Genetic Remodeling and Transcriptional Remodeling of Subtelomeric Heterochromatin Are Different ² Sabrina Venditti,* ,‡ Glauco Di Stefano, ‡ Manuela D’Eletto, ‡ and Ernesto Di Mauro ‡,§,| Centro di Studio per gli Acidi Nucleici (CNR), Fondazione Istituto Pasteur, Fondazione Cenci Bolognetti, and Dipartimento di Genetica e Biologia Molecolare, UniVersita’ di Roma La Sapienza, P. le A. Moro 5, 00185, Roma, Italy ReceiVed December 14, 2001; ReVised Manuscript ReceiVed February 14, 2002 ABSTRACT: The structure, the extension, and the regulatory functions of telomeric and subtelomeric heterochromatin are not completely understood partly due to the difficulty of separating structural from functional features. We have previously observed that genetic alterations of telomeric heterochromatin components relieve transcriptional silencing. We have developed an analytical system allowing the separate determination of the effects of transcription and of genetic alterations on the subtelomeric structures. The uncoupled analysis, performed on the left extremity of chromosome III of Saccharomyces cereVisiae, consists of genetic dissections, induction of transcription of a resident gene, and chromatin analysis. The results allow (i) the determination of the precise localization and of the extension of heterochromatin (here from 0.9 to 2.6 kb from the innermost extremity of the C 1-3 A tract) and (ii) the definition of the transcription and of the genetically induced chromatin remodelings and of their marked differences, thus allowing (iii) specific analyses of the structural effects of the genetic modification of the heterochromatin components. Although not cytologically detectable as in higher eukary- otes, Saccharomyces cereVisiae telomeric chromatin shows the functional characteristics of heterochromatin, such as late replication, resistance to nucleases, and position effects on the genes located in their vicinity (1). Coupled with the repetitive nature of telomeric and subtelomeric DNA se- quences (both locally and among different chromosomal ends), these two latter properties make the analysis of telomeric chromatin structure and of its function particularly difficult. The scarcity of data on the transcription of naturally occurring genes in subtelomeric regions adds to this dif- ficulty. Telomeric position effect (TPE) 1 is exerted through the involvement of the silent information regulators, Sir3p, Sir2p, and Sir4p. Deletion of each Sir protein causes loss of telomeric transcriptional silencing (2). From the structural point of view a number of studies have revealed that telomeres are covered with a complex aggregate of Sir proteins that in yeast is responsible for all of the properties mentioned above. This aggregate includes several components such as the SIR proteins, along with Rif1p and Rif2p (3, 4), and the Ku proteins involved in DNA repair and recombination (5, 6). The Sir complex starts its nucleation from the terminal telomeric repeats, due to the ability of the Sir proteins to bind Rap1p, which in turn directly binds DNA at sites encompassed in the sequence-repetitive region (7, 8). The Sir proteins spread along the subtelomeric regions interacting with each other and with the N-terminal tails of histones H3 and H4 (9). The diffusion by spreading is reinforced by the folding back of the chromosome end onto the subtelomeric region, according to a model based on chromatin immuno- precipitation analysis (7). Lack of any one of the Sir proteins causes disruption of the complex, indicating that its integrity is required for the correct organization of the heterochromatin structure (7). The individual functions of the Sir proteins have been clarified only partially, making it difficult to define how they affect telomeric heterochromatin from a biochemi- cal point of view. However, some evidence points to the involvement of the Sir proteins in the organization of the underlying repressive nucleosome structure. First, Sir2p, which participates in the complex through interaction with Sir4p, was recently described to have NAD- dependent histone deacetylase activity (10, 11). Substrates for this activity were shown to be Lys9 and Lys14 of histone H3 and Lys16 of histone H4, located in the histone domains involved in the interaction with Sir3p and Sir4p (9, 12). A second line of evidence comes from previous work on a S. cereVisiae Ty5-1 element present in subtelomeric position on the left arm of chromosome III. Being defective for transposition, this element is unique in the yeast genome. ² This work was supported by CNR Target Project on Biotechnology, by MURST 5% project Biomolecole per la Salute Umana, and by MURST 40% Projects. * To whom correspondence should be addressed. E-mail: sabrina.venditti@uniroma1.it. Phone: +11.39.06.49912659. Fax: +11.39.06.49912500. ‡ Dipartimento di Genetica e Biologia Molecolare. § Centro di Studio per gli Acidi Nucleici (CNR). | Fondazione Istituto Pasteur, Fondazione Cenci Bolognetti. 1 Abbreviations: SIR, silent information regulator; TPE, telomeric position effect; MNase, micrococcal nuclease; LTR, long terminal repeat; PRE, pheromone response element. 4901 Biochemistry 2002, 41, 4901-4910 10.1021/bi016052y CCC: $22.00 © 2002 American Chemical Society Published on Web 03/20/2002