Phosphorylation of histone H3 at serine 10 occurs during mitosis and meiosis in a wide range of eukaryotes and has been shown to be required for proper chromosome transmission in Tetrahymena. Here we report that Ipl1/aurora kinase and its genetically interacting phosphatase, Glc7/PP1, are responsible for the balance of H3 phosphorylation during mitosis in Saccharomyces cerevisiae and Caenorhabditis elegans. In these models, both enzymes are required for H3 phosphorylation and chromosome segregation, although a causal link between the two processes has not been demonstrated. Deregulation of human aurora kinases has been implicated in oncogenesis as a consequence of chromosome missegregation. Our findings reveal an enzyme system that regulates chromosome dynamics and controls histone phosphorylation that is conserved among diverse eukaryotes.

Recent studies of histone methylation have yielded fundamental new insights pertaining to the role of this modification in gene activation as well as in gene silencing. While a number of methylation sites are known to occur on histones, only limited information exists regarding the relevant enzymes that mediate these methylation events. We thus sought to identify native histone methyltransferase (HMT) activities from Saccharomyces cerevisiae. Here, we describe the biochemical purification and characterization of Set2, a novel HMT that is site-specific for lysine 36 (Lys36) of the H3 tail. Using an antiserum directed against Lys36 methylation in H3, we show that Set2, via its SET domain, is responsible for methylation at this site in vivo. Tethering of Set2 to a heterologous promoter reveals that Set2 represses transcription, and part of this repression is mediated through the HMT activity of the SET domain. These results suggest that Set2 and methylation at H3 Lys36 play a role in the repression of gene transcription.Eukaryotic DNA is complexed in cells by histone proteins to form the fundamental repeating unit of chromatin, the nucleosome. Stretches of nucleosomes are further folded upon themselves to create higher-order chromatin structures that are currently not well defined. Compaction of DNA in this manner imposes a severe impediment to proteins that require access to the DNA template. Clear examples of this impediment have been shown to exist for the machinery that drives DNA transcription (28,38,41). However, this same impediment faces all aspects of DNA metabolism, including replication, repair and recombination (18,40).Posttranslational modifications of histone amino termini are recognized to play a central role in the control of chromatin structure and function. A diverse array of covalent histone modifications have been documented that take place on the tail domains of histones which protrude away from the nucleosome (9, 39). We and others have proposed that these modifications form a histone code which directly regulates chromatin function either by altering the specific structure of the chromatin polymer itself and/or by recruiting proteins or protein complexes that uniquely recognize a single or combinatorial set of modifications on one or more histone tails (14,35,37). For example, recent evidence showing that the bromodomains of various histone acetyltransferases, including PCAF, GCN5 and TAF II 250, bind to acetylated lysines in the histone tails suggests that specific recruitment of the transcriptional apparatus to promoters is one likely mechanism to explain how histone modifications influence transcription (8,22). It appears that other histone modifications, including methylation, function in the same manner (see below).Histone methylation is a posttranslational modification that occurs on lysine and arginine residues in the H3 and H4 tail domains (reviewed in reference 42). In histone H3, lysines 4, 9, 27, and 36 are well-documented sites of methylation, while in histone H4, lysine methylati...

Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic ␤ cells. CD8 ؉ T cells are essential for this ␤ cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8 ؉ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.T he nonobese diabetic (NOD) mouse is a widely studied model of human type 1 diabetes, an autoimmune disease characterized by inflammation of pancreatic islets (insulitis) followed by T cell-mediated destruction of insulin (INS)-producing ␤ cells (1). Both CD4 ϩ and CD8 ϩ T cells are required for this pathogenic process (1); however, CD8 ϩ T cells appear to be responsible for the initial ␤ cell insult (1-3). Whereas the pathogenicity of B cells and autoantibodies is less clear, the autoantigens currently believed to contribute to autoimmune diabetes pathogenesis in NOD mice and humans all were originally identified based on the presence of specific autoantibodies rather than by T cell recognition (4-6). Little is known of the ␤ cell antigens targeted by the pathogenic CD8 ϩ T cells. Although one study identified an INS peptide as the antigenic target of the majority of islet-infiltrating CD8 ϩ T cells in NOD mice (7), the prevalence of these INS-reactive CD8 ϩ T cells was not confirmed in subsequent studies (8,9).A substantial proportion of ␤ cell-autoreactive CD8 ϩ T cells isolated from NOD islets express a shared T cell receptor ␣ (TCR␣) chain (V␣17-J␣42), suggesting recognition of a common ␤ cell peptide (3, 10). These T cells do not recognize the antigenic INS peptide mentioned above (11,12). The pathogenicity of this prevalent T cell population has been well established through studies of the 8.3 T cell clone (a representative T cell clone of the V␣17-J␣42-expressing T cell population) (13, 14). 8.3-Like T cells are present in the earliest islet infiltrates of NOD mice (3) and undergo avidity maturation as islet inflammation progresses to overt disease (8). At any given time, 8.3-like T cells can constitute up to 30-40% of the islet-associated CD8 ϩ T cells (9). Strikingly, quantification of 8.3-like T cells in peripheral blood predicts diabetes development in individual NOD mice (9), unlike any other single immune indicator identified to date. Although the prevalence and pathogenicity of 8.3-like T cells has been clearly established, the identity of their ligand has remained elusive. Materials and MethodsMice. NOD͞Lt mice were maintained by brother-sister mating. 8.3-TCR␣␤-transgenic NOD mice, designated 8.3-NOD, ha...

The fundamental unit of eukaryotic chromatin, the nucleosome, consists of genomic DNA wrapped around the conserved histone proteins H3, H2B, H2A and H4, all of which are variously modified at their amino- and carboxy-terminal tails to influence the dynamics of chromatin structure and function -- for example, conjugation of histone H2B with ubiquitin controls the outcome of methylation at a specific lysine residue (Lys 4) on histone H3, which regulates gene silencing in the yeast Saccharomyces cerevisiae. Here we show that ubiquitination of H2B is also necessary for the methylation of Lys 79 in H3, the only modification known to occur away from the histone tails, but that not all methylated lysines in H3 are regulated by this 'trans-histone' pathway because the methylation of Lys 36 in H3 is unaffected. Given that gene silencing is regulated by the methylation of Lys 4 and Lys 79 in histone H3, we suggest that H2B ubiquitination acts as a master switch that controls the site-selective histone methylation patterns responsible for this silencing.

Posttranslational modifications of histone amino termini play an important role in modulating chromatin structure and function. Lysine methylation of histones has been well documented, and recently this modification has been linked to cellular processes involving gene transcription and heterochromatin assembly. However, the existence of arginine methylation on histones has remained unclear. Recent discoveries of protein arginine methyltransferases, CARM1 and PRMT1, as transcriptional coactivators for nuclear receptors suggest that histones may be physiological targets of these enzymes as part of a poorly defined transcriptional activation pathway. Here we show by using mass spectrometry that histone H4, isolated from asynchronously growing human 293T cells, is methylated at arginine 3 (Arg-3) in vivo. In support, a novel antibody directed against histone H4 methylated at Arg-3 independently demonstrates the in vivo occurrence of this modification and reveals that H4 Arg-3 methylation is highly conserved throughout eukaryotes. Finally, we show that PRMT1 is the major, if not exclusive, H4 Arg-3 methyltransfase in human 293T cells. These findings suggest a role for arginine methylation of histones in the transcription process.

Minor histocompatibility antigens (mHAgs) present a significant impediment to organ and bone marrow transplantation between HLA-identical donor and recipient pairs. Here we report the identification of a new HLA-A*0201–restricted mHAg, HA-8. Designation of this mHAg as HA-8 is based on the nomenclature of Goulmy (Goulmy, E. 1996. Curr. Opin. Immunol. 8:75–81). This peptide, RTLDKVLEV, is derived from KIAA0020, a gene of unknown function located on chromosome 9. Polymorphic alleles of KIAA0020 encode the alternative sequences PTLDKVLEV and PTLDKVLEL. Genotypic analysis demonstrated that the HA-8–specific cytotoxic T lymphocyte (CTL) clone SKH-13 recognized only cells that expressed the allele encoding R at P1. However, when PTLDKVLEV was pulsed onto cells, or when a minigene encoding this sequence was used to artificially translocate this peptide into the endoplasmic reticulum, it was recognized by CTLs nearly as well as RTLDKVLEV. This indicates that the failure of CTLs to recognize cells expressing the PTLDKVLEV-encoding allele of KIAA0020 is due to a failure of this peptide to be appropriately proteolyzed or transported. Consistent with the latter possibility, PTLDKVLEV and its longer precursors were transported poorly compared with RTLDKVLEV by transporter associated with antigen processing (TAP). These studies identify a new human mHAg and provide the first evidence that minor histocompatibility differences can result from the altered processing of potential antigens rather than differences in interaction with the relevant major histocompatibility complex molecule or T cell receptor.

The Nek family of protein kinases in humans is composed of 11 members that share an amino-terminal catalytic domain related to NIMA, an Aspergillus kinase involved in the control of several aspects of mitosis, and divergent carboxyl-terminal tails of varying length. Nek6 (314AA) and Nek7 (303AA), 76% identical, have little noncatalytic sequence but bind to the carboxyl-terminal noncatalytic tail of Nercc1/Nek9, a NIMA family protein kinase that is activated in mitosis. Microinjection of anti-Nercc1 antibodies leads to spindle abnormalities and prometaphase arrest or chromosome missegregation. Herein we show that Nek6 is increased in abundance and activity during mitosis; activation requires the phosphorylation of Ser 206 on the Nek6 activation loop. This phosphorylation and the activity of recombinant Nek6 is stimulated by coexpression with an activated mutant of Nercc1. Moreover, Nercc1 catalyzes the direct phosphorylation of prokaryotic recombinant Nek6 at Ser 206 in vitro concomitant with 20 -25-fold activation of Nek6 activity; Nercc1 activates Nek7 in vitro in a similar manner. Nercc1/Nek9 is likely to be responsible for the activation of Nek6 during mitosis and probably participates in the regulation of Nek7 as well. These findings support the conclusion that Nercc1/Nek9 and Nek6 represent a novel cascade of mitotic NIMA family protein kinases whose combined function is important for mitotic progression.The NIMA family of protein kinases is named after the Aspergillus nidulans protein kinase encoded by the nimA gene (1). Mutation of nimA (never in mitosis A) arrests cells in G 2 without interfering with p34 cdc2 activation (2), suggesting that the NIMA protein has a central role in the G 2 /M transition. Moreover, if the G 2 arrest of nimA mutants is bypassed by additional mutations, the resulting mitotic cells show aberrant spindle and nuclear envelope organization (3, 4), pointing to functions of NIMA beyond the control of mitotic entry. NIMA can induce chromatin condensation and nuclear membrane breakdown in mammalian cells as it does in Aspergillus (3, 5, 6), suggesting that these functions are also regulated by protein kinases with similar specificity in vertebrate cells. Eleven protein kinases with a catalytic domain related to NIMA have been identified in the human genome (7), and a substantial fraction were first described very recently (8 -12). The functions of these NIMA family kinases, mostly referred to as Neks, are largely unknown. The best characterized of these kinases, Nek2, has been implicated in the regulation of the centrosome (13); Nek1 and Nek8 mutations have been related to cystic kidney disease (14, 15); Nek6/7 have been suggested to phosphorylate and activate p70 S6 kinase (16); and Nek9/Nercc1 has been implicated in the control of mitotic spindle formation and chromosome segregation (10).Nek6 together with its close homolog, Nek7, were purified from rat liver as the predominant kinases capable of phosphorylating in vitro the hydrophobic regulatory site (Thr 412 ) of the p70 S6 kina...

The Nercc1 protein kinase autoactivates in vitro and is activated in vivo during mitosis. Autoactivation in vitro requires phosphorylation of the activation loop at threonine 210. Mitotic activation of Nercc1 in mammalian cells is accompanied by Thr210 phosphorylation and involves a small fraction of total Nercc1. Mammalian Nercc1 coimmunoprecipitates ␥-tubulin and the activated Nercc1 polypeptides localize to the centrosomes and spindle poles during early mitosis, suggesting that active Nercc has important functions at the microtubular organizing center during cell division. To test this hypothesis, we characterized the Xenopus Nercc1 orthologue (XNercc). XNercc endogenous to meiotic egg extracts coprecipitates a multiprotein complex that contains ␥-tubulin and several components of the ␥-tubulin ring complex and localizes to the poles of spindles formed in vitro. Reciprocally, immunoprecipitates of the ␥-tubulin ring complex polypeptide Xgrip109 contain XNercc. Immunodepletion of XNercc from egg extracts results in delayed spindle assembly, fewer bipolar spindles, and the appearance of aberrant microtubule structures, aberrations corrected by addition of purified recombinant XNercc. XNercc immunodepletion also slows aster assembly induced by Ran-GTP, producing Ran-asters of abnormal size and morphology. Thus, Nercc1 contributes to both the centrosomal and the chromatin/Ran pathways that collaborate in the organization of a bipolar spindle.