Distinct roles of haspin in stem cell division and male gametogenesis.

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

Progressive Phosphorylation Modulates the Self-Association of a Variably Modified Histone H3 Peptide.

Protein phosphorylation is a key regulatory mechanism in eukaryotic cells. In the intrinsically disordered histone tails, phosphorylation is often a part of combinatorial post-translational modifications and an integral part of the "histone code" that regulates gene expression. Here, we study the association between two histone H3 tail peptides modified to different degrees, using fully atomistic molecular dynamics simulations. Assuming that the initial conformations are either α-helical or fully extended, we compare the propensity of the two peptides to associate with one another when both are unmodified, one modified and the other unmodified, or both modified. The simulations lead to the identification of distinct inter- and intramolecular interactions in the peptide dimer, highlighting a prominent role of a fine-tuned phosphorylation rheostat in peptide association. Progressive phosphorylation appears to modulate peptide charge, inducing strong and specific intermolecular interactions between the monomers, which do not result in the formation of amorphous or ordered aggregates, as documented by experimental evidence derived from Circular Dichroism and NMR spectroscopy. However, upon complete saturation of positive charges by phosphate groups, this effect is reversed: intramolecular interactions prevail and dimerization of zero-charge peptides is markedly reduced. These findings underscore the role of phosphorylation thresholds in the dynamics of intrinsically disordered proteins. Phosphorylation rheostats might account for the divergent effects of histone modifications on the modulation of chromatin structure.

Haspin-dependent and independent effects of the kinase inhibitor 5-Iodotubercidin on self-renewal and differentiation.

The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting "off-target" effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the "off-target" effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.

Heterochromatin remodeling in embryonic stem cells proceeds through stochastic de-stabilization of regional steady-states.

Cell differentiation is associated with progressive immobilization of chromatin proteins, expansion of heterochromatin, decrease of global transcriptional activity and induction of lineage-specific genes. However, how these processes relate to one another remains unknown. We show here that the heterochromatic domains of mouse embryonic stem cells (ESCs) are dynamically distinct and possesses a mosaic sub-structure. Although random spatio-temporal fluctuations reshuffle continuously the chromatin landscape, each heterochromatic territory maintains its dynamic profile, exhibiting robustness and resembling a quasi-steady state. Transitions towards less dynamic states are detected sporadically as ESCs downregulate Nanog and exit the self-renewal phase. These transitions increase in frequency after lineage-commitment, but evolve differently depending on cellular context and transcriptional status. We propose that chromatin remodeling is a step-wise process, which involves stochastic de-stabilization of regional steady states and formation of new dynamic ensembles in coordination to changes in the gene expression program.

Dynamics and Structure-Function Relationships of the Lamin B Receptor (LBR).

The lamin B receptor (LBR) is a multi-spanning membrane protein of the inner nuclear membrane that is often employed as a "reporter" of nuclear envelope dynamics. We show here that the diffusional mobility of full-length LBR exhibits significant regional variation along the nuclear envelope, consistent with the existence of discrete LBR microdomains and the occurrence of multiple, asymmetrically-spaced anastomoses along the nuclear envelope-endoplasmic reticulum interface. Interestingly, a commonly used fusion protein that contains the amino-terminal region and the first transmembrane domain of LBR exhibits reduced mobility at the nuclear envelope, but behaves similarly to full-length LBR in the endoplasmic reticulum. On the other hand, carboxy-terminally truncated mutants that retain the first four transmembrane domains and a part or the whole of the amino-terminal region of LBR are generally hyper-mobile. These results suggest that LBR dynamics is structure and compartment specific. They also indicate that native LBR is probably "configured" by long-range interactions that involve the loops between adjacent transmembrane domains and parts of the amino-terminal region.

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. Microsecond-long simulations show that the binding of the trimethylated K9 H3 peptide in the aromatic cage of HP1 is only slightly affected by S10 phosphorylation, because the modified K9 and S10 do not interact directly with one another. Instead, the phosphate group of S10 seems to form a persistent intramolecular salt bridge with R8, an interaction that can provoke a major structural change and alter the hydrogen-bonding regime in the H3-HP1 complex. These observations suggest that interactions between adjacent methyl-lysine and phosphoserine side chains do not by themselves provide a binary switch in the H3-HP1 system, but arginine-phosphoserine interactions, which occur in both histones and nonhistone proteins in the context of a conserved RKS motif, are likely to serve a key regulatory function.

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 ß-barrel Tudor fold in solution, which, however, features an incomplete aromatic cage. Removal of LBR-TD renders LBR more mobile at the plane of the nuclear envelope, but the isolated module does not bind to nuclear lamins, heterochromatin proteins (MeCP2), and nucleosomes, nor does it associate with methylated Arg/Lys residues through its aromatic cage. Instead, LBR-TD exhibits tight and stoichiometric binding to the "histone-fold" region of unassembled, free histone H3, suggesting an interesting role in histone assembly. Consistent with such a role, robust binding to native nucleosomes is observed when LBR-TD is extended toward its carboxyl terminus, to include an area rich in Ser-Arg residues. The Ser-Arg region, alone or in combination with LBR-TD, binds both unassembled and assembled H3/H4 histones, suggesting that the TD/RS interface may operate as a "histone chaperone-like platform."

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. The characteristic prophase pattern can be reproduced by expressing ectopically the kinase haspin at interphase, suggesting that the formation of the PMM signature does not require a pre-existing mitotic environment. On the other hand, the ;dissolution' and displacement of PMM clusters from a centric to distal position can be induced by partial dephosphorylation or chromosome unravelling, indicating that these changes reflect the regulated grouping and scrambling of PMM subdomains during cell division. Formation of PMM is prevented by haspin knockdown and leads to delayed exit from mitosis. However, PMM-negative cells do not exhibit major chromosomal defects, suggesting that the local structures formed by PMM chromatin may serve as a ;licensing system' that allows quick clearance through the metaphase-anaphase checkpoint.

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. Transfection assays and FRAP experiments reveal that GFP-tagged beta-tubulin shuttles between the cytoplasm and the cell nucleus. Nuclear import seems to occur by passive diffusion, whereas exit from the nucleus appears to rely on nuclear export signals (NESs). Several such motifs can be identified by sequence criteria along the beta-tubulin molecule and mutations in one of these (NES-1) cause a significant accumulation in the nuclear compartment. Under these conditions, the cells are arrested in the G0-G1 phase and eventually die, suggesting that soluble tubulin interferes with important nuclear functions. Consistent with this interpretation, soluble tubulin exhibits stoichiometric binding to recombinant, normally modified and hyper-phosphorylated/acetylated histone H3. Tubulin-bound H3 no longer interacts with heterochromatin protein 1 and lamin B receptor, which are known to form a ternary complex under in vitro conditions. Based on these observations, we suggest that nuclear accumulation of soluble tubulin is part of an intrinsic defense mechanism, which tends to limit cell proliferation under pathological conditions. This readily explains why nuclear tubulin has been detected so far only in cancer or in transformed cells, and why accumulation of this protein in the nucleus increases after treatment with chemotherapeutic agents.

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., in a transcriptionally hyperactive state following growth factor stimulation and in transcriptionally paused mitotic chromosomes. There are several indications that mitotic phosphorylation of histone H3 at serine-10 by the Aurora B kinase and trimethylation at lysine-9 by the methyl transferase Suvar3,9 operate as a "binary switch", which determines recruitment or eviction of heterochromatin-specific proteins from pericentromeric repeats. Moreover, threonine-3 phosphorylation of histone H3 by the newly identified haspin kinase seems to promote chromatid cohesion during mitosis. We discuss here emerging information and new ideas suggesting that these modifications, in combination to upstream and downstream marks, constitute a system of intrinsic folding determinants that facilitate chromatin condensation and confer topological specificity to mitotic chromosomes.

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. Furthermore, our observations show that targeting of HP1gamma to heterochromatic sites depends on HP1alpha and H1beta and that, on an architectural level, HP1alpha is the most polymorphic variant of the HP1 family. These data provide evidence for HP1 plasticity under shifting microenvironmental conditions and offer a new conceptual framework for understanding chromatin dynamics at the molecular level.

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. Yet, the most instructive example of HP1 plasticity is observed in the lymph nodes, where HP1alpha and HP1beta exhibit regional patterns that are exactly complementary to one another. Furthermore, whereas HP1alpha shows a dispersed sub-nuclear distribution in the majority of peripheral lymphocytes, it coalesces into large heterochromatic foci upon stimulation with various mitogens and IL-2. The effect of inductive signals on HP1alpha distribution is reproduced by coculture of immortalized T- and B-cells and can be confirmed using specific markers. These complex patterns reveal an unexpected plasticity in HP1 variant expression and strongly suggest that the sub-nuclear distribution of HP1 proteins is regulated by humoral signals and microenvironmental cues.

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, 41.5 or 43 degrees C for 2 h. Cell proliferation, cell cycle kinetics, necrosis and apoptosis were evaluated. RESULTS: Hyperthermia exerted a cytostatic effect to all cell lines and at 43 degrees C a cytotoxic effect on MCF-7 cells. MCF-7 and SKOV-3 cells treated under normothermic conditions with paclitaxel were arrested at G2/M or M phase for at least 3 days. Most of MCF-7 cells and approximately half of SKOV-3 cells were in interphase and became multinucleated without properly completing cytokinesis. Hyperthermia at 41.5 degrees C altered cell cycle distribution and affected the paclitaxel-related effect on cell cycle kinetics of MCF-7 and SKOV-3 cells. Analysis of the mode of cell death showed that cell necrosis prevailed over apoptosis. Hyperthermia at 43 degrees C increased paclitaxel-mediated cytotoxicity in MCF-7 cells and to a lesser extent in SKOV-3 and HEpG2 cells. CONCLUSIONS: Short-time treatment of carcinoma cells with high (micromolar) concentrations of paclitaxel in normothermic and hyperthermic conditions is highly efficient for cell growth arrest and could be of clinical relevance in locoregional chemotherapy.

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.

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. Cell viability was assessed via an MTT assay. Necrotic and apoptotic cell death was determined using Trypan blue staining and TUNNEL assay, respectively. Flow cytometry was used for the analysis of cell cycle kinetics and the counting of apoptotic cells. Mitotic index, nuclear morphology and nuclear envelope organization were analyzed by fluorescence microscopy. RESULTS: Cells exposed to micromolar doses of taxol for 2 h and then transferred to a drug-free medium for 24 h were arrested at G2/M or M phase. When treated cells were cultured in normal media for longer periods, most of them remained in a tetraploid state, became multinucleated without properly completing cytokinesis and died mostly by necrosis. Hyperthermia alone exerted a cytotoxic effect, inhibited proliferation and caused minor changes in cell cycle kinetics. When combined with taxol treatment, hyperthermia modified the cell cycle-arresting effects of the drug, but did not alter significantly taxol-mediated cytotoxicity. CONCLUSIONS: From these data we conclude that short time incubation of HeLa cells under normothermic or hyperthermic conditions with micromolar concentrations of taxol is sufficient enough to induce extended cell growth arrest and cell death by necrosis.

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.

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. 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.

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.R. Miller, J. Pearce, R. Kothary, R.D. Burton, R. Paro, T.C. James, and S.J. Gaunt, 1991, Nucleic Acids Res. 19, 789-794). Recent studies have confirmed this view, unveiling specific interactions of HP1 with a variety of histone and nonhistone proteins. We discuss here some of these observations, concentrating on structure-function relationships and intracellular dynamics. Integrating the available information, we also present a hypothetical model describing how HP1, acting as a bifunctional cross-linker, could organize peripheral heterochromatin and contribute in the compartmentalization of the cell nucleus.