Analysis Of Chromatin Structure Research Articles

Genome-Scale Analysis of Hematopoietic Regulatory Sequences by Digital Analysis of Chromatin Structure.

In vivo, cis-regulatory elements coincide with focal disruptions in chromatin structure that manifest as DNaseI hypersensitive sites (HSs). We developed a novel stochastic methodology for comprehensive, genome-wide mapping of DNaseI HSs in vivo. We show that 19–20bp genomic DNA tags can be used to effect genome-wide localization of individual DNaseI cutting events in nuclear chromatin, and that such tags can be generated and sequenced in large numbers from limited numbers of starting cells. We analyzed 257,000 tags from a hematopoietic cell line (K562) and applied a quantitative algorithm to discriminate statistically significant tag clustering events. Such tag clusters identified both known and novel functional elements of hematopoietic genes across the genome. A unique feature of this approach is that it permits unbiased evaluation of the chromatin context of regulatory sequences from disperse genomic loci. We observed surprisingly large differences in the chromatin structural configuration of a variety of active erythroid and hematopoietic regulatory sequences, suggesting a discrete hierarchy of nuclear organization that is not apparent with conventional assays. This approach can be applied readily to generate a comprehensive catalogue of cis-regulatory sequences active in hematopoietic stem cells.

Multiple interactions between BRG1 and MHC class II promoter binding proteins

The class II transactivator (CIITA) interacts with the chromatin remodeling factor brahma related gene 1 (BRG1) as a necessary component of the transcriptional activation of human major histocompatibility complex (MHC) class II genes. We report here that RFXAP, a subunit of the DNA-binding RFX complex, also binds BRG1 and therefore provides a mechanism by which MHC class II gene chromatin can be remodeled in the absence of CIITA. RFXAP and CIITA bind to different regions of BRG1. The region of the RFXAP protein that binds BRG1 coincides with the minimally required fragment of RFXAP previously reported to be necessary to mediate MHC class II gene transcription. For CIITA, we found that BRG1 interacts with both the N-terminal acidic amino acid rich transactivation domain and a central region of CIITA that contains GTP-binding motifs. Analysis of chromatin structure of MHC class II genes in cell lines lacking CIITA or RFXAP, suggests that the RFXAP–BRG1 interaction may, in some cell types, produce chromatin remodeling.

Granulocyte-macrophage colony-stimulating factor enhancer activation requires cooperation between NFAT and AP-1 elements and is associated with extensive nucleosome reorganization.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is activated by an NFAT-dependent enhancer forming an inducible DNase I hypersensitive (DH) site. The enhancer core comprising the DH site contains the GM330 and GM420 elements that bind NFAT and AP-1 cooperatively. Here we demonstrate that both elements are essential for enhancer activity and that Sp1 and AML1 sites in the enhancer become occupied in vivo only after activation. Chromatin structure analysis revealed that the GM-CSF enhancer core elements are divided between two adjacent nucleosomes that become destabilized and highly accessible after activation. Inducible chromatin reorganization was not restricted to the enhancer core but extended across a 3-kb domain of mobilized nucleosomes, within which the nucleosome repeat length was compressed from approximately 185 to 150 bp. The GM420 element is a high-affinity site that binds NFAT independently of AP-1 but depends on the linked AP-1 site for enhancer function. Nevertheless, just the NFAT motif from the GM420 element was sufficient to form a DH site within chromatin even in the absence of the AP-1 site. Hence, NFAT has the potential to cooperate with other transcription factors by promoting chromatin remodelling and increasing accessibility at inducible regulatory elements.

Identification of a Myb-responsive enhancer of the chicken C/EBPbeta gene.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) that disrupts myelomonocytic differentiation and transforms myelomonocytic cells. It is thought that the biological effects of v-Myb are caused by deregulation of specific target genes. The CCAAT box/enhancer binding protein beta (C/EBPbeta), a member of the basic region-leucine zipper (bzip) class of transcription factors, which itself plays an important role during myelomonocytic differentiation, has previously been shown to be regulated by Myb. Here we have addressed the mechanism by which v-Myb affects C/EBPbeta expression. We have employed the mapping of DNase I hypersensitive sites (DHSs) in chromatin as a tool to detect in vivo target sites of v-Myb. Our data identify a DHS downstream of the C/EBPbeta gene that appears to be specific for v-myb-transformed myeloblasts. We have confirmed by chromatin immunoprecipitation that v-Myb is bound to this region in vivo. Furthermore, we have found that ectopic expression of v-Myb in a myelomonocytic cell line is able to induce a DHS downstream of the C/EBPbeta gene, showing for the first time that v-Myb can affect chromatin structure. Reporter gene experiments demonstrate that the downstream DHS acts as a Myb-dependent enhancing element in transiently as well as in stably transfected myelomonocytic cells. Previous work has shown that v-Myb acts on the C/EBPbeta promoter; it now appears that Myb stimulates C/EBPbeta expression by acting on the promoter as well as on an enhancer of the C/EBPbeta gene. Interestingly, the mechanisms by which Myb acts on both elements differ; while Myb activation of the promoter requires the cooperation with C/EBPbeta, activation of the enhancer by Myb is independent of C/EBPbeta. Apart from the identification of a novel Myb-dependent enhancer, our work demonstrates the potential of chromatin structure analysis for the identification of Myb target sites.

The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote.

The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, we performed a genome-wide chromatin structure analysis in a higher eukaryote. We found a binary pattern of histone modifications among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity.

High-level activation of transcription of the yeast U6 snRNA gene in chromatin by the basal RNA polymerase III transcription factor TFIIIC.

Transcription of the U6 snRNA gene (SNR6) in Saccharomyces cerevisiae by RNA polymerase III (pol III) requires TFIIIC and its box A and B binding sites. In contrast, TFIIIC has little or no effect on SNR6 transcription with purified components in vitro due to direct recognition of the SNR6 TATA box by TFIIIB. When SNR6 was assembled into chromatin in vitro by use of the Drosophila melanogaster S-190 extract, transcription of these templates with highly purified yeast pol III, TFIIIC, and TFIIIB displayed a near-absolute requirement for TFIIIC but yielded a 5- to 15-fold-higher level of transcription relative to naked DNA (>100-fold activation over repressed chromatin). Analysis of chromatin structure demonstrated that TFIIIC binding leads to remodeling of U6 gene chromatin, resulting in positioning of a nucleosome between boxes A and B. The resulting folding of the intervening DNA into the nucleosome could bring the suboptimally spaced SNR6 box A and B elements into greater proximity and thus facilitate activation of transcription. In the absence of ATP, however, the binding of TFIIIC to box B in chromatin was not accompanied by remodeling and the transcription activation was approximately 35% of that seen in its presence, implying that both TFIIIC binding and ATP-dependent chromatin remodeling were required for the full activation of the gene. Our results suggest that TFIIIC, which is a basal transcription factor of pol III, also plays a direct role in remodeling chromatin on the SNR6 gene.

Analysis of chromatin structure on mitotic chromosomes by use of DNA damaging agents

Analysis of chromatin structure on mitotic chromosomes by use of DNA damaging agents

Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites

AbstractTo get insight into the regulation of human interleukin-12 (IL-12) synthesis, we determined the chromatin organization of the IL-12(p35) promoter region. First, we determined positioning of nucleosomes within the IL-12(p35) promoter using the indirect end-labeling technique in the THP-1 monocytic cell line. On stimulation with bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ), hypersensitivity to digestion with DNase I, micrococcal nuclease, and specific restriction enzymes was detected in the region encompassing nucleotide (nt) –310 to –160, indicating selective inducible chromatin remodeling involving disruption of a single nucleosome (named nuc-2). Using p35 promoter deletion mutants and reporter gene assays, we demonstrated that the –396/–241 region contained critical cis-acting elements. Within this latter region, we characterized physically and functionally 2 Sp1-binding sites, which were acting as key regulatory elements for both basal and LPS/IFN-γ–inducible p35 gene expression: Sp1#1 lies within the remodeled nuc-2 region and Sp1#2 is located in the nucleosome-free region immediately upstream of nuc-2. Finally, we extended the chromatin structure analysis to dendritic cells (DCs) derived from human monocytes and observed the same nucleosomal organization and remodeling as in the THP-1 cell line. Moreover, we found that in DCs, LPS and IFN-γ synergized in the induction of nucleosomal remodeling and that chromatin remodeling at the p35 locus immediately preceded IL-12(p35) mRNA synthesis. Taken together, our results demonstrate that IL-12(p35) gene activation in the course of DC maturation involves selective and rapid remodeling of a single positioned nucleosome within a region of the promoter containing critical Sp1-binding sites.

Histone deacetylase-dependent establishment and maintenance of broad low-level histone acetylation within a tissue-specific chromatin domain.

The murine beta-globin locus in adult erythroid cells is characterized by a broad pattern of erythroid-specific histone acetylation. The embryonic beta-globin genes Ey and betaH1 are located in a approximately 30 kb central subdomain characterized by low-level histone acetylation, while the fetal/adult genes betamajor and betaminor and the upstream locus control region reside in hyperacetylated chromatin. Histone deacetylase (HDAC) inhibitors induce H4 acetylation at the Ey promoter [Forsberg, E. C., Downs, K. M., Christensen, H. M., Im, H., Nuzzi, P. A., and Bresnick, E. H. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 14494-14499], indicating that HDACs maintain low-level H4 acetylation at this site. Since little is known about the establishment of broad histone modification patterns, we asked whether this mechanism applies only to the promoter or to the entire subdomain. We show that the HDAC inhibitor trichostatin A induces H4 hyperacetylation at multiple sites within the subdomain in erythroid cells. The hematopoietic factors p45/NF-E2, GATA-1, and erythroid kruppel-like factor (EKLF), which function through cis elements of the beta-globin locus, were not required for induction of H4 hyperacetylation. Analysis of chromatin structure within the subdomain revealed low accessibility to restriction endonucleases and nearly complete CpG dinucleotide methylation. Induction of H4 hyperacetylation did not restore hallmark features of transcriptionally active chromatin. We propose that an HDAC-dependent surveillance mechanism counteracts constitutive histone acetyltransferase (HAT) access, thereby maintaining low-level H4 acetylation throughout the subdomain.

Pyrophosphorolysis-activatable oligonucleotides may facilitate detection of rare alleles, mutation scanning and analysis of chromatin structures.

Pyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allele-specific PCR for detection of known mutations in the presence of a great excess of wild-type allele. The high specificity of PAP derives from the serial coupling of pyrophosphorolysis-mediated activation of a pyrophosphorolysis-activatable oligonucleotide (P*) followed by extension of the activated oligonucleotide. Herein, we demonstrate that genetically engineered DNA polymerases greatly improve the efficiency of PAP, making it a practical technique for detection of rare mutations. We also show that P* oligonucleotides have the novel and unexpected property of high sensitivity to mismatches throughout at least the 16 3'-terminal nucleotides. Thus, PAP constitutes a technology platform of potential utility whenever high specificity is required along the length of an oligonucleotide.

Widespread collaboration of Isw2 and Sin3-Rpd3 chromatin remodeling complexes in transcriptional repression.

The yeast Isw2 chromatin remodeling complex functions in parallel with the Sin3-Rpd3 histone deacetylase complex to repress early meiotic genes upon recruitment by Ume6p. For many of these genes, the effect of an isw2 mutation is partially masked by a functional Sin3-Rpd3 complex. To identify the full range of genes repressed or activated by these factors and uncover hidden targets of Isw2-dependent regulation, we performed full genome expression analyses using cDNA microarrays. We find that the Isw2 complex functions mainly in repression of transcription in a parallel pathway with the Sin3-Rpd3 complex. In addition to Ume6 target genes, we find that many Ume6-independent genes are derepressed in mutants lacking functional Isw2 and Sin3-Rpd3 complexes. Conversely, we find that ume6 mutants, but not isw2 sin3 or isw2 rpd3 double mutants, have reduced fidelity of mitotic chromosome segregation, suggesting that one or more functions of Ume6p are independent of Sin3-Rpd3 and Isw2 complexes. Chromatin structure analyses of two nonmeiotic genes reveals increased DNase I sensitivity within their regulatory regions in an isw2 mutant, as seen previously for one meiotic locus. These data suggest that the Isw2 complex functions at Ume6-dependent and -independent loci to create DNase I-inaccessible chromatin structure by regulating the positioning or placement of nucleosomes.

P53 targets chromatin structure alteration to repress alpha-fetoprotein gene expression.

Many of the functions ascribed to p53 tumor suppressor protein are mediated through transcription regulation. We have shown that p53 represses hepatic-specific alpha-fetoprotein (AFP) gene expression by direct interaction with a composite HNF-3/p53 DNA binding element. Using solid-phase, chromatin-assembled AFP DNA templates and analysis of chromatin structure and transcription in vitro, we find that p53 binds DNA and alters chromatin structure at the AFP core promoter to regulate transcription. Chromatin assembled in the presence of hepatoma extracts is activated for AFP transcription with an open, accessible core promoter structure. Distal (-850) binding of p53 during chromatin assembly, but not post-assembly, reverses transcription activation concomitant with promoter inaccessibility to restriction enzyme digestion. Inhibition of histone deacetylase activity by trichostatin-A (TSA) addition, prior to and during chromatin assembly, activated chromatin transcription in parallel with increased core promoter accessibility. Chromatin immunoprecipitation analyses showed increased H3 and H4 acetylated histones at the core promoter in the presence of TSA, while histone acetylation remained unchanged at the site of distal p53 binding. Our data reveal that p53 targets chromatin structure alteration at the core promoter, independently of effects on histone acetylation, to establish repressed AFP gene expression.

Relationship of bull fertility to sperm nuclear shape.

The relationship between sperm nuclear shape and bull fertility was determined. Two groups of bulls, 3 per group, were selected. Bulls differed in fertility based on lifetime nonreturn rates. Digital images of propidium iodide-stained sperm from each bull were collected and shape-evaluated by Fourier harmonic amplitudes 0 to 5. A discriminant function (P < .05) was constructed based on harmonic amplitudes and the 2 fertility groups. When individual sperm were classified as being of high or lower fertility, the percentage of each bull's sperm placed in the high-fertility group had a linear relationship (r = .89, P < .05) with fertility. To construct a plot of mean sperm shapes, a novel technique to automatically orient and identify the anterior tip of the sperm head was developed. The mean nuclear shape of high-fertility sperm was more elongated and tapered than those of lower fertility. A discriminant function (P < .05) was also constructed that separated the 6 bulls into 2 groups based only on the harmonic amplitudes or sperm nuclear shape. The bulls were correctly classified into the 2 fertility groups. A comparison of sperm chromatin structure analysis (SCSA) and harmonic amplitudes found that overall size variance, anterior roundness, and posterior taperedness of sperm nuclei were related to chromatin stability (P < .05). Some of the differences observed in sperm nuclear shape between the high- and lower-fertility bulls may be explained by varying levels of chromatin stability. However, sperm nuclear shape appears to contain additional information from chromatin stability alone. In this particular study, with 6 bulls, all with good chromatin quality, sperm nuclear shape was a better predictor of bull fertility.

Chromatin Structure and U2AF1-RS1 Gene Expression in Embryonic Stem Cells following RA-Induced Apoptosis.

INTRODUCTION. Apoptosis is a genetically controlled, selective cellular death mechanism, which occurs during processes of differentiation and normal cellular development. AB1 embryonic stem cells were employed in our assays, based on previous studies where it was demonstrated that in vitro treatment with retinoic acid (RA) gave rise to apoptosis as well as cellular differentiation (1). In order to support the hypothesis that apoptosis is an active process even when apoptotic bodies appear, we decided to study the chromatin conformation of DNA (2) and the corresponding gene expression. To this end, we analyzed the U2af1-rs1 gene, which takes part in mRNA splicing. METHODS. AB1 cells after in vitro incubation with 1 µM RA for 48 and 72 hours, were stained with propidium iodine and analyzed by flow cytometry. Electron microscopy was employed to verify the ultrastructure of differentiated and apoptotic cells. In order to perform chromatin structure analysis, nuclei were isolated from non-differentiated and differentiated AB1 cells and from apoptotic bodies. After incubation at increasing concentrations of DNase-I, DNA was extracted from the nuclei, digested with Bgl II, electrophoresized and hybridized with a mouse U2af1-rs1 probe. Densitometric measurements on scanned x-ray films were performed with a Bio-Rad computer-assisted system. Total RNAs from non-differentiated and differentiated cells and from apoptotic bodies were hybridized with a U2af1-rs1 probe and with a mouse Gadph control probe. RESULTS. Following RA treatment, a decrease of the proliferative population and an increase of the non-proliferative population of AB1 cells was observed. In addition, apoptotical cell death was observed, especially after 72 hours RA-treatment. The ultrastructural phenotype of differentiated AB1 cells after RA-treatment consisted of an increment in the quantity and variety of organelles and the presence of eucromatic nuclei with evident nucleoli. The apoptotical fraction showed a characteristic morphology with cytoplasmic condensation and margination of the hypercondensed chromatin towards the nuclear periphery. Chromatin structure analysis of the U2af1-rs1 region revealed that differentiated cell DNA was more sensitive to DNase-I than undifferentiated cell DNA. Furthermore, we detected DNase-I sensitivity in apoptotic bodies. Northern blot analysis demonstrated that undifferentiated and differentiated cells and, even apoptotic bodies, expressed the U2af1-rs1 mRNA.

In vivo DNA analysis.

The in vivo analysis of DNA-protein interactions and chromatin structure can provide several kinds of critical information regarding regulation of gene expression and gene function. For example, DNA sequences spanned by nuclease-hypersensitive sites or bound by transcription factors often correspond to genetic regulatory elements. Using the ligation-mediated polymerase chain reaction (LMPCR) technology it is possible to map such DNA sequences and to demonstrate the existence of unusual DNA structures directly in living cells. LMPCR analyses can thus be used as a primary investigative tool to identify the regulatory sequences involved in gene expression. Once specific promoter sequence sites are shown to be bound by transcription factors in living cells, it is often possible to establish the identity of these factors simply by comparison with the consensus binding sites of known factors such as Sp1, AP-1, NF-1, and so forth. The identity of each factor can then be confirmed using in vitro gel shift (electrophoretic mobility shift assay [EMSA]) or footprinting assays.

Ligation-mediated PCR for quantitative in vivo footprinting.

Ligation-mediated polymerase chain reaction (LM-PCR) is a genomic analysis technique for determination of (1) primary DNA nucleotide sequences (2) cytosine methylation patterns (3) DNA lesion formation and repair, and (4) in vivo protein-DNA footprints. However, LM-PCR can be limited by the multiple steps required and the relatively short stretch of sequence (usually <200 bp) that can be analyzed per reaction. We report here a simplified, one-day LM-PCR protocol in which all pipetting steps can be performed by a robotic workstation and which, moreover, provides longer reads (>350 bp) and enhanced signal quality by use of nonradioactive detection and a LI-COR DNA sequencing instrument. Sensitivity comparable to radiolabeling is achieved using oligonucleotide primers that are 5'-end labeled with infrared fluorochromes. We showed that the technique could be used for sensitive and reproducible in vivo photofootprinting of the human phosphoglycerate kinase 1 (PGK1) promoter, as well as providing good Maxam-Gilbert sequence information. The methods described here should allow high-throughput, high-resolution analysis of transcription factor binding and chromatin structure, and also may be useful for sequencing gaps that are refractory to cloning.

Fluorescence lifetime imaging of nuclear DNA: effect of fluorescence resonance energy transfer.

DNA fluorescence dyes have been used to study DNA dynamics, chromatin structure, and cell cycle analysis. However, most microscopic fluorescence studies of DNA use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. In this paper, we combine fluorescence imaging of DNA with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin. We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of DNA-bound donors and acceptors in fixed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-specific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinomycin D (7-AAD). The intensity images of Ho alone showed a spatially irregular distribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the Ho-stained nuclei was typically flat. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the Ho-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the Ho decay becomes nonexponential, as is expected for a resonance energy transfer (RET) with multiple acceptors located over a range of distances. In approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease in intensity and lifetime. The RET efficiency was higher in G(2)/M than in G(0/1) phase cells. Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly condensed regions of DNA in nuclei.

Human insulin receptor substrate-2: gene organization and promoter characterization.

Insulin receptor substrate-2 (IRS-2) belongs to a family of cytoplasmic adaptor proteins, which link insulin, IGF-1, and cytokine receptor tyrosine kinases to signaling pathways regulating metabolism, growth, and differentiation (1-3). IRS-2-deficient mice display all characteristics of type 2 diabetes, suggesting that dysfunction of the IRS-2 gene may contribute to the pathogenesis of human type 2 diabetes (4). Based on its progesterone inducibility, we have recently cloned and sequenced a full-length human IRS-2 cDNA containing an open reading frame (ORF) of 4,014 bp and 5'- and 3'-untranslated regions (UTRs) of 516 and 2,466 bp (5). Although the IRS-2 gene has previously been thought to lack introns within the coding region (6,7), the amino acid sequence predicted from our cDNA sequence differed at its very COOH-terminal end from an IRS-2 protein sequence derived from genomic IRS-2 sequences. Therefore, we carefully analyzed the genomic structure of the IRS-2 gene and found that the IRS-2 gene contains an intron that disrupts the ORF. Characterization of promoter and 5'-flanking regions of IRS-2 by sequencing, reporter gene assays, and chromatin structure analysis suggests that elements conferring progesterone inducibility are not located immediately upstream of the gene promoter.

Heterochromatic silencing of Drosophila heat shock genes acts at the level of promoter potentiation.

In a variety of organisms, genes placed near heterochromatin are transcriptionally silenced. In order to understand the molecular mechanisms responsible for this block in transcription, high resolution in vivo chromatin structure analysis was performed on two heat shock genes, hsp26 and hsp70. These genes normally reside in euchromatin where GAGA factor and RNA Pol II are present on the promoter prior to heat shock induction. P-element transformation experiments led to the identification of stocks in which these two genes were inserted within heterochromatin of the chromosome 4 telomeric region. These transgenes exhibit silencing that is partially suppressed by mutations in the gene encoding HP1. Micrococcal nuclease analysis revealed that the heterochromatic transgenes were packaged in a more regular nucleosome array than when located in euchromatin. High resolution DNase I analysis demonstrated that GAGA factor and TFIID were not associated with these promoters in heterochromatin; potassium permanganate experiments showed a loss of Pol II association. Taken together, these data suggest that occlusion of trans-acting factors from their cis- acting regulatory elements leading to a block in promoter potentiation is a mechanism for heterochromatin gene silencing.

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.