ER-associated degradation (ERAD): Novel components and cellular regulation

The majority of eukaryotic proteins are degraded by the ubiquitin-proteasome system. In this pathway, cytosolic substrates are first earmarked for degradation by modification with ubiquitin ('ubiquitylation') and subsequently degraded by the 26S pro-teasome, a large protease residing in both the cytosol and the nucleus. ER-resident proteins are similarly degraded but take the route of a specialized pathway coined ER-associated degradation (ERAD). In order to reach the cytosolic ubiquitin/proteasome system, these substrates must first relocate from the ER to the cytosol, possibly with the help of protein conducting membrane channels. Previous work has shown that specific ubiquitin-conjugating enzymes (e.g. Ubc6, Ubc7) and ubiquitin ligases (e.g. Hrd1) con-tribute to ERAD, but how the substrates reach the proteasome remained to be clarified. Besides its function as a quality control system in recognizing and eliminating aberrant proteins, ERAD appears also to play a part in regulatory pathways. This study focuses on the identification of novel components contributing to ERAD. It could be demonstrated that the yeast protein Cdc48 (p97 in mammals), to-gether with its co-factors Ufd1 and Npl4, plays a key role in this process. Cdc48 belongs to the large family of AAA-type ATPases and is believed to function as a chaperone-like enzyme. Previous work has shown that the Cdc48 complex specifically acts on ubiquitylated substrates. This study indicates that the Cdc48 complex takes part in mo-bilization of ERAD substrates from the ER membrane for proteasomal targeting. Fur-thermore, degradation of some ERAD substrates involves the multiubiquitylation factor E4/Ufd2 and proteasome targeting factors of the Rad23 protein family. Another aspect of this work addresses the regulatory functions of ERAD. The fatty acid desaturase Ole1, an integral membrane protein of the ER, was identified as a novel ERAD substrate. Intriguingly, ERAD of Ole1 is specifically regulated since the protein is particularly short lived in the presence of high levels of unsaturated fatty acids, the products of Ole1. Thus, this feedback loop provides an additional mechanism, by which the cell regulates the amount of unsaturated fatty acids. The t-SNARE (syntaxin) protein Ufe1 was characterized as another substrate of ERAD. This protein is required for homotypic membrane fusion of ER vesicles. Notably, Ufe1 degradation is negatively controlled by its binding partner Sly1, a member of the SM (Sec1/Munc18) protein fam-ily. Reciprocal mutations in the Ufe1-Sly1 interaction face result in rapid degradation of Ufe1 by ERAD. Conversely, strong overproduction of Ufe1 was found to be detrimental for cellular growth. These findings suggest that one important function of Sly1 is to con-trol Ufe1 SNARE levels in order to ensure cellular homeostasis. In conclusion, analysis of the degradation of Ole1 and Ufe1 revealed an important contribution of ERAD to es-sential regulatory pathways

Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability.

Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While heterochromatin and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. Rather, the gene-protective effect is strictly dependent on Set1's catalytic activity. H3K4 methylation, the Set1 product, antagonizes spreading in two ways: directly inhibiting catalysis by Suv39/Clr4 and locally disrupting nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion

Crosstalk between H2A variant-specific modifications impacts vital cell functions

Selection of C-terminal motifs participated in evolution of distinct histone H2A variants. Hybrid types of variants combining motifs from distinct H2A classes are extremely rare. This suggests that the proximity between the motif cases interferes with their function. We studied this question in flowering plants that evolved sporadically a hybrid H2A variant combining the SQ motif of H2A.X that participates in the DNA damage response with the KSPK motif of H2A.W that stabilizes heterochromatin. Our inventory of PTMs of H2A.W variants showed that in vivo the cell cycle-dependent kinase CDKA phosphorylates the KSPK motif of H2A. W but only in absence of an SQ motif. Phosphomimicry of KSPK prevented DNA damage response by the SQ motif of the hybrid H2A.W/X variant. In a synthetic yeast expressing the hybrid H2A.W/X variant, phosphorylation of KSPK prevented binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and impaired response to DNA damage. Our findings illustrate that PTMs mediate interference between the function of H2A variant specific C-terminal motifs. Such interference could explain the mutual exclusion of motifs that led to evolution of H2A variants.Fil: Schmücker, Anna. Austrian Academy Of Sciences (oaw);Fil: Lei, Bingkun. Austrian Academy Of Sciences (oaw);Fil: Lorkovic, Zdravko J.. Ludwig Maximilians Universitat; AlemaniaFil: Capella, Matias. Ludwig Maximilians Universitat; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Braun, Sigurd. Ludwig Maximilians Universitat; AlemaniaFil: Bourguet, Pierre. Gregor Mendel Institute Of Molecular Plant Biology; Austria. Université Clermont Auvergne; FranciaFil: Mathieu, Olivier. Université Clermont Auvergne; FranciaFil: Mechtler, Karl. Gregor Mendel Institute Of Molecular Plant Biology; AustriaFil: Berger, Frédéric. Gregor Mendel Institute Of Molecular Plant Biology; Austri

A Synthetic Approach to Reconstruct the Evolutionary and Functional Innovations of the Plant Histone Variant H2A.W

Diversification of histone variants is marked by the acquisition of distinct motifs and functional properties through convergent evolution.1–4 H2A variants are distinguished by specific C-terminal motifs and tend to be segregated within defined domains of the genome.5,6 Whether evolution of these motifs pre-dated the evolution of segregation mechanisms or vice versa has remained unclear. A suitable model to address this question is the variant H2A.W, which evolved in plants through acquisition of a KSPK motif7 and is tightly associated with heterochromatin.4 We used fission yeast, where chromatin is naturally devoid of H2A.W, to study the impact of engineered chimeras combining yeast H2A with the KSPK motif. Biochemical assays showed that the KSPK motif conferred nucleosomes with specific properties. Despite uniform incorporation of the engineered H2A chimeras in the yeast genome, the KSPK motif specifically affected heterochromatin composition and function. We conclude that the KSPK motif promotes chromatin properties in yeast that are comparable to the properties and function of H2A.W in plant heterochromatin. We propose that the selection of functional motifs confer histone variants with properties that impact primarily a specific chromatin state. The association between a new histone variant and a preferred chromatin state can thus provide a setting for the evolution of mechanisms that segregate the new variant to this state, thereby enhancing the impact of the selected properties of the variant on genome activity.Fil: Lei, Bingkun. Gregor Mendel Institute; AustriaFil: Capella, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Montgomery, Sean A.. Gregor Mendel Institute; AustriaFil: Borg, Michael. Gregor Mendel Institute; AustriaFil: Osakabe, Akihisa. Gregor Mendel Institute; AustriaFil: Goiser, Malgorzata. Gregor Mendel Institute; AustriaFil: Muhammad, Abubakar. Universitat Technical Zu Munich; AlemaniaFil: Braun, Sigurd. Universitat Technical Zu Munich; AlemaniaFil: Berger, Frédéric. Gregor Mendel Institute; Austri

Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity

Global regulation of heterochromatin spreading by Leo1

Heterochromatin plays important roles in eukaryotic genome regulation. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates robust mechanisms to contain heterochromatin within defined boundaries and thus prevent silencing of expressed genes. Here we show that loss of the PAF complex (PAFc) component Leo1 compromises chromatin boundaries, resulting in invasion of heterochromatin into flanking euchromatin domains. Similar effects are seen upon deletion of other PAFc components, but not other factors with related functions in transcription-associated chromatin modification, indicating a specific role for PAFc in heterochromatin regulation. Loss of Leo1 results in reduced levels of H4K16 acetylation at boundary regions, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1 Delta cells, suggesting that Leo1 antagonises heterochromatin spreading by promoting H4K16 acetylation. Our findings reveal a previously undescribed role for PAFc in regulating global heterochromatin distribution

Chromatin binding and silencing: Two roles of the same protein Lem2

Transcriptionally repressed chromatin localizes to specific areas within the eukaryotic nucleus and is often found at the nuclear periphery, which is thought to provide a specialized compartment for gene silencing. However, the molecular mechanisms that establish this spatial chromatin organization are still poorly understood. In our recent work (Barrales et al. 2016), we identified the nuclear envelope protein Lem2, a homolog of metazoan lamin-associated proteins (LAPs), as a relevant factor for heterochromatin silencing and perinuclear localization in the fission yeast Schizosaccharomyces pombe. Several other LAPs have previously been reported to associate with heterochromatin, and it has been proposed that this interaction might directly contribute to gene repression, perhaps through tethering via chromatin-binding domains like the LEM domain. We demonstrated that the LEM domain of Lem2 is indeed essential for centromere binding and perinuclear tethering. However, we made the surprising finding that tethering via the LEM domain is functionally independent of Lem2's role in silencing, which instead is mediated by a different part of the protein, the MSC domain. Our study demonstrates that tethering and silencing, although mediated by the same molecule, Lem2, can be mechanistically separated. This further unveils a complex function of this protein at the interface between the nuclear periphery and silent chromatin, which might be preserved among the other members of this conserved family of LEM proteins

Beyond tethering and the LEM domain: MSCellaneous functions of the inner nuclear membrane Lem2

The nuclear envelope plays a pivotal role in the functional organization of chromatin. Various inner nuclear membrane (INM) proteins associate with transcriptionally repressed chromatin, which is often found at the nuclear periphery. A prominent example is the conserved family of LEM (LAP2-Emerin-MAN1) domain proteins that interact with DNA-binding proteins and have been proposed to mediate tethering of chromatin to the nuclear membrane. We recently reported that the fission yeast protein Lem2, a homolog of metazoan LEM proteins, contributes to perinuclear localization and silencing of heterochromatin. We demonstrate that binding and tethering of centromeric chromatin depends on the LEM domain of Lem2. Unexpectedly, this domain is dispensable for heterochromatin silencing, which is instead mediated by a different structural domain of Lem2, the MSC (MAN1-Src1 C-terminal) domain. Hence, silencing and tethering by Lem2 can be mechanistically separated. Notably, the MSC domain has multiple functions beyond heterochromatic silencing. Here we discuss the implications of these novel findings for the understanding of this conserved INM protein.This work was supported by grants to S.B. by the European Union Network of Excellence EpiGeneSys (HEALTH-2010-257082), the German Research Foundation (BR 3511/3-1), and the Friedrich-Baur Stiftung.Peer Reviewe

Shaping the landscape: mechanistic consequences of ubiquitin modification of chromatin

The organization of eukaryotic chromosomes into transcriptionally active euchromatin and repressed heterochromatin requires mechanisms that establish, maintain and distinguish these canonical chromatin domains. Post-translational modifications are fundamental in these processes. Monoubiquitylation of histones was discovered more than three decades ago, but its precise function has been enigmatic until recently. It is now appreciated that the spectrum of chromatin ubiquitylation is not restricted to monoubiquitylation of histones, but includes degradatory ubiquitylation of histones, histone-modifying enzymes and non-histone chromatin factors. These occur in a spatially and temporally controlled manner. In this review, we summarize our understanding of these mechanisms with a particular emphasis on how ubiquitylation shapes the physical landscape of chromatin