Reduced DNA polytenization of a minichromosome region undergoing position-effect variegation in Drosophila

Karpen, Cary H.; Spradling, Allan C.

doi:10.1016/0092-8674(90)90291-l

Cited by 125 publications

(77 citation statements)

References 43 publications

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“…Pulsed-field electrophoresis and Southern blot hybridization: Salivary gland nuclei isolated from the third instar larvae were embedded into agarose and DNA was treated in agarose inserts as described in Karpen and Spradling (1990). The DNA samples were digested with NotI and fractionated by contour-clamped homogeneous electric field pulsed-field gel electrophoresis on a Bio-Rad (Hercules, CA) DRII apparatus.…”

Section: Methodsmentioning

confidence: 99%

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The SU(VAR)3-9/HP1 Complex Differentially Regulates the Compaction State and Degree of Underreplication of X Chromosome Pericentric Heterochromatin in Drosophila melanogaster

et al. 2007

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In polytene chromosomes of Drosophila melanogaster, regions of pericentric heterochromatin coalesce to form a compact chromocenter and are highly underreplicated. Focusing on study of X chromosome heterochromatin, we demonstrate that loss of either SU(VAR)3-9 histone methyltransferase activity or HP1 protein differentially affects the compaction of different pericentric regions. Using a set of inversions breaking X chromosome heterochromatin in the background of the Su(var)3-9 mutations, we show that distal heterochromatin (blocks h26-h29) is the only one within the chromocenter to form a big ''puff''-like structure. The ''puffed'' heterochromatin has not only unique morphology but also very special protein composition as well: (i) it does not bind proteins specific for active chromatin and should therefore be referred to as a pseudopuff and (ii) it strongly associates with heterochromatin-specific proteins SU(VAR)3-7 and SUUR, despite the fact that HP1 and HP2 are depleted particularly from this polytene structure. The pseudopuff completes replication earlier than when it is compacted as heterochromatin, and underreplication of some DNA sequences within the pseudopuff is strongly suppressed. So, we show that pericentric heterochromatin is heterogeneous in its requirement for SU(VAR)3-9 with respect to the establishment of the condensed state, time of replication, and DNA polytenization.

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Section: Methodsmentioning

confidence: 99%

“…After pulsed-field gel electrophoresis, DNA was transferred to the Hybond-N filter (Amersham-Pharmacia-Biotech) and analyzed by Southern blot hybridization as described earlier (Karpen and Spradling 1990).…”

Section: Methodsmentioning

confidence: 99%

The SU(VAR)3-9/HP1 Complex Differentially Regulates the Compaction State and Degree of Underreplication of X Chromosome Pericentric Heterochromatin in Drosophila melanogaster

et al. 2007

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“…These observations and model are consistent with a previous report of heterochromatic repair foci in endocycle cells of the ovary (Hong et al 2007). In fact, previous analyses had indicated that these chromatin junctions are fragile in the endocycle, and that fragility correlates with the degree of underreplication (Karpen and Spradling 1990;Leach et al 2000;B. Calvi, unpubl.).…”

Section: Damage At Chromatin Junctions In Both Normal and Rereplicatimentioning

confidence: 99%

Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress

Mehrotra¹,

Maqbool²,

Kolpakas³

et al. 2008

Genes Dev.

127

155

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Initiation of DNA replication at origins more than once per cell cycle results in rereplication and has been implicated in cancer. Here we use Drosophila to examine the checkpoint responses to rereplication in a developmental context. We find that increased Double-parked (Dup), the Drosophila ortholog of Cdt1, results in rereplication and DNA damage. In most cells, this rereplication triggers caspase activation and apoptotic cell death mediated by both p53-dependent and -independent pathways. Elevated Dup also caused DNA damage in endocycling cells, which switch to a G/S cycle during normal development, indicating that rereplication and the endocycling DNA reduplication program are distinct processes. Unexpectedly, however, endocycling cells do not apoptose regardless of tissue type. Our combined evidence suggests that endocycling apoptosis is repressed in part because proapoptotic gene promoters are silenced. Normal endocycling cells had DNA lesions near heterochromatin, which increased after rereplication, explaining why endocycling cells must constantly repress the genotoxic apoptotic response. Our results reveal a novel regulation of apoptosis in development and new insights into the little-understood endocycle. Similar mechanisms may operate during vertebrate development, with implications for cancer predisposition in certain tissues.[Keywords: DNA replication; DNA damage; endocycle; checkpoint; apoptosis] Supplemental material is available at http://www.genesdev.org. The timely duplication of the genome during S phase of every cell division cycle requires that DNA replication initiate from thousands of origins. If too few origins initiate, replication forks can collapse, resulting in DNA damage and incomplete replication of the genome. Initiation of DNA replication from origins more than once per cell cycle, however, results in "rereplication" and subsequent DNA damage (Arias and Walter 2007). In recent years, it has become increasingly apparent that problems with DNA replication are common in premalignant cells, with subsequent checkpoint defects leading to genome instability and cancer (Dutta 2007). It remains unclear, however, whether all cells in development are equivalent with respect to their regulation of DNA replication and checkpoint responses. Here, we use Drosophila to investigate the checkpoint responses to rereplication in a developmental context. Two important steps in the cell cycle regulation of DNA replication are the assembly and activation of a prereplicative complex (pre-RC) (Sivaprasad et al. 2006). The pre-RC assembles onto origins in early G1 and is subsequently activated in S phase. During pre-RC assembly, the hexameric origin recognition complex (ORC) serves as a scaffold for origin association of Cdc6 and Cdt1, which are both required to load the hexameric minichromosome maintenance complex (MCM), the replicative helicase (Randell et al. 2006;Sivaprasad et al. 2006). Once the MCM complex is tightly bound, the origins are considered to be "licensed" and competent to initiate replic...

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“…The antisense RNA model also cannot easily account for the chromosome-local nature of the phenomenon demonstrated here, nor for the persistence of dominant variegation following complete excision of the entire wild-type brown gene from which antisense RNA is presumably made. A somatic gene loss model for position-effect variegation, originally proposed by Schultz and recently reformulated by Karpen and Spradling, also does not easily account for dominant variegation (Schultz 1936;Karpen and Spradling 1990). If gene loss were responsible for the variegation in V21, it is difficult to see how V21[P(Abw)] deletions could lead to suppressed phenotypes in a size-dependent manner.…”

Section: Active Inactivementioning

confidence: 99%

A pairing-sensitive element that mediates trans-inactivation is associated with the Drosophila brown gene.

Dreesen¹,

Henikoff²,

Loughney³

1991

Genes Dev.

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Position-effect variegation in Drosophila is the mosaic expression of a gene juxtaposed to heterochromatin by chromosome rearrangement. The brown (bw +) gene is unusual in that variegating mutations are dominant, causing "trans-inactivation" of the homologous allele. We show that copies of bw + transposed to ectopic sites are not trans-inactivated by rearrangements affecting the endogenous gene. However, when position-effect variegation is induced on an ectopic copy by chromosome rearrangement, the allele on its paired homolog is trans-inactivated, whereas other copies of bw + are not. This confirms that trans-inactivation is "chromosome local" and maps the responsive element to the immediate vicinity of brown. Subsequent P-transposase-induced deletions within the ectopic copy in cis to the rearrangement breakpoint caused partial suppression of trans-inactivation. Surprisingly, the amount of suppression was correlated with deletion size, with some degree of trans-inactivation persisting even when the P[bw +] transposon was completely excised. The chromosome-local nature of the phenomenon and its extreme sensitivity to small disruptions of somatic pairing leads to a model in which a regulator of the brown gene is inactivated by direct contact with heterochromatic proteins.[Key Words: Position-effect variegation; Drosophila; somatic pairing; in vivo deletion mappingl Received November 21, 1990; accepted December 18, 1990.A common class of mutations induced by ionizing radiation in Drosophila are the variegating position effects. These are generally caused by chromosomal rearrangements placing euchromatic genes in the vicinity of heterochromatin, the compacted regions of chromosomes that remain condensed during most of the cell cycle (for review, see Spofford 1976; Henikoff 1990). The variegated phenotype is caused by reduction in gene expression in some cells but not in others, evidently as a result of transcriptional inactivation (Henikoff 1981;Rushlow et al. 1984;Henikoff and Dreesen 1989). Consistent with this interpretation, position-effect variegation of nearly all genes is recessive, implying that inactivation of one copy of a gene in cis has no effect on the copy in trans. However, variegated position effects on a few genes, including the brown (bw +) gene, are dominant (Muller 1932;Slatis 1955;Stem and Kodani 1955;Henikoff 1979;O'Donnell et al. 1989).Recently, we showed that dominant position-effect variegation of brown involves a sharp reduction in mRNA accumulation from both copies of the gene, the copy in cis to the rearrangement breakpoint (cis-inactivation) and the copy in trans (trans-inactivation). We proposed a somatic pairing model in which the brown gene region, inactivated by virtue of its juxtaposition to heterochromatin, could transmit the inactivated state to the copy of brown in trans. Evidence for this model was the sensitivity of trans-inactivation to chromosome configurations that are expected to disrupt somatic pairing in the vicinity of the brown gene.The somatic pairing model makes sev...

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Reduced DNA polytenization of a minichromosome region undergoing position-effect variegation in Drosophila

Cited by 125 publications

References 43 publications

The SU(VAR)3-9/HP1 Complex Differentially Regulates the Compaction State and Degree of Underreplication of X Chromosome Pericentric Heterochromatin in Drosophila melanogaster

The SU(VAR)3-9/HP1 Complex Differentially Regulates the Compaction State and Degree of Underreplication of X Chromosome Pericentric Heterochromatin in Drosophila melanogaster

Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress

A pairing-sensitive element that mediates trans-inactivation is associated with the Drosophila brown gene.

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