Upregulation of Reactive Oxygen Species During the Retrovirus Life Cycle and Their Roles in a Mutant of Moloney Murine Leukemia Virus, ts1-Mediated Neurodegeneration

Viral invasion of the central nervous system (CNS) and development of neurological symptoms is a characteristic of many retroviruses. The mechanism by which retrovirus infection causes neurological dysfunction has yet to be fully elucidated. Given the complexity of the retrovirus-mediated neuropathogenesis, studies using small animal models are extremely valuable. Our laboratory has used a mutant moloney murine leukemia retrovirus, ts1-mediated neurodegneration. We hypothesize that astrocytes play an important role in ts1-induced neurodegeneration since they are retroviral reservoirs and supporting cells for neurons. It has been shown that ts1 is able to infect astrocytes in vivo and in vitro. Astrocytes, the dominant cell population in the CNS, extend their end feet to endothelial cells and neuronal synapse to provide neuronal support. Signs of oxidative stress in the ts1-infected CNS have been well-documented from previous studies. After viral infection, retroviral DNA is generated from its RNA genome and integrated into the host genome. In this study, we identified the life cycle of ts1 in the infected astrocytes. During the infection, we observed reactive oxygen species (ROS) upregulations: one at low levels during the early infection phase and another at high levels during the late infection phase. Initially we hypothesized that p53 might play an important role in ts1-mediated astrocytic cell death. Subsequently, we found that p53 is unlikely to be involved in the ts1-mediated astrocytic cell death. Instead, p53 phosphorylation was increased by the early ROS upregulation via ATM, the protein encoded by the ataxia-telangiectasia (A-T) mutated gene. The early upregulation of p53 delayed viral gene expression by suppressing expression of the catalytic subunit of NADPH oxidase (NOX). We further demonstrated that the ROS upregulation induced by NOX activation plays an important role in establishing retroviral genome into the host. Inhibition of NOX decreased viral replication and delayed the onset of pathological symptoms in ts1-infected mice. These observations lead us to conclude that suppression of NOX not only prevents the establishment of the retrovirus but also decreases oxidative stress in the CNS. This study provides us with new perspectives on the retrovirus-host cell interaction and sheds light on retrovirus-induced neurodegeneration as a result of the astrocyte-neuron interaction.

ROLE OF THE GCN5 HISTONE ACETYLTRANSFERASE IN SPINOCEREBELLAR ATAXIA TYPE 7 AND IN IMMATURE NEURONS

Spinocerebellar Ataxia type 7 (SCA7) is a neurodegenerative disease caused by expansion of a CAG repeat encoding a polyglutamine tract in ATXN7, a component of the SAGA histone acetyltransferase (HAT) complex. Previous studies provided conflicting evidence regarding the effects of polyQ-ATXN7 on the activity of Gcn5, the HAT catalytic subunit of SAGA. Here I showed that reducing Gcn5 expression accelerates both cerebellar and retinal degeneration in a mouse model of SCA7. Deletion of Gcn5 in Purkinje cells in mice expressing wild type Atxn7, however, causes only mild ataxia and does not lead to the early lethality observed in SCA7 mice. Reduced Gcn5 expression strongly enhances retinopathy in SCA7 mice, but does not affect the transcriptional targets of Atxn7, as expression of these genes is not further altered by Gcn5 depletion. These findings demonstrate that loss of Gcn5 functions can contribute to the time of onset and severity of SCA7 phenotypes, but suggest that non-transcriptional functions of SAGA may play a role in neurodegeneration in this disease.

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. ^

cAMP-dependent protein kinase and heterologous desensitization of the adenylyl cyclase

Previous experiments had shown no differences in desensitization in cells with mutations of the adenylyl cyclase or the cAMP-dependent protein kinase and had ruled out this kinase as a mediator of desensitization; however, the assays of adenylyl cyclase had been made at high concentrations of free magnesium. The work presented in this dissertation documents a role for cAMP-dependent protein kinase which became apparent with assays at low concentrations of free magnesium. (1) The adenylyl cyclase in membranes from wild type S49 lymphoma cells showed substantial desensitization after incubation of the intact cells with low concentrations of epinephrine (5-20 nM). This desensitization was heterologous, that is it reduced the subsequent responses of the adenylyl cyclase to both epinephrine and prostaglandin-E$\sb1$. (2) The adenylyl cyclase in membranes of S49 cyc$\sp-$ cells, which do not make cAMP in response to hormones, and S49 kin$\sp-$ cells, which lack cAMP-dependent protein kinase activity, showed no heterologous desensitization following incubation of the intact cells with low concentrations of hormones. (3) Heterologous desensitization of the adenylyl cyclase was induced by incubations of wild type cells with forskolin, which activates the adenylyl cyclase downstream of the hormone receptors, or dibutyryl-cAMP, which activates the cAMP-dependent protein kinase directly. (4) Site-directed mutagenesis was used to delete the cAMP-dependent protein kinase consensus phosphorylation sequences on the $\beta$-adrenergic receptor. Heterologous desensitization occurred in intact L-cells expressing the wild type receptor or the receptor lacking the C-terminal phosphorylation site; however, only homologous desensitization occurred when the phosphorylation site on the third intracellular loop of the receptor was deleted. (5) To test directly the effects of cAMP-dependent protein kinase on the adenylyl cyclase the catalytic subunit of the kinase was purified from bovine heart and incubated with adenylyl cyclase in plasma membrane preparations. In this cell-free system the kinase caused rapid heterlogous reductions of the responsiveness of the S49 wild type adenylyl cyclase. Additionally, the adenylyl cyclase in kin$\sp-$ membranes, which showed only homologous desensitization in the intact cell, was desensitization by cell-free incubation with the kinase.^ The epinephrine responsiveness was not affected in L-cell membranes expressing the $\beta$-adrenergic receptor lacking the cAMP-dependent protein kinase consensus sequence on the third intracellular loop. ^

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. ^

Whole genome RNAi screens reveal a critical role of REV3 in coping with replication stress

REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR-inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3-deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU-induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU-treated S-phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.

Activating BRAF and PIK3CA mutations cooperate to promote anaplastic thyroid carcinogenesis

UNLABELLED Thyroid malignancies are the most common type of endocrine tumors. Of the various histologic subtypes, anaplastic thyroid carcinoma (ATC) represents a subset of all cases but is responsible for a significant proportion of thyroid cancer-related mortality. Indeed, ATC is regarded as one of the more aggressive and hard to treat forms of cancer. To date, there is a paucity of relevant model systems to critically evaluate how the signature genetic abnormalities detected in human ATC contribute to disease pathogenesis. Mutational activation of the BRAF protooncogene is detected in approximately 40% of papillary thyroid carcinoma (PTC) and in 25% of ATC. Moreover, in ATC, mutated BRAF is frequently found in combination with gain-of-function mutations in the p110 catalytic subunit of PI3'-Kinase (PIK3CA) or loss-of-function alterations in either the p53 (TP53) or PTEN tumor suppressors. Using mice with conditional, thyrocyte-specific expression of BRAF(V600E), we previously developed a model of PTC. However, as in humans, BRAF(V600E)-induced mouse PTC is indolent and does not lead to rapid development of end-stage disease. Here, we use mice carrying a conditional allele of PIK3CA to demonstrate that, although mutationally activated PIK3CA(H1047R) is unable to drive transformation on its own, when combined with BRAF(V600E) in thyrocytes, this leads to development of lethal ATC in mice. Combined, these data demonstrate that the BRAF(V600E) cooperates with either PIK3CA(H1074R) or with silencing of the tumor-suppressor PTEN, to promote development of anaplastic thyroid carcinoma. IMPLICATIONS This genetically relevant mouse model of ATC will be an invaluable platform for preclinical testing of pathway-targeted therapies for the prevention and treatment of thyroid carcinoma.

RNA binding characteristics and overall topology of the vaccinia virus polyadenylation complex

mRNA 3′ polyadenylation is central to mRNA biogenesis in prokaryotes and eukaryotes, and is implicated in numerous aspects of mRNA metabolism, including efficiency of mRNA export from the nucleus, message stability, and initiation of translation. However, due to the great complexity of the eukaryotic polyadenylation apparatus, the mechanisms of RNA 3 ′ end processing have remained elusive. Although the RNA processing reactions leading to polyadenylated messenger RNA have been studied in many systems, and much progress has been made, a complete understanding of the biochemistry of the poly(A) polymerase enzyme is still lacking. My research uses Vaccinia virus as a model system to gain a better understanding of this complicated polyadenylation process, which consist of RNA binding, catalysis and polymerase translocation. ^ Vaccinia virus replicates in the cytoplasm of its host cell, so it must employ its own poly(A) polymerase (PAP), a heterodimer of two virus encoded proteins, VP55 and VP39. VP55 is the catalytic subunit, adding 30 adenylates to a non-polyadenylated RNA in a rapid processive manner before abruptly changing to a slow, non-processive mode of adenylate addition and dissociating from the RNA. VP39 is the stimulatory subunit. It has no polyadenylation catalytic activity by itself, but when associated with VP55 it facilitates the semi-processive synthesis of tails several hundred adenylates in length. ^ Oligonucleotide selection and competition studies have shown that the heterodimer binds a minimal motif of (rU)2 (N)25 U, the “heterodimer binding motif”, within an oligonucleotide, and its primer selection for polyadenylation is base-type specific. ^ Crosslinking studies using photosensitive uridylate analogs show that within a VP55-VP39-primer ternary complex, VP55 comes into contact with all three required uridylates, while VP39 only contacts the downstream uridylate. Further studies, using a backbone-anchored photosensitive crosslinker show that both PAP subunits are in close proximity to the downstream −10 to −21 region of 50mer model primers containing the heterodimer binding motif. This equal crosslinking to both subunits suggests that the dimerization of VP55 and VP39 creates either a cleft or a channel between the two subunits through which this region of RNA passes. ^ Peptide mapping studies of VP39 covalently crosslinked to the oligonucleotide have identified residue R107 as the amino acid in close proximity to the −10 uridylate. This helps us project a conceptual model onto the known physical surface of this subunit. In the absence of any tertiary structural data for VP55, we have used a series of oligonucleotide selection assays, as well as crosslinking, nucleotide transfer assays, and gel shift assays to gain insight into the requirements for binding, polyadenylation and translocation. Collectively, these data allow us to put together a comprehensive model of the structure and function of the polyadenylation ternary complex consisting of VP39, VP55 and RNA. ^

ATP- and carbachol -stimulated 1,2-diacylglycerol production during sensitization of adenylyl cyclase

Stimulation of LM5 cells with the phorbol ester 4$\beta$-phorbol 12-myristate 13-acetate (PMA), causes a 2-4 fold sensitization of hormonally-stimulated adenylyl cyclase (AC) activity. This effect is thought to be due to protein kinase C (PKC)-mediated phosphorylation of either G$\sb{\rm i}$ or the catalytic subunit of AC. PKC are components of the phosphatidylinositol-4,5-bisphosphate phospholipase C (PIP$\sb2$-PLC) pathway. The currently accepted model of this pathway is that its activation by an agonist results in the production of inositol 1,4,5-triphosphate (IP$\sb3$) which causes Ca$\sp{++}$ mobilization, and 1,2-diacylglycerols (DAG) which activate PKC. Based on this model, we predicted that stimulation of purinergic and muscarinic receptors with the agonists ATP and carbachol (CCh), respectively in the LM5 cells, should sensitize AC. Surprisingly we found that only stimulation of the purinergic receptors in these cells caused a sensitization of PGE$\sb1$-stimulated AC measured in cell-free assays.^ We hypothesized that ATP-and CCh-stimulated differential DAG production contributes to the effectiveness of these two agonists to sensitize PGE$\sb1$-stimulated AC activity. To test this hypothesis directly, we performed a combined high-performance liquid chromatography and gas-liquid chromatography analysis of the DAG produced in the LM5 cells in response to stimulation with ATP and CCh.^ We found that both ATP and CCh increased levels of 23 species of DAG. Relative to the control levels (0.261 nmol DAG/100 nmol phospholipid) the CCh-induced increase in DAG levels was 280% (0.738 $\pm$ 0.051 nmol DAG/100 nmol phospholipid) whereas the ATP-induced levels increased 180% (0.441 t 0.006 nmol DAG/100 nmol phospholipid). Neither agonist created new species or eliminated the existing ones. The major species which comprised $\approx$50% of the total cellular DAG in all of the groups were 16:0-18:1, 18:0-18:1, 18:1-18:1, and 18:0-20:4. CCh was more effective than ATP at stimulating these major DAG species.^ It is concluded that factor(s) other than DAG contribute(s) to the differences between ATP-and CCh-sensitization of PGE$\sb1$-stimulated AC activity in the LM5 cells. ^

Modulation of high voltage -activated calcium channels by phosphorylation

High voltage-activated (HVA) calcium channels from rat brain and rabbit heart are expressed in Xenopus laevis oocytes and their modulation by protein kinases studied. A subtype of the HVA calcium current expressed by rat brain RNA is potentiated by the phospholipid- and calcium-dependent protein kinase (PKC). The calcium channel clone $\alpha\sb{\rm1C}$ from rabbit heart is modulated by the cAMP-dependent protein kinase (PKA), and another factor present in the cytoplasm.^ The HVA calcium channels from rat brain do not belong to the L-type subclass since they are insensensitive to dihydropyridine (DHP) agonists and antagonists. The expressed currents do contain a N-type fraction which is identified by inactivation at depolarized potentials, and a P-type fraction as defined by blockade by the venom of the funnel web spider Agelenopsis Aperta. A non N-type fraction of this current is potentiated, by using phorbol esters to activate PKC. This residual fraction of current resembles the newly described Q-type channel from cerebellar granule cells in its biophysical properties, and potentiation by activation of PKC.^ The $\alpha\sb{\rm1C}$ clone from rabbit heart is expressed in oocytes and single-channel currents are measured using the cell-attached and cell-excised patch clamp technique. The single-channel current runs down within two minutes after patch excision into normal saline bath solution. The catalytic subunit of PKA + MgATP is capable of reversing this rundown for over 15 minutes. There also appears to be an additional factor present in the cytoplasm necessary for channel activity as revealed in experiments where PKA failed to prevent rundown.^ These data are important in that these types of channels are involved in synaptic transmission at many different types of synapses. The mammalian synapse is not accessible for these types of studies, however, the oocyte expression system allows access to HVA calcium channels for the study of their modulation by phosphorylation. ^

Regulation of v-Mos kinase activity by phosphorylation

In this thesis, I investigated the effect of cylic AMP-dependent protein kinase (PKA) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of the PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37$\rm\sp{v-mos}$ in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Ser-263 was identified as a residue that is normally phosphorylated at a very low level but whose phosphorylation is dramatically increased upon forskolin treatment. Consistent with the inhibitory role of Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. Based on our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation could be explained at least in part by its inhibition of Mos kinase.^ Combining tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies, I identified Ser-56 as the major in vivo phosphorylation site on v-Mos. I studied the interrelationship between Ser-34 and Ser-56 phosphorylation in regulating v-Mos function. After site-directed mutagenesis to substitute serine residues with alanine or glutamic acid in different combinations to mimick unphosphorylated and phosphorylated serines respectively, various v-Mos mutants were expressed in COS-1 cells. As expected, Ala-34 mutant of v-Mos had very low (less 5% of wild type) kinase activity. The Ala-56 mutant had kinase activity 50% that of wild type. Surprisingly, the Ala-34 Ala-56 double mutant and the Ala-56 mutant exhibited identical kinase activity. On the other hand, Ala-34 Glu-56 double mutant had reduced kinase activity comparable to Ala-34 mutant. These results suggest that the phosphorylation at Ser-56 may serve to inhibit the activation of newly synthesized Mos protein. As predicted from Xenopus c-Mos studies, Glu-34 mutant of v-Mos was highly active (125% that of wild type). Interestingly, consistant with the model involving an inhibitory role of Ser-56 phosphorylation, the Glu-34 Glu-56 double mutant was totally inactive as a kinase. Moreover in my experiments, there was a perfect correlation between the level of v-Mos kinase activity of various mutants and their transforming activity. The latter is dependent upon MEK1 phosphorylation/ activation in v-mos transformed cells. Residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos. Therefore, the cytostatic factor function of c-Mos may be regulated in the same manner as v-Mos kinase activity.^ It has been known that v-mos transforms cells by affecting G1 phase progression of the cell cycle. Here I showed that mos induces cyclin D1 expression in mos transformed NIH 3T3 cells and NRK 6m2 cells, and this induced level was found to be unaffected by serum starvation. Consequently, cyclin D1-Cdk4 and cyclin E-Cdk2 activities increase, and retinoblastoma protein is hyperphosphorylated. Based on studies from several laboratories, these findings suggest that increased amount of cyclin D1-Cdk4 complexes ties up the limited amount of cyclin E-Cdk2 inhibitors (e.g. p27), causing the activation of cyclin E-Cdk2. My results indicate that activation of key cell cycle regulators of G1 phase may be important for cellular transformation by mos. (Abstract shortened by UMI.) ^

Regulations of SOX9 activity during chondrogenesis

Sox9 is a transcription factor required for chondrocyte differentiation and cartilage formation. In an effort to identify SOX9 interacting protein(s), we screened a chondrocyte cDNA library with a modified yeast two-hybrid method, Son of Sevenless (SOS) recruitment system (SRS). The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA-Cα) and a new long form of c-Maf transcription factor (Lc-Maf) were found to interact specifically with SOX9. We showed here that two PKA phosphorylation consensus sites of SOX9 could be phosphorylated by PKA in vitro as well as in vivo. PKA phosphorylation of SOX9 increases its DNA binding and transcriptional activities on a Col2a1 chondrocyte-specific enhancer. Mutations of these two PKA phosphorylation sites markedly decreased the activation of SOX9 by PKA. ^ To test whether parathyroid hormone-related peptide (PTHrP) signaling results in SOX9 phosphorylation, we generated a phosphospecific antibody that specifically recognizes SOX9 that is phosphorylated at serine 181 (S 181) one of the two consensus PKA phosphorylation sites. Addition of PTHrP to COS7 cells cotransfected with SOX9 and PTH/PTHrP receptor strongly increased phosphorylation of SOX9 at S181; this phosphorylation was blocked by a PKA-specific inhibitor. In similar experiments we showed that PTHrP increased the activity of a SOX9-dependent Col2a1 enhancer. This increase in activity was abolished when a SOX9 mutant was used containing serine-to-alanine substitution in the two consensus PKA phosphorylation sites of SOX9. Using our phosphospecific SOX9 antibody we showed by immunohistochemistry of mouse embryos that Sox9 phosphorylated at S181 was localized almost exclusively in the pre-hypertrophic zone of the growth plate, an area corresponding to the major site of expression of PTH/PTHrP receptor. In contrast, no phosphorylation of Sox9 at S181 was detected in growth plates of PTH/PTHrP receptor null mutant mice. Sox9, regardless of phosphorylation state, was present in all chondrocytes of both genotypes except in hypertrophic chondrocytes. Thus, Sox9 is a target of PTHrP signaling and the PTHrP-dependent phosphorylation of SOX9 enhances its transcriptional activity. ^ In order to investigate the in vivo function of Sox9 phosphorylation by PKA, we are generating a mouse model of mutant Sox9 harboring point mutations in two PKA phosphorylation sites. Preliminary results indicated that heterozygous mice containing half amount of mutant Sox9 that can not be phosphorylated by PKA have normal skeletal phenotype and homozygous mice are being generated. ^ Lc-Maf encodes an extra ten amino acids at the carboxyl terminus of c-Maf and contains a completely different 3′ untranslated region. The interaction between SOX9 and Lc-Maf was further confirmed by co-immunoprecipitation and GST-pull down assays, which mapped the interacting domains of SOX9 to HMG DNA binding domain and that of Lc-Maf to basic leusine zipper motif. In situ hybridizations showed that RNA of Lc-Maf coexpressed with those of Sox9 and Col2a1 in areas of mesenchymal condensation during the early stages of mouse embryo development. A DNA binding site of Lc-Maf was identified at the 5′ part of a 48-bp Col2a1 enhancer element near the HMG binding site of SOX9. Lc-Maf and SOX9 synergistically activated a luciferase reporter plasmid containing a Col2al enhancer and increased the transcription of endogenous Col2a1 gene. In summary, Lc-Maf is the first identified SOX9-interating protein during chondrogenesis and may be an important activator of Col2a1 gene. ^

Essential role of germinal vesicle material in the meiotic cell cycle of Xenopus oocytes

In almost all animal species, immature oocytes are arrested naturally in the first meiotic prophase, with a large nucleus called the germinal vesicle. A number of previous studies showed that both activation of maturation/M phase-promoting factor (MPF) (assayed by semiquantitative cytological methods) and some other maturational events occur essentially normally in enucleated oocytes from many amphibian species and mice. Hence, for nearly three decades, it has generally been believed that nuclear material is dispensable for MPF activation and the meiotic cell cycle in vertebrate oocytes. Here, we have challenged this view by examining the histone H1 kinase activities and the molecular forms of MPF in experimentally manipulated Xenopus oocytes. We show that oocytes injected with nuclear material undergo much more rapid MPF activation and maturation than uninjected control oocytes. Conversely, enucleated oocytes, unlike nucleated counterparts, undergo only weak MPF activation in meiosis I and no detectable MPF reactivation in meiosis II, the latter accompanying inhibitory tyrosine phosphorylation of cdc2 kinase, the catalytic subunit of MPF. These results argue strongly that nuclear material is indispensable for the meiotic cell cycle, particularly MPF reactivation (or cdc2 tyrosine dephosphorylation) on entry into meiosis II, in Xenopus oocytes. The classical and general view may thus need reconsideration.

Identification of 5′-deoxyribose phosphate lyase activity in human DNA polymerase γ and its role in mitochondrial base excision repair in vitro

Mitochondria have been proposed to possess base excision repair processes to correct oxidative damage to the mitochondrial genome. As the only DNA polymerase (pol) present in mitochondria, pol γ is necessarily implicated in such processes. Therefore, we tested the ability of the catalytic subunit of human pol γ to participate in uracil-provoked base excision repair reconstituted in vitro with purified components. Subsequent to actions of uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, human pol γ was able to fill a single nucleotide gap in the presence of a 5′ terminal deoxyribose phosphate (dRP) flap. We report here that the catalytic subunit of human pol γ catalyzes release of the dRP residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The heat sensitivity of this activity suggests the dRP lyase function requires a three-dimensional protein structure. The dRP lyase activity does not require divalent metal ions, and the ability to trap covalent enzyme-DNA complexes with NaBH4 strongly implicates a Schiff base intermediate in a β-elimination reaction mechanism.

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity

Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.