Histone modifying enzymes contribute to the activation or inactivation of transcription by ultimately catalyzing the unfolding or further compaction, respectively, of chromatin structure. Actively transcribed genes are typically hyperacetylated at Lys residues of histones H3 and H4 and hypermethylated at Lys-4 of histone H3 (H3-K4). To determine whether covalent histone modifications play a role in the ␤ cell-specific expression of the insulin gene, we performed chromatin immunoprecipitation assays using anti-histone antibodies and extracts from ␤ cell lines, non-␤ cell lines, and ES cells, and quantitated specific histone modifications at the insulin promoter by real-time PCR. Our studies reveal that the proximal insulin promoter is hyperacetylated at histone H3 only in ␤ cells. This hyperacetylation is highly correlated to recruitment of the histone acetyltransferase p300 to the proximal promoter in ␤ cells, and is consistent with the role of hyperacetylation in promoting euchromatin formation. We also observed that the proximal insulin promoter of ␤ cells is hypermethylated at H3-K4, and that this modification is correlated to the recruitment of the histone methyltransferase SET7/9 to the promoter. ES cells demonstrate a histone modification pattern intermediate between that of ␤ cells and non-␤ cells, and is consistent with their potential to express the insulin gene. We therefore propose a model in which insulin transcription in the ␤ cell is facilitated by a unique combination of transcription factors that acts in the setting of an open, euchromatic structure of the insulin gene.The pancreatic ␤ cell is exclusively responsible for the synthesis and secretion of insulin. The production of insulin appears to be governed by constraints imposed at the level of transcription of the gene encoding insulin (Ins), 1 and involves an intricate interplay between transcription factors that are known to function as transactivators of the gene. In recent years, specific DNA elements within the proximal ϳ400 base pairs (bp) of the Ins promoter have been mapped precisely and shown to be bound by several major classes of transactivating transcription factors, including homeodomain factors (Pdx1, Lmx1.1), basic helix loop helix factors (NeuroD1, E47), and bZip factors (mMafA) (see Ref. 1 for review). In addition, coactivators such as p300 (by virtue of its interaction with Pdx1, NeuroD1, and E47) have also been suggested to contribute to Ins gene regulation (2, 3). Thus, it is hypothesized that the unique combination of ubiquitous and cell type-specific factors within the ␤ cell results in the transcription of the Ins gene (3-5). However, this hypothesis alone cannot explain why heterologous expression of ␤ cell factors results in activation of the endogenous Ins gene in only limited, "responsive" cell types (e.g. liver, pancreatic ductal cells, and embryonic and intestinal stem cells) (6 -10). Although this finding suggests that "unresponsive" cell types are still missing some critical genetic component (transcription facto...

Expression of the insulin gene is nearly exclusive to the ␤ cells of the pancreatic islets. Although the sequence-specific transcription factors that regulate insulin expression have been well studied, the interrelationship between these factors, chromatin structure, and transcriptional elongation by RNA polymerase II (pol II) has remained undefined. In this regard, recent studies have begun to establish a role for the methylation of histone H3 in the initiation or elongation of transcription by pol II. To determine a role for the transcriptional activator Pdx-1 in the maintenance of chromatin structure and pol II recruitment at the insulin gene, we performed small interfering RNA-mediated knockdown of Pdx-1 in ␤TC3 cells and subsequently studied histone modifications and pol II recruitment by chromatin immunoprecipitation. We demonstrated here that the 50% fall in insulin transcription following knockdown of Pdx-1 is accompanied by a 60% fall in dimethylated histone H3-Lys-4 at the insulin promoter. H3-Lys-4 methylation at the insulin promoter may be mediated, at least partially, by the methyltransferase Set9. Immunohistochemical analysis revealed that Set9 is expressed in an islet-enriched pattern in the pancreas, similar to the pattern of Pdx-1 expression. The recruitment of Set9 to the insulin gene appears to be a consequence of its direct interaction with Pdx-1, and small interfering RNA-mediated knockdown of Set9 attenuates insulin transcription. Pdx-1 knockdown was also associated with an overall shift in the recruitment of pol II isoforms to the insulin gene, from an elongation isoform (Ser(P)-2) to an initiation isoform (Ser(P)-5). Our findings therefore suggest a model whereby Pdx-1 plays a novel role in linking H3-Lys-4 dimethylation and pol II elongation to insulin transcription.During pancreatic development, endocrine and exocrine cell types differentiate from a common endodermal precursor cell via a tightly coordinated sequence of transcriptional events (1, 2). The Hox-like transcription factor Pdx-1 is thought to initiate this cascade by regulating specific genes within the endodermal precursor cell (3, 4). The disruption of pdx-1 expression during development, either by targeted knockout in mice (5, 6) or homozygous mutations in humans (7), can be catastrophic, resulting in the failure of pancreas formation and consequent development of diabetes. Within the mature pancreas, Pdx-1 is necessary for the maintenance and function of endocrine cell types within the islets of Langerhans. In the islet ␤ cell, Pdx-1 is believed to activate a number of critical genes involved in glucose sensing and insulin production, including glucokinase, glut2, and insulin (3,8).Expression of the insulin gene is almost exclusive to the islet ␤ cells. By using small interfering RNA (siRNA) 2 and chromatin immunoprecipitation (ChIP) in isolated mouse islets, we recently demonstrated that Pdx-1 is a direct activator of insulin transcription upon binding to A box DNA elements in the 5Ј-regulatory region of the gene (the ins...

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αβ-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective β-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/β-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with impact on neuronal function and survival in the developing brain.

The Caenorhabditis elegans LSD1 H3K4me2 demethylase SPR-5 reprograms epigenetic transcriptional memory during passage through the germ line. Here we show that mutants in the H3K9me2 methyltransferase, met-2, result in transgenerational epigenetic effects that parallel spr-5 mutants. In addition, we find that spr-5;met-2 double mutants have a synergistic effect on sterility, H3K4me2, and spermatogenesis expression. These results implicate MET-2 as a second histone-modifying enzyme in germ-line reprogramming and suggest a model in which SPR-5 and MET-2 function cooperatively to reestablish an epigenetic ground state required for the continued immortality of the C. elegans germ line. Without SPR-5 and MET-2, we find that the ability to express spermatogenesis genes is transgenerationally passed on to the somatic cells of the subsequent generation. This indicates that H3K4me2 may act in the maintenance of cell fate. Finally, we demonstrate that reducing H3K4me2 causes a large increase in H3K9me2 added by the SPR-5;MET-2 reprogramming mechanism. This finding suggests a novel histone code interaction in which the input chromatin environment dictates the output chromatin state. Taken together, our results provide evidence for a broader reprogramming mechanism in which multiple enzymes coordinately regulate histone information during passage through the germ line. Since the discovery of histone modifications and the initial proposal of the histone code (1), a number of experiments have implicated histone modifications in the maintenance of transcriptional memory (2-8). Despite this implicit association, an understanding of how histone modifications are regulated developmentally, as well as the exact relationship between certain histone modifications, remains elusive. For example, at fertilization, the highly differentiated transcriptional program of the germ line may be reprogrammed by maternal factors in the oocyte to reestablish the epigenetic ground state of the zygote. This reprogramming capacity of the oocyte is implied by the ability of oocyte factors to reprogram a differentiated cell during somatic cell nuclear transfer (SCNT) (9, 10). However, the low efficiency observed in SCNT and in the similar induction of pluripotent stem cells highlights the complexity involved in reprogramming differentiated fates back to a ground pluripotent state (11,12). Moreover, embryos derived from SCNT have been shown to continue to express genes from their previous differentiated state (13). These results indicate that germ-line reprogramming requires the resetting of an epigenetic transcriptional memory, but the mechanism of this reprogramming is not well understood.One emerging player in transcriptional memory is dimethylation of histone 3 at lysine 4 (H3K4me2). During transcription, H3K4 methyltransferases, such as mixed-lineage leukemia (MLL), associate with a core COMPASS complex consisting of WDR5, RbBP5, and ASH2L (14). This complex promotes H3K4 methyltransferase activity and may enable the methyltransferase to interact w...

Edited by Velia M. FowlerMicrotubule dynamics involves the polymerization and depolymerization of tubulin dimers and is an essential and highly regulated process required for cell viability, architecture, and division. The regulation of the microtubule network also depends on the maintenance of a pool of ␣␤-tubulin heterodimers. These dimers are the end result of complex folding and assembly events, requiring the TCP1 Ring Complex (TriC or CCT) chaperonin and five tubulin-specific chaperones, tubulin binding cofactors A-E (TBCA-TBCE). However, models of the actions of these chaperones are incomplete or inconsistent. We previously purified TBCD from bovine tissues and showed that it tightly binds the small GTPase ARL2 but appears to be inactive. Here, in an effort to identify the functional form of TBCD and using non-denaturing gels and immunoblotting, we analyzed lysates from a number of mouse tissues and cell lines to identify the quaternary state(s) of TBCD and ARL2. We found that both proteins co-migrated in native gels in a complex of ϳ200 kDa that also contained ␤-tubulin. Using human embryonic kidney cells enabled the purification of the TBCD⅐ARL2⅐␤-tubulin trimer found in cell and tissue lysates as well as two other novel TBCD complexes. Characterization of ARL2 point mutants that disrupt binding to TBCD suggested that the ARL2-TBCD interaction is critical for proper maintenance of microtubule densities in cells. We conclude that the TBCD⅐ARL2⅐␤-tubulin trimer represents a functional complex whose activity is fundamental to microtubule dynamics.Microtubules are highly dynamic polymers that are best known for their roles as the central cytoskeletal structure in cells and in mitotic spindles during cell division. They are also the tracks on which organelles traffic, particularly important in polarized cells that generate great distances between parts of the cell. Additionally, they are the core of sensory and motile cilia or flagella. The formation and destruction of microtubules and microtubule bundles are orchestrated by a large number of proteins that include the microtubule-associated proteins. Microtubules are polymers of the ␣␤-tubulin dimer, with several genes encoding each ␣-or ␤-tubulin subunit (e.g. see Lewis et al. (1)), resulting in some diversity in composition. Tubulins can also be modified by posttranslational modifications, including acetylation, tyrosination, and phosphorylation, which can alter the dynamics of the polymerization and depolymerization reactions. Because of the essential role of microtubules in cell division, they have also been the target of many antitumor therapies, e.g. the taxanes and Vinca alkaloids (2). However, despite their importance to cells and in the clinic and decades of research, we still lack a complete molecular-level understanding of the biosynthesis and regulation of the formation of ␣␤-tubulin.Tubulins are typically the most abundant proteins in mammalian cells, but the generation of the ␣␤-tubulin dimer requires a complex series of biosynthetic steps to ...

The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism (binding to promoter elements and activating basal transcriptional machinery) or an indirect mechanism (via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA (mature mRNA) and a short halflife insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels (to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.Insulin is produced almost exclusively by the ␤ cells of the pancreatic islets of Langerhans. This restriction of insulin production derives primarily from constraints imposed at the level of transcription of the gene encoding preproinsulin (the insulin gene), rather than at the level of translation of the nascent mRNA (1, 2). Studies of the rodent insulin genes indicate that ϳ400 base pairs (bp) of DNA 5Ј of the transcriptional start site (the insulin promoter) are sufficient to confer cell type-specific expression of insulin (3-7). Multiple discrete sequence elements within the proximal promoter region contribute to both the specificity and magnitude of insulin expression, and these elements are believed to serve as binding sites for several islet transcription factors, including Pdx-1, MafA, and BETA2/NeuroD (see Ref. 8 for a review). In the prevailing hypothesis of insulin transcription, the association of these transcription factors with the promoter and their subsequent interaction with ubiquitously expressed factors (e.g. E47 and p300) (9, 10) leads to the recruitment of the basal transcriptional machinery to the insulin gene. This hypothesis, however, has never been rigorously tested for the endogenous insulin gene in islet ␤ cells.The Hox-like homeodomain protein...

ARL2 is among the most highly conserved proteins, predicted to be present in the last eukaryotic common ancestor, and ubiquitously expressed. Genetic screens in multiple model organisms identified ARL2, and its cytosolic binding partner cofactor D (TBCD), as important in tubulin folding and microtubule dynamics. Both ARL2 and TBCD also localize to centrosomes, making it difficult to dissect these effects. A growing body of evidence also has found roles for ARL2 inside mitochondria, as a regulator of mitochondrial fusion. Other studies have revealed roles for ARL2, in concert with its closest paralog ARL3, in the traffic of farnesylated cargos between membranes and specifically to cilia and photoreceptor cells. Details of each of these signaling processes continue to emerge. We summarize those data here and speculate about the potential for cross-talk or coordination of cell regulation, termed higher order signaling, based upon the use of a common GTPase in disparate cell functions.

Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αβ-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A-E (TBCA-E), and GTP are required for the folding of α- and β-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCD•ARL2•β-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each’s role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not β-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCD•ARL2•β-tubulin complex represents a functional intermediate in the β-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the β-tubulin in the trimer is also shown, with implications to modeling the pathway.