Tri-methylated lysine 20 on histone H4 (Me(3)K20H4) is a marker of constitutive heterochromatin in murine interphase and metaphase cells. Heterochromatin marked by Me(3)K20H4 replicates late during S phase of the cell cycle. Serum starvation increases the number of cells that exhibit high levels of Me(3)K20H4 at constitutive heterochromatin. Me(3)K20H4 is also present at the centromeric heterochromatin of most meiotic chromosomes during spermatogenesis and at the pseudoautosomal region, as well as at some telomeres. It is not present on the XY-body. During murine embryogenesis the maternal pronucleus contains Me(3)K20H4; Me(3)K20H4 is absent from the paternal pronucleus. On Drosophila polytene chromosomes Me(3)K20H4 is present in a 'punctate pattern' at many chromosomal bands, including the chromocenter. In coccids it is present on the facultatively heterochromatinised paternal chromosome set. We also present evidence that Me(3)K20H4 is dependent upon H3-specific Suv(3)9 histone methyltransferase activity, suggesting that there may be 'epigenetic cross-talk' between histones H3 and H4

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylationdependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and crosslink these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.

Using heterochromatin-enriched fractions, we have detected specific binding of mononucleosomes to the N-terminal domain of the inner nuclear membrane protein lamin B receptor. Mass spectrometric analysis reveals that LBR-associated particles contain complex patterns of methylated/acetylated histones and are devoid of "euchromatic" epigenetic marks. LBR binds heterochromatin as a higher oligomer and forms distinct nuclear envelope microdomains in vivo. The organization of these membrane assemblies is affected significantly in heterozygous ic (ichthyosis) mutants, resulting in a variety of structural abnormalities and nuclear defects.A significant proportion of heterochromatin is localized in the periphery of the cell nucleus and maintains a close spatial association with the inner nuclear membrane (1-5). This spatial association reflects a multiplicity of interactions between chromatin components and integral or peripheral proteins of the nuclear envelope (NE) 1 (6, 7).Because chromatin is extensively and differentially modified (8, 9), it is tempting to think that certain epigenetic marks or factors associated with histone modifying enzymes provide binding sites for NE proteins. However, it is equally possible that transcriptionally active, noncondensed chromatin is subjected to silencing and "heterochromatinization" upon contact to the NE. Both of these hypotheses receive experimental support: chromatin that is silenced through binding to SIRs can tether itself to the NE (10), whereas targeting of marker genes to the inner nuclear membrane suppresses their expression (11).One of the factors that have been implicated in chromatin anchorage to the NE is the lamin B receptor (12). LBR is a polytopic inner nuclear membrane protein consisting of a long, N-terminal domain, seven or eight hydrophobic transmembrane regions, and a C-terminal tail (13). The N-terminal part of the molecule protrudes to the nucleoplasm and contains multiple serine-arginine motifs that are phosphorylated by the SRPK1 and the cdc2 kinases (14, 15); the hydrophobic region represents, instead, a (functional) form of sterol reductase and is involved in cholesterol metabolism (16).Immunodepletion of LBR from detergent-solubilized NE vesicles results in proteoliposomes with a diminished ability to bind chromatin. Furthermore, direct binding of electrophoretically purified LBR to metaphase chromosomes can be demonstrated by in vitro assays (17). Corroborating these observations, anti-LBR antibodies block nuclear assembly in sea urchin egg extracts (18), whereas direct (19) and indirect (20) interactions with heterochromatin protein 1 (HP1) have been claimed in the literature.Two critical parameters in LBR-chromatin interactions are the physical state of LBR and the molecular features of LBRassociated chromatin. To address these issues, we have isolated fragments of peripheral heterochromatin attached to the inner nuclear membrane. These subcellular fractions were utilized to affinity select mononucleosomes that associate with LBR and investigate L...

Nuclear envelope-peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino-terminal sequencing show that one of these activities co-isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post-translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio-temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.

To study the dynamics of mammalian HP1 proteins we have microinjected recombinant forms of mHP1a, M31 and M32 into the cytoplasm of living cells. As could be expected from previous studies, the three fusion proteins were ef®ciently transported into the nucleus and targeted speci®c chromatin areas. However, before incorporation into these areas the exogenous proteins accumulated in a peripheral zone and associated closely with the nuclear envelope. This transient association did not occur when the cells were treated with deacetylase inhibitors, indicating an acetylation-inhibited interaction. In line with these observations, recombinant HP1 proteins exhibited saturable binding to puri®ed nuclear envelopes and stained the nuclei of detergent-permeabilized cells in a rim-like fashion. Competition experiments with various M31 mutants allowed mapping of the nuclear envelope-binding site within an N-terminal region that includes the chromodomain. A His 6 -tagged peptide representing this region inhibited recruitment of LAP2b and B-type lamins around the surfaces of condensed chromosomes, suggesting involvement of HP1 proteins in nuclear envelope reassembly.

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We have examined HP1␤-chromatin interactions in different molecular contexts in vitro and in vivo. Employing purified components we show that HP1␤ exhibits selective, stoichiometric, and salt-resistant binding to recombinant histone H3, associating primarily with the helical "histone fold" domain. Furthermore, using "bulk" nucleosomes released by MNase digestion, S-phase extracts, and fragments of peripheral heterochromatin, we demonstrate that HP1␤ associates more tightly with destabilized or disrupted nucleosomes (H3/H4 subcomplexes) than with intact particles. Western blotting and mass spectrometry data indicate that HP1␤-selected H3/H4 particles and subparticles possess a complex pattern of posttranslational modifications but are not particularly enriched in me 3 K9-H3. Consistent with these results, mapping of HP1␤ and me 3 K9-H3 sites in vivo reveals overlapping, yet spatially distinct patterns, while transient transfection assays with synchronized cells show that stable incorporation of HP1␤-gfp into heterochromatin requires passage through the S-phase. The data amassed challenge the dogma that me 3 K9H3 is necessary and sufficient for HP1 binding and unveil a new mode of HP1-chromatin interactions.Histone modifications are thought to provide specific readouts that are selectively utilized in DNA transactions or chromatin state transitions (1). Given the multiplicity of modification sites and the diverse chemistries of post-translational modifications, the combinatorial repertoire of this putative "histone code" might have enormous dimensions; for instance, methylation of the five lysine residues that are located at the amino-terminal tail of histone H3 could yield alone over 15 ϫ 10 3 distinct patterns, while "saturation marking" of all lysines, arginines, serines, and threonines that are found in the same region would result in ϳ256 ϫ 10 6 combinations. Clearly then, even if 1% of the predicted patterns were materialized in vivo, this voluminous "instruction manual" could not be functionally interpreted without the aid of specific de-coding factors. Consistent with this idea, recent studies have identified a set of chromatin-associated proteins that bind specifically modified histones and could, at least in theory, fulfil such a de-coding role. As it turns out, these "effector" molecules are often components of large enzymatic assemblies and possess specialized modules known as bromo-, tudor-, or chromodomains (2).A classic example of a chromodomain-containing protein is HP1, a conserved constituent of eukaryotic cells, which, in metazoans, comprises three distinct variants: ␣, ␤, and ␥ (3). HP1␣ and HP1␤ are localized in compact heterochromatic regions, while HP1␥ is more abundant in euchromatic territories (reviewed in Refs. 4 and 5). All HP1 variants have the same molecular architecture: they contain an amino-terminal chromodomain (CD), 5 an intervening region ("hinge") and a carboxylterminal chromoshadow domain (CSD). The CD is thought to be responsible for chromatin association, whereas the CSD rep...

We have previously shown that the mouse heterochromatin protein 1 homologue M31 interacts dynamically with the nuclear envelope. Using quantitative in vitro assays, we now demonstrate that this interaction is potently inhibited by soluble factors present in mitotic and interphase cytosol. As indicated by depletion and orderof-addition experiments, the inhibitory activity co-isolates with a 55-kDa protein, which binds avidly to the nuclear envelope and presumably blocks M31-binding sites. Purification of this protein and microsequencing of tryptic peptides identify it as ␣2/6:␤2-tubulin. Consistent with this observation, bona fide tubulin, isolated from rat brain and maintained in a nonpolymerized state, abolishes binding of M31 to the nuclear envelope and aborts M31-mediated nuclear envelope reassembly in an in vitro system. These observations provide a new example of "moonlighting," a process whereby multimeric proteins switch function when their aggregation state or localization is altered. Heterochromatin protein 1 (HP1)1 represents the founding member of a large protein family, which includes the Polycomb group and other gene regulators (for recent reviews see Refs. 1 and 2). This molecule possesses a dimeric, quasi-symmetrical structure and contains two sequence-related and similarly folded domains: the N-terminal chromodomain (3, 4), and the C-terminal chromo shadow domain (5). These two domains consist of anti-parallel, three-stranded ␤-sheets packed against one or two ␣-helices and are separated from one another by a flexible hinge region. Dimerization of HP1 involves intermolecular interactions between chromo shadow domains that tether two polypeptide chains at their C-terminal ends but leave the chromodomains unconstrained (6, 7).A single HP1 species was originally identified in Drosophila melanogaster (8). However, subsequent studies have revealed multiple variants of this protein in higher eukaryotes. Mammalian HP1 includes three distinct isotypes termed hHP1␣, ␤, and ␥ in humans and mHP1␣, M31, and M32 in mice (3, 9 -11). Although these proteins are structurally similar, they are distributed in different territories of the cell nucleus (12-15).HP1 binds to several chromatin-remodeling factors and transcriptional regulators. Among these are CAF-1, BRG1/SNF2␤, and the transcriptional intermediary factors ␣ and ␤ (7, 10, 15, 16). Physical or spatial associations between HP1 and elements of the origin recognition complex, actin-related proteins (Arp4), and SET or chromodomain-containing proteins, such as Su(var)3-9 and Su(var)3-7 have also been described (14,(17)(18)(19). Finally, interactions with the centromeric protein inner centromere protein, the nuclear autoantigen SP100, and the inner nuclear membrane protein LBR have been reported recently (10, 11, 20 -23).The interactions between HP1 proteins and elements of the nuclear envelope are particularly intriguing. First, peripheral heterochromatin is in physical contact with the inner nuclear membrane during interphase and could be directly linked to ...