Position effect variegation in Drosophila is associated with an altered chromatin structure

A euchromatic gene placed in the vicinity of heterochromatin by a chromosomal rearrangement generally exhibits position effect variegation (PEV), a clonally inherited pattern showing gene expression in some somatic cells but not in others. The mechanism responsible for this loss of gene expression is investigated here using fly lines carrying a P element containing the Drosophila melanogaster white and hsp26 genes. Following mobilization of the P element, a screen for variegation of white expression recovered inserts at pericentric, telomeric, and fourth chromosome regions. Previously identified suppressors of PEV suppressed white variegation of pericentric and fourth chromosome inserts but not telomeric inserts on the second and third chromosomes. This implies a difference in the mechanism for gene repression at telomeres. Heat shock-induced hsp26 expression was reduced from pericentric and fourth chromosome inserts but not from telomeric inserts. Chromatin structure analysis revealed that the variegating inserts showed a reduction in accessibility to restriction enzyme digestion in the hsp26 regulatory region in isolated nuclei. Micrococcal nuclease digests showed that pericentric inserts were packaged in a more regular nucleosome array than that observed for euchromatic inserts. These data suggest that altered chromatin packaging plays a role in PEV. KeyWords: Heterochromatin; chromatin; transgenes; centromeres; telomere

DNA representation of variegating heterochromatic P-element inserts in diploid and polytene tissues of Drosophila melanogaster

Position-effect variegation (PEV) is the mosaic expression of a euchromatic gene brought into juxtaposition with heterochromatin. Fourteen different transformed Drosophila melanogaster lines with variegating P-element inserts were used to examine the DNA levels of these transgenes. Insert sites include pericentric, telomeric and fourth chromosome regions. Southern blot analyses showed that the heterochromatic hsp26 transgenes are underrepresented 1.3- to 33-fold in polytene tissue relative to the endogenous euchromatic hsp26 gene. In contrast, the heterochromatic hsp26 transgenes are present in approximately the same copy number as the endogenous euchromatic hsp26 gene in diploid tissue. It appears unlikely that DNA loss could account for the lack of gene expression in diploid tissues seen with these examples of PEV

Insensitivity of the present hsp26 chromatin structure to a TATA box mutation in Drosophila

The role of the TATA element in establishing the chromatin structure and inducible transcription of the Drosophila melanogaster hsp26 gene has been analyzed. An hsp26/lacZ fusion gene with a mutant promoter, in which the TATA box sequence TATAAA was changed to CCCAAA, was introduced into Drosophila by P-element transformation. The mutation had little effect on formation of the preset chromatin structure observed prior to induction. However, the mutation dramatically reduced transcription levels following heat shock. Northern analysis indicated that weak, inducible expression of the mutant promoter occurred within the same period of heat shock as for the normal promoter, suggesting that TFIID was associated with the mutant promoter prior to heat shock. Biochemical analysis showed that the mutant promoter still bound TFIID in vitro, but with 3-5-fold less affinity than the normal promoter. DNase I footprinting revealed that the conformation of the TFIID-DNA complex differed significantly from that of the normal promoter. These results indicate that alterations in the conformation or the stability of the TFIID-DNA complex drastically reduce the level of induction, but do not dramatically affect chromatin structure formation. Formation of the requisite chromatin structure is either independent of, or highly tolerant of, changes in the TFIID-DNA complex

Promoter sequence containing (CT)n.(GA)n repeats is critical for the formation of the DNase I hypersensitive sites in the Drosophila hsp26 gene

We have analyzed P-element-transformed lines carrying hsp26/lacZ transgenes with various deletions and substitutions within the Drosophila melanogaster hsp26 promoter region in order to identify the sequences required for the formation of the DNase I hypersensitive sites (DH sites). DH sites are generally found associated with promoters and enhancer elements of active and inducible eukaryotic genes, and are thought to be nucleosome-free regions of DNA that interact with regulatory proteins and the transcriptional machinery. There are two major DH sites located within the promoter region of the hsp26 gene, centered at -50 and at -350 (relative to the hsp26 transcription start site). The sequences from -135 to -85, which contain (CT)n.(GA)n repeats, contribute significantly to the formation of the DH sites in the hsp26 promoter region. Deletion or substitution of this (CT)n region drastically reduces the accessibility of the DNA at these sites to DNase I. This reduction in accessibility was quantified by measuring the susceptibility of the DNA within nuclei to cleavage at a restriction site within the DH site. In addition to the (CT)n region and the promoter at -85 to +11 (region P), one of two other regions must be present for effective creation of the DH sites: sequences between -351 and -135 (region A), or sequences between +11 and +632 (region D). Disruption of the wild-type chromatin structure, as assayed by the loss of accessibility to the DH sites, is correlated with a decrease in inducible transcriptional activity, even when the TATA box and heat shock regulatory elements are present in their normal positions

Myopathic lamin mutations impair nuclear stability in cells and tissue and disrupt nucleo-cytoskeletal coupling

Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotyp

LMNA variants cause cytoplasmic distribution of nuclear pore proteins in Drosophila and human muscle

Mutations in the human LMNA gene, encoding A-type lamins, give rise to laminopathies, which include several types of muscular dystrophy. Here, heterozygous sequence variants in LMNA, which result in single amino-acid substitutions, were identified in patients exhibiting muscle weakness. To assess whether the substitutions altered lamin function, we performed in vivo analyses using a Drosophila model. Stocks were generated that expressed mutant forms of the Drosophila A-type lamin modeled after each variant. Larvae were used for motility assays and histochemical staining of the body-wall muscle. In parallel, immunohistochemical analyses were performed on human muscle biopsy samples from the patients. In control flies, muscle-specific expression of the wild-type A-type lamin had no apparent affect. In contrast, expression of the mutant A-type lamins caused dominant larval muscle defects and semi-lethality at the pupal stage. Histochemical staining of larval body wall muscle revealed that the mutant A-type lamin, B-type lamins, the Sad1p, UNC-84 domain protein Klaroid and nuclear pore complex proteins were mislocalized to the cytoplasm. In addition, cytoplasmic actin filaments were disorganized, suggesting links between the nuclear lamina and the cytoskeleton were disrupted. Muscle biopsies from the patients showed dystrophic histopathology and architectural abnormalities similar to the Drosophila larvae, including cytoplasmic distribution of nuclear envelope proteins. These data provide evidence that the Drosophila model can be used to assess the function of novel LMNA mutations and support the idea that loss of cellular compartmentalization of nuclear proteins contributes to muscle disease pathogenesis

A Comparative Study of Drosophila and Human A-Type Lamins

Nuclear intermediate filament proteins, called lamins, form a meshwork that lines the inner surface of the nuclear envelope. Lamins contain three domains: an N-terminal head, a central rod and a C-terminal tail domain possessing an Ig-fold structural motif. Lamins are classified as either A- or B-type based on structure and expression pattern. The Drosophila genome possesses two genes encoding lamins, Lamin C and lamin Dm0, which have been designated A- and B-type, respectively, based on their expression profile and structural features. In humans, mutations in the gene encoding A-type lamins are associated with a spectrum of predominantly tissue-specific diseases known as laminopathies. Linking the disease phenotypes to cellular functions of lamins has been a major challenge. Drosophila is being used as a model system to identify the roles of lamins in development. Towards this end, we performed a comparative study of Drosophila and human A-type lamins. Analysis of transgenic flies showed that human lamins localize predictably within the Drosophila nucleus. Consistent with this finding, yeast two-hybrid data demonstrated conservation of partner-protein interactions. Drosophila lacking A-type lamin show nuclear envelope defects similar to those observed with human laminopathies. Expression of mutant forms of the A-type Drosophila lamin modeled after human disease-causing amino acid substitutions revealed an essential role for the N-terminal head and the Ig-fold in larval muscle tissue. This tissue-restricted sensitivity suggests a conserved role for lamins in muscle biology. In conclusion, we show that (1) localization of A-type lamins and protein-partner interactions are conserved between Drosophila and humans, (2) loss of the Drosophila A-type lamin causes nuclear defects and (3) muscle tissue is sensitive to the expression of mutant forms of A-type lamin modeled after those causing disease in humans. These studies provide new insights on the role of lamins in nuclear biology and support Drosophila as a model for studies of human laminopathies involving muscle dysfunction

RSF Governs Silent Chromatin Formation via Histone H2Av Replacement

Human remodeling and spacing factor (RSF) consists of a heterodimer of Rsf-1 and hSNF2H, a counterpart of Drosophila ISWI. RSF possesses not only chromatin remodeling activity but also chromatin assembly activity in vitro. While no other single factor can execute the same activities as RSF, the biological significance of RSF remained unknown. To investigate the in vivo function of RSF, we generated a mutant allele of Drosophila Rsf-1 (dRsf-1). The dRsf-1 mutant behaved as a dominant suppressor of position effect variegation. In dRsf-1 mutant, the levels of histone H3K9 dimethylation and histone H2A variant H2Av were significantly reduced in an euchromatic region juxtaposed with heterochromatin. Furthermore, using both genetic and biochemical approaches, we demonstrate that dRsf-1 interacts with H2Av and the H2Av-exchanging machinery Tip60 complex. These results suggest that RSF contributes to histone H2Av replacement in the pathway of silent chromatin formation