Nucleoporins directly stimulate expression of developmental and cell-cycle genes inside the nucleoplasm.

Nuclear pore complexes (NPCs) mediate transport across the nuclear envelope. In yeast, they also interact with active genes, attracting or retaining them at the nuclear periphery. In higher eukaryotes, some NPC components (nucleoporins) are also found in the nucleoplasm, with a so far unknown function. We have functionally characterized nucleoporin-chromatin interactions specifically at the NPC or within the nucleoplasm in Drosophila. We analyzed genomic interactions of full-length nucleoporins Nup98, Nup50, and Nup62 and nucleoplasmic and NPC-tethered forms of Nup98. We found that nucleoporins predominantly interacted with transcriptionally active genes inside the nucleoplasm, in particular those involved in developmental regulation and the cell cycle. A smaller set of nonactive genes interacted with the NPC. Genes strongly interacting with nucleoplasmic Nup98 were downregulated upon Nup98 depletion and activated on nucleoplasmic Nup98 overexpression. Thus, nucleoporins stimulate developmental and cell-cycle gene expression away from the NPC by interacting with these genes inside the nucleoplasm.

Maternal regulation of chromosomal imprinting in animals.

Chromosomal imprinting requires an epigenetic system that "imprints" one of the two parental chromosomes such that it results in a heritable (cell-to-cell) change in behavior of the "imprinted" chromosome. Imprinting takes place when the parental genomes are separate, which occurs during gamete formation in the respective germ-lines and post-fertilization during the period when the parental pro-nuclei lie separately within the ooplasm of the zygote. In the mouse, chromosomal imprinting is regulated by germ-line specific DNA methylation. But the methylation machinery in the respective germ-lines does not discriminate between imprinted and non-imprinted regions. As a consequence, the mouse oocyte nucleus contains over a thousand oocyte-specific germ-line differentially methylated regions (gDMRs). Upon fertilization, the sperm provides a few hundred sperm-specific gDMRs of its own. Combined, there are around 1600 imprinted and non-imprinted gDMRs in the pro-nuclei of the newly fertilized zygote. It is a remarkable fact that beginning in the maternal ooplasm, there are mechanisms that manage to preserve DNA methylation at ~ 26 known imprinted gDMRs in the face of the ongoing genome-wide DNA de-methylation that characterizes pre-implantation development. Specificity is achieved through the binding of KRAB-zinc finger proteins to their cognate recognition sequences within the gDMRs of imprinted genes. This in turn nucleates the assembly of localized heterochromatin-like complexes that preserve methylation at imprinted gDMRs through recruitment of the maintenance methyl transferase Dnmt1. These studies have shown that a germ-line imprint may cause parent-of-origin-specific behavior only if "licensed" by mechanisms that operate post-fertilization. Study of the germ-line and post-fertilization contributions to the imprinting of chromosomes in classical insect systems (Coccidae and Sciaridae) show that the ooplasm is the likely site where imprinting takes place. By comparing molecular and genetic studies across these three species, we suggest that mechanisms which operate post-fertilization play a key role in chromosomal imprinting phenomena in animals and conserved components of heterochromatin are shared by these mechanisms.

DNA copy-number control through inhibition of replication fork progression.

Proper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell-cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates the repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass spectrometry identification of SUUR-associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through the inhibition of replication fork progression.

Interbands behave as decompacted autonomous units in Drosophila melanogaster polytene chromosomes.

We studied whether interbands can be ectopically formed in Drosophila melanogaster polytene chromosomes. For comparative purposes, two types of P-element constructs were used. The first type was represented by P-element based insertions into compact bands. Sequences of these insertions or adjacent genomic sequences could be activated ectopically either by GAL4 or by dosage compensation machinery. In the second type, the DNA from transcriptionally silent interbands was positioned between the FRT sites, and was flanked by DNA sequences of genes that were also inactive in salivary glands. Electron microscopy analysis of salivary gland polytene chromosomes demonstrated that both types of constructs formed distinct, yet morphologically similar interbands. Notably, the second class of transposon insertions appeared in polytene chromosomes as two bands separated by one interband. Excision of interband material from such insertions resulted in fusion of newly appeared bands into a single band. We were able to confirm by molecular means that the DNA sequences in integrated constructs were intact, that chromatin organization of this DNA mimicked that of native interbands, and that it was accurately excised from the constructs by FLP. Thus, we demonstrate that transfer of interband DNA into a silent genetic environment does not compromise interband formation. Our results do not support the idea of the existence of distinct cytogenetic "band + interband" units, furthermore, they suggest the autonomy of the decompacted state of interbands.

Interaction between the Drosophila heterochromatin proteins SUUR and HP1.

SUUR (Suppressor of Under-Replication) protein is responsible for late replication and, as a consequence, for DNA underreplication of intercalary and pericentric heterochromatin in Drosophila melanogaster polytene chromosomes. However, the mechanism by which SUUR slows down the replication process is not clear. To identify possible partners for SUUR we performed a yeast two-hybrid screen using full-length SUUR as bait. This identified HP1, the well-studied heterochromatin protein, as a strong SUUR interactor. Furthermore, we have determined that the central region of SUUR is necessary and sufficient for interaction with the C-terminal part of HP1, which contains the hinge and chromoshadow domains. In addition, recruitment of SUUR to ectopic HP1 sites on chromosomes provides evidence for their association in vivo. Indeed, we found that the distributions of SUUR and HP1 on polytene chromosomes are interdependent: both absence and overexpression of HP1 prevent SUUR from chromosomal binding, whereas SUUR overexpression causes redistribution of HP1 to numerous sites occupied by SUUR. Finally, HP1 binds to intercalary heterochromatin when histone methyltransferase activity of SU(VAR)3-9 is increased. We propose that interaction with HP1 is crucial for the association of SUUR with chromatin.

Overexpression of the SuUR gene induces reversible modifications at pericentric, telomeric and intercalary heterochromatin of Drosophila melanogaster polytene chromosomes.

The SuUR (suppressor of underreplication) gene controls late replication and underreplication of DNA in Drosophila melanogaster polytene chromosomes: its mutation suppresses DNA underreplication whereas additional doses of the normal allele strongly enhances underreplication. The SuUR protein is localized in late replicating and underreplicating regions. The N-terminal part of the SuUR protein shares modest similarity with the ATPase/helicase domain of SWI2/SNF2 chromatin remodeling factors, suggesting a role in modification of chromatin structure. Here we describe novel structural modifications of polytene chromosomes (swellings) and show that SuUR controls chromatin organization in polytene chromosomes. The swellings develop as the result of SuUR ectopic expression in the transgene system Sgs3-GAL4; UAS-SuUR(+). They are reminiscent of chromosome puffs and appear in approximately 190 regions of intercalary, pericentric and telomeric heterochromatin; some of them attain tremendous size. The swellings are temperature sensitive: they are maximal at 29 degrees C and are barely visible at 18 degrees C. Shifting from 29 degrees C to 18 degrees C results in the complete recovery of the normal structure of chromosomes. The swellings are transcriptionally inactive, since they do not incorporate [(3)H]uridine. The SuUR protein is not visualized in regions of maximally developed swellings. Regular ecdysone-inducible puffs are not induced in cells where these swellings are apparent.