The cellular response to ocean warming in Emiliania huxleyi.

Marine phytoplankton contribute substantially to the global flux of carbon from the atmosphere to the deep ocean. Sea surface temperatures will inevitably increase in line with global climate change, altering the performance of marine phytoplankton. Differing sensitivities of photosynthesis and respiration to temperature, will likely shift the strength of the future oceanic carbon sink. To further clarify the molecular mechanisms driving these alterations in phytoplankton function, shotgun proteomic analysis was carried out on the globally-occurring coccolithophore Emiliania huxleyi exposed to moderate- (23°C) and elevated- (28°C) warming. Compared to the control (17°C), growth of E. huxleyi increased under elevated temperatures, with higher rates recorded under moderate- relative to elevated- warming. Proteomic analysis revealed a significant modification of the E. huxleyi cellular proteome as temperatures increased: at lower temperature, ribosomal proteins and photosynthetic machinery appeared abundant, as rates of protein translation and photosynthetic performance are restricted by low temperatures. As temperatures increased, evidence of heat stress was observed in the photosystem, characterized by a relative down-regulation of the Photosystem II oxygen evolving complex and ATP synthase. Acclimation to elevated warming (28°C) revealed a substantial alteration to carbon metabolism. Here, E. huxleyi made use of the glyoxylate cycle and succinate metabolism to optimize carbon use, maintain growth and maximize ATP production in heat-damaged mitochondria, enabling cultures to maintain growth at levels significantly higher than those recorded in the control (17°C). Based on the metabolic changes observed, we can predict that warming may benefit photosynthetic carbon fixation by E. huxleyi in the sub-optimal to optimal thermal range. Past the thermal optima, increasing rates of respiration and costs of repair will likely constrain growth, causing a possible decline in the contribution of this species to the oceanic carbon sink depending on the evolvability of these temperature thresholds.

Cerebrospinal Fluid and Brain Proteoforms of the Granin Neuropeptide Family in Alzheimer's Disease.

The granin neuropeptide family is composed of acidic secretory signaling molecules that act throughout the nervous system to help modulate synaptic signaling and neural activity. Granin neuropeptides have been shown to be dysregulated in different forms of dementia, including Alzheimer's disease (AD). Recent studies have suggested that the granin neuropeptides and their protease-cleaved bioactive peptides (proteoforms) may act as both powerful drivers of gene expression and as a biomarker of synaptic health in AD. The complexity of granin proteoforms in human cerebrospinal fluid (CSF) and brain tissue has not been directly addressed. We developed a reliable nontryptic mass spectrometry assay to comprehensively map and quantify endogenous neuropeptide proteoforms in the brain and CSF of individuals diagnosed with mild cognitive impairment and dementia due to AD compared to healthy controls, individuals with preserved cognition despite AD pathology ("Resilient"), and those with impaired cognition but no AD or other discernible pathology ("Frail"). We drew associations between neuropeptide proteoforms, cognitive status, and AD pathology values. Decreased levels of VGF proteoforms were observed in CSF and brain tissue from individuals with AD compared to controls, while select proteoforms from chromogranin A showed the opposite effect. To address mechanisms of neuropeptide proteoform regulation, we showed that the proteases Calpain-1 and Cathepsin S can cleave chromogranin A, secretogranin-1, and VGF into proteoforms found in both the brain and CSF. We were unable to demonstrate differences in protease abundance in protein extracts from matched brains, suggesting that regulation may occur at the level of transcription.

The Role of luxS in Campylobacter jejuni Beyond Intercellular Signaling.

The full role of the luxS gene in the biological processes, such as essential amino acid synthesis, nitrogen and pyruvate metabolism, and flagellar assembly, of Campylobacter jejuni has not been clearly described to date. Therefore, in this study, we used a comprehensive approach at the cellular and molecular levels, including transcriptomics and proteomics, to investigate the key role of the luxS gene and compared C. jejuni 11168ΔluxS (luxS mutant) and C. jejuni NCTC 11168 (wild type) strains. Transcriptomic analysis of the luxS mutant grown under optimal conditions revealed upregulation of luxS mutant metabolic pathways when normalized to wild type, including oxidative phosphorylation, carbon metabolism, citrate cycle, biosynthesis of secondary metabolites, and biosynthesis of various essential amino acids. Interestingly, induction of these metabolic pathways was also confirmed by proteomic analysis, indicating their important role in energy production and the growth of C. jejuni. In addition, genes important for the stress response of C. jejuni, including nutrient starvation and oxidative stress, were upregulated. This was also evident in the better survival of the luxS mutant under starvation conditions than the wild type. At the molecular level, we confirmed that metabolic pathways were upregulated under optimal conditions in the luxS mutant, including those important for the biosynthesis of several essential amino acids. This also modulated the utilization of various carbon and nitrogen sources, as determined by Biolog phenotype microarray analysis. In summary, transcriptomic and proteomic analysis revealed key biological differences in tricarboxylic acid (TCA) cycle, pyruvate, nitrogen, and thiamine metabolism as well as lipopolysaccharide biosynthesis in the luxS mutant. IMPORTANCE Campylobacter jejuni is the world's leading foodborne bacterial pathogen of gastrointestinal disease in humans. C. jejuni is a fastidious but widespread organism and the most frequently reported zoonotic pathogen in the European Union since 2005. This led us to believe that C. jejuni, which is highly sensitive to stress factors (starvation and oxygen concentration) and has a low growth rate, benefits significantly from the luxS gene. The role of this gene in the life cycle of C. jejuni is well known, and the expression of luxS regulates many phenotypes, including motility, biofilm formation, host colonization, virulence, autoagglutination, cellular adherence and invasion, oxidative stress, and chemotaxis. Surprisingly, this study confirmed for the first time that the deletion of the luxS gene strongly affects the central metabolic pathway of C. jejuni, which improves its survival, showing its role beyond the intercellular signaling system.

Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation.

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with ß-alanine buildup. Raising ß-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.

Ubiquitin-based pathway acts inside chloroplasts to regulate photosynthesis.

Photosynthesis is the energetic basis for most life on Earth, and in plants it operates inside double membrane-bound organelles called chloroplasts. The photosynthetic apparatus comprises numerous proteins encoded by the nuclear and organellar genomes. Maintenance of this apparatus requires the action of internal chloroplast proteases, but a role for the nucleocytosolic ubiquitin-proteasome system (UPS) was not expected, owing to the barrier presented by the double-membrane envelope. Here, we show that photosynthesis proteins (including those encoded internally by chloroplast genes) are ubiquitinated and processed via the CHLORAD pathway: They are degraded by the 26S proteasome following CDC48-dependent retrotranslocation to the cytosol. This demonstrates that the reach of the UPS extends to the interior of endosymbiotically derived chloroplasts, where it acts to regulate photosynthesis, arguably the most fundamental process of life.

KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity.

The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PALB2 binding to chromatin. In unperturbed cells, the 7K-patch is targeted for KAT2A/2B-mediated acetylation, which in turn enhances the direct association of PALB2 with nucleosomes. Importantly, DNA damage triggers a rapid deacetylation of ChAM and increases the overall mobility of PALB2. Distinct missense mutations of the 7K-patch render the mode of PALB2 chromatin binding, making it either unstably chromatin-bound (7Q) or randomly bound with a reduced capacity for mobilisation (7R). Significantly, both of these mutations confer a deficiency in RAD51 foci formation and increase DNA damage in S phase, leading to the reduction of overall cell survival. Thus, our study reveals that acetylation of the ChAM 7K-patch acts as a molecular switch to enable dynamic PALB2 shuttling for HR repair while protecting active genes during DNA replication.

CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation.

CDK9 is a kinase critical for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.

CAPTURE of the Human U2 snRNA Genes Expands the Repertoire of Associated Factors.

In order to identify factors involved in transcription of human snRNA genes and 3' end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with these human snRNA genes including RNA polymerase II (pol II), Cyclin-Dependent Kinase 7 (CDK7), Negative Elongation Factor (NELF), Suppressor of Ty 5 (SPT5), Mediator 23 (MED23) and several subunits of the Integrator Complex. Suppressor of Ty 6 (SPT6); Cyclin K, the partner of Cyclin-Dependent Kinase 12 (CDK12) and Cyclin-Dependent Kinase 13 (CDK13); and SWI/SNF chromatin remodelling complex-associated SWI/SNF-related, Matrix-associated, Regulator of Chromatin (SMRC) factors were also enriched. Several polyadenylation factors, including Cleavage and Polyadenylation Specificity Factor 1 (CPSF1), Cleavage Stimulation Factors 1 and 2 (CSTF1,and CSTF2) were enriched by U2 gene CAPTURE. We have already shown by chromatin immunoprecipitation (ChIP) that CSTF2-and Pcf11 and Ssu72, which are also polyadenylation factors-are associated with the human U1 and U2 genes. ChIP-seq and ChIP-qPCR confirm the association of SPT6, Cyclin K, and CDK12 with the U2 genes. In addition, knockdown of SPT6 causes loss of subunit 3 of the Integrator Complex (INTS3) from the U2 genes, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes and helped to identify a functional role for SPT6.

Alkaline nucleoplasm facilitates contractile gene expression in the mammalian heart.

Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated "striated muscle contraction" as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.

The C-Terminal Domains of the PB2 Subunit of the Influenza A Virus RNA Polymerase Directly Interact with Cellular GTPase Rab11a.

Influenza A virus (IAV) contains a segmented RNA genome that is transcribed and replicated by the viral RNA polymerase in the cell nucleus. Replicated RNA segments are assembled with viral polymerase and oligomeric nucleoprotein into viral ribonucleoprotein (vRNP) complexes which are exported from the nucleus and transported across the cytoplasm to be packaged into progeny virions. Host GTPase Rab11a associated with recycling endosomes is believed to contribute to this process by mediating the cytoplasmic transport of vRNPs. However, how vRNPs interact with Rab11a remains poorly understood. In this study, we utilized a combination of biochemical, proteomic, and biophysical approaches to characterize the interaction between the viral polymerase and Rab11a. Using pulldown assays, we showed that vRNPs but not complementary RNPs (cRNPs) from infected cell lysates bind to Rab11a. We also showed that the viral polymerase directly interacts with Rab11a and that the C-terminal two-thirds of the PB2 polymerase subunit (PB2-C) comprising the cap-binding, mid-link, 627, and nuclear localization signal (NLS) domains mediate this interaction. Small-angle X-ray scattering (SAXS) experiments confirmed that PB2-C associates with Rab11a in solution forming a compact folded complex with a 1:1 stoichiometry. Furthermore, we demonstrate that the switch I region of Rab11a, which has been shown to be important for binding Rab11 family-interacting proteins (Rab11-FIPs), is also important for PB2-C binding, suggesting that IAV polymerase and Rab11-FIPs compete for the same binding site. Our findings expand our understanding of the interaction between the IAV polymerase and Rab11a in the cytoplasmic transport of vRNPs. IMPORTANCE The influenza virus RNA genome segments are replicated in the cell nucleus and are assembled into viral ribonucleoprotein (vRNP) complexes with viral RNA polymerase and nucleoprotein (NP). Replicated vRNPs need to be exported from the nucleus and trafficked across the cytoplasm to the cell membrane, where virion assembly takes place. The host GTPase Rab11a plays a role in vRNP trafficking. In this study, we showed that the viral polymerase directly interacts with Rab11a mediating the interaction between vRNPs and Rab11a. We mapped this interaction to the C-terminal domains of the PB2 polymerase subunit and the switch I region of Rab11a. Identifying the exact site of Rab11a binding on the viral polymerase could uncover a novel target site for the development of an influenza antiviral drug.

Acyl carrier protein promotes MukBEF action in Escherichia coli chromosome organization-segregation.

Structural Maintenance of Chromosomes (SMC) complexes act ubiquitously to compact DNA linearly, thereby facilitating chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, which is essential for its in vivo function, requires its interaction with the dimeric kleisin, MukF that in turn interacts with the KITE protein, MukE. Here we demonstrate that, in addition, MukB interacts specifically with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction at the joint of the MukB coiled-coil and show that the interaction is necessary for MukB ATPase and for MukBEF function in vivo.

Cardiomyocyte Na+/H+ Exchanger-1 Activity Is Reduced in Hypoxia.

Fully-activated Na+/H+ exchanger-1 (NHE1) generates the cardiomyocyte's largest trans-membrane extrusion of H+ ions for an equimolar influx of Na+ ions. This has the desirable effect of clearing excess intracellular acidity, but comes at a large energetic premium because the exchanged Na+ ions must ultimately be extruded by the sodium pump, a process that consumes the majority of the heart's non-contractile ATP. We hypothesize that the state of NHE1 activation depends on metabolic resources, which become limiting in periods of myocardial hypoxia. To test this functionally, NHE1 activity was measured in response to in vitro and in vivo hypoxic treatments. NHE1 flux was interrogated as a function of intracellular pH by fluorescence imaging of rodent ventricular myocytes loaded with pH-sensitive dyes BCECF or cSNARF1. Anoxic superfusates promptly inhibited NHE1, tracking the time-course of mitochondrial depolarization. Mass spectrometry of NHE1 immuno-precipitated from Langendorff-perfused anoxic hearts identified Tyr-581 dephosphorylation and Tyr-561 phosphorylation. The latter residue is part of the domain that interacts with phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid that becomes depleted under metabolic inhibition. Tyr-561 phosphorylation is expected to electrostatically weaken this activatory interaction. To test if a period of hypoxia produces a persistent inhibition of NHE1, measurements under normoxia were performed on myocytes that had been incubated in 2% O2 for 4 h. NHE1 activity remained inhibited, but the effect was ablated in the presence of Dasatinib, an inhibitor of Abl/Src-family tyrosine kinases. Chronic tissue hypoxia in vivo, attained in a mouse model of anemic hypoxia, also resulted in persistently slower NHE1. In summary, we show that NHE1 responds to oxygen, a physiologically-relevant metabolic regulator, ostensibly to divert ATP for contraction. We describe a novel mechanism of NHE1 inhibition that may be relevant in cardiac disorders featuring altered oxygen metabolism, such as myocardial ischemia and reperfusion injury.

The WHHERE coactivator complex is required for retinoic acid-dependent regulation of embryonic symmetry.

Bilateral symmetry is a striking feature of the vertebrate body plan organization. Vertebral precursors, called somites, provide one of the best illustrations of embryonic symmetry. Maintenance of somitogenesis symmetry requires retinoic acid (RA) and its coactivator Rere/Atrophin2. Here, using a proteomic approach we identify a protein complex, containing Wdr5, Hdac1, Hdac2 and Rere (named WHHERE), which regulates RA signaling and controls embryonic symmetry. We demonstrate that Wdr5, Hdac1, and Hdac2 are required for RA signaling in vitro and in vivo. Mouse mutants for Wdr5 and Hdac1 exhibit asymmetrical somite formation characteristic of RA-deficiency. We also identify the Rere-binding histone methyltransferase Ehmt2/G9a, as a RA coactivator controlling somite symmetry. Upon RA treatment, WHHERE and Ehmt2 become enriched at RA target genes to promote RNA polymerase II recruitment. Our work identifies a protein complex linking key epigenetic regulators acting in the molecular control of embryonic bilateral symmetry.Retinoic acid (RA) regulates the maintenance of somitogenesis symmetry. Here, the authors use a proteomic approach to identify a protein complex of Wdr5, Hdac1, Hdac2 that act together with RA and coactivator Rere/Atrophin2 and a histone methyltransferase Ehmt2 to regulate embryonic symmetry.

The TAF10-containing TFIID and SAGA transcriptional complexes are dispensable for early somitogenesis in the mouse embryo.

During development, tightly regulated gene expression programs control cell fate and patterning. A key regulatory step in eukaryotic transcription is the assembly of the pre-initiation complex (PIC) at promoters. PIC assembly has mainly been studied in vitro, and little is known about its composition during development. In vitro data suggest that TFIID is the general transcription factor that nucleates PIC formation at promoters. Here we show that TAF10, a subunit of TFIID and of the transcriptional co-activator SAGA, is required for the assembly of these complexes in the mouse embryo. We performed Taf10 conditional deletions during mesoderm development and show that Taf10 loss in the presomitic mesoderm (PSM) does not prevent cyclic gene transcription or PSM segmental patterning, whereas lateral plate differentiation is profoundly altered. During this period, global mRNA levels are unchanged in the PSM, with only a minor subset of genes dysregulated. Together, our data strongly suggest that the TAF10-containing canonical TFIID and SAGA complexes are dispensable for early paraxial mesoderm development, arguing against the generic role in transcription proposed for these fully assembled holo-complexes.

MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress.

The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.

ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo.

Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron-sulfur cluster (Fe-S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial Fe4S4 proteins. Here we report that mouse ISCA1 and ISCA2 are Fe2S2-containing proteins that combine all features of Fe-S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe-S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe4S4 proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1-ISCA2 complex seem to exist.

KAT2-mediated PLK4 acetylation contributes to genomic stability by preserving centrosome number.

We have recently identified the first human lysine (K) acetyltransferase 2A and 2B (called KAT2A/2B; known also as GCN5/PCAF, respectively)-dependent acetylome and revealed a mechanism by which KAT2A/2B-mediated acetylation of serine/threonine polo-like kinase 4 (PLK4) maintains correct centrosome number in human cells, therefore contributing to the maintenance of genome stability.1.

KAT2A/KAT2B-targeted acetylome reveals a role for PLK4 acetylation in preventing centrosome amplification.

Lysine acetylation is a widespread post-translational modification regulating various biological processes. To characterize cellular functions of the human lysine acetyltransferases KAT2A (GCN5) and KAT2B (PCAF), we determined their acetylome by shotgun proteomics. One of the newly identified KAT2A/2B substrate is polo-like kinase 4 (PLK4), a key regulator of centrosome duplication. We demonstrate that KAT2A/2B acetylate the PLK4 kinase domain on residues K45 and K46. Molecular dynamics modelling suggests that K45/K46 acetylation impairs kinase activity by shifting the kinase to an inactive conformation. Accordingly, PLK4 activity is reduced upon in vitro acetylation of its kinase domain. Moreover, the overexpression of the PLK4 K45R/K46R mutant in cells does not lead to centrosome overamplification, as observed with wild-type PLK4. We also find that impairing KAT2A/2B-acetyltransferase activity results in diminished phosphorylation of PLK4 and in excess centrosome numbers in cells. Overall, our study identifies the global human KAT2A/2B acetylome and uncovers that KAT2A/2B acetylation of PLK4 prevents centrosome amplification.

Cytoplasmic TAF2-TAF8-TAF10 complex provides evidence for nuclear holo-TFIID assembly from preformed submodules.

General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic cells. How human TFIID-a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)-assembles into a functional transcription factor is poorly understood. Here we describe a heterotrimeric TFIID subcomplex consisting of the TAF2, TAF8 and TAF10 proteins, which assembles in the cytoplasm. Using native mass spectrometry, we define the interactions between the TAFs and uncover a central role for TAF8 in nucleating the complex. X-ray crystallography reveals a non-canonical arrangement of the TAF8-TAF10 histone fold domains. TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core-TFIID complex that exists in the nucleus. Our results provide evidence for a stepwise assembly pathway of nuclear holo-TFIID, regulated by nuclear import of preformed cytoplasmic submodules.