No abstract

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.

BackgroundCTCs expressing variable levels of epithelial and mesenchymal markers in breast cancer have previously been reported. However, no information exists for keratin expression levels of CTCs in association with disease status, whereas assays for the characterization of transitional EMT phenotypes of CTCs in breast cancer are rather lacking. We investigated the correlation between keratin expression of CTCs and patients’ outcome and characterized the EMT status of CTCs via the establishment of a numerical “ratio” value of keratin and vimentin expression levels on a single cell basis.MethodsKeratin expression was evaluated in 1262 CTCs from 61 CTC-positive patients with metastatic breast cancer, using analysis of images obtained through the CellSearch System. For the determination of vimentin/keratin (vim/K) ratios, expression levels of keratin and vimentin were measured in cytospin preparations of luminal (MCF-7 and T47D) and basal (MDA.MB231 and Hs578T) breast cancer cell lines and 110 CTCs from 5 CTC-positive patients using triple immunofluorescence laser scanning microscopy and image analysis.ResultsMCF-7 and T47D displayed lower vim/K ratios compared to MDA.MB231 and Hs578T cells, while MCF-7 cells that had experimentally undergone EMT were characterized by varying intermediate vim/K ratios. CTCs were consisted of an heterogeneous population presenting variable vim/K values with 46% of them being in the range of luminal breast cancer cell lines. Keratin expression levels of CTCs detected by the CellSearch System correlated with triple negative (p = 0.039) and ER-negative (p = 0.025) breast cancer, and overall survival (p = 0.038).ConclusionsKeratin expression levels of CTCs correlate with tumor characteristics and clinical outcome. Moreover, CTCs display significant heterogeneity in terms of the degree of EMT phenotype that probably reflects differential invasive potential. The assessment of the vim/K ratios as a surrogate marker for the EMT status of CTCs merits further investigation as a prognostic tool in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1386-7) contains supplementary material, which is available to authorized users.