Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation-dependent 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 cross-link 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.     

M33 condenses chromatin through nuclear body formation and methylation of both histone H3 lysine 9 and lysine 27

Heterochromatin, a type of condensed DNA in eukaryotic cells, has two main categories: Constitutive heterochromatin, which contains H3K9 methylation, and facultative heterochromatin, which contains H3K27 methylation. Methylated H3K9 and H3K27 serve as docking sites for chromodomain-containing proteins that compact chromatin. M33 (also known as CBX2) is a chromodomain-containing protein that binds H3K27me3 and compacts chromatin in vitro. However, whether M33 mediates chromatin compaction in cellulo remains unknown. Here we show that M33 compacts chromatin into DAPI-intense heterochromatin domains in cells. The formation of these heterochromatin domains requires H3K27me3, which recruits M33 to form nuclear bodies. G9a and SUV39H1 are sequentially recruited into M33 nuclear bodies to create H3K9 methylated chromatin in a process that is independent of HP1α. Finally, M33 decreases progerin-induced nuclear envelope disruption caused by loss of heterochromatin. Our findings demonstrate that M33 mediates the formation of condensed chromatin by forming nuclear bodies containing both H3K27me3 and H3K9me3. Our model of M33-dependent chromatin condensation suggests H3K27 methylation corroborates with H3K9 methylation during the formation of facultative heterochromatin and provides the theoretical basis for developing novel therapies to treat heterochromatin-related diseases.     

p66alpha and p66beta of the Mi-2/NuRD complex mediate MBD2 and histone interaction

The Mi-2/NuRD complex is a multi-subunit protein complex with enzymatic activities involving chromatin remodeling and histone deacetylation. Targeting of Mi-2/NuRD to methylated CpG sequences mediates gene repression. The function of p66α and of p66β within the multiple subunits has not been addressed. Here, we analyzed the in vivo function and binding of both p66-paralogs. Both factors function in synergy, since knocking-down p66α affects the repressive function of p66β and vice versa. Both proteins interact with MBD2 functionally and biochemically. Mutation of a single amino acid of p66α abolishes in vivo binding to MBD2 and interferes with MBD2-mediated repression. This loss of binding results in a diffuse nuclear localization in contrast to wild-type p66α that shows a speckled nuclear distribution. Furthermore, wild-type subnuclear distribution of p66α and p66β depends on the presence of MBD2. Both proteins interact with the tails of all octamer histones in vitro, and acetylation of histone tails interferes with p66 binding. The conserved region 2 of p66α is required for histone tail interaction as well as for wild-type subnuclear distribution. These results suggest a two-interaction forward feedback binding mode, with a stable chromatin association only after deacetylation of the histones has occurred.     

Heterochromatin protein 2 interacts with Nap-1 and NURF: a link between heterochromatin-induced gene silencing and the chromatin remodeling machinery in Drosophila

Heterochromatin protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster that binds to heterochromatin protein 1 (HP1) and has been implicated in heterochromatin-induced gene silencing. Heretofore, HP1 has been the only known binding partner of HP2, a large protein devoid of sequence motifs other than a pair of AT hooks. In an effort to identify proteins that interact with HP2 and assign functions to its various domains, nuclear proteins were fractionated under nondenaturing conditions. On separation of nuclear proteins, nucleosome assembly protein 1 (Nap-1) has an overlapping elution profile with HP2 (assayed by Western blot) and has been identified by mass spectrometry in fractions with HP2. Upon probing fractions in which HP2 and Nap-1 are both present, we find that the nucleosome remodeling factor (NURF), an ISWI-dependent chromatin remodeling complex, is also present. Results from coimmunoprecipitation experiments suggest that HP2 interacts with Nap-1 as well as with NURF; NURF appears to interact directly with both HP2 and Nap-1. Three distinct domains within HP2 mediate the interaction with NURF, allowing us to assign NURF binding domains in addition to the AT hooks and HP1 binding domains already mapped in HP2. Mutations in Nap-1 are shown to suppress position effect variegation, suggesting that Nap-1 functions to help to assemble chromatin into a closed form, as does HP2. On the basis of these interactions, we speculate that HP2 may cooperate with these factors in the remodeling of chromatin for silencing.     

The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

The Epstein-Barr virus transactivator Zta triggers lytic gene expression and is essential for replication of the lytic origin, oriLyt. Previous analysis indicated that the Zta activation domain contributed a replication-specific function. We now show that the Zta activation domain interacts with components of the EBV helicase-primase complex. The three helicase-primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc epitope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (primase subcomplex) were separately transfected, the proteins localized to the cytoplasm. Interaction between Zta and the components of the helicase-primase complex was tested by examining the ability of Zta to alter the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; similarly, cotransfection of Zta with the primase subcomplex led to nuclear translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This relocalization provides evidence for an interaction between Zta and the helicase and Zta and the primase subcomplex. An affinity assay using glutathione S-transferase–Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino acids 1 to 133). In the nuclear relocalization assay, the amino-terminal 25 amino acids of Zta were required for efficient interaction with the primase subcomplex but not for interaction with BBLF4. Evidence for interaction between oriLyt bound Zta and the helicase-primase complex was obtained in a superactivation assay using an oriLyt-chloramphenicol acetyltransferase (CAT) reporter. Zta activated expression from a CAT reporter containing the complete oriLyt region and regulated by the oriLyt BHLF1 promoter. Cotransfection of the helicase-primase proteins, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression. This assay also provided evidence for an interaction between the single-stranded DNA binding protein, BALF2, and the Zta-tethered helicase-primase complex. The helicase-primase interaction is consistent with a role for Zta in stabilizing the formation of an origin-bound replication complex.     

Proteasome subunit Rpn1 binds ubiquitin-like protein domains

The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain. This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes. Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome. A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain. A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome. We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding. We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes.     

Heterochromatin protein 1 binds to nucleosomes and DNA in vitro

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein primarily associated with the pericentric heterochromatin and telomeres in Drosophila. The molecular mechanism by which HP1 specifically recognizes and binds to chromatin is unknown. The purpose of this study was to test whether HP1 can bind directly to nucleosomes. HP1 binds nucleosome core particles and naked DNA. HP1-DNA complex formation is length-dependent and cooperative but relatively sequence-independent. We show that histone H4 amino-terminal peptides bind to monomeric and dimeric HP1 in vitro. Acetylation of lysine residues had no significant effect on in vitro binding. The C-terminal chromo shadow domain of HP1 specifically binds H4 N-terminal peptide. Neither the chromo domain nor chromo shadow domain alone binds DNA; intact native HP1 is required for such interactions. Together, these observations suggest that HP1 may serve as a cross-linker in chromatin, linking nucleosomal DNA and nonhistone protein complexes to form higher order chromatin structures.     

Components of coated vesicles and nuclear pore complexes share a common molecular architecture

Numerous features distinguish prokaryotes from eukaryotes, chief among which are the distinctive internal membrane systems of eukaryotic cells. These membrane systems form elaborate compartments and vesicular trafficking pathways, and sequester the chromatin within the nuclear envelope. The nuclear pore complex is the portal that specifically mediates macromolecular trafficking across the nuclear envelope. Although it is generally understood that these internal membrane systems evolved from specialized invaginations of the prokaryotic plasma membrane, it is not clear how the nuclear pore complex could have evolved from organisms with no analogous transport system. Here we use computational and biochemical methods to perform a structural analysis of the seven proteins comprising the yNup84/vNup107–160 subcomplex, a core building block of the nuclear pore complex. Our analysis indicates that all seven proteins contain either a β-propeller fold, an α-solenoid fold, or a distinctive arrangement of both, revealing close similarities between the structures comprising the yNup84/vNup107–160 subcomplex and those comprising the major types of vesicle coating complexes that maintain vesicular trafficking pathways. These similarities suggest a common evolutionary origin for nuclear pore complexes and coated vesicles in an early membrane-curving module that led to the formation of the internal membrane systems in modern eukaryotes.     

Novel Drosophila heterochromatin protein 1 (HP1)/origin recognition complex-associated protein (HOAP) repeat motif in HP1/HOAP interactions and chromocenter associations

Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Here we show direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain) (47). We find heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin.     

Antagonistic functions of SET-2/SET1 and HPL/HP1 proteins in C. elegans development

Cellular identity during metazoan development is maintained by epigenetic modifications of chromatin structure brought about by the activity of specific proteins which mediate histone variant incorporation, histone modifications, and nucleosome remodeling. HP1 proteins directly influence gene expression by modifying chromatin structure. We previously showed that the Caenorhabditis elegans HP1 proteins HPL-1 and HPL-2 are required for several aspects of post-embryonic development. To gain insight into how HPL proteins influence gene expression in a developmental context, we carried out a candidate RNAi screen to identify suppressors of hpl-1 and hpl-2 phenotypes. We identified SET-2, the homologue of yeast and mammalian SET1, as an antagonist of HPL-1 and HPL-2 activity in growth and somatic gonad development. Yeast Set1 and its mammalian counterparts SET1/MLL are H3 lysine 4 (H3K4) histone methyltransferases associated with gene activation as part of large multisubunit complexes. We show that the nematode counterparts of SET1/MLL complex subunits also antagonize HPL function in post-embryonic development. Genetic analysis is consistent with SET1/MLL complex subunits having both shared and unique functions in development. Furthermore, as observed in other species, we find that SET1/MLL complex homologues differentially affect global H3K4 methylation. Our results suggest that HP1 and a SET1/MLL-related complex may play antagonistic roles in the epigenetic regulation of specific developmental programs.     

Drosophila RISC Component VIG and Its Homolog Vig2 Impact Heterochromatin Formation

Heterochromatin formation plays an important role in gene regulation and the maintenance of genome integrity. Here we present results from a study of the D. melanogaster gene vig, encoding an RNAi complex component and its homolog vig2 (CG11844) that support their involvement in heterochromatin formation and/or maintenance. Protein null mutations vig^EP812 and vig2^PL470 act as modifiers of Position Effect Variegation (PEV). VIG and Vig2 are present in polytene chromosomes and partially overlap with HP1. Quantitative immunoblots show depletion of HP1 and HP2 (large isoform) in isolated nuclei from the vig^EP812 mutant. The vig2^PL470 mutant strain demonstrates a decreased level of H3K9me2. Pull-down experiments using antibodies specific to HP1 recovered both VIG and Vig2. The association between HP1 and both VIG and Vig2 proteins depends on an RNA component. The above data and the developmental profiles of the two genes suggest that Vig2 may be involved in heterochromatin targeting and establishment early in development, while VIG may have a role in stabilizing HP1/HP2 chromatin binding during later stages.