% of the input material bound specifically to the proteins, which generated positive results. Migration of molecular mass standards (in kilodaltons) is indicated on the left. A schematic diagram of HP1Hsalpha showing the chromodomain and chromo shadow domain and the positions of some amino acids, diagrams of the in vitro translated polypeptides, and an autoradiogram showing the translated polypeptides can be found in Fig. 3A.
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HCA of the Sequence of the Nucleoplasmic Domain of LBR

We have previously demonstrated that the nucleoplasmic amino-terminal domain of LBR binds to human HP1-type proteins (8). The sequence of this segment of LBR was analyzed by HCA (Fig. 6). This analysis suggested that the amino-terminal domain of LBR contained two globular domains separated by a hinge region of ~40 amino acids. The first globular domain of LBR is located between amino acids 1 and 60. The second globular domain is roughly located between amino acids 105 and 210. The hinge region between these two globular domains is highly charged between amino acids 70 and 100. The globular domains of LBR are distinct from the chromodomain and chromo shadow domains in HP1-type proteins.


Fig. 6. General organization of the nucleoplasmic amino-terminal domain of LBR as predicted from the analysis of its HCA plot. HCA detects two globular domains (boxed) separated by a hinge region that is highly charged between amino acids 70 and 100. The first putative transmembrane segment of LBR that follows the nucleoplasmic domain stretches from approximately amino acids 210 to 230 (shaded). Details on the presentation of the data and the symbols used can be found in the legend to Fig. 1.
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Identification of the Domain of LBR That Interacts with HP1Hsalpha

We examined the interactions of various portions of the amino-terminal domain of LBR with HP1Hsalpha in the yeast two-hybrid assay (Fig. 7). Full-length LBR interacted with full-length HP1Hsalpha in this assay. The first 100 amino acids of LBR that contained the first globular domain detected by HCA plus the hinge region did not interact with HP1Hsalpha . The portion of LBR from amino acids 97 to 208 that contained the second globular domain interacted with HP1Hsalpha . LBR from amino acids 1 to 174 also interacted with HP1Hsalpha , as did the stretch between amino acids 97 and 174, which contained only the first portion of the second globular domain. The region of LBR from amino acids 124 to 208 did not interact with HP1Hsalpha . These results show that the first portion of the second globular domain identified by HCA in the amino-terminal domain of LBR is responsible for its binding to HP1Hsalpha in the yeast two-hybrid assay.


Fig. 7. The first part of the second globular domain of LBR binds to HP1Hsalpha in the yeast two-hybrid assay. Schematic diagrams of the various LBR polypeptides (hatched rectangles) fused to the DNA-binding domain of GAL4 (white rectangles) used in the two-hybrid assay are shown. The names of the plasmids that encoded each construct are given to the right of each schematic diagram. Plasmid pGBT9, which expresses the GAL4 DNA-binding domain alone, was used as a negative control. These plasmids were cotransformed with pACT2-HP1Hsalpha expressing amino acids 2-191 of HP1Hsalpha fused to the transcription activation domain of GAL4. The panel on the right shows the results of filter assays for beta -galactosidase activity; Blue indicates detectable beta -galactosidase activity, and White indicates no detectable enzyme activity. The results of a liquid culture assay to measure beta -galactosidase activities are also shown, with the values given as means ± S.E. in arbitrary units (n = 3).
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To confirm the results obtained in the yeast two-hybrid assay, we examined the binding of domains of LBR synthesized by in vitro translation to a GST-HP1Hsalpha fusion protein (Fig. 8). The 35S-labeled LBR amino-terminal domain and several portions were synthesized by in vitro translation in reticulocyte lysates (Fig. 8A). The LBR amino-terminal domain was precipitated by the full-length GST-HP1Hsalpha fusion protein (Fig. 8B, lane 1), as was the portion from amino acids 97 to 208 that contained the second globular domain (Fig. 8B, lane 2). A polypeptide that contained the first globular domain plus the hinge region from amino acids 1 to 100 of LBR was not precipitated from reticulocyte lysates by the HP1Hsalpha fusion protein (Fig. 8B, lane 3). LBR from amino acids 1 to 174 and from amino acids 97 to 174 (Fig. 8B, lanes 4 and 5) were also precipitated from lysates by the HP1Hsalpha fusion protein, but the region of LBR between amino acids 124 and 208 was not precipitated (Fig. 8B, lane 6). These findings are consistent with those obtained in the yeast two-hybrid assay and show that the first portion of the second globular domain identified by HCA in the nucleoplasmic domain of LBR mediates its binding to human HP1-type proteins.


Fig. 8. The first part of the second globular domain of LBR binds to HP1Hsalpha in vitro. A, cDNAs for different domains of LBR were cloned into pBFT4, and the corresponding in vitro translated products of each are shown in the autoradiogram. The 35S-labeled polypeptides synthesized were the full-length nucleoplasmic amino-terminal domain (lane 1) and the portions from amino acids 1 to 100 (lane 2), from amino acids 97 to 208 (lane 3), from amino acids 1 to 174 (lane 4), from amino acids 97 to 174 (lane 5), and from amino acids 124 to 208 (lane 6). Migration of molecular mass standards (in kilodaltons) is indicated on the left. B, 35S-labeled portions of LBR shown in A were synthesized in reticulocyte lysates by in vitro translation and incubated with GST-HP1Hsalpha coupled to glutathione-Sepharose. After washing, the proteins that remained bound to glutathione-Sepharose were eluted with SDS, separated by SDS-PAGE, and detected by autoradiography. Approximately 10-15% of the input material bound specifically to the proteins, which generated positive results. Migration of molecular mass standards (in kilodaltons) is indicated on the left.
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DISCUSSION

Domain-specific Interactions of HP1-type Chromodomain Proteins

We have used HCA to analyze the structures of human HP1-type proteins and have shown that the chromo shadow domain mediates the self-associations of these proteins as well as their binding to the inner nuclear membrane protein LBR. In both the yeast two-hybrid and in vitro binding assays, the chromo shadow domain was necessary and sufficient for the self-association of HP1Hsalpha and for its binding to HP1Hsgamma . In the yeast two-hybrid assay, the chromo shadow domain of HP1Hsalpha was also necessary and sufficient for its interaction with LBR. However, in the in vitro binding assay, ~30 additional amino acids in the hinge region at the amino-terminal side were required for the chromo shadow domain to bind to LBR. The portion of HP1Hsalpha containing the chromodomain and these same 30 amino acids of the hinge region did not bind to LBR in either the yeast two-hybrid or in vitro binding assay. These additional amino acids may have been necessary for the initial portion of the chromo shadow domain to achieve a proper conformation. In the yeast two-hybrid assay, the chromodomain also contained a portion of yeast GAL4 at its amino-terminal side.

The chromo superfamily of proteins is generally divided into several classes with distinct structural features (3, 4). Members of all classes in this superfamily contain at least one classical chromodomain, which was first shown to be common to the Drosophila proteins HP1 and Polycomb (5). The HP1-type proteins compose one class (Class A) of the chromo superfamily. Our finding that the HP1-type proteins contain a second globular domain that is similar to the classical chromodomain was also reported by others while this work was in progress (3, 4). This second globular domain, called the chromo shadow domain, is not present in other classes of the chromo superfamily. The Polycomb-type proteins contain only one classical chromodomain, and some other superfamily members, such as CHD-1, contain two classical chromodomains. Proteins with one or two classical chromodomains and no chromo shadow domain can be considered as members of separate classes (Classes B and C, respectively). Using HCA, we have recently demonstrated that Tetrahymena Pdd1p contains three chromo-like domains and that it may represent a fourth class in the chromo superfamily.2

The chromodomain and chromo shadow domain demonstrate their greatest sequence divergence in their carboxyl-terminal regions (3, 4).2 As aromatic amino acids play important roles in protein-protein interactions (34), differences in such residues may explain the specific interactions of the chromo shadow domains with themselves and with proteins such as LBR (8) and the TIF transcriptional coactivators (17). In particular, Phe-167 and Tyr-168 are highly conserved in chromo shadow domains, but not in chromodomains of different proteins (3, 4).2 These residues may therefore be critical in the specificity of protein-protein interactions of the chromo shadow domain. On the other hand, Trp-41 of the chromodomain, which corresponds to Trp-142 of the chromo shadow domain, is conserved in almost all chromo-type domains and could be significant in their general interacting properties. The chromodomain, and various other domains in different members of the chromo superfamily of proteins, may mediate protein-protein (and possibly protein-nucleic acid) interactions different from those mediated by the chromo shadow domain. Hence, chromodomain-containing proteins may function in determining overall chromatin organization by mediating a series of hierarchical modular interactions with many other proteins and possibly DNA.

Domain-specific Interactions of LBR

LBR was originally identified by its in vitro binding to B-type nuclear lamins (11), which has been confirmed in several subsequent studies (13, 35-37). The amino-terminal domain of LBR has also been shown to bind to double-stranded DNA in vitro (13) and more recently to HP1-type proteins (8). Using HCA, we have predicted that the amino-terminal domain of LBR can be divided into distinct subdomains. This suggests that distinct domains may mediate different protein-protein and possibly protein-nucleic acid interactions.

Indirect evidence suggests that the first 60 amino acids of LBR, which correspond to the first globular domain identified by HCA, may play an important role in its association with B-type lamins. LBR autoantibodies present in occasional patients with primary biliary cirrhosis have been shown to be anti-idiotypic to some B-type lamin autoantibodies, suggesting that they recognize a region of LBR involved in its binding to B-type lamins (35). We have mapped the minimal epitope of LBR recognized by these autoantibodies to the region of the protein between amino acids 1 and 60 (38), a region that corresponds to the first globular domain identified by HCA. Although the entire amino-terminal domain of LBR is necessary for binding to B-type lamins in vitro (13), the data obtained on the anti-idiotypic autoantibodies suggest that its first globular domain may be critical for this interaction.

We have also shown that the stretch of LBR from amino acids 70 to 100 can bind to double-stranded DNA in vitro. Sequence-specific DNA binding has not been identified, and the physiological significance of the in vitro protein-DNA interaction is not yet known. HCA shows that the DNA-binding domain of LBR is the hinge region of the amino-terminal domain. This region does not have predicted secondary structure, is highly basic, and contains many proline residues, features that would be expected in polypeptides that can bind to DNA.

In this study, we have shown that part of the second globular domain of LBR mediates its binding to HP1-type proteins. A threonine residue in this second globular domain of LBR is phosphorylated by p34cdc2-type protein kinase during mitosis (33). Although this residue is outside of the minimal binding domain, it is located within the globular domain, and phosphorylation may alter the structure of the entire domain, including the HP1-binding region. Hence, mitotic phosphorylation of LBR on a threonine residue in the second globular domain may disrupt its binding to HP1-type proteins at the start of mitosis when the inner nuclear membrane dissociates from chromatin. Dephosphorylation at the mitosis to G1 interphase (33) may activate the binding capacity, allowing for the targeting of membrane vesicles to chromatin early in nuclear envelope reassembly (15, 16).


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant CA66974, the March of Dimes Birth Defects Foundation, and United States-France Cooperative Research Grant 9415591 from the National Science Foundation.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.
§   Supported in part by a postdoctoral research fellowship from the American Liver Foundation. Present address: Lab. of Developmental Hematopoiesis, Sloan-Kettering Inst. for Cancer Research, New York, NY 10021.
**   Supported by INSERM and by a United States-France cooperative research grant from CNRS.
Dagger Dagger    An Irma T. Hirschl Scholar. To whom correspondence should be addressed: Dept. of Medicine, College of Physicians and Surgeons, Columbia University, 630 West 168th St., 10th Floor, Rm. 508, New York, NY 10032. Tel.: 212-305-8156; Fax: 212-305-6443; E-mail: hjw14{at}columbia.edu.
1   The abbreviations used are: HCA, hydrophobic cluster analysis; PAGE, polyacrylamide slab gel electrophoresis; GST, glutathione S-transferase.
2   I. Callebaut, J.-C. Courvalin, H. J. Worman, and J.-P. Mornon, submitted for publication.

ACKNOWLEDGEMENTS

We thank P. Hossenlopp for invaluable suggestions; J.-P. Mornon for discussions on protein structure analysis; and S. J. Elledge, S. Fields, and J. Licht for reagents.


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