Substrate specificity analysis and novel substrates of the protein lysine methyltransferase NSD1.

The nuclear receptor binding SET [su(var) 3-9, enhancer of zeste, trithorax] domain-containing protein 1 (NSD1) protein lysine methyltransferase (PKMT) was known to methylate histone H3 lysine 36 (H3K36). We show here that NSD1 prefers aromatic, hydrophobic, and basic residues at the -2, -1 and +2, and +1 sites of its substrate peptide, respectively. We show methylation of 25 nonhistone peptide substrates by NSD1, two of which were (weakly) methylated at the protein level, suggesting that unstructured protein regions are preferred NSD1 substrates. Methylation of H4K20 and p65 was not observed. We discovered strong methylation of H1.5 K168, which represents the best NSD1 substrate protein identified so far, and methylation of H4K44 which was weaker than H3K36. Furthermore, we show that Sotos mutations in the SET domain of NSD1 inactivate the enzyme. Our results illustrate the importance of specificity analyses of PKMTs for understanding protein lysine methylation signaling pathways.

Expression of p16INK4A in gastrointestinal stromal tumours (GISTs): two different forms exist that independently correlate with poor prognosis.

AIMS: To determine the prognostic impact of p16INK4A expression in gastrointestinal stromal tumours (GISTs), which is currently being questioned, with both loss and overexpression said to be correlated with poor prognosis. METHODS AND RESULTS: Two different forms of p16INK4A were identified, presenting with predominantly nuclear and cytoplasmic expression pattern, respectively. The immunohistochemical expression of the two forms and their correlation with E2F1 and prognosis were analysed in a series of 120 GISTs with clinical follow-up. Low nuclear p16INK4A expression correlated with E2F1 up-regulation, higher mitotic counts, and tumour progression. The prognostic value of nuclear p16INK4A expression was only marginally significant (P=0.05). Strong expression of the cytoplasmic p16INK4A form was significantly associated with shorter disease-free survival (P=2x10(-5)). The prognostic impact of strong expression of the cytoplasmic p16INK4A form was independent of anatomical localization, tumour size and mitotic counts, and significant even among the cohort of tumours with high malignant potential. CONCLUSIONS: Low expression of the nuclear p16INK4A form and strong expression of the cytoplasmic p16INK4A form both represent two independent parameters each associated with tumour progression in GISTs. Low nuclear p16INK4A expression enables E2F1 up-regulation and consecutive accelerated cell proliferation. In contrast, strong cytoplasmic p16INK4A expression probably reflects a negative feedback loop as a result of (as yet unknown) oncogenic events.

Tieg1/Klf10 is upregulated by NGF and attenuates cell cycle progression in the pheochromocytoma cell line PC12.

The transcription factor Tieg1/Klf10 belongs to a family of Sp1/Klf proteins that have been shown to play important roles during development and maintenance of various tissues and cell types. Upregulation of Tieg1/Klf10 has been reported for TGF-beta, BMP2, BMP4, ActivinA and GDNF as members of the TGF-beta superfamily. Moreover, estrogen, the cytostatic drugs homoharringtonine and velcade as well as nitric oxide are also able to trigger Tieg1/Klf10 transcription. Recent studies suggest a role for members of the neurotrophin family in regulating Tieg1/Klf10 transcriptional upregulation. Using semi-quantitative RT-PCR and immunoblotting, we present data describing that nerve growth factor (NGF) regulates the expression of Tieg1/Klf10 in the pheochromocytoma cell line PC12 in a TrkA-dependent manner. Moreover, we provide evidence for the existence of NGF-responsive elements in the 5'-regulatory region of Tieg1/Klf10 that contain binding sites for the transcription factors Sp1 and CREB. After treatment with NGF PC12 cells exit the cell cycle and start to differentiate towards a neuron-like phenotype indicated by neurite outgrowth. Using flow cytometry and differentiation assays we demonstrate that Tieg1/Klf10 reduces cell cycle progression in PC12 cells but fails to promote their terminal differentiation. Together, our results identify Tieg1/Klf10 as a new NGF target gene and substantiate its anti-proliferative function in the NGF signaling pathway in PC12 cells.

Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI/SNF or NURF.

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.

H1 subtype expression during cell proliferation and growth arrest.

H1 histone subtype genes differ in their expression patterns during the different stages of the cell cycle interphase. While the group of replication-dependent H1 histone subtypes is synthesized during S phase, the replacement histone subtype H1.0 is also expressed replication-independently in non-proliferating cells. The present study is the first report about the analysis of the cell cycle-dependent expression of all five replication-dependent H1 subtypes, the replacement histone H1.0 and the ubiquitously expressed subtype H1x. The expression of these H1 histone subtypes in HeLa cells was analyzed on mRNA level by quantitative real-time RT-PCR as well as on protein level by immunoblotting. We found that after arrest of HeLa cells in G(1) phase by treatment with sodium butyrate, the mRNA levels of all replication-dependently expressed H1 subtypes decreased, but to very different extent. During S phase the individual replication-dependently expressed H1 subtypes show similar kinetics regarding their mRNA levels. However, the variations in their protein amounts partially differ from the respective RNA levels which especially applies to histone H1.3. In contrast, the mRNA as well as the protein level of H1x remained nearly unchanged in G(1) as well as during S phase progression. The results of the present study demonstrate that the cell cycle-dependent mRNA and protein expression of various H1 subtypes is differentially regulated, supporting the hypothesis of a functional heterogeneity.

M phase-specific phosphorylation of histone H1.5 at threonine 10 by GSK-3.

H1 histones are progressively phosphorylated during the cell cycle. The number of phosphorylated sites is zero to three in late S phase and increases to five or six in late G2 phase and M phase. It is assumed that this phosphorylation modulates chromatin condensation and decondensation, but its specific role remains unclear. Recently, it was shown that the somatic H1 histone subtype H1.5 becomes pentaphosphorylated during mitosis, with phosphorylated threonine 10 being the last site to be phosphorylated. We have generated an antiserum specific for human H1.5 phosphorylated at threonine 10. Immunofluorescence labeling of HeLa cells with this antiserum revealed that the phosphorylation at this site appears in prometaphase and disappears in telophase, and that this hyperphosphorylated form of H1.5 is mainly chromatin-bound in metaphase when chromatin condensation is maximal. In search of the kinase responsible for the phosphorylation at this site, we found that threonine 10 of H1.5 can be phosphorylated by glycogen synthase kinase-3 in vitro, but not by cyclin-dependent kinase 1/cyclin B and cyclin-dependent kinase 5/p35, respectively. Furthermore, addition of specific glycogen synthase kinase-3 inhibitors led to a reduction in phosphorylation at this site both in vivo and in vitro.

Histone H1 and its isoforms: contribution to chromatin structure and function.

The lysine-rich H1 histone family in mammals includes eleven different subtypes, and thus it is the most divergent class of histone proteins. The central globular H1 domain asymmetrically interacts with DNA at the exit or entry end of the nucleosomal core DNA, and the C-terminal domain has a major impact on the linker DNA conformation and chromatin condensation. H1 histones are thus involved in the formation of higher order chromatin structures, and they modulate the accessibility of regulatory proteins, chromatin remodeling factors and histone modification enzymes to their target sites. The major posttranslational modification of H1 histones is phosphorylation, which reaches a peak during G2 and mitosis. Phosphorylation is, however, also involved in the control of DNA replication and it contributes to the regulation of gene expression. Disruption of linker histone genes, initially performed in order to delineate subtype-specific functions, revealed that disruption of one or two H1 subtype genes is quantitatively compensated by an increased expression of other subtypes. This suggests a functional redundancy among H1 subtypes. However, the inactivation of three subtypes and the reduction of the H1 moiety in half finally resulted in a phenotypic effect. On the other hand, studies on the role of particular subtypes at specific developmental stages in lower eukaryotes, but also in vertebrates suggest that specific subtypes of H1 participate in particular systems of gene regulation.

Histone H1x is highly expressed in human neuroendocrine cells and tumours.

BACKGROUND: Histone H1x is a ubiquitously expressed member of the H1 histone family. H1 histones, also called linker histones, stabilize compact, higher order structures of chromatin. In addition to their role as structural proteins, they actively regulate gene expression and participate in chromatin-based processes like DNA replication and repair. The epigenetic contribution of H1 histones to these mechanisms makes it conceivable that they also take part in malignant transformation. METHODS: Based on results of a Blast data base search which revealed an accumulation of expressed sequence tags (ESTs) of H1x in libraries from neuroendocrine tumours (NETs), we evaluated the expression of H1x in NETs from lung and the gastrointestinal tract using immunohistochemisty. Relative protein and mRNA levels of H1x were analysed by Western blot analysis and quantitative real-time RT-PCR, respectively. Since several reports describe a change of the expression level of the replacement subtype H1.0 during tumourigenesis, the analysis of this subtype was included in this study. RESULTS: We found an increased expression of H1x but not of H1.0 in NET tissues in comparison to corresponding normal tissues. Even though the analysed NETs were heterogenous regarding their grade of malignancy, all except one showed a considerably higher protein amount of H1x compared with corresponding non-neoplastic tissue. Furthermore, double-labelling of H1x and chromogranin A in sections of pancreas and small intestine revealed that H1x is highly expressed in neuroendocrine cells of these tissues. CONCLUSION: We conclude that the high expression of histone H1x in NETs is probably due to the abundance of this protein in the cells from which these tumours originate.

Diversity in mammalian chiasmatic architecture: ipsilateral axons are deflected at glial arches in the prechiasmatic optic nerve of the eutherian Tupaia belangeri.

Permanent ipsilaterally projecting axons approach the chiasmatic midline in rodents but are confined to lateral parts of the optic chiasm in marsupials. Hence, principally different mechanisms were thought to underlie axon pathway choice in eutherian (placental) and marsupial mammals. First evidence of diversity in eutherian chiasmatic architecture came from studies in the newborn and adult tree shrew Tupaia belangeri (Jeffery et al. [1998] J. Comp. Neurol. 390:183-193). Here, as in marsupials, ipsilaterally projecting axons do not approach the midline. The present study aims to clarify how the developing tree shrew chiasm is organized, how glial cells are arranged therein, and the extent to which the tree shrew chiasm is similar to that of marsupials or other eutherians. By using routinely stained serial sections as well as immunohistochemistry with antibodies against glial fibrillary acidic protein, vimentin, and medium-molecular-weight neurofilament protein, we investigated chiasm formation from embryonic day 18 (E18) to birth (E43). From E22 onward, ipsilaterally projecting axons diverged from contralaterally projecting axons in prechiasmatic parts of the optic nerve. They made sharp turns when arriving at glial arches found at the transition from the optic nerve to the chiasm. Thus, during the ingrowth period of axons, Tupaia belangeri and marsupials have specialized glial arrays in common, which probably help to deflect ipsilaterally projecting axons to lateral parts of the chiasm. Our observations provide new evidence of diversity in eutherian chiasmatic architecture and identify Tupaia belangeri as an appropriate animal model for studies on the mechanisms underlying axon guidance in the developing chiasm of higher primates.

H1 histone subtype constitution and phosphorylation state of the ageing cell system of human peripheral blood lymphocytes.

Until a few years ago, the H1 histones were exclusively considered to be the architectural proteins of chromatin involved in chromatin condensation. However there is now increasing data to support the hypothesis that the H1 subtypes are involved in genomic integrity and that they may have unexpected functional roles in various biological processes such as in differentiation and DNA repair, apoptosis and lifespan. Moreover, the H1 histones are phosphorylated to a great extent. Recent work has implicated phosphorylation of H1 in the regulation of chromatin remodeling. In light of the fact that chromatin reorganization and heterochromatin formation has been shown to take place during ageing and senescence, in the present investigation, we have analyzed the changes that take place in the somatic H1 linker histone subtype profile and their phosphorylation states in human peripheral blood lymphocytes as a function of donor age. Results from this work show that there is a significant age-related dephosphorylation of H1.4 and H1.5 and an increase in the heterochromatin protein HP1alpha as a function of donor age. These results indicate that dephosphorylation of H1 histones may be related to an increase in senescence-associated heterochromatin formation during the in vivo ageing of human peripheral blood lymphocytes.

G1 phase-dependent nucleolar accumulation of human histone H1x.

BACKGROUND INFORMATION: H1 histones are a protein family comprising several subtypes. Although specific functions of the individual subtypes could not be determined so far, differential roles are indicated by varied nuclear distributions as well as differential expression patterns of the H1 subtypes. Although the group of replication-dependent H1 subtypes is synthesized during S phase, the replacement H1 subtype, H1 degrees , is also expressed in a replication-independent manner in non-proliferating cells. Recently we showed, by protein biochemical analysis, that the ubiquitously expressed subtype H1x is enriched in the micrococcal nuclease-resistant part of chromatin and that, although it shares common features with H1 degrees , its expression is differentially regulated, since, in contrast to H1 degrees , growth arrest or induction of differentiation did not induce an accumulation of H1x. RESULTS: In the present study, we show that H1x exhibits a cell-cycle-dependent change of its nuclear distribution. This H1 subtype showed a nucleolar accumulation during the G(1) phase, and it was evenly distributed in the nucleus during S phase and G(2). Immunocytochemical analysis of the intranucleolar distribution of H1x indicated that it is located mainly in the condensed nucleolar chromatin. In addition, we demonstrate that the amount of H1x protein remained nearly unchanged during S phase progression, which is in contrast to the replication-dependent subtypes. CONCLUSION: These results suggest that the differential localization of H1x provides a mechanism for a control of H1x activity by means of shuttling between nuclear subcompartments instead of a controlled turnover of the protein.

Site-dependent differential KIT and PDGFRA expression in gastric and intestinal gastrointestinal stromal tumors.

In gastrointestinal stromal tumors (GISTs), mutually exclusive gain-of-function mutations of KIT and PDGFRA are associated with different mutation-dependent clinical behavior. Taking into account the well-known different clinical behavior of GISTs from the stomach or the intestine, the aim of the current study is to evaluate the mutation- and site-dependent effects on mRNA and protein expression of KIT and PDGFRA in a large series of primary GISTs. Fresh-frozen tissue of 53 primary GISTs from gastric (75%) or intestinal (25%) sites were analyzed for mutation of KIT or PDGFRA using direct sequencing. Furthermore, KIT and PDGFRA mRNA and protein expression were determined using quantitative RT-PCR and quantitative densitometric evaluation of Western blot data. Each tumor either had a mutation of KIT (79%) or PDGFRA (21%). All GISTs with PDGFRA mutation were from gastric sites. Mutation-dependently, GISTs with KIT mutation had a significantly higher expression of KIT and at the same time a significantly lower expression of PDGFRA compared to GISTs with PDGFRA mutation. Site-dependently, gastric GISTs had a significantly higher expression of PDGFRA and a significantly lower expression of KIT compared to intestinal GISTs. Additionally, even if the KIT-mutated GISTs alone were considered, a significantly higher expression of PDGFRA could be observed in gastric than in intestinal tumors. We also found a significant correlation between a higher protein expression of PDGFRA and longer disease-free survival. The correlation of gastric site and PDGFRA mutation with higher PDGFRA expression and longer disease-free survival suggests different regulatory roles of KIT and PDGFRA gene expression on the control of cell proliferation, and, thereby on clinical behavior. The higher PDGFRA expression in gastric GISTs possibly contributes to the well-known site-dependent clinical behavior.

Regulation of geminin functions by cell cycle-dependent nuclear-cytoplasmic shuttling.

The geminin protein functions both as a DNA rereplication inhibitor through association with Cdt1 and as a repressor of Hox gene transcription through the polycomb pathway. Here, we report that the functions of avian geminin are coordinated with and regulated by cell cycle-dependent nuclear-cytoplasmic shuttling. In S phase, geminin enters nuclei and inhibits both loading of the minichromosome maintenance (MCM) complex onto chromatin and Hox gene transcription. At the end of mitosis, geminin is exported from nuclei by the exportin protein Crm1 and is unavailable in the nucleus during the next G(1) phase, thus ensuring proper chromatin loading of the MCM complex and Hox gene transcription. This mechanism for regulating the functions of geminin adds to distinct mechanisms, such as protein degradation and ubiquitination, applied in other vertebrates.

Development of starburst cholinergic amacrine cells in the retina of Tupaia belangeri.

"Starburst" cholinergic amacrines specify the response of direction-selective ganglion cells to image motion. Here, development of cholinergic amacrines was studied in the tree shrew Tupaia belangeri (Scandentia) by immunohistochemistry with antibodies against choline acetyltransferase (ChAT) and neurofilament proteins. Starburst amacrines expressed ChAT much earlier than previously thought. From embryonic day 34 (E34) onward, orthotopic and displaced subpopulations segregated from a single cluster of immunoreactive precursor cells. Orthotopic starburst amacrines rapidly took up positions in the inner nuclear layer. Displaced starburst amacrines were first arranged in a monocellular row in the inner plexiform layer, and, with a delay of 1 week, they descended to the ganglion cell layer. Conversely, dendritic stratification of displaced amacrines slightly preceded that of orthotopic ones. Starburst amacrines expressed the medium-molecular-weight neurofilament protein (NF-M) from E34 to postnatal day 11 (P11) and coexpressed alpha-internexin from E36.5 to P11. Consequently, neurofilaments composed of alpha-internexin and NF-M may stabilize developing dendrites of starburst amacrines. During the first 2 postnatal weeks, subpopulations of anti-NF-M-labeled ganglion cells costratified with the preexisting dendritic strata of starburst amacrines in the ON sublamina, OFF sublamina, or both. Hence, anti-NF-M-labeled ganglion cells may include direction-selective ones. Thereafter, NF-M and alpha-internexin proteins disappeared from starburst amacrines, and NF-M immunoreactivity was lost in the dendrites of ganglion cells. Our findings suggest that NF-M and alpha-internexin are important for starburst amacrines and ganglion cells to recognize each other and, thus, contribute to the formation of early developing retinal circuits in the inner plexiform layer.

Surgical management after neoadjuvant imatinib therapy in gastrointestinal stromal tumours (GISTs) with respect to imatinib resistance caused by secondary KIT mutations.

BACKGROUND: In metastasized GISTs, resistance to imatinib after initial tumour response has been associated with observation of secondary mutations in the activation loop of KIT. The aim of the current study was to evaluate the tumour response and observance of secondary KIT mutations in a case of GIST undergoing neoadjuvant imatinib therapy. METHODS: We report on a case of an initially unresectable gastric GIST with curative resection after 10 months of neoadjuvant imatinib therapy. Mutation analysis of KIT was performed on a pretherapeutic biopsy specimen, as well as on the resected tumour specimen. RESULTS: The pretherapeutic biopsy revealed cKit positive tumour cells with mutation of KIT exon 11 Del 560-576. The remaining tumour mass after neoadjuvant imatinib therapy almost exclusively consisted of hypocellular myxohyalinale stroma with rare microfoci of cKit positive tumour cells. Laser microdissection of several tumour microfoci revealed two additional point mutations located in the activation loop of KIT exon 17, C809G and N822Y, each observed separately in a distinct microfocus. Neither of these two point mutations has been reported in a GIST so far. CONCLUSIONS: Neoadjuvant imatinib therapy successfully reduces tumour size in GISTs. Since resistance relevant secondary mutations of the activation loop of KIT may be observed after neoadjuvant imatinib therapy, the time elapse with preoperative imatinib therapy should be chosen as short as curative tumour resection or function sparing surgery can be carried out. The determination of the optimal time point for surgery is therefore a critical event and will be discussed.

HP1 binds specifically to Lys26-methylated histone H1.4, whereas simultaneous Ser27 phosphorylation blocks HP1 binding.

Histone lysine methylation can have positive or negative effects on transcription, depending on the precise methylation site. According to the "histone code" hypothesis these methylation marks can be read by proteins that bind them specifically and then regulate downstream events. Hetero-chromatin protein 1 (HP1), an essential component of heterochromatin, binds specifically to methylated Lys(9) of histone H3 (K9/H3). The linker histone H1.4 is methylated on Lys(26) (K26/H1.4), but the role of this methylation in downstream events remains unknown. Here we identify HP1 as a protein specifically recognizing and binding to methylated K26/H1.4. We demonstrate that the Chromo domain of HP1 is mediating this binding and that phosphorylation of Ser(27) on H1.4 (S27/H1.4) prevents HP1 from binding. We suggest that methylation of K26/H1.4 could have a role in tethering HP1 to chromatin and that this could also explain how HP1 is targeted to those regions of chromatin where it does not colocalize with methylated K9/H3. Our results provide the first experimental evidence for a "phospho switch" model in which neighboring phosphorylation reverts the effect of histone lysine methylation.

Characterisation of human histone H1x.

The members of the H1 histone family can be classified into three groups, which are the main class subtypes expressed in somatic cells, the developmental- and tissue-specific subtypes, and the replacement subtype H1(o). Until now, the subtype H1x was not classified, since it has not yet been thoroughly examined. The results of this study show that H1x shares similarities but also exhibits slight differences in its biochemical behaviour in comparison to the main class H1 histones. In HeLa cells it is located in the nucleus and partially associated with nucleosomes. Nevertheless, it is, like H1(o), mainly located in chromatin regions that are not affected by micrococcal nuclease digestion. Further common features of H1x and the replacement histone H1(o) are that the genes of both subtypes are solitarily located and give rise to polyadenylated mRNA. However, comparison of the inducibility of their expression revealed that their genes are regulated differentially.

Topoisomerase inhibitor induced dephosphorylation of H1 and H3 histones as a consequence of cell cycle arrest.

Posttranslational modifications of histones have an integral function in the structural and functional organization of chromatin. Several changes in the modification state of histones could be observed after induction of apoptosis with topoisomerase inhibitors and other inducers. Most of these studies include the analysis of the state of phosphorylation of histones, and the results are to some extent controversial, depending on cell lines and agents used. In the present study we compared the kinetics of the dephosphorylation of H1 and H3 histones with apoptosis markers after treatment of leukemic cell lines with topoisomerase inhibitors. In parallel, we determined cell cycle parameters in detail. Dephosphorylation of both histone classes started within 1 h of induction, and no direct correlation with timing and intensity of the investigated apoptotic features could be observed. In contrast, we show that the effect of topoisomerase inhibitors on the state of H1 and H3 phosphorylation is not directly related to apoptosis, but reflects the changes in the cell cycle distribution of cells treated with these inducers.

Arabidopsis mu A-adaptin interacts with the tyrosine motif of the vacuolar sorting receptor VSR-PS1.

In receptor-mediated transport pathways in mammalian cells, clathrin-coated vesicle (CCV) mu-adaptins are the main binding partners for the tyrosine sorting/internalization motif (YXXØ). We have analyzed the function of the mu A-adaptin, one of the five mu-adaptins from Arabidopsis thaliana, by pull-down assays and plasmon resonance measurements using its receptor-binding domain (RBD) fused to a histidine tag. We show that this adaptin is able to bind the consensus tyrosine motif YXXØ from the pea vacuolar sorting receptor (VSR)-PS1, as well as from the mammalian trans-Golgi network (TGN)38 protein. Moreover, the tyrosine residue was revealed to be crucial for binding of the complete cytoplasmic tail of VSR-PS1 to the plant mu A-adaptin. The trans-Golgi localization of the mu A-adaptin strongly suggests its involvement in Golgi- to vacuole-trafficking events.