Gcn5 and Sirtuins Regulate Acetylation of the Ribosomal Protein Transcription Factor Ifh1

SummaryBackgroundIn eukaryotes, ribosome biosynthesis involves the coordination of ribosomal RNA and ribosomal protein (RP) production. In S. cerevisiae, the regulation of ribosome biosynthesis occurs largely at the level of transcription. The transcription factor Ifh1 binds at RP genes and promotes their transcription when growth conditions are favorable. Although Ifh1 recruitment to RP genes has been characterized, little is known about the regulation of promoter-bound Ifh1.ResultsWe used a novel whole-cell-extract screening approach to identify Spt7, a member of the SAGA transcription complex, and the RP transactivator Ifh1 as highly acetylated nonhistone species. We report that Ifh1 is modified by acetylation specifically in an N-terminal domain. These acetylations require the Gcn5 histone acetyltransferase and are reversed by the sirtuin deacetylases Hst1 and Sir2. Ifh1 acetylation is regulated by rapamycin treatment and stress and limits the ability of Ifh1 to act as a transactivator at RP genes.ConclusionsOur data suggest a novel mechanism of regulation whereby Gcn5 functions to titrate the activity of Ifh1 following its recruitment to RP promoters to provide more than an all-or-nothing mode of transcriptional regulation. We provide insights into how the action of histone acetylation machineries converges with nutrient-sensing pathways to regulate important aspects of cell growth

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Rif1 prolongs the embryonic S phase at the <i>Drosophila</i> mid-blastula transition

<div><p>In preparation for dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the mid-blastula transition (MBT). In <i>Drosophila melanogaster</i> embryos, down-regulation of cyclin-dependent kinase 1 (Cdk1) activity initiates this slowing by delaying replication of heterochromatic satellite sequences and extending S phase. We found that Cdk1 activity inhibited the chromatin association of Rap1 interacting factor 1 (Rif1), a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 down-regulation at the MBT. In the next S phase, Rif1 dissociated from different satellites in an orderly schedule that anticipated their replication. Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. Loss of Rif1 in mutant embryos shortened the post-MBT S phase and rescued embryonic cell cycles disrupted by depletion of the S phase–promoting kinase, cell division cycle 7 (Cdc7). Our work shows that Rif1 and S phase kinases compose a replication timer controlling first the developmental onset of late replication and then the precise schedule of replication within S phase. In addition, we describe how onset of late replication fits into the progressive maturation of heterochromatin during development.</p></div

Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila

Acquisition of chromatin modifications during embryogenesis distinguishes different regions of an initially naïve genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is marked by di/trimethylation of histone H3K9 and the associated protein HP1a. These modifications enforce the unique epigenetic properties of heterochromatin. However, in the early Drosophila melanogaster embryo, the heterochromatin lacks these modifications, which appear only later, when rapid embryonic cell cycles slow down at the midblastula transition (MBT). Here we focus on the initial steps restoring heterochromatic modifications in the embryo. We describe the JabbaTrap, a technique for inactivating maternally provided proteins in embryos. Using the JabbaTrap, we reveal a major requirement for the methyltransferase Eggless/SetDB1 in the establishment of heterochromatin. In contrast, other methyltransferases contribute minimally. Live imaging reveals that endogenous Eggless gradually accumulates on chromatin in interphase but then dissociates in mitosis, and its accumulation must restart in the next cell cycle. Cell cycle slowing as the embryo approaches the MBT permits increasing accumulation and action of Eggless at its targets. Experimental manipulation of interphase duration shows that cell cycle speed regulates Eggless. We propose that developmental slowing of the cell cycle times embryonic heterochromatin formation

Sequential appearance at satellite sequences suggests that Rif1 influences late replication prior to an impact of HP1a.

<p>(A) Still images from time-lapse imaging of Rif1-GFP and RFP-HP1a during cycle 14 (early S phase through mitosis). During S phase, the number of Rif1 foci decline as the number of HP1a foci increase. G2 nuclei lack Rif1 foci but retain strong HP1a foci (74 min). During the asynchronous mitosis 14 (74/90/96 min), both proteins are lost but are rapidly recruited to late anaphase chromosomes (90/96 min). (B) Still images from time-lapse imaging of GFP-HP1a protein injected into WT embryos (above) and <i>rif1</i> embryos (below) during S phase 14. HP1a recruitment to the heterochromatin proceeded similarly in control and mutant embryos. (C) Still images from time-lapse imaging of the replication of the 359-satellite progressing from G2 of cycle 14 until completion of its replication in cycle 15. Note that the TALE-light signal is not immediately visible following mitosis (4:00 min frame). We previously described how HP1a binds to the 359 bp repeat following its replication during S phase 14 and subsequently delays its replication in S phase 15. In <i>rif1</i> embryos, the 359 repeat also replicated late in S phase 15 (in frame 30:00, yellow arrow indicates where the 359 TALE signal overlapped with PCNA). GFP, green fluorescent protein; HP1a, heterochromatin protein 1a; PCNA, proliferating cell nuclear antigen; RFP, red fluorescent protein; Rif1, Rap1 interacting factor 1; TALE, transcription activator-like effector; WT, wild-type.</p

Two stages of Rif1 recruitment, their contributions to specificity, and reliance on origin licensing and the replication checkpoint.

<p>Time-lapse confocal microscopy on Rif1-GFP, His2Av-RFP embryos showing the initial binding of Rif1 during transit from one cell cycle to the next. (A) During late anaphase 13, on the approach to cycle 14, chromosomes first exhibit faint and ubiquitous Rif1 staining that accumulates for about 1 min. During the following minute as S phase begins, accumulation continues but is now localized to foci that become clearer as the nuclei swell. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005687#pbio.2005687.s006" target="_blank">S2 Movie</a>. (B) During late anaphase 14, on the approach to cycle 15, the early stage of Rif1 staining shows some specificity for the pericentric regions of the chromosomes and forming chromocenter. This specificity is amplified as the interphase 15 nucleus forms and S phase begins. (C) Time-lapse imaging of Rif1-GFP during the normal transition from cycle 13 to 14 and in an embryo injected with purified geminin protein in interphase of 13. Times are indicated with reference to the start of S phase 14. The geminin block to pre-RC formation prevented the recruitment of Rif1 to foci but did not block the initial generalized binding during mitotic exit. (D) Imaging of Rif1-GFP during cell cycle 13 in control (<i>mei41/+</i>) and <i>mei41</i>-null embryos. The Mei41-dependent replication checkpoint is essential during cycle 13 to prevent premature entry into mitosis (10:20 frame). Rif1 foci still form in the absence of a replication checkpoint, but the Rif1 foci are lost earlier, and the premature mitosis leads to bridging and defective cycle 14 nuclei. GFP, green fluorescent protein; His2Av, histone 2A variant; pre-RC, pre-replicative complex; RFP, red fluorescent protein; Rif1, Rap1 interacting factor 1.</p