Unlike higher eukaryotes, where an inverse correlation has been generally observed between gene expression and methylation of CpG sites, the budding yeast Saccharomyces cerevisiae lacks DNA methylation. Gene regulatory mechanisms can function independently of DNA methylation in yeast, and yeast strains expressing foreign DNA methylases that modify adenine and CpG residues have been found to be viable. We have used such strains to determine whether the transcriptional status of genes can influence the level of their DNA methylation in vivo. Several genes were tested, for example, GALl, -7, and -10, PH05, HMRa and HMLa, and STE2 and STE3. Surprisingly, we found that all the genes displayed severalfold more methylation in the expressed state as compared to the repressed state. This procedure serves as a novel in vivo probe for the chromatin structure of yeast and potentially for higher eukaryotes.

Analyses of frequency profiles of markers on disease or drug-response related genes in diverse populations are important for the dissection of common diseases. We report the results of analyses of data on 405 SNPs from 75 such genes and a 5.2 Mb chromosome, 22 genomic region in 1871 individuals from diverse 55 endogamous Indian populations. These include 32 large (>10 million individuals) and 23 isolated populations, representing a large fraction of the people of India. We observe high levels of genetic divergence between groups of populations that cluster largely on the basis of ethnicity and language. Indian populations not only overlap with the diversity of HapMap populations, but also contain population groups that are genetically distinct. These data and results are useful for addressing stratification and study design issues in complex traits especially for heterogeneous populations.

High mobility group (HMG) proteins 1 and 2 are thought to be associated with chromatin enriched in active gene sequences, to stimulate endogenous transcription of class II and III genes using HMG-depleted nuclei, and to bind specific DNA sequences upstream of the coding regions of trout HMG-T and human beta-globin genes. In testing the possibility that these proteins may act as general transcription factors, the run-off transcription of trout protamine, human beta-globin, adenovirus 2 major late promoter, and herpes simplex virus (HSV) thymidine kinase genes was found to be inhibited by affinity-purified HMG-1 and -2 antibodies. The inhibition was partially relieved by exogenously added HMG-1 or -2. A complementation assay showed that the 0.15 M KCl flowthrough of HeLa nuclear extract fractionated by anion-exchange chromatography (DE-52) could be replaced by purified HMG-1 and/or -2 to complement transcription of the trout protamine gene by the 0.5 M KCl eluate fraction. Inhibition studies with heparin showed that HMG-1 and -2 were required for initiation of transcription. These results indicate an absolute requirement of HMG-1 and -2 for class II gene transcription. Western blotting and transcription reconstituted with purified factors show a copurification of HMG-1 and -2 with factor II B, described earlier by Reinberg and Roeder [(1987) J. Biol. Chem. 262, 3310-3321].

In the fission yeast Schizosaccharomyces pombe, the double-stranded chromosomal break (DSB) at the mating-type locus (mat1) initiates recombination during mating-type switching. A constant DSB level is maintained throughout the cell-cycle. In the strand-segregation model for mating-type switching, it was postulated that if the DSB is generated during or soon after mat1 replication, one of the chromatids could be repaired and switched during replication in the next cell cycle, while the other chromatid inherits the break. Here we report a molecular characterization of swi7, one of the genes required for DSB formation. Surprisingly, a gene complementing the swi7 mutation maps to chromosome I and encodes S. pombe DNA polymerase-alpha. Disruption of this gene is lethal in both switching and non-switching strains, as expected. S. pombe DNA polymerase-alpha must therefore play a role in generating the DSB at mat1, suggesting that DSB formation is coupled with DNA replication.

Although DNA replication has been thought to play an important role in the silencing of mating type loci in Saccharomyces cerevisiae, recent studies indicate that silencing can be decoupled from replication. In Schizosaccharomyces pombe, mating type silencing is brought about by the trans-acting proteins, namely Swi6, Clr1-Clr4, and Rhp6, in cooperation with the cis-acting silencers. The latter contain an autonomous replication sequence, suggesting that DNA replication may be critical for silencing in S. pombe. To investigate the connection between DNA replication and silencing in S. pombe, we analyzed several temperature-sensitive mutants of DNA polymerase ␣. We find that one such mutant, swi7H4, exhibits silencing defects at mat, centromere, and telomere loci. This effect is independent of the checkpoint and replication defects of the mutant. Interestingly, the extent of the silencing defect in the swi7H4 mutant at the silent mat2 locus is further enhanced in absence of the cis-acting, centromere-proximal silencer. The chromodomain protein Swi6, which is required for silencing and is localized to mat and other heterochromatin loci, interacts with DNA polymerase ␣ in vivo and in vitro in wild type cells. However, it does not interact with the mutant pol␣ and is delocalized away from the silent mat loci in the mutant. Our results demonstrate a role of DNA polymerase ␣ in the establishment of silencing. We propose a recruitment model for the coupling of DNA replication with the establishment of silencing by the chromodomain protein Swi6, which may be applicable to higher eukaryotes.The well studied system of mating type silencing in the budding yeast Saccharomyces cerevisiae has served as a paradigm for developmental regulation of gene regulation. Although the mating type phenotype of a homothallic strain is dictated by the MAT locus depending on whether it harbors the a-or ␣-specific alleles, two copies of the same genetic information are located at distant sites on the same chromosome, namely HML and HMR, which harbor ␣ and a alleles, respectively. However, these alleles are transcriptionally silent. The silencing is achieved by the cis-acting sequences E (essential) and I (important) that flank both HML and HMR loci (1, 2). In addition, several genes encode factors named mating type regulator/silent information regulator (MAR/SIR) that function in trans through the cis-acting sequences in keeping the HML and HMR loci silent. Extensive studies in S. cerevisiae have suggested that DNA replication is important for repression of the silent mating type loci HML and HMR (see Refs. 1 and 2 for reviews). These findings include a requirement of passage through S phase, a functional autonomous replication sequence (ARS) flanking the silent locus HMR, and a functional origin recognition complex for silencing (reviewed in Ref. 2). However, the requirement of DNA replication for silencing is obviated if the SIR1 silencing protein is recruited by alternative means, although passage through S phase is still essential (3, 4)....

Swi6/HP1, an evolutionarily conserved protein, is critical for heterochromatin assembly in fission yeast and higher eukaryotes. In fission yeast, histone deacetylation by histone deacetylases is thought to be followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/Suv39H1. H3-Lys-9-Me2 interacts with the chromodomain of Swi6/HP1. Swi6/HP1 is thought to act downstream of Clr4/Suv39, and further self-association of Swi6/HP1 is assumed to stabilize the heterochromatin structure. Here, we show that the self-association-defective mutant of Swi6 does not interact with Clr4. It not only fails to localize to heterochromatin loci but also interferes with heterochromatic localization of H3-Lys-9-Me2 (and thereby Clr4) and the endogenous Swi6 in a dominant negative manner. Thus, self-association of Swi6/HP1 helps in binding to and recruitment of Clr4 and thereby in establishment and maintenance of heterochromatin by a concerted rather than a sequential mechanism.Distinct sets of histone modifications organize chromatin into transcriptionally expressed or repressed structures. As in higher eukaryotes, fission yeast euchromatic regions are associated with hyperacetylated histones, particularly Lys-9 and Lys-14 acetylated and Lys-4 methylated histone H3. Conversely, heterochromatin assembly (silent mating type, centromere, rDNA, and telomere loci) involves deacetylation of histone H3 at Lys-9 and Lys-14 by Clr3, Clr6, and Sir2, followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/ Suv39H1. H3-Lys-9-Me2 is recognized by the chromodomain in Swi6/HP1 (1, 2). The property of Swi6 to form multimers is thought to cause folding of chromatin into a transcriptionally inactive structure (3). The RNAi pathway is also involved in heterochromatin assembly. The dcr1⌬, rdp1⌬, and ago1⌬ mutants are defective in H3-Lys-9 methylation and Swi6 recruitment at heterochromatin loci (4).Structural studies show that the chromoshadow domain (CSD) 3 in Swi6/HP1 contains a dimerization motif that creates a cleft for binding a pentapeptide (5, 6). Mutations in the conserved residues in the cleft inhibit interaction with the pentapeptide motif in proteins (6, 7). We found that mutation of one such residue (L315E) severely compromises the self-association property of Swi6. is not only defective in complementing the silencing defect in the swi6⌬ mutant but also exerts a dominant negative effect on silencing, accompanied by loss of heterochromatic localization not only of itself but also that of native Swi6 and H3-Lys-9-Me2 and, by implication, of Clr4. These results support a concerted rather than a sequential action of Clr4 and Swi6 in establishment and maintenance of heterochromatin. EXPERIMENTAL PROCEDURESStrains and Plasmids Used-All strains and plasmids used are listed in supplemental Tables 1 and 2, respectively.Media Compositions-All fission yeast media were prepared according to Moreno et al. (8). The serial dilution plate assay has been described earlier (9). Iodine staining assays for switching and silencing have ...

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