Chromatin Modifications and Their Function

High-resolution profiling of histone methylations in the human genome.

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

Polycomb group (PcG) proteins play important roles in maintaining the silent state of HOX genes. Recent studies have implicated histone methylation in long-term gene silencing. However, a connection between PcG-mediated gene silencing and histone methylation has not been established. Here we report the purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. We demonstrate that the complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, we show that H3-K27 methylation colocalizes with, and is dependent on, E(Z) binding at an Ultrabithorax (Ubx) Polycomb response element (PRE), and that this methylation correlates with Ubx repression. Methylation on H3-K27 facilitates binding of Polycomb (PC), a component of the PRC1 complex, to histone H3 amino-terminal tail. Thus, these studies establish a link between histone methylation and PcG-mediated gene silencing.

https://science.sciencemag.org/content/sci/298/5595/1039.full.pdf

Role of Histone H3 Lysine 27 Methylation in Polycomb-Group Silencing

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.

/pdf/designer-deletion-strains-derived-from-saccharomyces-2djselfyo7.pdf

Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.

Genes located near telomeres in Saccharomyces cerevisiae undergo position-effect variegation; their transcription is subject to reversible but mitotically heritable repression. This position effect and the finding that telomeric DNA is late replicating suggest that yeast telomeres exist in a heterochromatin-like state. Mutations in genes that suppress the telomeric position effect suggest that a special chromatin structure exists near chromosomal termini. Thus transcriptional repression may be explained by the inability of DNA binding proteins to access the DNA near telomeres. To test this hypothesis, the Escherichia coli Dam DNA methyltransferase, which modifies the sequence GATC, was introduced into S. cerevisiae cells. DNA sequences near the telomere were highly refractive to Dam methylation but were modified when located at positions more internal on the chromosome. Telomeric sequences were accessible to methyltransferase activity in strains that contained a mutation that suppressed the telomeric position effect. These data support the model that sequence-specific DNA binding proteins are excluded from telomere-proximal sequences in vivo and show that expression of DNA methyltransferase activity may serve as a useful tool for mapping chromosomal structural domains in vivo.

Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo.

There is a strong correlation between age and cancer, but the mechanism by which this phenomenon occurs is unclear. We chose Saccharomyces cerevisiae to examine one of the hallmarks of cancer--genomic instability--as a function of cellular age. As diploid yeast mother cells aged, an approximately 100-fold increase in loss of heterozygosity (LOH) occurred. Extending life-span altered neither the onset nor the frequency of age-induced LOH; the switch to hyper-LOH appears to be on its own clock. In young cells, LOH occurs by reciprocal recombination, whereas LOH in old cells was nonreciprocal, occurring predominantly in the old mother's progeny. Thus, nuclear genomes may be inherently unstable with age.

An age-induced switch to a hyper-recombinational state.

The replicative life span (RLS) of Saccharomyces cerevisiae has been established as a model for the genetic regulation of longevity despite the inherent difficulty of the RLS assay, which requires separation of mother and daughter cells by micromanipulation after every division Here we present the mother enrichment program (MEP), an inducible genetic system in which mother cells maintain a normal RLS—a median of 36 generations in the diploid MEP strain—while the proliferative potential of daughter cells is eliminated Thus, the viability of a population over time becomes a function of RLS, and it displays features of a survival curve such as changes in hazard rate with age We show that viability of mother cells in liquid culture is regulated by SIR2 and FOB1, two opposing regulators of RLS in yeast We demonstrate that viability curves of these short- and long-lived strains can be easily distinguished from wild type, using a colony formation assay This provides a simplified screening method for identifying genetic or environmental factors that regulate RLS Additionally, the MEP can provide a cohort of cells at any stage of their life span for the analysis of age-associated phenotypes These capabilities effectively remove the hurdles presented by RLS analysis that have hindered S cerevisiae aging studies since their inception 50 years ago

https://www.genetics.org/content/genetics/183/2/413.full.pdf

The Mother Enrichment Program: A Genetic System for Facile Replicative Life Span Analysis in Saccharomyces cerevisiae

https://www.cell.com/molecular-cell/pdf/S1097-2765(10)00960-3.pdf

Disorder targets misorder in nuclear quality control degradation: a disordered ubiquitin ligase directly recognizes its misfolded substrates.

Different regions of the eukaryotic genome are known to replicate at distinct, reproducible times within the period of S phase. This is observed in plants, insects, and vertebrates, as well as in the yeast Saccharomyces cerevisiae (Hand 1978; Fangman and Brewer 1992). In species with large genomes, extensive regions of the chromosomes replicate early in S phase, whereas other domains do not initiate replication until after the early domains have completed synthesis. Frequently, late replication of DNA is associated with its assembly into heterochromatin, which is highly condensed chromatin that often contains repeated DNA sequences such as those found at centromeres and telomeres (John 1988). This condensed chromosome replicates much later in S phase than its transcriptionally active homolog.

Telomeric chromatin in S. cerevisiae has several traits typical of heterochromatin (Grunstein 1998). In particular, telomeres confer epigenetic silencing of nearby genes (position effect variegation), and they replicate late in S phase (McCarroll and Fanagman 1988; Gottschling et al. 1990). The special chromatin found near telomeres is composed of hypoacetylated core histones as well as the SIR proteins, which are required for silencing telomeric genes (Grunstein 1998). Of the SIR proteins, Sir3p is probably the key component that defines a telomeric domain of transcriptional repression. It interacts with the tails of histones H3 and H4, spreading from the telomere inward along the chromosome, and its abundance in the cell determines how far a silent domain extends from the telomere (Renauld et al. 1993; Hecht et al. 1996).

In yeast, chromosome replication initiates at ARS (autonomous replicating sequences) elements (Fangman and Brewer 1991). ARS elements were originally identified as sequences that permit high-efficiency transformation of plasmids in yeast by serving as origins of DNA replication. However, in their normal chromosomal context, only a subset of the ARS elements initiate DNA replication within the ∼30 min duration of S phase (Fangman and Brewer 1992). Specific chromosomal origins of S. cerevisiae, like ARS1 on chromosome IV, initiate replication relatively early in S phase. Other ARS elements initiate later in S phase, such as ARS501 on chromosome V, which replicates ∼10 min after ARS1 (Ferguson et al. 1991).

It appears that telomeres can confer late replication on proximal origins. For instance, telomere-proximal middle repetitive sequences replicate relatively late in S phase (McCarroll and Fangman 1988; Louis 1995). In addition, whereas ARS elements on circular plasmids initiate replication early in S phase, they replicate late in S phase when the plasmid is linearized and telomeric sequences are added to its ends (Ferguson and Fangman 1992). In this study we investigated the role of silent chromatin in imposing late replication on origins near telomeres.

/pdf/telomeric-chromatin-modulates-replication-timing-near-2wg8ky60r2.pdf

Daniel E. Gottschling

Papers

Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo.

An age-induced switch to a hyper-recombinational state.

The Mother Enrichment Program: A Genetic System for Facile Replicative Life Span Analysis in Saccharomyces cerevisiae

Disorder targets misorder in nuclear quality control degradation: a disordered ubiquitin ligase directly recognizes its misfolded substrates.

Telomeric chromatin modulates replication timing near chromosome ends