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.
Although subtelomeric regions in humans are heterochromatic, the epigenetic nature of human telomeres remains controversial. This controversy might have been influenced by the confounding effect of subtelomeric regions and interstitial telomeric sequences (ITSs) on telomeric chromatin structure analyses. In addition, different human cell lines might carry diverse epigenetic marks at telomeres. We have developed a reliable procedure to study the chromatin structure of human telomeres independently of subtelomeres and ITSs. This procedure is based on the statistical analysis of multiple ChIP-seq experiments. We have found that human telomeres are not enriched in the heterochromatic H3K9me3 mark in most of the common laboratory cell lines, including embryonic stem cells. Instead, they are labeled with H4K20me1 and H3K27ac, which might be established by p300. These results together with previously published data argue that subtelomeric heterochromatin might control human telomere functions. Interestingly, U2OS cells that exhibit alternative lengthening of telomeres have heterochromatic levels of H3K9me3 in their telomeres.
Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp.Synechococcus sp. is a strict photoautotrophic cyanobacterium which is able to use nitrate, nitrite, or ammonium as a nitrogen source. Nitrate assimilation involves nitrate uptake into the cell (8) and its intracellular reduction, via nitrite, to ammonium, which is the inorganic nitrogen source incorporated into carbon skeletons (10). In Synechococcus sp., a nitrate transport element has been identified as a ca. 48-kilodalton (kDa) cytoplasmic membrane protein (19,23,30), and nitrate reductase (a molybdenum-containing enzyme) and nitrite reductase have been isolated and characterized and represent single-polypeptide enzymes of about 75 and 50 kDa, respectively (10). An ammonium transport system has been characterized in Synechococcus sp. by means of studying methylammonium transport (1). Ammonium, which either results from the reduction of nitrate or nitrite or is taken up from the external medium, is mainly assimilated via glutamine synthetase (10).In Synechococcus sp., ammonium acts as a repressor of a number of activities related to nitrogen nutrition, including nitrate reductase (13), nitrite reductase (15), the 48-kDa nitrate transport protein (19,23,30), glutamine synthetase (7), and methylammonium transport (1, 2). The cellular levels of these proteins are higher in nitrate-than in ammonium-grown cells. However, nitrate is not required as an inducer, since the increase in the activities and proteins takes place in response to ammonium deprivation (2,13,15,19). This behavior is similar to that of nitrogenase synthesis and heterocyst development in filamentous, nitrogen-fixing cyanobacteria (31). In the case of nitrate reductase and nitrite reductase, it has been established that ammonium metabolism through glutamine synthetase is required for ammonium repression to take place (13,15 With the aim of gaining insight into the genetics of nitrate assimilation in a Synechococcus sp., we isolated, from strain PCC 7942, a new set of mutants impaired in nitrate assimilation and characterized them by looking for new phenotypes. Here we report the identification and cloning of a gene involved in the regulation of the synthesis not only of the nitrate assimilatory system but also of glutamine synthetase and of the ammonium (methylammonium) permease. MATERIALS AND METHODSStrains and growth conditions. Th...
The chromatin structure of eukaryotic telomeres plays an essential role in telomere functions. However, their study might be impaired by the presence of interstitial telomeric sequences (ITSs), which have a widespread distribution in different model systems. We have developed a simple approach to study the chromatin structure of Arabidopsis telomeres independently of ITSs by analyzing ChIP-seq data. This approach could be used to study the chromatin structure of telomeres in some other eukaryotes. The analysis of ChIP-seq experiments revealed that Arabidopsis telomeres have higher density of histone H3 than centromeres, which might reflects their short nucleosomal organization. These experiments also revealed that Arabidopsis telomeres have lower levels of heterochromatic marks than centromeres (H3K9Me2 and H3K27Me), higher levels of some euchromatic marks (H3K4Me2 and H3K9Ac) and similar or lower levels of other euchromatic marks (H3K4Me3, H3K36Me2, H3K36Me3 and H3K18Ac). Interestingly, the ChIP-seq experiments also revealed that Arabidopsis telomeres exhibit high levels of H3K27Me3, a repressive mark that associates with many euchromatic genes. The epigenetic profile of Arabidopsis telomeres is closely related to the previously defined chromatin state 2. This chromatin state is found in 23% of Arabidopsis genes, many of which are repressed or lowly expressed. At least, in part, this scenario is similar in rice.
The functions of telomeres and, probably, of interstitial telomeric sequences (ITSs) are influenced by their chromatin structure and organization. Telomeres in higher eukaryotes fold into nucleosomes that are about 20-40 bp shorter than the nucleosomes associated with bulk chromatin. Although the functional relevance of this short nucleosomal organization remains unknown, it is believed that short nucleosomes should contribute to telomere function. Whereas telomeric nucleosomes have been widely studied in different organisms, very little is known about the nucleosomal organization of ITSs. Chinese hamster ITSs have been found to associate with short nucleosomes. However, we have found that Arabidopsis thaliana ITSs fold into nucleosomes that have a repeat length similar to bulk chromatin. We discuss how the primary sequence of telomeres and ITSs could influence their nucleosomal organization.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.