In Saccharomyces cerevisiae, heterochromatin-like regions are found near telomeres and at the silent mating-type loci, where they can repress genes in an epigenetic manner. Several proteins are involved in telomeric heterochromatin structure including Rap1, Sir2, Sir3, Sir4, yKu70 (Hdf1), yKu80 (Hdf2), and the N termini of histones H3 and H4. By recognizing cis-acting DNA-binding sites, Rap1 is believed to recruit Sir and other silencing proteins and determine where heterochromatin forms. The integrity of heterochromatin also requires the binding of Sir proteins to histones that may form a scaffold for Sir protein interactions with chromatin. In this study we describe how the heterochromatin complex may form initially and how it differs from the complex that spreads along the chromosome. We found that close to the telomere end, Sir4 can bind Rap1 independently of Sir2, Sir3, yKu70/yKu80, and the intact H4 N terminus. In contrast, Sir4 binding requires all of the silencing factors further along telomeric heterochromatin. These data indicate that Sir4 binding to Rap1 initiates the sequential association of Sir and other proteins, allowing the subsequent spreading of the heterochromatin proteins along the chromosome. In complex eukaryotes, such as Drosophila melanogaster, heterochromatin at centromeres and telomeres is cytologically condensed throughout the cell cycle, located near the nuclear periphery, and represses gene activity in an epigenetic manner. Such repression occurs by the spreading of the heterochromatin complex into adjacent genes (Henikoff 1990;Wakimoto 1998). Yeast, Saccharomyces cerevisiae, also contains heterochromatin-like regions that include telomeres and the silent matingtype loci (HML␣ and HMRa). These regions, that are found near the nuclear periphery, use a number of known proteins that include Rap1; Silencing Information Regulators Sir2, Sir3, Sir4; and histones H3 and H4 to form heterochromatin (Laurenson and Rine 1992;Grunstein 1997;Cockell and Gasser 1999). Among these proteins Rap1 has a special role in the initiation of heterochromatin because it directly binds cis-acting DNA elements such as C 1-3 A repeat sequences at telomeres and the E and I silencers at the silent HM mating-type loci. Because of the interaction of Rap1 with both Sir3 and Sir4 in two-hybrid experiments and in vitro (Moretti et al. 1994), and interactions in cell extracts and in vitro among Sir3-Sir4-Sir2 (Moazed et al. 1997;Strahl-Bolsinger et al. 1997), it has been proposed that Rap1 may initiate the formation of heterochromatin by recruiting the Sir complex (Moretti et al. 1994;Hecht et al. 1996). The yKu70/yKu80 heterodimer, the yeast homolog of mammalian Ku70/Ku80 that binds to the ends of doublestranded DNA with high affinity (Mimori and Hardin 1986), is also present at the telomeres and is required for the integrity of telomeric heterochromatin (Laroche et al. 1998;Mishra and Shore 1999). Recent studies have shown that the yKu70/yKu80 heterodimer participates in silencing by counteracting Rif1, a Rap1-bindi...

The gene ntcA is required for full expression of proteins subject to ammonium repression in the cyanobacterium Synechococcus. A 3.1 kb DNA fragment able to complement an ntcA mutant was digested with exonuclease III, and deleted fragments of different size were tested for complementation of that mutant, allowing the localization of its mutation within a BamHI-HindIII genomic fragment of c. 0.4 kb. Insertion of a chloramphenicol-resistance-encoding gene cassette into both the BamHI and the HindIII sites of wild-type Synechococcus resulted in a pleiotropic, nitrogen-assimilation-minus phenotype, corroborating the presence of the ntcA gene in that genomic region. Sequencing of DNA in this region showed the presence of an open reading frame that included both the BamHI and the HindIII sites. The ntcA gene product, NtcA, is a protein of 24817 Da which belongs to a family of bacterial transcriptional activators that, among others, includes Crp and Fnr from Escherichia coli. Of special biological significance, it appears, is the presence of a conserved helix-turn-helix motif in the sequence close to the C-terminal end of all the proteins in the family. The gene ntcA is proposed to encode a transcriptional activator of genes subject to nitrogen control in Synechococcus.

Telomere function is influenced by chromatin structure and organization, which usually involves epigenetic modifications. We describe here the chromatin structure of Arabidopsis thaliana telomeres. Based on the study of six different epigenetic marks we show that Arabidopsis telomeres exhibit euchromatic features. In contrast, subtelomeric regions and telomeric sequences present at interstitial chromosomal loci are heterochromatic. Histone methyltransferases and the chromatin remodeling protein DDM1 control subtelomeric heterochromatin formation. Whereas histone methyltransferases are required for histone H3K92Me and non-CpG DNA methylation, DDM1 directs CpG methylation but not H3K92Me or non-CpG methylation. These results argue that both kinds of proteins participate in different pathways to reinforce subtelomeric heterochromatin formation.

The 32 telomeres in the budding yeast genome cluster in three to seven perinuclear foci. Although individual telomeres and telomeric foci are in constant motion, preferential juxtaposition of some telomeres has been scored. To examine the principles that guide such long-range interactions, we differentially tagged pairs of chromosome ends and developed an automated three-dimensional measuring tool that determines distances between two telomeres. In yeast, all chromosomal ends terminate in TG 1-3 and middle repetitive elements, yet subgroups of telomeres also share extensive homology in subtelomeric coding domains. We find that up to 21 kb of >90% sequence identity does not promote telomere pairing in interphase cells. To test whether unique sequence elements, arm length, or chromosome territories influence juxtaposition, we reciprocally swapped terminal domains or entire chromosomal arms from one chromosome to another. We find that the distal 10 kb of Tel6R promotes interaction with Tel6L, yet only when the two telomeres are present on the same chromosome. By manipulating the length and sequence composition of the right arm of chr 5, we confirm that contact between telomeres on opposite chromatid arms of equal length is favored. These results can be explained by the polarized Rabl arrangement of yeast centromeres and telomeres, which promote to telomere pairing by allowing contact between chromosome arms of equal length in anaphase.