Simulation of Drosophila circadian oscillations, mutations, and light responses by a model with VRI, PDP-1, and CLK.

A model of Drosophila circadian rhythm generation was developed to represent feedback loops based on transcriptional regulation of per, Clk (dclock), Pdp-1, and vri (vrille). The model postulates that histone acetylation kinetics make transcriptional activation a nonlinear function of [CLK]. Such a nonlinearity is essential to simulate robust circadian oscillations of transcription in our model and in previous models. Simulations suggest that two positive feedback loops involving Clk are not essential for oscillations, because oscillations of [PER] were preserved when Clk, vri, or Pdp-1 expression was fixed. However, eliminating positive feedback by fixing vri expression altered the oscillation period. Eliminating the negative feedback loop in which PER represses per expression abolished oscillations. Simulations of per or Clk null mutations, of per overexpression, and of vri, Clk, or Pdp-1 heterozygous null mutations altered model behavior in ways similar to experimental data. The model simulated a photic phase-response curve resembling experimental curves, and oscillations entrained to simulated light-dark cycles. Temperature compensation of oscillation period could be simulated if temperature elevation slowed PER nuclear entry or PER phosphorylation. The model makes experimental predictions, some of which could be tested in transgenic Drosophila.

DEFINING THE ROLE OF EGFR ACETYLATION IN CELLULAR PROCESSES: CLINICAL IMPLICATIONS

Epidermal growth factor receptor (EGFR) is a cell membrane tyrosine kinase receptor and plays a pivotal role in regulating cell growth, differentiation, cell cycle, and tumorigenesis. Deregulation of EGFR causes many diseases including cancers. Intensive investigation of EGFR alteration in human cancers has led to profound progress in developing drugs to target EGFR-mediated cancers. While exploring possible synergistic enhancement of therapeutic efficacy by combining EGFR tyrosine kinase inhibitors (TKI) with other anti-cancer agents, we observed that suberoylanilide hydroxamic acid (SAHA, a deacetylase inhibitor) enhanced TKI-induced cancer cell death, which further led us to question whether SAHA-mediated sensitization to TKI was associated with EGFR acetylation. What we know so far is that SAHA can inhibit class I and II histone deacetylases (HDACs), which could possibly preserve acetylation of underlying HDAC-targeted proteins including both histone and non-histone proteins. In addition, it has been reported that an HDAC inhibitor, TSA, enhanced EGFR phosphorylation in ovarian cancer cells. EGFR acetylation has also been reported to play a role in the regulation of EGFR endocytosis recently. These observations indicate that there might be an intrinsic correlation between acetylation and phosphorylation of EGFR. In other words, the interplay between EGFR acetylation and phosphorylation may contribute to HDAC inhibitors (HDACi)-augmented EGFR phosphorylation. In this investigation, we showed that CBP acetyltransferase acetylated EGFR in vivo. In response to EGF stimulation, CBP rapidly translocated from the nucleus to the cytoplasm. We also demonstrated protein-protein interaction between CBP and EGFR as well as the enhancement of EGFR acetylation by CBP. Moreover, EGFR acetylation enhanced EGFR tyrosine phosphorylation and augmented its association with Src kinase. Acetylation-deficient EGFR mutant (EGFR-K3R) significantly reduced the function and activity of EGFR. Furthermore, ectopic expression of EGFR-K3R mutant abrogated its ability to respond to EGF-induced cell proliferation, DNA synthesis, and anchorage-independent growth using cell-based assays and tumor growth in nude mice. In addition, we demonstrated that EGFR expression was associated with SAHA resistance in the treatment of cancer cells that overexpress EGFR. The knockdown of EGFR in MDA-MB-468 breast cancer cells could sensitize the cells to respond to SAHA. The overexpression of EGFR in SAHA-sensitive MDA-MB-453 breast cancer cells rendered the cells resistant to SAHA. Together, these findings suggest that EGFR plays an important role in SAHA resistance in breast carcinoma cells that we tested. The combination therapy of HDACi with TKI has been proposed for treating cancers with aberrant expression of EGFR. The evidence from pre-clinical or clinical trials demonstrated significant enhancement of therapeutic efficacy by using such a combination therapy. Our in vivo study also demonstrated that the combination of SAHA and TKI for the treatment of breast cancer significantly reduced tumor burden compared with either SAHA or TKI alone. The significance of our study elucidated another possible underlying molecular mechanism by which HDACi mediated sensitization to TKI. Our results unveiled a critical role of EGFR acetylation that regulates EGFR tyrosine phosphorylation and may further provide an experiment-based rationale for combinatorial targeted therapy.

Identification of a SIRT1 mutation in a family with type 1 diabetes.

Type 1 diabetes is caused by autoimmune-mediated β cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to β cell antigens, and a rapid dependence on insulin. Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations identify a role for SIRT1 in human autoimmunity and unveil a monogenic form of type 1 diabetes.

Rubisco in complex with Rubisco large subunit methyltransferase.

SET domain protein lysine methyltransferases (PKMT) are a structurally unique class of enzymes that catalyze the specific methylation of lysine residues in a number of different substrates. Especially histone-specific SET domain PKMTs have received widespread attention because of their roles in the regulation of epigenetic gene expression and the development of some cancers. Rubisco large subunit methyltransferase (RLSMT) is a chloroplast-localized SET domain PKMT responsible for the formation of trimethyl-lysine-14 in the large subunit of Rubisco, an essential photosynthetic enzyme. Here, we have used cryoelectron microscopy to produce an 11-A density map of the Rubisco-RLSMT complex. The atomic model of the complex, obtained by fitting crystal structures of Rubisco and RLSMT into the density map, shows that the extensive contact regions between the 2 proteins are mainly mediated by hydrophobic residues and leucine-rich repeats. It further provides insights into potential conformational changes that may occur during substrate binding and catalysis. This study presents the first structural analysis of a SET domain PKMT in complex with its intact polypeptide substrate.

CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes--including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia--have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCalpha by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum- and glucocorticoid-induced kinase-1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCalpha promoter and derepression of ENaCalpha transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCalpha and likely other aldosterone-induced genes.

Regulation of Set1-mediated methylation of Dam1

Eukaryotic genomes exist within a dynamic structure named chromatin in which DNA is wrapped around an octamer of histones forming the nucleosome. Histones are modified by a range of posttranslational modifications including methylation, phosphorylation, and ubiquitination, which are integral to a range of DNA-templated processes including transcriptional regulation. A hallmark for transcriptional activity is methylation of histone H3 on lysine (K) 4 within active gene promoters. In S. cerevisiae, H3K4 methylation is mediated by Set1 within the COMPASS complex. Methylation requires prior ubiquitination of histone H2BK123 by the E2-E3 ligases Rad6 and Bre1, as well as the Paf1 transcriptional elongation complex. This regulatory pathway exemplifies cross-talk in trans between posttranslational modifications on distinct histone molecules. Set1 has an additional substrate in the kinetochore protein Dam1, which is methylated on K233. This methylation antagonizes phosphorylation of adjacent serines by the Ipl1 Aurora kinase. The discovery of a second Set1 substrate raised the question of how Set1 function is regulated at the kinetochore. I hypothesized that transcriptional regulatory factors essential for H3K4 methylation at gene promoters might also regulate Set1-mediated methylation of Dam1K233. Here I show that the regulatory factors essential for COMPASS activity at gene promoters is also indispensable for the methylation of Dam1K233. Deletion of members of the COMPASS complex leads to loss of Dam1K233 methylation. In addition, deletion of Rad6, Bre1, or members of the Paf1 complex abolishes Dam1 methylation. The role of Rad6 and Bre1 in Dam1 methylation is dependent on H2BK123 ubiquitination, as mutation of K123 within H2B results in complete loss of Dam1 methylation. Importantly, methylation of Dam1K233 is independent of transcription and occurs at the kinetochore. My results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription at the kinetochore. My data provide the first example of cross-talk in trans between modifications on a histone and a non-histone protein. Additionally, my results indicate that several factors previously thought to be required for Set1 function at gene promoters are more generally required for the catalytic activity of the COMPASS complex regardless of substrate or cellular process.

ROLE OF SYNAPSIN IN LONG-TERM SYNAPTIC FACILITATION IN APLYSIA

Enhanced expression of the presynaptic protein synapsin has been correlated with certain forms of long-term plasticity and learning and memory. However, the regulation and requirement for enhanced synapsin expression in long-term memory remains unknown. In the present study the technical advantages of the marine mollusc Aplysia were exploited in order to address this issue. In Aplysia, learning-induced enhancement in synaptic strength is modulated by serotonin (5-HT) and treatment with 5-HT in vitro of the sensorimotor synapse induces long-term facilitation (LTF) of synaptic transmission, which lasts for days, as well as the formation of new connections between the sensory and motor neuron. Results from immunofluorescence analysis indicated that 5-HT treatment upregulates synapsin protein levels within sensory neuron varicosities, the presumed site of neurotransmitter release. To investigate the mechanisms underlying increased synapsin expression, the promoter region of the Aplysia synapsin gene was cloned and a cAMP response element (CRE) was identified, raising the possibility that the transcriptional activator cAMP response element-binding protein-1 (CREB1) mediates the 5-HT-induced regulation of synapsin. Results from Chromatin Immunoprecipitation (ChIP) assays indicated that 5-HT treatment enhanced association of CREB1 surrounding the CRE site in the synapsin promoter and led to increased acetylation of histones H3 and H4 and decreased association of histone deacetylase 5 surrounding the CRE site in the synapsin promoter, a sign of transcriptional activation. In addition, sensory neurons injected with an enhanced green fluorescent protein (EGFP) reporter vector driven by the synapsin promoter exhibited a significant increase in EGFP expression following treatment with 5-HT. These results suggest that synapsin expression is regulated by 5-HT in part through transcriptional activation of the synapsin gene and through CREB1 association with the synapsin promoter. Furthermore, RNA interference that blocks 5-HT-induced elevation of synapsin expression also blocked long-term synaptic facilitation. These results indicate that 5-HT-induced regulation of synapsin is necessary for LTF and that synapsin is part of the cascade of synaptic events involved in the consolidation of memory.

MOLECULAR MECHANISMS UNDERLYING THE TRANSCRIPTIONAL REGULATION OF T HELPER 17 AND REGULATORY T CELLS

CD4+ T helper (Th) lymphocytes are vital for integrating immune responses by orchestrating the function of other immune cell types. Naïve Th cells can differentiate into different effector subsets that are characterized by their cytokine profile and immune regulatory functions. These subsets include Th1, Th2, Th17, natural and inducible regulatory T cells (nTreg and iTreg respectively), among others. We focused our investigation on two Th lineages, Th17 and regulatory T cells, with opposing functions in the immune system. These subsets have been suggested to be reciprocally regulated since they both require TGF-b for their development. We investigated the role of the Treg-associated master transcription factor Foxp3, and found that Foxp3 inhibits Th17 cell generation by preventing the transcriptional activity of the two main Th17-specific transcription factors, nuclear orphan receptors RORa and RORgt. At the molecular level, we identified two different functional domains in Foxp3 required for such inhibition: the LQALL sequence in exon 2 and the TIP60/HDAC7 binding domain. These domains could be crucial to either prevent the association of the nuclear receptors to coactivators or to recruit histone deacetylases to RORa- or RORgt-target genes. Since TGF-b is a common cytokine required for the commitment towards both Th lineages, we determined the role of the TGF-b-dependent signaling pathway in the generation of each subset. By using mice with deficiencies in signaling molecules downstream of TGF-b, we found that while Smad2, Smad3 and Smad4 are required for the generation of iTreg cells, only Smad2 is indispensable for the induction of IL-17-producing cells, suggesting that TGF-b induces these T helper lineages through differential signaling pathways. Thus, our findings describe novel transcriptional regulatory mechanisms that control the generation of two T helper lineages with opposing functions. These findings could provide novel therapeutic targets to treat diseases where the balance of these T cells is dysregulated, such as in autoimmunity, chronic infectious diseases and cancer.

IDENTIFICATION OF FACTORS INVOLVED IN DNA METHYLATION OF CPG-ISLAND-PROMOTERS

Repression of many tumor suppressor genes (TSGs) in cancer is mediated by aberrantly increased DNA methylation levels at promoter CpG islands (CGI). About one-fourth of empirically defined human promoters are surrounded by or contain clustered repetitive elements. It was previously observed that a sharp transition of methylation occurs between highly methylated repetitive elements (SINE or LINE) and unmethylated CGI-promoters (e.g. P16, VHL, CDH and RIL) in normal tissues. The functions that lead to increased CGI methylation in cancer remain poorly understood. We propose that CGI-promoters contain cis-elements for triggering de novo DNA methylation. In the first part of our project, we established a site-specific integration system with enforced local transcriptional repression in colorectal cancer cells and monitored the occurrence of de novo DNA methylation in exogenous fragments containing a CGI-promoter and repetitive elements. Initial de novo methylation was seeded at specific CG sites in a repetitive element, and accelerated by persistent binding of a KRAB-containing transcriptional repressor. Furthermore, additional repetitive elements (LINE and SINE) located adjacent to the promoter could confer DNA methylation spreading into the CGI particularly in the setting of KRAB-factor binding. However, a repressive chromatin alone was not sufficient to initiate DNA methylation, which required specific DNA sequences and was integration-site (and/or cell-line) specific. In addition, all the methylation observed showed slow and gradual accumulation over several months of culture. Overall, these results demonstrate a requirement for specific DNA sequences to trigger de novo DNA methylation, and repetitive elements as cis-regulatory factors to cooperate with strengthened transcriptional repression in promoting methylation spreading. In the second part, we re-introduced disrupted DNMT3B or DNMT1 into HCT116 DKO cells and mapped the remethylation pattern through a profiling method (DREAM). Moderate remethylation occurred when DNMT3B was re-expressed with a preference toward non-CGI and non-promoter regions. Hence, there exists a set of genomic regions with priority to be targets for DNMT3B in somatic cells.

FUNCTION OF ZNF668 IN CANCER DEVELOPMENT

Human cancer develops as a result of accumulation of mutations in oncogenes and tumor suppressor genes. Zinc finger protein 668 (ZNF668) has recently been identified and validated as one of the highly mutated genes in breast cancer, but its function is entirely unknown. Here, we report two major functions of ZNF668 in cancer development. (1) ZNF668 functions as a tumor suppressor by regulating p53 protein stability and function. We demonstrate that ZNF668 is a nucleolar protein that physically interacts with both MDM2 and p53. By binding to MDM2, ZNF668 regulates MDM2 autoubiquitination and prevents MDM2-mediated p53 ubiquitination and degradation; ZNF668 deficiency impairs DNA damage-induced p53 stabilization. Notably, ZNF668 effectively suppresses breast cancer cell proliferation and transformation in vitro and tumorigenicity in vivo. Consistently, ZNF668 knockdown readily transforms normal mammary epithelial cells. Together, our studies identify ZNF668 as a novel breast tumor suppressor gene that acts at least in part by regulating the stability and function of p53. (2) ZNF668 functions as a DNA repair protein by regulating histone acetylation. DNA repair proteins need to access the chromatin by chromatin modification or remodeling to use DNA template within chromatin. Dynamic posttranslational modifications of histones are critical for cells to relax chromatin in DNA repair. However, the precise underlying mechanism mediating enzymes responsible for these modifications and their recruitment to DNA lesions remains poorly understood. We observed ZNF668 depletion causes impaired chromatin relaxation as a result of impaired DNA-damage induced histone H2AX hyper-acetylation. This results in the decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after DNA damage, albeit with the presence of a functional ATM/ATR dependent DNA-damage signaling cascade. Importantly, the impaired loading of repair proteins and the defect in DNA repair in ZNF668-deficient cells can be counteracted by chromatin relaxation, indicating that the DNA-repair defect that was observed in the absence of ZNF668 is due to impeded chromatin accessibility at sites of DNA breaks. Our findings therefore identify ZNF668 as a key molecule that links chromatin relaxation with response to DNA damage in the control of DNA repair.

DEFINITION OF THE LANDSCAPE OF CHROMATIN STRUCTURE AT THE FRATAXIN GENE IN FRIEDREICH’S ATAXIA

Friedreich’s ataxia (FRDA) is caused by the transcriptional silencing of the frataxin (FXN) gene. FRDA patients have expansion of GAA repeats in intron 1 of the FXN gene in both alleles. A number of studies demonstrated that specific histone deacetylase inhibitors (HDACi) affect either histone modifications at the FXN gene or FXN expression in FRDA cells, indicating that the hyperexpanded GAA repeat may facilitate heterochromatin formation. However, the correlation between chromatin structure and transcription at the FXN gene is currently limited due to a lack of more detailed analysis. Therefore, I analyzed the effects of the hyperexpanded GAA repeats on transcription status and chromatin structure using lymphoid cell lines derived from FRDA patients. Using chromatin immunoprecipitation and quantitative PCR, I observed significant changes in the landscape of histone modifications in the vicinity of the GAA tract in FRDA cells relative to control cells. Similar epigenetic changes were observed in GFP reporter construct containing 560 GAA repeats. Further, I detected similar levels of FXN pre-mRNA at a region upstream of hyperexpanded GAA repeats in FRDA and control cells, indicating similar efficiency of transcription initiation in FRDA cells. I also showed that histone modifications associated with hyperexpanded GAA repeats are independent of transcription progression using the GFP reporter system. My data strongly support evidence that FXN deficiency in FRDA patients is consequence of defective transition from initiation to elongation of FXN transcription due to heterochromatin-like structures formed in the proximity of the hyperexpanded GAAs.

THE ROLE OF E2F1 IN THE RESPONSE TO DNA DOUBLE STRAND BREAKS

The importance of E2F transcription factors in the processes of proliferation and apoptosis are well established. E2F1, but not other E2F family members, is also phosphorylated and stabilized in response to various forms of DNA damage to regulate the expression of cell cycle and pro-apoptotic genes. E2F1 also relocalizes and forms foci at sites of DNA double-strand breaks but the function of E2F1 at sites of damage is still unknown. Here I reveal that E2F1 deficiency leads to increased spontaneous DNA break and impaired recovery following exposure to ionizing radiation. In response to DNA double-strand breaks, NBS1 phosphorylation and foci formation are defective in cells lacking E2F1, but NBS1 expression levels are unaffected. Moreover, it was observed that an association between NBS1 and E2F1 is increased in response to DNA damage, suggesting that E2F1 may promote NBS1 foci formation through a direct or indirect interaction at sites of DNA breaks. E2F1 deficient cells also display impaired foci formation of RPA and Rad51, which suggests a defect in DNA end resection and formation of single-stranded DNA at DNA double-strand breaks. I also found E2F1 status affects foci formation of the histone acetyltransferase GCN5 in response to DNA double-strand breaks. E2F1 is phosphorylated at serine 31 (serine 29 in mouse) by the ATM kinase as part of the DNA damage response. To investigate the importance of this event, our lab developed an E2F1 serine 29 mutant mouse model. I find that E2F1 serine 29 mutant cells show loss of E2F1 foci formation in response to DNA double-strand breaks. Furthermore, DNA repair and NBS1 foci formation are impaired in E2f1S29A/S29A cells. Taken together, my results indicate novel roles for E2F1 in the DNA damage response, which may directly promote DNA repair and genome maintenance.

Molecular basis of maturation promoting factor

Maturation promoting factor (MPF), which is functionally defined by its ability to induce Xenopus oocyte maturation, is an M phase (meiosis and mitosis) specific activity that is present in all species tested. It was hypothesized that MPF is a universal trigger of the interphase to M phase transition during the cell cycle. The current model for the molecular basis of MPF is that MPF is a protein kinase having the cdc2 protein as its catalytic subunit and is identical to the M phase-specific histone H1 kinase. In the present study, I have shown that more than just cdc2 kinase contributes to MPF activity, and M phase-specific H1 kinase is composed of at least two entities, instead of just cdc2 kinase. Therefore, the simple model of MPF = cdc2 kinase = M phase-specific H1 kinase should be ruled out.^ My study began with the characterization of the mitosis-specific monoclonal antibody MPM-2. MPM-2 reacts specifically with M phase cells from different species by recognizing a discrete set of proteins once they are phosphorylated at the G$\sb2$/M transition. I found that phosphorylation of MPM-2 antigens coincided with the appearance of MPF activity during oocyte maturation stimulated by progesterone. If MPM-2 was injected into oocytes before the stimulation, MPF activity failed to appear, and the oocytes could not mature. Furthermore, MPM-2 was able to deplete MPF activity from M phase extracts. These results identified MPM-2 as a probe that recognizes either MPF itself or a regulator of MPF.^ Since M phase-specific H1 kinase was believed to be identical to cdc2 kinase and MPF, I proceeded to determine whether MPM-2 recognized the M phase-specific H1 kinase. I found that MPM-2 did recognize an M phase-specific H1 kinase. However, this kinase was not cdc2 kinase. This kinase (MPM-2 kinase) is present in a latent form in immature oocytes and is activated in tandem with the activation of MPF during oocyte maturation. It appears to accelerate progesterone-induced oocyte maturation. Therefore, MPM-2 kinase may be a novel positive regulator of MPF activation.^ MPM-2 depletes MPF activity, but not cdc2 kinase activity. This discrepancy caused me to question the equivalency of MPF with cdc2 kinase. I found that when a high percentage of MPF activity was recovered from gel filtration of mature oocyte extract, the recovered MPF activity was due to two factors, cdc2 kinase and a factor recognized by MPM-2. This factor might activate and stabilize cdc2 kinase. Identification of this factor in the present study may contribute to the understanding of the autoactivation of MPF. ^

Functional analysis of histones H3 and H4 in Tup1 repression and in GCN5 activation

Tup1 forms a complex with Ssn6 in yeast. Ssn6-Tup1 complex is recruited via direct interactions with specific DNA binding proteins to a specific promoter region and mediates repression of several sets of genes including a-cell specific genes (asg) in $\alpha$ cells. It has been shown that repression of asgs also requires histone H4 and that Tup1 can directly interact with H3 and H4 in vitro. To address whether histone H3 is required for the repression of asgs, I have examined the effect of H3 and H4 mutations on the expression of a $\alpha$2-controlled LacZ reporter. Assay of $\beta$-glactosidase shows that mutations in either H3 or H4 cause a weak derepression of the reporter gene. Some double mutations result in a stronger derepression, while others do not. The H3 N-terminal deletion also leads to a slightly decreased expression of the reporter gene in $\alpha$ cells. Our data suggest that the N-termini of both H3 and H4 are cooperatively involved in the repression of a-cell specific genes in $\alpha$ cells, possibly through their interaction with Tup1.^ GCN5 was originally identified as a transcriptional regulator required to activate a subset of genes in yeast. Recently, it has been shown that GCN5 encodes the catalytic subunit of a nuclear histone acetyltransferase, providing the first direct link between histone acetylation and gene transcription. Recombinant Gcn5p (rGcn5p) exhibits a limited substrate specificity in vitro. However, neither the specificity of this enzyme in vivo nor the importance of particular acetylated residues to transcription or cell growth are well defined. In order to define the sites of histone acetylation mediated by Gcn5p in vivo and assess the significance of histone acetylation, more than 30 yeast strains have been constructed to bear specific H3 and/or H4 mutations in the presence or absence of GCN5 function. Our genetic data suggest that Gcn5p may have additional targets in vivo that are not identified as the targets of rGcn5p by previous studies. Western analysis using antibodies specifically recognizing particular acetylated isoforms of H3 and H4 led us to conclude that Gcn5p is necessary for full acetylation of multiple sites in both H3 and H4 in vivo. Consistent with these observations, rGcn5p still acetylates histones H3 and H4 bearing mutations either in H3 K14 or H4 K8,16, sites previously identified as the targets of acetylation by rGcn5p in H3 and H4. Our data also demonstrated that Gcn5p-mediated acetylation events are important for normal progression of the cell cycle and for transcriptional activation. Furthermore, a critical overall level of acetylation is essential for cell viability. ^