Epigenetic regulation of 5α reductase-1 underlies adaptive plasticity of reproductive function and pubertal timing.

BACKGROUND: Women facing increased energetic demands in childhood commonly have altered adult ovarian activity and shorter reproductive lifespan, possibly comprising a strategy to optimize reproductive success. Here, we sought to understand the mechanisms of early-life programming of reproductive function, by integrating analysis of reproductive tissues in an appropriate mouse model with methylation analysis of proxy tissue DNA in a well-characterized population of Bangladeshi migrants in the UK. Bangladeshi women whose childhood was in Bangladesh were found to have later pubertal onset and lower age-matched ovarian reserve than Bangladeshi women who grew-up in England. Subsequently, we aimed to explore the potential relevance to the altered reproductive phenotype of one of the genes that emerged from the screens. RESULTS: Of the genes associated with differential methylation in the Bangladeshi women whose childhood was in Bangladesh as compared to Bangladeshi women who grew up in the UK, 13 correlated with altered expression of the orthologous gene in the mouse model ovaries. These mice had delayed pubertal onset and a smaller ovarian reserve compared to controls. The most relevant of these genes for reproductive function appeared to be SRD5A1, which encodes the steroidogenic enzyme 5α reductase-1. SRD5A1 was more methylated at the same transcriptional enhancer in mice ovaries as in the women's buccal DNA, and its expression was lower in the hypothalamus of the mice as well, suggesting a possible role in the central control of reproduction. The expression of Kiss1 and Gnrh was also lower in these mice compared to controls, and inhibition of 5α reductase-1 reduced Kiss1 and Gnrh mRNA levels and blocked GnRH release in GnRH neuronal cell cultures. Crucially, we show that inhibition of this enzyme in female mice in vivo delayed pubertal onset. CONCLUSIONS: SRD5A1/5α reductase-1 responds epigenetically to the environment and its downregulation appears to alter the reproductive phenotype. These findings help to explain diversity in reproductive characteristics and how they are shaped by early-life environment and reveal novel pathways that might be targeted to mitigate health issues caused by life-history trade-offs.

Epigenetic repression of gonadotropin gene expression via a GnRH-mediated DNA delivery system.

The reproductive axis is activated by gonadotropin-releasing hormone (GnRH), which stimulates the pituitary gonadotropes to secrete hormones that drive gonadal function and steroidogenesis. Thus repression of this axis, which is conserved across mammals and sexes, can reduce steroid levels and/or prevent reproduction. Steroid-dependent pathologies, including various cancers, are commonly treated with GnRH super-analogs which have long-term side-effects, while humane solutions for controlling reproduction in domestic and wild animal populations are lacking. GnRH-conjugated toxins are undergoing clinical trials for GnRHR-expressing cancer cells, and have been examined for gonadotrope ablation in animals, but showed low and/or transient effects and administration of toxins has many potential complications. Here we exploit GnRH targeting to gonadotropes to deliver DNA encoding an effector that induces gonadotropin gene repressive epigenetic modifications which are perpetuated over time. Several layers of specificity are endowed through targeting to GnRHR-expressing cells and due to local cleavage of the peptide packaging the DNA; the DNA-encoded effector is expressed and directed to the target genes by the DNA binding domain of a highly specific transcription factor. This design has multiple advantages over existing methods of shutting down the reproductive axis, and its modular design should allow adaptation for broad applications.

Sensitivity of pituitary gonadotropes to hyperglycemia leads to epigenetic aberrations and reduced follicle-stimulating hormone levels.

The connection between metabolism and reproductive function is well recognized, and we hypothesized that the pituitary gonadotropes, which produce luteinizing hormone and follicle-stimulating hormone (FSH), mediate some of the effects directly via insulin-independent glucose transporters, which allow continued glucose metabolism during hyperglycemia. We found that glucose transporter 1 is the predominant glucose transporter in primary gonadotropes and a gonadotrope precursor-derived cell line, and both are responsive to culture in high glucose; moreover, metabolite levels were altered in the cell line. Several of the affected metabolites are cofactors for chromatin-modifying enzymes, and in the gonadotrope precursor-derived cell line, we recorded global changes in histone acetylation and methylation, decreased DNA methylation, and increased hydroxymethylation, some of which did not revert to basal levels after cells were returned to normal glucose. Despite this weakening of epigenetic-mediated repression seen in the model cell line, FSH ß-subunit ( Fshb) mRNA levels in primary gonadotropes were significantly reduced, apparently due in part to increased autocrine/paracrine effects of inhibin. However, unlike thioredoxin interacting protein and inhibin subunit α, Fshb mRNA levels did not recover after the return of cells to normal glucose. The effect on Fshb expression was also seen in 2 hyperglycemic mouse models, and levels of circulating FSH, required for follicle growth and development, were reduced. Thus, hyperglycemia seems to target the pituitary gonadotropes directly, and the likely extensive epigenetic changes are sensed acutely by Fshb. This scenario would explain clinical findings in which, even after restoration of optimal blood glucose levels, fertility often remains adversely affected. However, the relative accessibility of the pituitary provides a possible target for treatment, particularly crucial in the young in which hyperglycemia is increasingly common and fertility most relevant.-Feldman, A., Saleh, A., Pnueli, L., Qiao, S., Shlomi, T., Boehm, U., Melamed, P. Sensitivity of pituitary gonadotropes to hyperglycemia leads to epigenetic aberrations and reduced follicle-stimulating hormone levels.

An epigenetic switch repressing Tet1 in gonadotropes activates the reproductive axis.

The TET enzymes catalyze conversion of 5-methyl cytosine (5mC) to 5-hydroxymethyl cytosine (5hmC) and play important roles during development. TET1 has been particularly well-studied in pluripotent stem cells, but Tet1-KO mice are viable, and the most marked defect is abnormal ovarian follicle development, resulting in impaired fertility. We hypothesized that TET1 might play a role in the central control of reproduction by regulating expression of the gonadotropin hormones, which are responsible for follicle development and maturation and ovarian function. We find that all three TET enzymes are expressed in gonadotrope-precursor cells, but Tet1 mRNA levels decrease markedly with completion of cell differentiation, corresponding with an increase in expression of the luteinizing hormone gene, Lhb We demonstrate that poorly differentiated gonadotropes express a TET1 isoform lacking the N-terminal CXXC-domain, which represses Lhb gene expression directly and does not catalyze 5hmC at the gene promoter. We show that this isoform is also expressed in other differentiated tissues, and that it is regulated by an alternative promoter whose activity is repressed by the liganded estrogen and androgen receptors, and by the hypothalamic gonadotropin-releasing hormone through activation of PKA. Its expression is also regulated by DNA methylation, including at an upstream enhancer that is protected by TET2, to allow Tet1 expression. The down-regulation of TET1 relieves its repression of the methylated Lhb gene promoter, which is then hydroxymethylated and activated by TET2 for full reproductive competence.

Mitogen- and stress-activated protein kinase 1 is required for gonadotropin-releasing hormone-mediated activation of gonadotropin α-subunit expression.

Pituitary gonadotropin hormones are regulated by gonadotropin-releasing hormone (GnRH) via MAPK signaling pathways that stimulate gene transcription of the common α-subunit (Cga) and the hormone-specific ß-subunits of gonadotropin. We have reported previously that GnRH-induced activities at these genes include various histone modifications, but we did not examine histone phosphorylation. This modification adds a negative charge to residues of the histone tails that interact with the negatively charged DNA, is associated with closed chromatin during mitosis, but is increased at certain genes for transcriptional activation. Thus, the functions of this modification are unclear. We initially hypothesized that GnRH might induce phosphorylation of Ser-10 in histone 3 (H3S10p) as part of its regulation of gonadotropin gene expression, possibly involving cross-talk with H3K9 acetylation. We found that GnRH increases the levels of both modifications around the Cga gene transcriptional start site and that JNK inhibition dramatically reduces H3S10p levels. However, this modification had only a minor effect on Cga expression and no effect on H3K9ac. GnRH also increased H3S28p and H3K27ac levels and also those of activated mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 inhibition dramatically reduced H3S28p levels in untreated and GnRH-treated cells and also affected H3K27ac levels. Although not affecting basal Cga expression, MSK1/2 inhibition repressed GnRH activation of Cga expression. Moreover, ChIP analysis revealed that GnRH-activated MSK1 targets the first nucleosome just downstream from the TSS. Given that the elongating RNA polymerase II (RNAPII) stalls at this well positioned nucleosome, GnRH-induced H3S28p, possibly in association with H3K27ac, would facilitate the progression of RNAPII.

RNA transcribed from a distal enhancer is required for activating the chromatin at the promoter of the gonadotropin α-subunit gene.

Since the discovery that many transcriptional enhancers are transcribed into long noncoding RNAs termed "enhancer RNAs" (eRNAs), their putative role in enhancer function has been debated. Very recent evidence has indicted that some eRNAs play a role in initiating or activating transcription, possibly by helping recruit and/or stabilize binding of the general transcription machinery to the proximal promoter of their target genes. The distal enhancer of the gonadotropin hormone α-subunit gene, chorionic gonadotropin alpha (Cga), is responsible for Cga cell-specific expression in gonadotropes and thyrotropes, and we show here that it encodes two bidirectional nonpolyadenylated RNAs whose levels are increased somewhat by exposure to gonadotropin-releasing hormone but are not necessarily linked to Cga transcriptional activity. Knockdown of the more distal eRNA led to a drop in Cga mRNA levels, initially without effect on the forward eRNA levels. With time, however, the repression on the Cga increased, and the forward eRNA levels were suppressed also. We demonstrate that the interaction of the enhancer with the promoter is lost after eRNA knockdown. Dramatic changes also were seen in the chromatin, with an increase in total histone H3 occupancy throughout this region and a virtual loss of histone H3 Lys 4 trimethylation at the promoter following the eRNA knockdown. Moreover, histone H3 Lys 27 (H3K27) acetylation, which was found at both enhancer and promoter in wild-type cells, appeared to have been replaced by H3K27 trimethylation at the enhancer. Thus, the Cga eRNA mediates the physical interaction between these genomic regions and determines the chromatin structure of the proximal promoter to allow gene expression.

Gonadotropin gene transcription is activated by menin-mediated effects on the chromatin.

The genes encoding luteinizing hormone and follicle stimulating hormone are activated by gonadotropin-releasing hormone (GnRH), and we hypothesized that this involves GnRH-induction of various histone modifications. At basal conditions in an immature gonadotrope-derived cell line, the hormone-specific ß-subunit gene promoters are densely packed with histones, and contain low levels of H3K4 trimethylation (H3K4me3). GnRH both induces this modification and causes histone loss, creating a more active chromatin state. The H3K4me3 appears to be mediated by menin and possibly catalyzed by the menin-mixed-lineage leukemia (MLL) 1/2 methyl transferase complex, as inhibition of MLL recruitment or menin knockdown reduced gene expression and the levels of H3K4me3 on all three promoters. Menin recruitment to the ß-subunit gene promoters is increased by GnRH, possibly involving transcription factors such as estrogen receptor α and/or steroidogenic factor 1, with which menin interacts. Menin also interacts with ring finger protein 20, which ubiquitylates H2BK120 (H2BK120ub), which was reported to be a pre-requisite for H3K4me3 at various gene promoters. Although levels of H2BK120ub are increased by GnRH in the coding regions of these genes, levels at the promoters do not correlate with those of H3K4me3, nor with gene expression, suggesting that H3K4me3 is not coupled to H2BK120ub in transcriptional activation of these genes.

The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner.

BACKGROUND: mRNA binding proteins (RBPs) constitute 10-15% of the eukaryotic proteome and play important part in post-transcriptional regulation of gene expression. Due to the instability of RNA and the transient nature its interaction with RBPs, identification of novel RBPs is a significant challenge. Recently, a novel methodology for RBP purification and identification (termed RaPID) was presented, which allows high affinity purification of RBPs while associated with mRNA in vivo. RESULTS: We performed a RaPID screen for proteins that interact with PMP1 mRNA in order to identify novel mRNA binding proteins. PMP1 mRNA was tagged in its 3' UTR with multiple MS2 loops and co-expressed with MS2-binding protein fused to streptavidin binding protein (SBP). RNA-protein complexes were cross-linked in vivo and isolated through streptavidin beads. The eluted proteins were subjected to mass spectroscopy analysis. The screen identified many proteins, about half of them were previously shown to bind RNA. We focused on eEF3 (YEF3), an essential translation elongation factor that interacts with ribosomes. Purification of TAP-tagged Yef3 with its associated RNAs confirmed that the native PMP1 transcript is associated with it. Intriguingly, high association with Yef3-TAP was observed when purification was performed in the presence of EDTA, and with PMP1 that contains stop codons immediately downstream to the initiation codon. Furthermore, high association was observed with a transcript containing only the 3' UTR of PMP1. Complementary, RaPID isolation of MS2-tagged 3' UTRs with their associated proteins revealed that Yef3 can efficiently interact with these regions. CONCLUSIONS: This study identifies many novel proteins that interact with PMP1 mRNA. Importantly, the elongation factor Yef3 was found to interact with mRNA in non-coding regions and in a translation independent manner. These results suggest an additional, non-elongation function for this factor.

Srd5a1 is Differentially Regulated and Methylated During Prepubertal Development in the Ovary and Hypothalamus.

5α-reductase-1 catalyzes production of various steroids, including neurosteroids. We reported previously that expression of its encoding gene, Srd5a1, drops in murine ovaries and hypothalamic preoptic area (POA) after early-life immune stress, seemingly contributing to delayed puberty and ovarian follicle depletion, and in the ovaries the first intron was more methylated at two CpGs. Here, we hypothesized that this CpG-containing locus comprises a methylation-sensitive transcriptional enhancer for Srd5a1. We found that ovarian Srd5a1 mRNA increased 8-fold and methylation of the same two CpGs decreased up to 75% between postnatal days 10 and 30. Estradiol (E2) levels rise during this prepubertal stage, and exposure of ovarian cells to E2 increased Srd5a1 expression. Chromatin immunoprecipitation in an ovarian cell line confirmed ESR1 binding to this differentially methylated genomic region and enrichment of the enhancer modification, H3K4me1. Targeting dCas9-DNMT3 to this locus increased CpG2 methylation 2.5-fold and abolished the Srd5a1 response to E2. In the POA, Srd5a1 mRNA levels decreased 70% between postnatal days 7 and 10 and then remained constant without correlation to CpG methylation levels. Srd5a1 mRNA levels did not respond to E2 in hypothalamic GT1-7 cells, even after dCas9-TET1 reduced CpG1 methylation by 50%. The neonatal drop in POA Srd5a1 expression occurs at a time of increasing glucocorticoids, and treatment of GT1-7 cells with dexamethasone reduced Srd5a1 mRNA levels; chromatin immunoprecipitation confirmed glucocorticoid receptor binding at the enhancer. Our findings on the tissue-specific regulation of Srd5a1 and its methylation-sensitive control by E2 in the ovaries illuminate epigenetic mechanisms underlying reproductive phenotypic variation that impact life-long health.

SpRET: highly sensitive and reliable spectral measurement of absolute FRET efficiency.

Contemporary research aims to understand biological processes not only by identifying participating proteins, but also by characterizing the dynamics of their interactions. Because Förster's Resonance Energy Transfer (FRET) is invaluable for the latter undertaking, its usage is steadily increasing. However, FRET measurements are notoriously error-prone, especially when its inherent efficiency is low, a not uncommon situation. Furthermore, many FRET methods are either difficult to implement, are not appropriate for observation of cellular dynamics, or report instrument-specific indices that hamper communication of results within the scientific community. We present here a novel comprehensive spectral methodology, SpRET, which substantially increases both the reliability and sensitivity of FRET microscopy, even under unfavorable conditions such as weak fluorescence or the presence of noise. While SpRET overcomes common pitfalls such as interchannel crosstalk and direct excitation of the acceptor, it also excels in removal of autofluorescence or background contaminations and in correcting chromatic aberrations, often overlooked factors that severely undermine FRET experiments. Finally, SpRET quantitatively reports absolute rather than relative FRET efficiency values, as well as the acceptor-to-donor molar ratio, which is critical for full and proper interpretation of FRET experiments. Thus, SpRET serves as an advanced, improved, and powerful tool in the cell biologist's toolbox.

Proliferating primary pituitary cells as a model for studying regulation of gonadotrope chromatin and gene expression.

The chromatin organization of the gonadotropin gene promoters in the pituitary gonadotropes plays a major role in determining how these gene are activated, but is difficult to study because of the low numbers of these cells in the pituitary gland. Here, we set out to create a cell model to study gonadotropin chromatin, and found that by optimizing cell culture conditions, we can maintain stable proliferating cultures of primary non-transformed gonadotrope cells over weeks to months. Although expression of the gonadotropin genes drops very low, these cells are enriched in gonadotrope markers and respond to GnRH. Furthermore, >85% of the cells contained Lhb and/or Fshb mature transcripts; though these were virtually restricted to the nuclei. The gonadotropes were harvested initially due to expression of dTOMATO, following activation of Cre recombinase by the Gnrhr promoter. Over 6 mo in culture, a similar proportion of the recombined DNA was maintained (i.e. cells derived from the original gonadotropes or having acquired Gnrhr-promoter activity), together with cells of a distinct origin. The cells are enriched with markers of proliferating pituitary and stem cells, including Sox2, suggesting that multipotent precursor cells might have proliferated and differentiated into gonadotrope-like cells. These cell cultures offer a new and versatile methodology for research in gonadotrope differentiation and function, and can provide enough primary cells for chromatin immunoprecipitation and epigenetic analysis, while our initial studies also indicate a possible regulatory mechanism that might be involved in the nuclear export of gonadotropin gene mRNAs.

Yeast translational response to high salinity: global analysis reveals regulation at multiple levels.

Genome-wide studies of steady-state mRNA levels revealed common principles underlying transcriptional changes in response to external stimuli. To uncover principles that govern other stages of the gene-expression response, we analyzed the translational response and its coordination with transcriptome changes following exposure to severe stress. Yeast cells were grown for 1 h in medium containing 1 M NaCl, which elicits a maximal but transient translation inhibition, and nonpolysomal or polysomal mRNA pools were subjected to DNA-microarray analyses. We observed a strong repression in polysomal association for most mRNAs, with no simple correlation with the changes in transcript levels. This led to an apparent accumulation of many mRNAs as a nontranslating pool, presumably waiting for recovery from the stress. However, some mRNAs demonstrated a correlated change in their polysomal association and their transcript levels (i.e., potentiation). This group was enriched with targets of the transcription factors Msn2/Msn4, and the translational induction of several tested mRNAs was diminished in an Msn2/Msn4 deletion strain. Genome-wide analysis of a strain lacking the high salinity response kinase Hog1p revealed that the group of translationally affected genes is significantly enriched with motifs that were shown to be associated with the ARE-binding protein Pub1. Since a relatively small number of genes was affected by Hog1p deletion, additional signaling pathways are likely to be involved in coordinating the translational response to severe salinity stress.

The 3'-UTR mediates the cellular localization of an mRNA encoding a short plasma membrane protein.

Cotranslational synthesis of proteins into the endoplasmic reticulum is preceded by targeting of the translating mRNA once a signal peptide emerges from the ribosome exit tunnel. Many mRNAs, however, are unlikely to be targeted by this process because they encode proteins that do not contain a signal peptide or because they are too short to be recognized by the signal recognition particle. Herein we tested the possible involvement of the 3'-UTR in the localization of an mRNA that encodes a very short Saccharomyces cerevisiae protein (Pmp1). We found by ribosome density mapping, sedimentation analysis, differential centrifugation, and fluorescent in situ hybridization that the 3'-UTR is essential for the association of the transcript with membrane compartments. Fusion of the 3'-UTR to heterologous open reading frames conferred on them a sedimentation and cellular localization pattern resembling that of PMP1. Mutation analysis revealed that a repeating UG-rich sequence within the 3'-UTR is important for membrane association. Taken together, our results reveal an essential role for elements within the 3'-UTR in the localization of an mRNA that is likely to be ignored by the standard signal-dependant mechanism.

Unravelling the role of epigenetics in reproductive adaptations to early-life environment.

Reproductive function adjusts in response to environmental conditions in order to optimize success. In humans, this plasticity includes age of pubertal onset, hormone levels and age at menopause. These reproductive characteristics vary across populations with distinct lifestyles and following specific childhood events, and point to a role for the early-life environment in shaping adult reproductive trajectories. Epigenetic mechanisms respond to external signals, exert long-term effects on gene expression and have been shown in animal and cellular studies to regulate normal reproductive function, strongly implicating their role in these adaptations. Moreover, human cohort data have revealed differential DNA methylation signatures in proxy tissues that are associated with reproductive phenotypic variation, although the cause-effect relationships are difficult to discern, calling for additional complementary approaches to establish functionality. In this Review, we summarize how adult reproductive function can be shaped by childhood events. We discuss why the influence of the childhood environment on adult reproductive function is an important consideration in understanding how reproduction is regulated and necessitates consideration by clinicians treating women with diverse life histories. The resolution of the molecular mechanisms responsible for human reproductive plasticity could also lead to new approaches for intervention by targeting these epigenetic modifications.

Tet Enzymes, Variants, and Differential Effects on Function.

Discovery of the ten-eleven translocation 1 (TET) methylcytosine dioxygenase family of enzymes, nearly 10 years ago, heralded a major breakthrough in understanding the epigenetic modifications of DNA. Initially described as catalyzing the oxidation of methyl cytosine (5mC) to hydroxymethyl cytosine (5hmC), it is now clear that these enzymes can also catalyze additional reactions leading to active DNA demethylation. The association of TET enzymes, as well as the 5hmC, with active regulatory regions of the genome has been studied extensively in embryonic stem cells, although these enzymes are expressed widely also in differentiated tissues. However, TET1 and TET3 are found as various isoforms, as a result of utilizing alternative regulatory regions in distinct tissues. Some of these isoforms, like TET2, lack the CXXC domain which probably has major implications on their recruitment to specific loci in the genome, while in certain contexts TET1 is seen paradoxically to repress transcription. In this review we bring together these novel aspects of the differential regulation of these Tet isoforms and the likely consequences on their activity.

Multifaceted Targeting of the Chromatin Mediates Gonadotropin-Releasing Hormone Effects on Gene Expression in the Gonadotrope.

Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility.

Genome-wide polysomal analysis of a yeast strain with mutated ribosomal protein S9.

BACKGROUND: The yeast ribosomal protein S9 (S9) is located at the entrance tunnel of the mRNA into the ribosome. It is known to play a role in accurate decoding and its bacterial homolog (S4) has recently been shown to be involved in opening RNA duplexes. Here we examined the effects of changing the C terminus of S9, which is rich in acidic amino acids and extends out of the ribosome surface. RESULTS: We performed a genome-wide analysis to reveal effects at the transcription and translation levels of all yeast genes. While negligible relative changes were observed in steady-state mRNA levels, a significant number of mRNAs appeared to have altered ribosomal density. Notably, 40% of the genes having reliable signals changed their ribosomal association by more than one ribosome. Yet, no general correlations with physical or functional features of the mRNA were observed. Ribosome Density Mapping (RDM) along four of the mRNAs with increased association revealed an increase in ribosomal density towards the end of the coding region for at least two of them. Read-through analysis did not reveal any increase in read-through of a premature stop codon by the mutant strain. CONCLUSION: The ribosomal protein rpS9 appears to be involved in the translation of many mRNAs, since altering its C terminus led to a significant change in ribosomal association of many mRNAs. We did not find strong correlations between these changes and several physical features of the mRNA, yet future studies with advanced tools may allow such correlations to be determined. Importantly, our results indicate an accumulation of ribosomes towards the end of the coding regions of some mRNAs. This suggests an involvement of S9 in ribosomal dissociation during translation termination.

Glucose and nitrogen regulate the switch from histone deacetylation to acetylation for expression of early meiosis-specific genes in budding yeast.

In eukaryotes, the switch between alternative developmental pathways is mainly attributed to a switch in transcriptional programs. A major mode in this switch is the transition between histone deacetylation and acetylation. In budding yeast, early meiosis-specific genes (EMGs) are repressed in the mitotic cell cycle by active deacetylation of their histones. Transcriptional activation of these genes in response to the meiotic signals (i.e., glucose and nitrogen depletion) requires histone acetylation. Here we follow how this regulated switch is accomplished, demonstrating the existence of two parallel mechanisms. (i) We demonstrate that depletion of glucose and nitrogen leads to a transient replacement of the histone deacetylase (HDAC) complex on the promoters of EMG by the transcriptional activator Ime1. The occupancy by either component occurs independently of the presence or absence of the other. Removal of the HDAC complex depends on the protein kinase Rim15, whose activity in the presence of nutrients is inhibited by protein kinase A phosphorylation. (ii) In the absence of glucose, HDAC loses its ability to repress transcription, even if this repression complex is directly bound to a promoter. We show that this relief of repression depends on Ime1, as well as on the kinase activity of Rim11, a glycogen synthase kinase 3beta homolog that phosphorylates Ime1. We further show that the glucose signal is transmitted through Rim11. In cells expressing the constitutive active rim11-3SA allele, HDAC repression in glucose medium is impaired.

The in vivo activity of Ime1, the key transcriptional activator of meiosis-specific genes in Saccharomyces cerevisiae, is inhibited by the cyclic AMP/protein kinase A signal pathway through the glycogen synthase kinase 3-beta homolog Rim11.

Phosphorylation is the main mode by which signals are transmitted to key regulators of developmental pathways. The glycogen synthase kinase 3 family plays pivotal roles in the development and well-being of all eukaryotic organisms. Similarly, the budding yeast homolog Rim11 is essential for the exit of diploid cells from the cell cycle and for entry into the meiotic developmental pathway. In this report we show that in vivo, in cells grown in a medium promoting vegetative growth with acetate as the sole carbon source (SA medium), Rim11 phosphorylates Ime1, the master transcriptional activator required for entry into the meiotic cycle and for the transcription of early meiosis-specific genes. We demonstrate that in the presence of glucose, the kinase activity of Rim11 is inhibited. This inhibition could be due to phosphorylation on Ser-5, Ser-8, and/or Ser-12 because in the rim11S5AS8AS12A mutant, Ime1 is incorrectly phosphorylated in the presence of glucose and cells undergo sporulation. We further show that this nutrient signal is transmitted to Rim11 and consequently to Ime1 by the cyclic AMP/protein kinase A signal transduction pathway. Ime1 is phosphorylated in SA medium on at least two residues, Tyr-359 and Ser-302 and/or Ser-306. Ser-302 and Ser-306 are part of a consensus site for the mammalian homolog of Rim11, glycogen synthase kinase 3-beta. Phosphorylation on Tyr-359 but not Ser-302 or Ser-306 is essential for the transcription of early meiosis-specific genes and sporulation. We show that Tyr-359 is phosphorylated by Rim11.

The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication.

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.