REGULATION (part 2/2)

Protein Interactions

Mapping of regions of Lamin that are required for its binding to chromosomes

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo, and lamins bind specifically to chromatin in vitro. Deletion mutants of Drosophila lamin Dm0 are expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm0 tail domain. The apparent Kd of binding of the lamin Dm0 tail domain is found to be approximately 1 microM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm0. Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displace the binding of lamin Dm0 tail to chromosomes. This displacement is specific, because polyamines or proteins such as histones H1, H3, or H4 do not displace the binding of the lamin Dm0 tail to chromosomes. In addition, DNA sequences, including M/SARs, do not interfere with the binding of lamin Dm0 tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm0 and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomes in vivo (Goldberg, 1999).

Previous studies have shown that lamin Dm0 can specifically bind decondensed sperm chromatin and nuclei assembled in vitro in Drosophila embryonic extracts. Here it is shown that lamin Dm0 can also bind mitotic chromosomes in a similar fashion to vertebrate lamins A/C. The binding of lamin Dm0 is mostly peripheral, with few lamin aggregates on each chromosome. Such lamin aggregates can also be detected outside the chromosomes. The binding of lamin Dm0 to chromosomes does not require lamin polymerization, since lamin Dm0 mutated in Arg-64 (R64 -> H), which is unable to polymerize, can bind chromosomes with an overall pattern of binding similar to that of wild-type lamin Dm0. To analyze whether the binding of lamin Dm0 to chromosomes is mediated by its tail domain, 50- and 100-fold excess amounts of T425-622 were used to displace the binding of either wild-type lamin or lamin R64 -> H to chromosomes, and lamin Dm0 binding was analyzed with affinity purified polyclonal antibodies directed against the lamin Dm0 rod domain. The binding of lamin R64 -> H to chromosomes can be displaced by T425-622. With the exception of a few aggregates, wild-type lamin Dm0 is displaced from mitotic chromosomes by T425-622 with similar efficiency to that of R64 -> H (Goldberg, 1999).

To identify the regions in the Dm0 lamin protein that are required for the interaction with chromatin, substitution and deletion mutants of lamin Dm0 were expressed in bacteria and purified to near homogeneity. The purified lamin Dm0 constructs were analyzed for their ability to bind mitotic chromosomes. The headless lamin Dm0 (rod + tail; amino acids 55-622) binds to chromosomes. The isolated lamin Dm0 tail domain (amino acids 411-622) and T425-622 can bind to the periphery of the mitotic chromosomes with an intensity similar to that of the wild-type lamin Dm0. In contrast, under similar conditions, the isolated lamin Dm0 rod domain (amino acids 55-413) does not bind mitotic chromosomes. The T425-572 protein binds chromosomes with an intensity similar to that of the complete tail domain. The T425-522 protein binds chromosomes with lower immunofluorescence intensity than T425-622 protein, but with significantly higher intensity than T473-622, T523-622, and T473-572 proteins. A 50-fold molar excess of T473-622, T523-622, or T473-572 can not compete with the T425-622 protein for its binding to chromosomes. In addition, the intensity of the binding of T473-572 to chromosomes is close to background levels. Taken together, these data indicate that sequences within amino acids 425-473 and amino acids 572-622 are required for efficient binding of lamin Dm0 to chromosomes (Goldberg, 1999). It is worth noting that sequences within amino acids 425-473 share homology to sequences that are involved in the binding of Xenopus lamin B2 and human lamin A/C to chromatin, as well as to Drosophila lamin C (Bossie, 1993 and Goldberg, 1999).

To identify the target molecules for the binding of lamin Dm0 to chromosomes, the following were all tested for their ability to displace the binding of the lamin Dm0 tail domain to chromosomes: DNA sequences, polyamines, nonrelevant proteins, and histones. Previous reports have shown a strong affinity of lamin Dm0 to S/MAR DNA sequences. However, these DNA sequences are not the target of lamin tail binding to chromosomes, because neither a 100-fold molar excess of the Drosophila ftz S/MAR sequence nor yeast ARS sequences, as well as sequences adjacent to the M/SARs and plasmid DNA, will displace the binding of the T425-622 protein to chromosomes. A 100-fold molar excess of isolated nucleosome core particles displaces lamin Dm0 tail from chromosomes. In addition, a 100-fold molar excess of commercially available crude preparation of core histones and histone H1 (Sigma) efficiently displaces the lamin tail's binding to chromosomes (Goldberg, 1999).

To identify specific histone(s) that interact with the lamin Dm0 tail domain, the individual core histones and histone H1 were tested for their ability to displace the binding of the T425-622 protein to chromosomes. A molar excess concentration of in vitro-assembled histone octamers blocks the binding of the T425-622 protein. Rough estimation of the efficiency of the blocking showed that greater that 95% of the binding is displaced with purified histone octamers. Because the binding of the T425-622 protein in the presence of histone octamers was performed at 70 mM KCl and 10-20 mM NaCl, these histone octamers must have disintegrated into H2A/H2B dimers and H3/H4 tetramers when placed in the binding reaction. Indeed, a 12-fold molar excess of purified H2A/H2B dimers also efficiently displaces the binding of the T425-622 protein to chromosomes. Purified histones of both H2A or H2B can block the binding of the T425-622 protein to chromosomes. In contrast, a 12-fold molar excess of H3/H4 histone tetramer or 30- to 120-fold molar excess of individual histones H3 or H4 does not displace the T425-622 protein's binding to chromosomes. The role of histone H1 in lamin Dm0 binding to chromosomes was investigated by performing the binding reaction in the presence of a 32-fold molar excess of purified histone H1 (amino acids 1-142). This histone H1 fragment does not displaced the binding of lamin tail to chromosomes (Goldberg, 1999).

Like all core histones, histone H2A has a structurally defined central domain and a labile amino-terminal domain in which the structure and molecular interactions are not well defined. The amino-terminal domain of histone H2A is thought to be involved in the regulation of replication and transcription. It is, therefore, interesting to analyze the possible involvement of the histone H2A amino-terminal domain in the lamin tail binding. Preliminary results suggest that the binding of lamin Dm0 tail domain to chromosomes does not require the histone H2A amino-terminal domain. Additional competition experiments utilizing mutant histones will be required to further map the lamin-histone interaction. Mutant core histones lacking specific domains, histone H2A/H2B heterodimer that lacks the amino terminal domains of both histones H2A and H2B, and histone octamers assembled from mutant and wild-type histone combinations should prove to be useful for this purpose (Goldberg, 1999).

Lamin interactions with Otefin, a nuclear lamina associated protein involved in the assembly of the nuclear envelope and fs(1)Ya, a nuclear lamina associated protein involved in chromatin condensation

Otefin is a 45-kDa nuclear envelope protein with no apparent homology to other known proteins. It includes a large hydrophilic domain, a single carboxyl-terminal hydrophobic sequence of 17 amino acids, and a high content of serine and threonine residues. Cytological labeling located otefin on the nucleoplasmic side of the nuclear envelope. Chemical extraction of nuclei from Drosophila embryos reveals that Otefin is a peripheral protein whose association with the nuclear envelope is stronger than that of lamin. Deletion mutants of otefin were expressed in order to identify regions that direct Otefin to the nuclear envelope. These experiments revealed that the hydrophobic sequence at the carboxyl terminus is essential for correct targeting to the nuclear envelope, whereas additional regions in the hydrophilic domain of Otefin are required for its efficient targeting and stabilization in the nuclear envelope (Ashery-Padan, 1997a).

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila. During nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, Otefin colocalizes with Lamin derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, Otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of Lamin derivatives. There is a relatively large nonnuclear pool of Otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, Otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of Otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of Otefin. Together, these data suggest an essential role for Otefin in the assembly of the Drosophila nuclear envelope (Ashery-Padan, 1997b).

Lamin, Otefin, and YA are the three Drosophila nuclear envelope proteins that have been characterized in early embryos. The yeast two-hybrid system was used to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, Lamin Dm, interacts with both Otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, Lamin and Otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and will colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among Lamin, Otefin, and YA. Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between Lamin and YA shows that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA's COOH-terminal region is necessary and sufficient for interaction with lamin. These results suggest that interactions with lamin might mediate or stabilize the localization of Otefin and YA in the nuclear lamina. They also suggest that the need for both Otefin and Lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins. Since the hydrophobic COOH terminus of Otefin is required for targeting to the inner nuclear membrane, Otefin may connect with the inner nuclear membrane through its COOH terminus and with the nuclear lamina through other regions of otefin. Interaction between Otefin and Lamin may stabilize the localization of Otefin. This could be similar to the case of the lamin B receptor (LBR) in vertebrates, which has a hydrophilic NH2 terminus and a hydrophobic COOH terminus that is capable of targeting the LBR to the inner nuclear membrane. Since the NH2 terminus of the LBR alone targets a cytosolic protein to the nucleus but a type II integral protein to the inner nuclear membrane, this suggests that targeting a protein to the inner nuclear membrane requires a special domain, such as one mediating interaction with other nuclear envelope proteins (Goldberg, 1998)

The Drosophila protein fs(1)Ya (for Young arrest) is an essential component of the early embryonic nuclear lamina. Mutant zygotes lacking functional Ya arrest in the first division cycles following fertilization, hence the 'young arrest' noted in their development. The nuclear lamina is thought to act as the structural backbone for the nucleus and to provide anchoring sites for interphase chromosomes. Ya is not required for the de novo formation of nuclear structures. Since Ya's sequence predicts potential DNA binding motifs, this protein may instead function to connect the lamina and chromosomes, and thus aid in organizing the nucleus. Ya was ectopically expressed in polytene cells and it associates with polytene chromosomes, preferentially at interbands. Furthermore, embryonic Ya protein is capable of associating with decondensed chromatin. These observations suggest that Ya may be required for the interaction between chromatin and the nuclear envelope during early embryogenesis (Lopez, 1997).

A strong Ya mutation results in early arrest of embryos. To define the function of Ya protein in the nuclear envelope during early embryonic development, the phenotypes of four Ya mutants alleles were characterized and their molecular lesions determined. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division; a proportion of embryos from two leaky Ya mutants proceed beyond this but arrest after several abnormal divisions. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that Ya function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Two leaky Ya alleles that partially complement have lesions at opposite ends of the Ya protein, suggesting that the N- and C-termini are important for Ya function and that Ya might interact with itself either directly or indirectly (Liu, 1995).

Upon fertilization, a sperm nucleus reorganizes to become a male pronucleus. This reorganization includes breakdown and reformation of the nuclear envelope of the male pronucleus. A maternally encoded nuclear lamina protein, Ya and another lamina protein, Lamin Dm, were used in parallel as probes to study the formation of the male pronuclear lamina in Drosophila. Ectopically expressed Ya is present in the nuclear envelopes of spermatocytes, but not in mature sperm, similar to endogenous Lamin Dm. This suggests that the nuclear envelope of Drosophila sperm differs from that of somatic cells. Upon fertilization, Ya and Lamin Dm are recruited to the periphery of the male-derived nucleus before or during the early stages of migration by the male pronucleus. Using a paternal effect mutation, snky, it was found that recruitment of lamina proteins to the male pronucleus requires, and probably accompanies, reorganization of the sperm nucleus. In order to identify factors that affect the recruitment of nuclear lamina proteins to the male pronucleus, the subcellular localization of Ya and Lamin Dm were studied in mutant embryos defective for the function of either the male pronucleus (mh, K81, and pal) or both pronuclei (gnu, png, and plu). None of these mutations affect the recruitment of Ya or Lamin Dm to the male pronuclear envelope, suggesting that the mutations affect processes independent of, or after, reorganization of the nuclear envelope. Double mutant analyses between Ya and gnu suggest that Ya plays a role in the nuclear envelope permissive for rounds of DNA replication (Liu, 1997).

The Drosophila YA protein is a nuclear lamina component whose function is essential to initiate embryonic development. To identify regions of YA required for its action in its normal cellular context, targeted mutations were made in the YA protein and their consequences tested in flies and embryos in vivo. Critical amino acids are distributed along the length of the YA molecule, with functionally important regions including the N- and the C-terminal ends, the cysteine residues in YA's two potential zinc fingers, a serine/threonine-rich region, and a potential maturation-promoting factor or mitogen-activated protein kinase phosphorylation target site, ITPIR. In addition, several Ya mutations showed intragenic complementation, with N-terminal mutations complementing C-terminal mutations, suggesting that YA proteins interact with one another. In support of this interaction, immunoprecipitation experiments demonstrate that YA molecules are present in complexes with one another. The C-terminal 179 amino acids of YA are necessary to target, or retain, YA in the nuclear envelope (Liu, 1998).

The YA protein is a maternally provided nuclear lamina component that is essential during the transition from meiosis to mitosis at the beginning of embryogenesis. Localization of YA to the nuclear envelope is required for its function; this localization is cell cycle-dependent during embryogenesis. The ability of YA to enter nuclei is modulated during development. In developing egg chambers, YA protein is made but excluded from nuclei of nurse cells and oocytes; upon egg activation (but independent of fertilization) YA acquires the ability to enter nuclei and becomes incorporated into the nuclear lamina in unfertilized eggs and cleavage-stage embryos. This localization switch correlates with changes in the phosphorylation state of YA. YA in ovaries is hyperphosphorylated relative to YA in unfertilized eggs and embryos. Through site-directed mutagenesis, 443Thr (a potential phosphorylation site for both cyclin-dependent protein kinase and mitogen-activated-protein kinase) was identified as one of the sites likely involved in this developmental control. YA is specifically dephosphorylated at 443Thr upon egg activation. These results suggest that phosphorylation plays a role in modulating the localization of YA during development. A model for developmental regulation of the nuclear entry of YA is proposed and implications for understanding Drosophila egg activation are discussed. It is suggested that dephosphorylation at or near YA's nuclear localization signal (NLS), might activate or unmask the NLS. Perhaps the cell cycle-dependent nuclear envelope localization of YA results from the cell cycle-dependent availability of YA-binding sites in the nuclear lamina. These YA-binding molecules in the nuclear envelope may be modified during the cell cycle to lose or gain YA-binding sites. YA may be involved in the coordination of chromosome condensation and nuclear fusion after egg activation. Alternatively, the nuclear lamina may play a role in mitotic spindle formation (Yu, 1999).

The nuclear lamina provides an architectural framework for the nuclear envelope and an attachment site for interphase chromatin. In Drosophila eggs and early embryos its major constituent, lamin Dm0, interacts with a lamina protein called YA. When the lamin-interaction region of YA is deleted, YA still enters nuclei but fails to localize to nuclear envelopes, suggesting that lamin interaction targets YA to the nuclear envelope. The C-terminal lamin-interacting region of YA is sufficient to target the heterologous soluble protein GFP-NLS to the nuclear periphery in Drosophila tissue culture cells. Yeast two-hybrid analysis and transient transfection assays further defined this domain: residues 556-696 of YA are sufficient for both lamin Dm0 interaction and the targeting of GFP-NLS to the nuclear periphery. This region of YA is hydrophilic and lacks any transmembrane domain or known membrane-targeting motifs. It is proposed that the localization of YA to the nuclear lamina involves interaction with polymerized lamin Dm0 mediated by the lamin-targeting domain of YA. This hydrophilic YA domain might provide a useful molecular tool for targeting heterologous non-membrane-associated proteins to the nuclear envelope (Mani, 2003).

The cell cycle dynamics of YA are consistent with this model. Lamin Dm0 is present in the nuclei of all cells in which YA is in the nuclear envelope. At metaphase, the two proteins disappear from the nuclear periphery simultaneously, as one would expect if the presence of YA at the nuclear periphery depended on the presence of polymerized lamin. After anaphase, both proteins reappear at the nuclear periphery, but lamin Dm0 is detected there before YA reappears, consistent with the idea that lamin polymerizes to form a scaffold to which YA becomes attached. This model is further supported by the finding that the YA-lamin interaction in yeast is abolished if either the head or tail domain of lamin is deleted, suggesting that YA interacts with a multimeric form of lamin Dm0 (Mani, 2003).

The 90 amino-acid region of YA defined in this study also provides a useful tool for further studies of nuclear structure and function in the Drosophila model system. It could be used to bring to the nuclear envelope hydrophilic proteins that are not normally found at the nuclear periphery, for studies of nuclear assembly, replication or transcriptional silencing of chromatin. It would also be interesting to see if this domain can compensate for loss of a nuclear targeting domain from nuclear envelope proteins that normally use other mechanisms to target to this cellular compartment (Mani, 2003).

M/SARs, DNA sites that associate with Lamins

The nuclear matrix maintains specific interactions with genomic DNA at sites known as matrix attachment regions (M/SARs). M/SARs bind in vitro to lamin polymers. The polymerized alpha-helical rod domain of Lamin provides by itself the specific binding to the fushi tarazu M/SAR. In contrast, an unpolymerized rod domain does not bind specifically to this M/SAR. Non-specific binding to DNA is also observed, either with Lamin containing a point mutation that impairs its ability to polymerize or with the isolated tail domain. These data suggest that the specific binding of lamins to M/SARs requires the rod domain and depends on the lamin polymerization state (Zhao, 1996).

M/SAR of Drosophila melanogaster DNA clone gamma 20p 1.4 was localized on chromosomes by in situ hybridization of the gamma 20p 1.4 DNA fragment and its deletion mutants with salivary gland chromosomes of third instar larvae. The M/SAR sequence, capable of specifically binding to Lamin in vitro, hybridizes with the pericentric regions 20CD, 40EF, 41AB, the proximal part of region 81 of chromosome 3, and region 101 of chromosome 4. These DNA regions attach to the nuclear envelope in cells of Drosophila salivary glands. Hybridization is also observed to region 49D, which does not come into contact with the nuclear periphery. This is the first M/SAR localized in pericentric chromatin of Drosophila (Sharakhov, 1997).

This paper presents a molecular biological characteristic of a DNA fragment (delta 20p1.4, a moderately repetitive sequence of the Drosophila melanogaster genome). The fragment is present in about 120 copies per haploid genome. The main pool of the delta 20p1.4 homologous DNA can be isolated, along with the nuclear matrix DNA, and consists of Hind III-EcoR I monomers 1.4-1.6 kb in length. The monomers may occur in the genome as both single copies and tandem clusters forming chromosome fragments up to 6-10 kb in length. The region of the delta 20p1.4 fragment between nucleotides 350 and 905 polymerizes with purified Lamin from Drosophila. The sites of the fragment that have physical contact with Lamin in vitro were determined using an Exo III protection. It is demonstrated that ATATTT, A, and T boxes located in four nonperfect tandem repeats were involved in the contact, both DNA strains reacting with lamin (Bogachev, 1996).

A DNA fragment designated lambda 20p1.4 binds in vitro to polymerized Drosophila melanogaster Lamin. In situ hybridization of lambda 20p1.4 to isolated polytene chromosomes reveals localization at the chromocenter and to the 49 CD region on the right arm of chromosome 2. About 120 copies of sequences homologous to lambda 20p1.4 are detected per haploid genome. Nucleotide (nt) sequence analysis demonstrates that lambda 20p1.4 is an A + T-rich, 1327-bp fragment containing four repeated units between nt 595 and 919. Results suggest that lamin interacts with a region of lambda 20p1.4 between nt 300 and 1000. Confocal immunofluorescence co-localization demonstrates that in situ, the major locus of lambda 20p1.4 hybridization, the chromocenter, is found juxtaposed to the nuclear envelope (lamina). This is the first demonstration that a DNA sequence that binds specifically to nuclear lamins in vitro, is located at or near the nuclear envelope in situ and, presumably, in vivo (Baricheva, 1996).

back to Regulation part 1/2

Lamin: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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