473
COMMENT Heterochromatin and genome size in Drosophila1
Genome Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/22/14 For personal use only.
Arthur J. Hilliker and Rhodri W. Taylor-Kamall
Concomitant with the explosive rise of genomics in recent years, there has been much discussion centering on the role of noncoding, or “junk”, DNA. With the recent sequencing of the “minimalist genome” (82 megabases) of the bladderwort plant Utricularia gibba (Ibarra-Laclette et al. 2013), the role played by noncoding DNA once again comes into question. Despite the existence of minimalist genomes such as the aforementioned U. gibba, in which noncoding DNA accounts for only 3% of the genome size, compared to the usual 10%–60% for other plants (Ibarra-Laclette et al. 2013), we argue that noncoding DNA is certainly not all junk DNA, and it can play an important role in the genomes of higher organisms. Noncoding DNA is often found in heterochromatin, which is located at the pericentromeric regions and, in plants, also at the telomeric regions of chromosomes. The term heterochromatin was introduced by Emil Heitz in 1928 to describe the morphology of those regions of the chromosome that remain condensed throughout the cell cycle. These regions are now known as constitutive heterochromatin to avoid confusion with facultative heterochromatin (euchromatin that has been heterochromatinized)— the most famous example being the mammalian Barr body. For the proceeding discussion, heterochromatin refers exclusively to constitutive heterochromatin, which is a ubiquitous feature of the genomes of higher eukaryotes. Distinguishing features of heterochromatin include the following: a high degree of compaction relative to euchromatin throughout the cell cycle, late replication during S-phase, and enrichment in highly repetitive nontranscribed tandem arrays of DNA known as satellite sequences (reviewed in Hilliker et al. 1980). Heterochromatic regions are often found associated with each other in composite chromocenters. The molecular aspects of the highly repeated sequences can vary among species on the basis of repeat length, size of the blocks of tandem arrays, and GC content. The organism in which heterochromatin has been most thoroughly studied is Drosophila melanogaster. For many years, it was thought that heterochromatin was genetically inert, its main function being to silence transcription due to the phenomenon of position-effect variegation (PEV). PEV occurs when a euchromatic gene is placed, by chromosomal rearrangement, next to heterochromatin, leading to patchy, or variegating, expression of the euchromatic gene (reviewed in Spofford 1976). The view that heterochromatin was merely used to silence expression (which still persists in many university level textbooks) had to be revised in the 1970s with the mapping of two unique sequence genes, rolled and light, to autosomal heterochromatin (Hilliker 1976). Since then, still further heterochromatic genes have been discovered in our laboratory in D. melanogaster chromosome 2 (Coulthard et al. 2010). It was later shown that rolled was dependent on its heterochromatic location for expression (Eberl et al. 1993), and that a heterochromatin-associated protein (Heterochromatin Protein 1)
was required for the expression of both light and rolled (Lu et al. 2000). These studies show that heterochromatin is required for the expression of at least some genes in heterochromatin. Further analysis is needed to determine whether this is true for all heterochromatic genes. What is most likely underlying the importance of heterochromatin is not necessarily the specific sequences of the satellite DNA, per se, but rather simply, their repetitive nature independent of the sequence (as discussed below). The heterochromatic regions of eukaryotes share the commonality of being mainly comprised of blocks of long tandem arrays of highly repeated sequences that can vary greatly among different species, and they can often account for some of the size differences in the genomes of closely related species in some genera (however, in other genera, euchromatic transposable elements are primarily responsible for variation in genome size). In many species, the main components of heterochromatin are the satellite sequences, long tandem repeats that can be as simple as the 2-bp repeat ((AT)n) found in the crab species Cancer borealis and Gecarcinus lateralis (Gray and Skinner 1974) or even as complex as the 359-bp repeat found in D. melanogaster (Hsieh and Brutlag 1979). In mapping the satellite sequences of D. melanogaster, it was found that many of the satellite sequences throughout the genome were the same, i.e., the same simple sequences appear in different parts of the genome, but the number of repeats varied per location (Lohe et al. 1993). Furthermore, when looking at sibling species within the genus Drospohila, the story gets a little more interesting. Lohe (1981) found that the sibling species D. melanogaster and D. simulans have very different overall complements of their major satellite DNA sequences. Through hybridization melt analysis with 10 known satellite sequences of D. melanogaaster, it was shown that identical sequences appeared in D. erecta (all 10) and D. simulans (7 of the 10); however, large variations were found between the sister species of Drosophila in the quantity of these specific satellite sequences (Lohe and Brutlag 1987). That they all have satellite sequences, but highly variable amounts of specific satellites, points to the notion that the sequences themselves may not be important, but rather the repetitive nature of the satellites. This is likely related to establishing the environment necessary for the function of heterochromatic genes. In some genera, the difference in the satellite content plays a major role in the genome size differences. Bosco et al. (2007), using flow cytometry to compare the 2C value (total amount of DNA contained within the diploid nucleus) of different Drosophila species, found that there were very small differences among strains of the same species, but rather large differences between species. Much of this difference was found to be in the content of heterochromatic satellite DNA. Similar findings have been reported in other genera, such as the voles. In a cytogenetic com-
Received 30 August 2013. Accepted 4 September 2013. Corresponding Editor: Jillian Bainard. A.J. Hilliker and R.W. Taylor-Kamall. Department of Biology, York University, Toronto, ON M3J 1P3, Canada. Corresponding author: Arthur J. Hilliker (e-mail: [email protected]). 1This article is one of a selection of papers published in this Special Issue on Genome Size Evolution.
Genome 56: 473–474 (2013) dx.doi.org/10.1139/gen-2013-0157
Published at www.nrcresearchpress.com/gen on 26 September 2013.
474
parison of two closely related species of vole, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus, the G-banded (euchromatic) karyotypes were shown to be very similar, but marked differences were seen in the C-banding patterns of the giant sex chromosomes, largely overlapping with the hybridization pattern of a satellite DNA probe (Gornung et al. 2011). Thus, although heterochromatin has functions (which may not be due to sequence specificity, but rather sequence repetition) it is often responsible for at least some portion of variations in genome size among closely related species.
Genome Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/22/14 For personal use only.
References Bosco, G., Campbell, P., Leiva-Neto, J.T., and Markow, T.A. 2007. Analysis of Drosophila species genome size and satellite DNA content reveals significant differences among strains as well as between species. Genetics, 177(3): 1277– 1290. doi:10.1534/genetics.107.075069. PMID:18039867. Coulthard, A.B., Alm, C., Cealiac, I., Sinclair, D.A., Honda, B.M., Rossi, F., et al. 2010. Essential loci in centromeric heterochromatin of Drosophila melanogaster. I: The right arm of chromosome 2. Genetics, 185(2): 479–495. doi:10.1534/ genetics.110.117259. PMID:20382826. Eberl, D.F., Duyf, B.J., and Hilliker, A.J. 1993. The role of heterochromatin in the expression of a heterochromatic gene, the rolled locus of Drosophila melanogaster. Genetics, 134(1): 277–292. PMID:8514136. Gornung, E., Castiglia, R., Rovatsos, M., Marchal, J.A., Díaz de la Guardia-Quiles, R., and Sanchez, A. 2011. Comparative cytogenetic study of two sister species of Iberian ground voles, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus (rodentia, cricetidae). Cytogenet. Genome Res. 132(3): 144–150. doi:10.1159/ 000321572. PMID:21042006. Gray, D.M., and Skinner, D.M. 1974. A circular dichroism study of the primary
Genome Vol. 56, 2013
structures of three crab satellite DNA’s rich in A:T base pairs. Biopolymers, 13(4): 843–852. doi:10.1002/bip.1974.360130417. PMID:4847589. Hilliker, A.J. 1976. Genetic analysis of the centromeric heterochromatin of chromosome 2 of Drosophila melanogaster: deficiency mapping of EMS-induced lethal complementation groups. Genetics, 83(4): 765–782. PMID:823073. Hilliker, A.J., Appels, R., and Schalet, A. 1980. The genetic analysis of D. melanogaster heterochromatin. Cell, 21(3): 607–619. doi:10.1016/00928674(80)90424-9. PMID:6777044. Hsieh, T., and Brutlag, D. 1979. Sequence and sequence variation within the 1.688 g/cm3 satellite DNA of Drosophila melanogaster. J. Mol. Biol. 135(2): 465– 481. doi:10.1016/0022-2836(79)90447-9. PMID:231676. Ibarra-Laclette, E., Lyons, E., Hernández-Guzmán, G., Pérez-Torres, C.A., Carretero-Paulet, L., Chang, T.-H., et al. 2013. Architecture and evolution of a minute plant genome. Nature, 498(7452): 94–98. doi:10.1038/nature12132. PMID:23665961. Lohe, A.R. 1981. The satellite DNAs of Drosophila simulans. Genet. Res. 38(3): 237– 250. doi:10.1017/S0016672300020589. PMID:7333458. Lohe, A.R., and Brutlag, D.L. 1987. Identical satellite DNA sequences in sibling species of Drosophila. J. Mol. Biol. 194(2): 161–170. doi:10.1016/0022-2836(87) 90365-2. PMID:3112413. Lohe, A.R., Hilliker, A.J., and Roberts, P.A. 1993. Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics, 134(4): 1149–1174. doi:10.1016/0168-9525(93)90135-5. PMID:8375654. Lu, B.Y., Emtage, P.C., Duyf, B.J., Hilliker, A.J., and Eissenberg, J.C. 2000. Heterochromatin protein 1 is required for the normal expression of two heterochromatin genes in Drosophila. Genetics, 155(2): 699–708. PMID:10835392. Spofford, J.B. 1976. Position-effect variegation in Drosophila. In The genetics and biology of Drosophila. Vol. 1c. Edited by M. Ashburner and E. Novitsky. Academic Press Inc., New York. pp. 955–1018.
Published by NRC Research Press
COMMENT Heterochromatin and genome size in Drosophila1
Genome Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/22/14 For personal use only.
Arthur J. Hilliker and Rhodri W. Taylor-Kamall
Concomitant with the explosive rise of genomics in recent years, there has been much discussion centering on the role of noncoding, or “junk”, DNA. With the recent sequencing of the “minimalist genome” (82 megabases) of the bladderwort plant Utricularia gibba (Ibarra-Laclette et al. 2013), the role played by noncoding DNA once again comes into question. Despite the existence of minimalist genomes such as the aforementioned U. gibba, in which noncoding DNA accounts for only 3% of the genome size, compared to the usual 10%–60% for other plants (Ibarra-Laclette et al. 2013), we argue that noncoding DNA is certainly not all junk DNA, and it can play an important role in the genomes of higher organisms. Noncoding DNA is often found in heterochromatin, which is located at the pericentromeric regions and, in plants, also at the telomeric regions of chromosomes. The term heterochromatin was introduced by Emil Heitz in 1928 to describe the morphology of those regions of the chromosome that remain condensed throughout the cell cycle. These regions are now known as constitutive heterochromatin to avoid confusion with facultative heterochromatin (euchromatin that has been heterochromatinized)— the most famous example being the mammalian Barr body. For the proceeding discussion, heterochromatin refers exclusively to constitutive heterochromatin, which is a ubiquitous feature of the genomes of higher eukaryotes. Distinguishing features of heterochromatin include the following: a high degree of compaction relative to euchromatin throughout the cell cycle, late replication during S-phase, and enrichment in highly repetitive nontranscribed tandem arrays of DNA known as satellite sequences (reviewed in Hilliker et al. 1980). Heterochromatic regions are often found associated with each other in composite chromocenters. The molecular aspects of the highly repeated sequences can vary among species on the basis of repeat length, size of the blocks of tandem arrays, and GC content. The organism in which heterochromatin has been most thoroughly studied is Drosophila melanogaster. For many years, it was thought that heterochromatin was genetically inert, its main function being to silence transcription due to the phenomenon of position-effect variegation (PEV). PEV occurs when a euchromatic gene is placed, by chromosomal rearrangement, next to heterochromatin, leading to patchy, or variegating, expression of the euchromatic gene (reviewed in Spofford 1976). The view that heterochromatin was merely used to silence expression (which still persists in many university level textbooks) had to be revised in the 1970s with the mapping of two unique sequence genes, rolled and light, to autosomal heterochromatin (Hilliker 1976). Since then, still further heterochromatic genes have been discovered in our laboratory in D. melanogaster chromosome 2 (Coulthard et al. 2010). It was later shown that rolled was dependent on its heterochromatic location for expression (Eberl et al. 1993), and that a heterochromatin-associated protein (Heterochromatin Protein 1)
was required for the expression of both light and rolled (Lu et al. 2000). These studies show that heterochromatin is required for the expression of at least some genes in heterochromatin. Further analysis is needed to determine whether this is true for all heterochromatic genes. What is most likely underlying the importance of heterochromatin is not necessarily the specific sequences of the satellite DNA, per se, but rather simply, their repetitive nature independent of the sequence (as discussed below). The heterochromatic regions of eukaryotes share the commonality of being mainly comprised of blocks of long tandem arrays of highly repeated sequences that can vary greatly among different species, and they can often account for some of the size differences in the genomes of closely related species in some genera (however, in other genera, euchromatic transposable elements are primarily responsible for variation in genome size). In many species, the main components of heterochromatin are the satellite sequences, long tandem repeats that can be as simple as the 2-bp repeat ((AT)n) found in the crab species Cancer borealis and Gecarcinus lateralis (Gray and Skinner 1974) or even as complex as the 359-bp repeat found in D. melanogaster (Hsieh and Brutlag 1979). In mapping the satellite sequences of D. melanogaster, it was found that many of the satellite sequences throughout the genome were the same, i.e., the same simple sequences appear in different parts of the genome, but the number of repeats varied per location (Lohe et al. 1993). Furthermore, when looking at sibling species within the genus Drospohila, the story gets a little more interesting. Lohe (1981) found that the sibling species D. melanogaster and D. simulans have very different overall complements of their major satellite DNA sequences. Through hybridization melt analysis with 10 known satellite sequences of D. melanogaaster, it was shown that identical sequences appeared in D. erecta (all 10) and D. simulans (7 of the 10); however, large variations were found between the sister species of Drosophila in the quantity of these specific satellite sequences (Lohe and Brutlag 1987). That they all have satellite sequences, but highly variable amounts of specific satellites, points to the notion that the sequences themselves may not be important, but rather the repetitive nature of the satellites. This is likely related to establishing the environment necessary for the function of heterochromatic genes. In some genera, the difference in the satellite content plays a major role in the genome size differences. Bosco et al. (2007), using flow cytometry to compare the 2C value (total amount of DNA contained within the diploid nucleus) of different Drosophila species, found that there were very small differences among strains of the same species, but rather large differences between species. Much of this difference was found to be in the content of heterochromatic satellite DNA. Similar findings have been reported in other genera, such as the voles. In a cytogenetic com-
Received 30 August 2013. Accepted 4 September 2013. Corresponding Editor: Jillian Bainard. A.J. Hilliker and R.W. Taylor-Kamall. Department of Biology, York University, Toronto, ON M3J 1P3, Canada. Corresponding author: Arthur J. Hilliker (e-mail: [email protected]). 1This article is one of a selection of papers published in this Special Issue on Genome Size Evolution.
Genome 56: 473–474 (2013) dx.doi.org/10.1139/gen-2013-0157
Published at www.nrcresearchpress.com/gen on 26 September 2013.
474
parison of two closely related species of vole, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus, the G-banded (euchromatic) karyotypes were shown to be very similar, but marked differences were seen in the C-banding patterns of the giant sex chromosomes, largely overlapping with the hybridization pattern of a satellite DNA probe (Gornung et al. 2011). Thus, although heterochromatin has functions (which may not be due to sequence specificity, but rather sequence repetition) it is often responsible for at least some portion of variations in genome size among closely related species.
Genome Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/22/14 For personal use only.
References Bosco, G., Campbell, P., Leiva-Neto, J.T., and Markow, T.A. 2007. Analysis of Drosophila species genome size and satellite DNA content reveals significant differences among strains as well as between species. Genetics, 177(3): 1277– 1290. doi:10.1534/genetics.107.075069. PMID:18039867. Coulthard, A.B., Alm, C., Cealiac, I., Sinclair, D.A., Honda, B.M., Rossi, F., et al. 2010. Essential loci in centromeric heterochromatin of Drosophila melanogaster. I: The right arm of chromosome 2. Genetics, 185(2): 479–495. doi:10.1534/ genetics.110.117259. PMID:20382826. Eberl, D.F., Duyf, B.J., and Hilliker, A.J. 1993. The role of heterochromatin in the expression of a heterochromatic gene, the rolled locus of Drosophila melanogaster. Genetics, 134(1): 277–292. PMID:8514136. Gornung, E., Castiglia, R., Rovatsos, M., Marchal, J.A., Díaz de la Guardia-Quiles, R., and Sanchez, A. 2011. Comparative cytogenetic study of two sister species of Iberian ground voles, Microtus (Terricola) duodecimcostatus and M. (T.) lusitanicus (rodentia, cricetidae). Cytogenet. Genome Res. 132(3): 144–150. doi:10.1159/ 000321572. PMID:21042006. Gray, D.M., and Skinner, D.M. 1974. A circular dichroism study of the primary
Genome Vol. 56, 2013
structures of three crab satellite DNA’s rich in A:T base pairs. Biopolymers, 13(4): 843–852. doi:10.1002/bip.1974.360130417. PMID:4847589. Hilliker, A.J. 1976. Genetic analysis of the centromeric heterochromatin of chromosome 2 of Drosophila melanogaster: deficiency mapping of EMS-induced lethal complementation groups. Genetics, 83(4): 765–782. PMID:823073. Hilliker, A.J., Appels, R., and Schalet, A. 1980. The genetic analysis of D. melanogaster heterochromatin. Cell, 21(3): 607–619. doi:10.1016/00928674(80)90424-9. PMID:6777044. Hsieh, T., and Brutlag, D. 1979. Sequence and sequence variation within the 1.688 g/cm3 satellite DNA of Drosophila melanogaster. J. Mol. Biol. 135(2): 465– 481. doi:10.1016/0022-2836(79)90447-9. PMID:231676. Ibarra-Laclette, E., Lyons, E., Hernández-Guzmán, G., Pérez-Torres, C.A., Carretero-Paulet, L., Chang, T.-H., et al. 2013. Architecture and evolution of a minute plant genome. Nature, 498(7452): 94–98. doi:10.1038/nature12132. PMID:23665961. Lohe, A.R. 1981. The satellite DNAs of Drosophila simulans. Genet. Res. 38(3): 237– 250. doi:10.1017/S0016672300020589. PMID:7333458. Lohe, A.R., and Brutlag, D.L. 1987. Identical satellite DNA sequences in sibling species of Drosophila. J. Mol. Biol. 194(2): 161–170. doi:10.1016/0022-2836(87) 90365-2. PMID:3112413. Lohe, A.R., Hilliker, A.J., and Roberts, P.A. 1993. Mapping simple repeated DNA sequences in heterochromatin of Drosophila melanogaster. Genetics, 134(4): 1149–1174. doi:10.1016/0168-9525(93)90135-5. PMID:8375654. Lu, B.Y., Emtage, P.C., Duyf, B.J., Hilliker, A.J., and Eissenberg, J.C. 2000. Heterochromatin protein 1 is required for the normal expression of two heterochromatin genes in Drosophila. Genetics, 155(2): 699–708. PMID:10835392. Spofford, J.B. 1976. Position-effect variegation in Drosophila. In The genetics and biology of Drosophila. Vol. 1c. Edited by M. Ashburner and E. Novitsky. Academic Press Inc., New York. pp. 955–1018.
Published by NRC Research Press
