KdmA, a histone H3 demethylase with bipartite function, differentially regulates primary and secondary in Aspergillus nidulans metabolism
ABSTRACT: A. nidulans kdmA encodes a member of the KDM4 family of jumonji histone demethylase proteins, highly similar to metazoan orthologues both within functional domains and in domain architecture. This family of proteins exhibits demethylase activity toward lysines 9 and 36 of histone H3 and plays a prominent role in gene expression and chromosome structure in many species. Mass spectrometry mapping of A. nidulans histones revealed that around 3% of bulk histone H3 carried trimethylated H3K9 (H3K9me3) but more than 90% of histones carried either H3K36me2 or H3K36me3. KdmA functions as H3K36me3 demethylase and has roles in transcriptional regulation. Genetic manipulation of KdmA levels is tolerated without obvious effect in most conditions, but strong phenotypes are evident under various conditions of stress. Transcriptome analysis revealed that – in submerged early and late cultures – between 25% and 30% of the genome is under KdmA influence, respectively. Transcriptional imbalance in the kdmA deletion mutant may contribute to the lethal phenotype observed upon exposure of mutant cells to low-density visible light on solid medium. While KdmA acts as transcriptional co-repressor of primary metabolism (PM) genes it is required for full expression of several genes involved in biosynthesis of secondary metabolites (SM). Two strains, wild type and kdmA deletion, at two conditions, growth at primary (17h) and secondary (48h), were analyzed. Each sample was replicated.
Project description:Histone posttranslational modifications (HPTMs) are involved in regulating the synthesis of fungal bioactive compounds. The exact molecular mechanisms of the silencing/activation of secondary metabolism (SM) clusters by these epigenetic events however are not yet fully understood. This work applies a combined approach of quantitative mass spectrometry (LC-MS/MS) and chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq) to identify the chromatin landscape in two metabolic states: primary and secondary metabolism. Furthermore, to link the particular chromatin states to the expression of condition specific genes, genome wide transcriptome (RNA-seq) was performed. Strikingly, we found that silent A. nidulans SM clusters are free of repressive H3K9me3 though this heterochromatic mark forms distinguished peaks flanking the many SM clusters. In addition, silent SM clusters do not contain detectable levels of activating histone marks such as H3K4me3, H3K36me3 or H3Ac, which, to some extent, are established upon activation of the clusters. In order to investigate the function of dynamic H3K4 methylation/demethylation in transcription, we characterized the KdmB- Jarid1 family histone demethylase. The in vitro assay using heterologously expressed KdmB showed that it is an active demethylase; moreover, MS/MS as well ChIP-seq approaches revealed that it targets H3K4me3 in vivo mediating transcriptional repression. KdmB positively regulates the expression of 40% of A. nidulans SM genes and this function appears to be independent of its demethylase activity. Our bioinformatics approach revealed two states of H3K4me3 in A. nidulans genome: loci with low levels of this mark are more disposed to differential expression in response to environmental clues, while the genes marked by high H3K4me3 levels are constitutively transcribed in our experimental conditions. Taken together our data reveal important role of H3K4 methylation/demethylation in transcription regulation. Furthermore, this study presents the first genome-wide map of H3K4me3, H3K9me3, H3K36me3 and H3Ac in A. nidulans in different metabolic conditions. Two strains, wild type and kdmB deletion, at two conditions, growth at primary (17h) and secondary (48h), were analysed. Each sample was replicated.
Project description:This model is described in the article:
Kinetics of histone gene expression during early development of Xenopus laevis.
Koster JG, Destrée OH and Westerhoff HV. J Theor Biol.
1988 Nov 21;135(2):139-67. PMID: 3267765
Using literature data for transcriptional and translational rate constants, gene copy numbers, DNA concentrations, and stability constants, we have calculated the expected concentrations of histones and histone mRNA during embryogenesis of Xenopus laevis. The results led us to conclude that: (i) for X. laevis the gene copy number of the histone genes is too low to ensure the synthesis of sufficient histones during very early development, inheritance from the oocyte of either histone protein or histone mRNA (but not necessarily both) is necessary; (ii) from the known storage of histones in the oocyte and the rates of histone synthesis determined by Adamson and Woodland (1977), there would be sufficient histones to structure the newly synthesized DNA up to gastrulation but not thereafter (these empirical rates of histone synthesis may be underestimates); (iii) on the other hand, the amount of H3 mRNA recently observed during early embryogenesis (Koster, 1987, Koster et al., 1988) could direct a higher and sufficient synthesis of H3 protein, also after gastrulation. We present a quantitative model that accounts both for the observed H3 mRNA concentration as a function of time during embryogenesis and for the synthesis of sufficient histones to structure the DNA throughout early embryogenesis. The model suggests that X. laevis exhibits a major (i.e. some 14-fold) reduction in transcription of histone genes approximately 11 hours after fertilization. This reduction could be due to a decrease in the number of transcribed histone genes, a decreased rate constant of transcription with continued transcription of all the histone genes, and/or a reduction in the time during the cell cycle in which histone mRNA synthesis takes place. Alternatively, the histone mRNA stability might decrease approximately 16-fold 11 hours after fertilization.
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Project description:Histone H3 lysine 4 tri-methylation (H3K4me3) is a hallmark of transcription initiation, but how H3K4me3 is demethylated during gene repression is poorly understood. Jhd2, a JmjC domain protein, was recently identified as the major H3K4me3 histone demethylase (HDM) in S. cerevisiae. While JHD2 is required for removal of methylation upon gene repression, deletion of JHD2 does not result in increased levels of H3K4me3 in bulk histones, indicating that this HDM is unable to demethylate histones during steady state conditions. In this study, we showed that this was due to the negative regulation of Jhd2 activity by histone H3 lysine 14 acetylation, which co-localizes with H3K4me3 across the yeast genome. We demonstrated that loss of the histone H3-specific acetyltransferases (HATs) resulted in genome-wide-depletion of H3K4me3, and this was not due to a transcription defect. Moreover, H3K4me3 levels were reestablished in HAT mutants following loss of JHD2, which suggested that H3-specific HATs and Jhd2 served opposing functions in regulating H3K4me3 levels. We revealed the molecular basis for this suppression by demonstrating that histone H3K14 acetylation negatively regulated Jhd2 demethylase activity on an acetylated peptide in vitro. These results revealed the existence of a general mechanism for removal of H3K4me3 following gene repression. Examination of H3K4me3 in WT, ada2sas3, ada2sas3jhd2, and jhd2 strains.
Project description:Epigenetic mechanisms including histone post-translational modifications control longevity in diverse organisms. Relatedly, loss of proper transcriptional regulation on a global scale is an emerging aspect of shortened lifespan, but the specific mechanisms linking these observations remain to be uncovered. Here, we describe a lifespan screen in S. cerevisiae, designed to identify altered amino acid residues of histones that alter yeast replicative aging. Our results reveal that lack of sustained H3K36 methylation is commensurate with increased cryptic transcription in a set of genes in old cells and shorter lifespan. Deletion of the K36me2/3 demethylase Rph1 increases H3K36me3 within these genes and suppresses cryptic transcript initiation to extend lifespan. We show that this aging phenomenon is conserved, as cryptic transcription also increases in old worms. We propose that epigenetic misregulation in aging cells leads to an increase in transcriptional noise that is detrimental to lifespan, and, importantly, this acceleration in aging can be reversed by restoring transcriptional fidelity. Overall design: This study examines H3K36me3 in yeast aging using a WT or Rph1 mutant background over a sequence of time-dependent FACS sorts of old cells. There are three sorts: S2Y (young yeast), S2O or S3O (both old yeast). The study has two replicates, F1 and F2. F2 is the combination of two flowcells of sequenced tags. For each sample, we sequenced H3K36me3, H3, and input, and we use H3 as a sonication efficiency normalization in the associated study.
Project description:The methylation of histone H3 lysine 79 (H3K79) acts as an active chromatin marker and is enriched in actively transcribed regions of the genome. However, demethylase of this mark remains unknown despite intensive research. Here, we show that KDM2B (FBXL10), a member of the Jumonji C (JmjC) family of proteins and previously known for its histone H3K36 demethylase activity, is a di- and trimethyl H3K79 demethylase. We demonstrate that KDM2B induces transcriptional repression of HOXA7 and MEIS1 via occupancy of promoters and demethylation of H3K79. Furthermore, genome-wide analysis suggests that H3K79 methylation levels are increased when KDM2B is depleted, suggesting that KDM2B functions as an H3K79 demethylase in vivo. Finally, stable KDM2B-knockdown cell lines exhibited displacement of SIRT1, with concomitant increases in H3K79 methylation and H4K16 acetylation. Our findings identify KDM2B as an H3K79 demethylase and link its function to transcriptional repression via SIRT1-mediated chromatin silencing. Overall design: We profiled genomic occupancy of H3K79me2 and H3K79me3 via ChIP-seq for KDM2B KD and control in human 293T stable cell lines.
Project description:We present here evidence that histones H3.1 and H4 can be imported into the nucleus as monomers in human cells. Using a tether-and-release system to study the cytosolic phase and import dynamics of newly synthesised histones, we find that H3.1 and H4 can be maintained as stable monomers in the cytosol in a tethered state. Cytosolically tethered histones are bound tightly to Importin- proteins (predominantly IPO4), but not to the histone specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, dissociation with IPO4 and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones under steady-state conditions reveals an excess of H3 specifically associated with sNASP, suggesting that NASP can maintain a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol, with sNASP acting as a potential nuclear receptor for monomeric histone H3.
Project description:Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as anti-silencing factors and prevent silencing of genes next to TEs. Whether TE silencing is counterbalanced by the activity of anti-silencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 auto-ubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3 and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anti-correlated with histone H3 lysine 9 dimethylation (H3K9me2) levels at AtMu1c. Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2, and requires H3K9 methyltransferases for its activity on AtMu1c. Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs. Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 [with the mutated JmjC domain] is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity. Overall design: 9 samples, three mutant replicates, three overexpresser replicates, three wild type replicates.
Project description:Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the requirements for this critical factor, we have performed genome-wide analyses of transcription, chromatin structure, and histone modifications in an S. pombe spt6 mutant. Our results demonstrate several dramatic changes to transcription and chromatin structure in the spt6 mutant, including an elevation of antisense transcripts at over 70 percent of all genes and general loss of the +1 nucleosome. Furthermore, Spt6 is required for the trimethylation of histone H3 on lysines 4 and 36, marks associated with active transcription. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones. ChIP-seq experiments were performed on wild type and spt6-1 strains on the following proteins: RNA polymerase II (Rpb1), Paf1 Complex (Ctr9), COMPASS (Swd1), Set2, Spt6, histones H2B and H3, histone modifications H3K4me3 and H3K36me3. Experiments were performed in replicates and matching inputs were also sequenced.