The Role of Heterochromatin in the Expression of a Heterochromatic Gene , the rolled Locus of Drosophila melanogaster

Constitutive heterochromatic regions of chromosomes are those that remain condensed through most or all of the cell cycle. In Drosophila melanogaster, the constitutive heterochromatic regions, located around the centromere, contain a number of gene loci, but at a much lower density than euchromatin. In the autosomal heterochromatin, the gene loci appear to be unique sequence genes interspersed among blocks of highly repeated sequences. Euchromatic genes do not function well when brought into the vicinity of heterochromatin (position-effect variegation). We test the possibility that the blocks of centromeric heterochromatin provide an environment essential for heterochromatic gene function. To assay directly the functional requirement of autosomal heterochromatic genes to reside in heterochromatin, the rolled ( r l ) gene, which is normally located deep in chromosome 2R heterochromatin, was relocated within small blocks of heterochromatin to a variety of euchromatic positions by successive series of chromosomal rearrangements. The function of the rl gene is severely affected in rearrangements in which the rl gene is isolated in a small block of heterochromatin, and these position effects can be reverted by rearrangements which bring the rl gene closer to any large block of autosomal or X chromosome heterochromatin. There is some evidence that five other 2R heterochromatic genes are also affected among these rearrangements. These findings demonstrate that the heterochromatic genes, in contrast to euchromatic genes whose function is inhibited by relocation to heterochromatin, require proximity to heterochromatin to function properly, and they argue strongly that a major function of the highly repeated satellite DNA, which comprises most of the heterochromatin, is to provide this heterochromatic environment. C ONSTITUTIVE heterochromatin refers to those regions of chromosomes which remain heteropyknotic through most or all of the cell cycle, and the remaining parts of chromosomes, those that disappear at telophase, are termed euchromatin (HEITZ 1928). Heterochromatin is phylogenetically a very widespread phenomenon, nearly ubiquitous among higher eukaryotes. In the Drosophila melanogaster chromosomes, the subject of the present study, constitutive heterochromatin flanks the centromeres and is detectable cytologically at all stages of development except in the early embryonic cleavage divisions (HUETTNER 1933; RABINOWITZ 1941). In contrast, facultative heterochromatin (BROWN 1966) involves the heteropyknosis of an entire chromosome relative to its homolog. Facultative heterochromatin is typified by the heterochromatinized X chromosome (Barr body) in placental mammals (BARR and BERTRAM 1949; MITTWOCH 1964) and by the entire heterochromatinized set of chromosomes in mealy bugs (HUGHES-SCHRADER 1948). Facultative heterochromatinization of chromosomes is correlated ’ Current address: Department of Biochemical Pharmacology, State Uni’ Author to whom all correspondence should be addressed. versity of New York, Buffalo, New York 14260. Genetics 134: 277-292 (May, 1993) with genetic inactivation; however, the homologous chromosome is not affected. From this point the term “heterochromatin” will refer exclusively to “constitutive heterochromatin” and facultative heterochromatin will not be discussed further. T h e cytological discovery of constitutive heterochromatin immediately raised the question of its biological significance. Three lines of evidence correlated heterochromatin with genetic inactivity. First, few genes mapped to heterochromatic regions in D. melanogaster (HEITZ 1933, MULLER and PAINTER 1932). Second, position-effect variegation showed that most euchromatic genes are partially suppressed if brought near heterochromatin by chromosome rearrangement (SCHULTZ 1936; DEMEREC 1941; SPOFFORD 1976). Third, constitutive heterochromatin appeared to be composed largely of highly repeated DNA sequences (GALL, COHEN and POLAN 197 1; PEACOCK et al. 1973, 1977; STEFFENSEN, APPELS and PEACOCK 1981), which most likely lack coding functions. All such evidence argued that heterochromatin was a location, and indicator and an agent of genetic inactivity. In spite of this strong apparent correlation between heterochromatin and genetic inactivity, it is now clear, at least in D. melanogaster, that there are in fact active 278 D. F. Eberl, B. J. Duyf and A. J. Hilliker genes within heterochromatin, although these genes are present at a much lower density than genes in euchromatin [see HILLIKER and SHARP (1988) for a review]. The Y chromosome is completely heterochromatic and carries a minimum of 12 genetic elements, including six male-fertility genes and an rDNA locus. The X chromosome heterochromatin (Xh) carries six genetic elements including the rDNA locus. There are 19 genes in chromosome 2 heterochromatin, as shown in Figure 1. Chromosome 3 heterochromatin carries at least 1 1 vital genes (MARCHANT and HOLM How is it that some 50 genes are located in heterochromatin and are still able to function even though euchromatic genes cannot function when put into this environment? Are these heterochromatic genes able to function regardless of their location, or do they actually require a heterochromatic environment to function, as speculated by HILLIKER (1976)? This latter question can be answered directly by assaying position effects on heterochromatic genes in chromosomal rearrangements which move these genes into euchromatic environments. Position effects on heterochromatic genes have heretofore been most thoroughly studied on the light (It) locus, a gene in the heterochromatin of chromosome arm 2L near the euchromatic boundary (HILLIKER and HOLM 1975; DIMITRI 1991). Rearrangements which break anywhere between the It locus and the chromosome 2 centromere, and which relocate It and its associated heterochromatin to distal euchromatic positions, can show position effects on It (HESSLER 1958; HILLIKER 1980; WAKIMOTO and HEARN 1990). Evidence of position effects on other genes in 2L heterochromatin (2Lh) in rearrangements with position effects on It has also been found. SHARP (1988) saw viability effects on the group VI1 locus EMS 56-4 and on the group IX locus EMS 40-18 in translocations that variegate for It. WAKIMOTO and HEARN (1990) describe position effects on these genes and on the EMS 40-2, concertina (cta) and EMS 40-6 genes in 2Lh in It-variegating rearrangements. It is noteworthy that all these genes are located in distal heterochromatin (see Figure l) , close to the euchromatic boundary. This localization is based on detachment frequencies of compound autosomes (HILLIKER and HOLM 1975) and on cytological mapping (DIMITRI 1991). It has been suggested that the heterochromatin near the euchromatic boundary, P-heterochromatin, is qualitatively different from the more proximal a-heterochromatin, which constitutes most of the mitotic heterochromatin, and that It (and presumably also many of the other 2Lh genes) is located in P-heterochromatin (DEVLIN, BINGHAM and WAKIMOTO 1990; DEVLIN et al. 1990; WAKIMOTO and HEARN 1990). I t is postulated that P-heterochromatin is quite unlike a-het1988). A 2L rl 2R Group IX Vlll VI1 VI I II Ill IV v