Chromatin assembly factor-1 preserves genome stability in ctf4∆ cells by promoting sister chromatid cohesion

Chromatin assembly and the establishment of sister chromatid cohesion are intimately connected to the progression of DNA replication forks. Here we examined the genetic interaction between the heterotrimeric chromatin assembly factor-1 (CAF-1), a central component of chromatin assembly during replication, and the core replisome component Ctf4. We find that CAF-1 deficient cells as well as cells affected in newly-synthesized H3-H4 histones deposition during DNA rep-lication exhibit a severe negative growth with ctf4∆ mutant. We dissected the role of CAF-1 in the maintenance of genome stability in ctf4∆ yeast cells. In the absence of CTF4, CAF-1 is essential for viability in cells experiencing replication problems, in cells lacking functional S-phase checkpoint or functional spindle checkpoint, and in cells lacking DNA repair pathways involving homologous recombination. We present evidence that CAF-1 affects cohesin association to chromatin in a DNA-damage-dependent manner and is essential to maintain cohesion in the absence of CTF4. We also show that Eco1-catalyzed Smc3 acetylation is reduced in absence of CAF-1. Furthermore, we describe genetic interactions between CAF-1 and essential genes involved in cohesin loading, cohesin stabilization, and cohesin component indicating that CAF-1 is crucial for viability when sister chromatid cohesion is affected. Finally, our data indicate that the CAF-1-dependent pathway required for cohesion is functionally distinct from the Rtt101-Mms1-Mms22 pathway which functions in replicated chromatin assembly. Collectively, our results suggest that the deposition by CAF-1 of newly-synthesized H3-H4 histones during DNA replication creates a chromatin environment that favors sister chromatid cohesion and maintains genome integrity

Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription

International audienceHistone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3'-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms

Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5′ end of active coding regions but a decrease of H3K4 dimethylation at the 3′ end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3′ end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.Acciones Integradas Hispano-Francesas HF2003-0170Ministerio de Educación y Ciencia BFU2005-02603Ministerio de Ciencia y Tecnología BMC2003- 07072-C03-0

FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication

Insertion of Proteins into Membranes A Survey

International audienceIntegral membrane proteins are defined as proteins that span the membrane at least once. Until now, hundreds of coding sequences have been obtained for integral membrane proteins, but by contrast only a limited amount of information about the atomic structure of detergent solubilized proteins has been reported. So far, four kinds of structures have been observed for integral membrane proteins whose structures have been determined either by X-ray crystallography or electron crystallography. The structures known with high resolution are the photo-synthetic reaction centers, the porins, bacteriorhodopsin, and the light harvesting complex II. Determination of these three-dimensional (3-D) structures has provided the information upon which the extensively used prediction methods for the arrangement of membrane proteins have been based. In the absence of three-dimensional structure information, computational methods based on the analysis and comparison of amino-acid sequences have been used to predict the topology of membrane proteins. These methods give a two-dimensional picture of the arrangement of the protein in the membrane. In the meantime, new experimental procedures have been developed, increasing the possibilities to probe membrane topology, and thus the validity of the computational methods

Histone purification from Saccharomyces cerevisiae

International audienceThe nucleosome structure consists of a histone octamer made by a tetramer of H3-­‐H4 histones and two dimers of H2A-­‐H2B. Nucleosomes undergo extensive post-­‐ translational modifications that regulate nucleosome interactions, position, and stability. We describe a protocol allowing the robust purification of histones from the yeast Saccharomyces cerevisiae. This method appears to be suitable to quantitatively analyse specific posttranslational histone modifications

Insertion of Proteins into Membranes A Survey

International audienceIntegral membrane proteins are defined as proteins that span the membrane at least once. Until now, hundreds of coding sequences have been obtained for integral membrane proteins, but by contrast only a limited amount of information about the atomic structure of detergent solubilized proteins has been reported. So far, four kinds of structures have been observed for integral membrane proteins whose structures have been determined either by X-ray crystallography or electron crystallography. The structures known with high resolution are the photo-synthetic reaction centers, the porins, bacteriorhodopsin, and the light harvesting complex II. Determination of these three-dimensional (3-D) structures has provided the information upon which the extensively used prediction methods for the arrangement of membrane proteins have been based. In the absence of three-dimensional structure information, computational methods based on the analysis and comparison of amino-acid sequences have been used to predict the topology of membrane proteins. These methods give a two-dimensional picture of the arrangement of the protein in the membrane. In the meantime, new experimental procedures have been developed, increasing the possibilities to probe membrane topology, and thus the validity of the computational methods

RAP1 moonlights to activate NF-κB and Notch in ALT

International audienceSUMOylated RAP1 dissociates from telomeres to promote their maintenance by ALT (Robinson et al. , in 29 June 2021 issue)