Structural role of RKS motifs in chromatin interactions: a molecular dynamics study of HP1 bound to a variably modified histone tail.

The current understanding of epigenetic signaling assigns a central role to post-translational modifications that occur in the histone tails. In this context, it has been proposed that methylation of K9 and phosphorylation of S10 in the tail of histone H3 represent a binary switch that controls its reversible association to heterochromatin protein 1 (HP1). To test this hypothesis, we performed a comprehensive molecular dynamics study in which we analyzed a crystallographically defined complex that involves the HP1 chromodomain and an H3 tail peptide.

Solution structure and molecular interactions of lamin B receptor Tudor domain.

Lamin B receptor (LBR) is a polytopic protein of the nuclear envelope thought to connect the inner nuclear membrane with the underlying nuclear lamina and peripheral heterochromatin. To better understand the function of this protein, we have examined in detail its nucleoplasmic region, which is predicted to harbor a Tudor domain (LBR-TD). Structural analysis by multidimensional NMR spectroscopy establishes that LBR-TD indeed adopts a classical β-barrel Tudor fold in solution, which, however, features an incomplete aromatic cage.

Phosphorylation of histone H3 at Thr3 is part of a combinatorial pattern that marks and configures mitotic chromatin.

We have previously shown that histone H3 is transiently phosphorylated at Thr3 during mitosis. Extending these studies, we now report that phosphorylated Thr3 is always in cis to trimethylated Lys4 and dimethylated Arg8, forming a new type of combinatorial modification, which we have termed PMM. PMM-marked chromatin emerges at multiple, peripheral sites of the prophase nucleus, then forms distinct clusters at the centric regions of metaphase chromosomes, and finally spreads (as it wanes) to the distal areas of segregating chromatids.

Nucleocytoplasmic shuttling of soluble tubulin in mammalian cells.

We have investigated the subcellular distribution and dynamics of soluble tubulin in unperturbed and transfected HeLa cells. Under normal culture conditions, endogenous alpha/beta tubulin is confined to the cytoplasm. However, when the soluble pool of subunits is elevated by combined cold-nocodazole treatment and when constitutive nuclear export is inhibited by leptomycin B, tubulin accumulates in the cell nucleus.

Chromatin remodeling during mitosis: a structure-based code?

Histone modifications have been associated with particular states of transcriptional activity and are thought to serve as an "information code". However, this principle does not apply to histone phosphorylation, which can be detected in two, seemingly contrasting situations, i.e.

Plasticity of HP1 proteins in mammalian cells.

We have compared the distribution of endogenous heterochromatin protein 1 (HP1) proteins (alpha, beta and gamma) in different epithelial lines, pluripotent stem cells and embryonic fibroblasts. In parallel, we have interrogated assembly and dynamics of newly expressed HP1-GFP proteins in cells lacking both HP1alpha and HP1beta alleles, blocked at the G1-S boundary, or cultured in the presence of HDAC and HAT inhibitors. The results reveal a range of cell type and differentiation state-specific patterns that do not correlate with 'fast' or 'slow' subunit exchange in heterochromatin.

Variant-specific patterns and humoral regulation of HP1 proteins in human cells and tissues.

We have examined the occurrence and distribution of HP1alpha and HP1beta under in vivo, ex vivo and in vitro conditions. Consistent with a non-essential role in heterochromatin maintenance, both proteins are diminished or undetectable in several types of differentiated cells and are universally downregulated during erythropoiesis. Variant-specific patterns are observed in almost all human and mouse tissues examined.

Short-term exposure of cancer cells to micromolar doses of paclitaxel, with or without hyperthermia, induces long-term inhibition of cell proliferation and cell death in vitro.

Background: During intraoperative hyperthermic intraperitoneal chemotherapy for primary or secondary peritoneal malignancies, tumor cells are exposed to high drug concentrations for a relatively short period of time. We investigated in vitro the effect of paclitaxel and hyperthermia on cell proliferation, cell cycle kinetics and cell death under conditions resembling those during intraoperative hyperthermic intraperitoneal chemotherapy.

Methods: Human breast MCF-7, ovarian SKOV-3 and hepatocarcinoma HEpG2 cells were exposed to 10 and 20 microM paclitaxel at 37, 41.

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.

Micromolar taxol, with or without hyperthermia, induces mitotic catastrophe and cell necrosis in HeLa cells.

Purpose: Although the mode of action of taxol, when used in nanomolar or micromolar concentrations during long periods, is extensively studied, there are few data available on taxol-mediated cytotoxicity when the drug is applied for a short time alone or in combination with hyperthermia. We studied the effect of taxol and hyperthermia on cell cycle kinetics, proliferation, and mode of cell death in human cervical carcinoma HeLa cells, following a scheme which resembles the one currently used in regional chemotherapy.

Methods: Cells were incubated with micromolar doses of taxol for two h under normothermic or hyperthermic conditions and then cultured in drug-free medium for several days.

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.

Mitotic phosphorylation of histone H3 at threonine 3.

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.

HP1: facts, open questions, and speculation.

The demonstration, over a decade ago, that HP1 is a highly conserved constituent of heterochromatin was accompanied by the explicit view that this protein plays a pivotal role in epigenetic regulation (P.B. Singh, J.