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.     

The multi-domain protein Np95 connects DNA methylation and histone modification

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ß. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.     

Structural insight into recognition of methylated histone tails by retinoblastoma-binding protein 1

Retinoblastoma-binding protein 1 (RBBP1), also named AT-rich interaction domain containing 4A (ARID4A), is a tumor and leukemia suppressor involved in epigenetic regulation in leukemia and Prader-Willi/Angelman syndromes. Although the involvement in epigenetic regulation is proposed to involve its chromobarrel and/or Tudor domains because of their potential binding to methylated histone tails, the structures of these domains and their interactions with methylated histone tails are still uncharacterized. In this work, we first found that RBBP1 contains five domains by bioinformatics analysis. Three of the five domains, i.e. chromobarrel, Tudor, and PWWP domains, are Royal Family domains, which potentially bind to methylated histone tails. We further purified these domains and characterized their interaction with methylated histone tails by NMR titration experiments. Among the three Royal Family domains, only the chromobarrel domain could recognize trimethylated H4K20 (with an affinity of ～3 mm), as well as recognizing trimethylated H3K9, H3K27, and H3K36 (with lower affinities). The affinity could be further enhanced up to 15-fold by the presence of DNA. The structure of the chromobarrel domain of RBBP1 determined by NMR spectroscopy has an aromatic cage. Mutagenesis analysis identified four aromatic residues of the cage as the key residues for methylated lysine recognition. Our studies indicate that the chromobarrel domain of RBBP1 is responsible for recognizing methylated histone tails in chromatin remodeling and epigenetic regulation, which presents a significant advance in our understanding of the mechanism and relationship between RBBP1-related gene suppression and epigenetic regulation.     

Inner nuclear membrane protein LBR preferentially interacts with DNA secondary structures and nucleosomal linker

The lamin B receptor (LBR) is an integral protein of inner nuclear membrane whose nucleoplasmic amino-terminal domain contributes to the attachment of the membrane to chromatin. Here we analyzed the interactions of a recombinant GST protein containing the amino-terminal domain of the protein with in vitro reconstituted nucleosomes and short DNA fragments. Data show that the LBR amino-terminal domain (AT) binds linker DNA but does not interact with the nucleosome core. Titration and competition studies revealed that the interaction between LBR AT and DNA is saturable, of high affinity (K(D) approximately 4 nM), independent of DNA sequence, and enhanced by DNA curvature and supercoiling. In this respect, LBR amino-terminal domain binding to nucleosomes is similar to that of histone H1 and non histone proteins HMG1/2 which both bind preferentially to linker DNA and present a significant affinity for DNA secondary structures.     

Interactions of HP1 Bound to H3K9me3 Dinucleosome by Molecular Simulations and Biochemical Assays

Heterochromatin protein 1 (HP1), associated with heterochromatin formation, recognizes an epigenetically repressive marker, trimethylated lysine 9 in histone H3 (H3K9me3), and generally contributes to long-term silencing. How HP1 induces heterochromatin is not fully understood. Recent experiments suggested that not one, but two nucleosomes provide a platform for this recognition. Integrating previous and new biochemical assays with computational modeling, we provide near-atomic structural models for HP1 binding to the dinucleosomes. We found that the dimeric HP1α tends to bind two H3K9me3s that are in adjacent nucleosomes, thus bridging two nucleosomes. We identified, to our knowledge, a novel DNA binding motif in the hinge region that is specific to HP1α and is essential for recognizing the H3K9me3 sites of two nucleosomes. An HP1 isoform, HP1γ, does not easily bridge two nucleosomes in extended conformations because of the absence of the above binding motif and its shorter hinge region. We propose a molecular mechanism for chromatin structural changes caused by HP1.     

Recognition of multivalent histone states associated with heterochromatin by UHRF1 protein

Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16(INK4A), when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression.     

The inner nuclear membrane protein lamin B receptor forms distinct microdomains and links epigenetically marked chromatin to the nuclear envelope

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.     

Retinoblastoma-binding Protein 1 Has an Interdigitated Double Tudor Domain with DNA Binding Activity

Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10–100 μm; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.     

Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins

Members of the heterochromatin protein 1 (HP1) family are silencing nonhistone proteins. Here, we show that in P19 embryonal carcinoma (EC) nuclei, HP1 alpha, beta, and gamma form homo- and heteromers associated with nucleosomal core histones. In vitro, all three HP1s bind to tailed and tailless nucleosomes and specifically interact with the histone-fold of histone H3. Furthermore, HP1alpha interacts with the linker histone H1. HP1alpha binds to H3 and H1 through its chromodomain (CD) and hinge region, respectively. Interestingly, the Polycomb (Pc1/M33) CD also interacts with H3, and HP1alpha and Pc1/M33 binding to H3 is severely impaired by CD mutations known to abrogate HP1 and Polycomb silencing in Drosophila. These results define a novel function for the conserved CD and suggest that HP1 self-association and histone binding may play a crucial role in HP1-mediated heterochromatin assembly.     

Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4

We used a novel single-cell strategy to examine the fate of histones during G₂-phase. Consistent with previous results, we find that in G₂-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly.     

Methylation-independent binding to histone H3 and cell cycle-dependent incorporation of HP1beta into heterochromatin

We have examined HP1beta-chromatin interactions in different molecular contexts in vitro and in vivo. Employing purified components we show that HP1beta 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 HP1beta associates more tightly with destabilized or disrupted nucleosomes (H3/H4 subcomplexes) than with intact particles. Western blotting and mass spectrometry data indicate that HP1beta-selected H3/H4 particles and subparticles possess a complex pattern of posttranslational modifications but are not particularly enriched in me3K9-H3. Consistent with these results, mapping of HP1beta and me3K9-H3 sites in vivo reveals overlapping, yet spatially distinct patterns, while transient transfection assays with synchronized cells show that stable incorporation of HP1beta-gfp into heterochromatin requires passage through the S-phase. The data amassed challenge the dogma that me3K9H3 is necessary and sufficient for HP1 binding and unveil a new mode of HP1-chromatin interactions.     

Chromosomal protein HMGN1 modulates the phosphorylation of serine 1 in histone H2A

Here we demonstrate that HMGN1, a nuclear protein that binds specifically to nucleosomes, modulates the level of histone H2A phosphorylation. In Hmgn1-/- cells, loss of HMGN1 elevates the steady-state levels of H2AS1ph throughout the cell cycle. In vitro, HMGN1 reduces the rate of Rsk2- and Msk1-mediated phosphorylation of nucleosomal, but not free, histone H2A. HMGN1 inhibits H2A phosphorylation by binding to nucleosomes since an HMGN mutant, which cannot bind to chromatin, does not inhibit the Rsk2- mediated H2A phosphorylation. HMGN2 also inhibits H2A phosphorylation, suggesting that the inhibition of H2A phosphorylation is not specific to only one member of this protein family. Thus, the present data add modifications of histone H2A to the list of histone modifications affected by HMGN proteins. It supports the suggestion that structural chromatin binding proteins can modify the whole profile of post-translational modifications of core histones.