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Transcriptional memory of gene expression enables adaptation to repeated stimuli across many organisms. However, the regulation and heritability of transcriptional memory in single cells and through divisions remains poorly understood. Here, we combined microfluidics with single-cell live imaging to monitor Saccharomyces cerevisiae galactokinase 1 (GAL1) expression over multiple generations. By applying pedigree analysis, we dissected and quantified the maintenance and inheritance of transcriptional reinduction memory in individual cells through multiple divisions. We systematically screened for loss- and gain-of-memory knockouts to identify memory regulators in thousands of single cells. We identified new loss-of-memory mutants, which affect memory inheritance into progeny. We also unveiled a gain-of-memory mutant, elp6Δ, and suggest that this new phenotype can be mediated through decreased histone occupancy at the GAL1 promoter. Our work uncovers principles of maintenance and inheritance of gene expression states and their regulators at the single-cell level. 78 5 915 925.e7

Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N-alpha-terminal acetyltransferase Nat4 and loss of its associated H4 N-terminal acetylation (N-acH4) extend yeast replicative lifespan. Notably, nat4Δ-induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N-acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N-acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress-response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ-mediated longevity. Collectively, these findings establish histone N-acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity. © 2016 The Authors. Published under the terms of the CC BY 4.0 license 17 1829 1843 Cited By :2

Gene ontology (GO) terms enriched by the negative genetic interactions of SUTs and of their flanking genes. Heatmap of GO terms enriched with Pâ â ¤â 0.05 (generated using the Fisher exact test) by genes classified as negative genetic interactions for the indicated SUTs and for their corresponding adjacent protein-coding genes. The negative genetic interactions of the flanking protein-coding genes were extracted from the Drygin database ( http://drygin.ccbr.utoronto.ca/ ). (PDF 95 kb)

Lack of SUT457 accelerates senescence in tlc1Δ cells. a, b Senescence assays performed using liquid cultures of the indicated isogenic wild-type and mutant strains. The strains were generated through tetrad dissection of the heterozygous diploid double mutant SUT457/sut457ΔTLC1/tlc1Δ. Each plot represents one of three independent tetrads examined (see also Fig. 3e). (PDF 65 kb)

Lack of SUT457 accelerates senescence in tlc1Δ cells. a, b Senescence assays performed using liquid cultures of the indicated isogenic wild-type and mutant strains. The strains were generated through tetrad dissection of the heterozygous diploid double mutant SUT457/sut457ΔTLC1/tlc1Δ. Each plot represents one of three independent tetrads examined (see also Fig. 3e). (PDF 65 kb)

Deletion of EXO1 rescues the growth arrest of gbp2Î sut457Î mutant cells. Isogenic strains of the indicated genotype (top panels) were streaked repeatedly on solid rich medium and their growth at passage 5 is shown. (PDF 68 kb)

Detection of SUT457 RNA. Total RNA was isolated from wild-type and sut457Î cells and analysed by northern blotting using a probe (Additional file 14: Table S7) against the SUT457 sequence (Additional file 6: Table S5). A band of 345Â bp corresponding to the size of SUT457 is detected only in wild-type cells (left panel). A probe against actin (Additional file 14: Table S7) was used as a loading control (right panel). (PDF 26 kb)

Detection of SUT457 RNA. Total RNA was isolated from wild-type and sut457Î cells and analysed by northern blotting using a probe (Additional file 14: Table S7) against the SUT457 sequence (Additional file 6: Table S5). A band of 345Â bp corresponding to the size of SUT457 is detected only in wild-type cells (left panel). A probe against actin (Additional file 14: Table S7) was used as a loading control (right panel). (PDF 26 kb)

Background Transcriptome studies have revealed that many eukaryotic genomes are pervasively transcribed producing numerous long non-coding RNAs (lncRNAs). However, only a few lncRNAs have been ascribed a cellular role thus far, with most regulating the expression of adjacent genes. Even less lncRNAs have been annotated as essential hence implying that the majority may be functionally redundant. Therefore, the function of lncRNAs could be illuminated through systematic analysis of their synthetic genetic interactions (GIs). Results Here, we employ synthetic genetic array (SGA) in Saccharomyces cerevisiae to identify GIs between long intergenic non-coding RNAs (lincRNAs) and protein-coding genes. We first validate this approach by demonstrating that the telomerase RNA TLC1 displays a GI network that corresponds to its well-described function in telomere length maintenance. We subsequently performed SGA screens on a set of uncharacterised lincRNAs and uncover their connection to diverse cellular processes. One of these lincRNAs, SUT457, exhibits a GI profile associating it to telomere organisation and we consistently demonstrate that SUT457 is required for telomeric overhang homeostasis through an Exo1-dependent pathway. Furthermore, the GI profile of SUT457 is distinct from that of its neighbouring genes suggesting a function independent to its genomic location. Accordingly, we show that ectopic expression of this lincRNA suppresses telomeric overhang accumulation in sut457Δ cells assigning a trans-acting role for SUT457 in telomere biology. Conclusions Overall, our work proposes that systematic application of this genetic approach could determine the functional significance of individual lncRNAs in yeast and other complex organisms. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0325-7) contains supplementary material, which is available to authorized users.

Gene ontology (GO) terms enriched by the negative genetic interactions of SUTs and of their flanking genes. Heatmap of GO terms enriched with Pâ â ¤â 0.05 (generated using the Fisher exact test) by genes classified as negative genetic interactions for the indicated SUTs and for their corresponding adjacent protein-coding genes. The negative genetic interactions of the flanking protein-coding genes were extracted from the Drygin database ( http://drygin.ccbr.utoronto.ca/ ). (PDF 95 kb)

Degradation of telomeric ssDNA by bacterial Exonuclease I. Genomic DNA extracted from four individual sut457Δ strains at passage five was either untreated (-) or digested with bacterial exonuclease I (+), which degrades telomeric overhang DNA in the 3’ to 5’ direction, before being subjected to native southern analysis using a biotinylated probe against telomeric repeats (upper blot). The DNA on the membrane was then denatured with 0.4 N NaOH and re-probed to monitor equal loading (lower blot). (PDF 48 kb)

Overlap between TLC1 and EST1 negative genetic interactions. a Venn diagram showing the overlap (P = 0.001 generated using the hypergeometric test) between genes whose deletions result in negative genetic interactions with TLC1 in this study and with EST1 as reported in [43]. GO terms enriched by common negative genetic interactions with P

Growth curve analysis validates the positive genetic interaction between sut457Δ and yku70Δ. The indicated isogenic strains were diluted to OD600 of 0.1 and cultured at 30 °C in rich YPD medium until saturation. The OD600 of each strain was measured every hour for a total of 24 hours using Infinite M200 (Tecan Trading AG). Error bars represent SEM of eight independent tetrads. (PDF 52 kb)

G protein-coupled receptors (GPCRs) are a large family of trans-membrane receptors that transmit signals from extracellular stimuli to target intracellular signal transduction pathways. The gonadotropin-releasing hormone receptor (GnRH-R) is a GPCR which binds the decapeptide GnRH. In the pituitary gonadotrope, GnRH stimulates gonadotropin (LH and FSH) biosynthesis and secretion to regulate reproduction. GnRH and the GnRH-Rs are also present in many extra-pituitary tissues, although their role at these sites remains largely undetermined. GnRH-Rs are known to recruit a diverse array of signalling pathway mediators in different cell-types. These include; Gq/11-PLCβ-IP3/DAG-Ca2+/PKC signalling, monomeric G-proteins and integrins to mediate cell adhesion and migration, the activation of the major members of the mitogen-activated protein kinase (MAPK) super-family (extracellular signal-regulated kinase (ERK), c-Jun N-terminal Kinase (JNK) and p38MAPK), and β-catenin and other mediators of the canonical Wnt signalling pathway. This thesis describes the regulation of Forkhead Box O (FOXO) transcription factors by GnRH. The mammalian FOXO transcription factors, FOXO1, FOXO3a and FOXO4, are emerging as an important family of proteins that modulate the expression of genes involved in cell-cycle regulation, induction of apoptosis, DNA damage repair and response to oxidative stress. In this thesis, emphasis is placed on delineating the novel role of FOXO transcription factors in mediating two important and widely-researched areas of GnRH biology. Firstly, the role of FOXO transcription factors in mediating cell-growth inhibition in response to GnRH treatment is assessed in a heterologous HEK293/GnRH-R expressing cell line. Secondly, the role of transcription factors in regulating luteinising hormone-β (LHβ)-subunit expression is investigated in the LβT2 gonadotrope cell line. Activation of the GnRH-R can inhibit cell proliferation and induce apoptosis in certain tumour-derived cell lines. Several studies have reported that these events can occur as a result of changes in the expression profiles of specific cell-cycle regulatory and apoptotic genes, many of which are FOXO-target genes, including GADD45, FasL, p21Cip1 and p27Kip1. In this thesis, a role for FOXOs in targeting the expression of several of these genes in response to GnRH is assessed, highlighting a specific role for FOXO3a in mediating GADD45 and FasL expression. The signalling mechanisms through which FOXO3a regulates GADD45 expression in response to GnRH is also described. Finally, a stable FOXO3a-knock-down cell line was generated in order to further examine FOXO3a involvement in GnRH-induced cell-growth inhibition. GnRH is an essential regulator of the reproductive process by stimulating the synthesis of LH and FSH in pituitary gonadotropes, thereby regulating gametogenesis and steroidogenesis. Diverse signalling pathways have been reported to regulate LHβ-subunit expression in response to GnRH, including the ERK/JNK/p38MAPK cascades and factors such as Egr1, SF1 and β-catenin. In the second part of this thesis, the role of FOXOs in regulating LHβ-subunit expression in response to GnRH is described. The data presented suggests that GnRH can regulate LHβ-subunit expression through both indirect and direct FOXO3a-mediated mechanisms. Firstly, FOXO3a was found to regulate Egr1 expression to indirectly target LHβ-promoter activity. Secondly, a role for β-catenin as a FOXO3a co-factor to directly regulate LHβ-subunit expression, together with Egr1 and SF1, is also proposed. FOXO3a expression and sub-cellular localisation was assessed and demonstrated in LβT2 cells and in adult human male pituitary sections. The research presented in this thesis adds to the diversity of signalling pathways and mediators that GnRH can target in different cellular backgrounds in order to mediate a variety of cellular processes. The antiproliferative and apoptotic effects of GnRH on tumour-derived cell lines are well-documented, and this research highlights a novel role for FOXO3a in mediating these events. The regulation of gonadotropin synthesis remains an important topic of research, and the novel implication of FOXO3a in mediating LHβ-subunit expression adds further complexity to gonadotrope physiology.