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.

Hat1p and Hat2p are the two subunits of a type B histone acetyltransferase from Saccharomyces cerevisiae that acetylates free histone H4 on lysine 12 in vitro. However, the role for these gene products in chromatin function has been unclear, as deletions of the HAT1 and/or HAT2 gene displayed no obvious phenotype. We have now identified a role for Hat1p and Hat2p in telomeric silencing. Telomeric silencing is the transcriptional repression of telomere-proximal genes and is mediated by a special chromatin structure. While there was no change in the level of silencing on a telomeric gene when the HAT1 or HAT2 gene was deleted, a significant silencing defect was observed when hat1Δ or hat2Δ was combined with mutations of the histone H3 NH2-terminal tail. Specifically, when at least two lysine residues were changed to arginine in the histone H3 tail, a hat1Δ-dependent telomeric silencing defect was observed. The most dramatic effects were seen when one of the two changes was in lysine 14. In further analysis, we found that a single lysine out of the five in the histone H3 tail was sufficient to mediate silencing. However, K14 was the best at preserving silencing, followed by K23 and then K27; K9 and K18 alone were insufficient. Mutational analysis of the histone H4 tail indicated that the role of Hat1p in telomeric silencing was mediated solely through lysine 12. Thus, in contrast to other histone acetyltransferases, Hat1p activity was required for transcriptional repression rather than gene activation.

Type B histone acetyltransferase Hat1p participates in telomeric silencing.

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.

/pdf/the-ubiquitin-conjugating-enzyme-rad6-ubc2-is-required-for-3ju20ddpe9.pdf

The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae.

The good and the bad of being connected: the integrons of aging.

Replicative aging in yeast is asymmetric-mother cells age but their daughter cells are rejuvenated Here we identify an asymmetry in pH between mother and daughter cells that underlies aging and rejuvenation Cytosolic pH increases in aging mother cells, but is more acidic in daughter cells This is due to the asymmetric distribution of the major regulator of cytosolic pH, the plasma membrane proton ATPase (Pma1) Pma1 accumulates in aging mother cells, but is largely absent from nascent daughter cells We previously found that acidity of the vacuole declines in aging mother cells and limits lifespan, but that daughter cell vacuoles re-acidify We find that Pma1 activity antagonizes mother cell vacuole acidity by reducing cytosolic protons However, the inherent asymmetry of Pma1 increases cytosolic proton availability in daughter cells and facilitates vacuole re-acidification and rejuvenation

Mother-daughter asymmetry of pH underlies aging and rejuvenation in yeast.

http://www.kaganovichlab.com/uploads/7/2/5/6/7256268/nihms83888.pdf

Daniel E. Gottschling

Papers

Type B histone acetyltransferase Hat1p participates in telomeric silencing.

The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae.

The good and the bad of being connected: the integrons of aging.

Mother-daughter asymmetry of pH underlies aging and rejuvenation in yeast.

A mother's sacrifice: what is she keeping for herself?