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Paul Schedl

Publications and source records attributed to Paul Schedl.

17 recordsLinked to original sources
PubMed2026

Metadomain and metaloop genome interactions in mammalian T cells.

Recent studies have advanced understanding of chromosomal organization and its role in gene regulation, yet most analyses focus on short-range interactions (<2 Mb), limiting insight into broader architecture. The relationships between topologically associating domains (TADs), sub-TAD loops, cross-TAD interactions, and chromosomal compartmentalization remain poorly understood. Here, using high-resolution Hi-C analysis, we identify extensive multi-megabase and interchromosomal interactions (metaloops) in T lymphocytes that organize into meta-TAD associations (metadomains). These metaloops connect distal promoters and regulatory elements of genes functionally important in T cells, including Ctla4, Ikzf2, Il2ra, Ets1, and Foxo1. Reanalysis of mouse and human datasets confirms their reproducibility and dependence on superenhancers. Genome-wide clustering reveals three distinct interchromosomal hubs, including a superenhancer-enriched hub linked to T cell-specific gene activation. Integrative analysis of regulatory genomics data identifies factors associated with short- versus long-range interactions. This study introduces a broadly applicable computational framework and reveals features of T cell genome organization.

Animals↗
PubMed2006

Architecture of a polycomb nucleoprotein complex.

Polycomb group (PcG) epigenetic silencing proteins act through cis-acting DNA sequences, named Polycomb response elements (PREs). Within PREs, Pleiohomeotic (PHO) binding sites and juxtaposed Pc binding elements (PBEs) function as an integrated DNA platform for the synergistic binding of PHO and the multisubunit Polycomb core complex (PCC). Here, we analyzed the architecture of the PHO/PCC/PRE nucleoprotein complex. DNase I footprinting revealed extensive contacts between PHO/PCC and the PRE. Scanning force microscopy (SFM) in combination with DNA topological assays suggested that PHO/PCC wraps the PRE DNA around its surface in a constrained negative supercoil. These features are difficult to reconcile with the simultaneous presence of nucleosomes at the PRE. Indeed, chromatin immunoprecipitations (ChIPs) and nuclease mapping demonstrated that PREs are nucleosome depleted in vivo. We discuss the implications of these findings for models explaining PRE function.

Animals↗
PubMed2006

The drosophila fragile X protein dFMR1 is required during early embryogenesis for pole cell formation and rapid nuclear division cycles.

The FMR family of KH domain RNA-binding proteins is conserved from invertebrates to humans. In humans, inactivation of the X-linked FMR gene fragile X is the most common cause of mental retardation and leads to defects in neuronal architecture. While there are three FMR family members in humans, there is only a single gene, dfmr1, in flies. As in humans, inactivation of dfmr1 causes defects in neuronal architecture and in behavior. dfmr1 has other functions in the fly in addition to neurogenesis. Here we have analyzed its role during early embryonic development. We found that dfmr1 embryos display defects in the rapid nuclear division cycles that precede gastrulation in nuclear migration and in pole cell formation. While the aberrations in nuclear division are correlated with a defect in the assembly of centromeric/centric heterochromatin, the defects in pole cell formation are associated with alterations in the actin-myosin cytoskeleton.

Animals↗
PubMed2005

Drosophila argonaute-2 is required early in embryogenesis for the assembly of centric/centromeric heterochromatin, nuclear division, nuclear migration, and germ-cell formation.

The RNA-induced silencing complex (RISC) or the RISC complex mediates RNAi and is comprised of proteins belonging to the dicer and Argonaute family proteins. Here we show that Argonaute-2 (ago-2) is required for proper nuclear migration, pole cell formation, and cellularization during the early stages of embryonic development in Drosophila. We have traced these defects back to the nuclear division cycles. Unlike wild type, nuclear division is asynchronous in ago-2 embryos and there are defects in chromosome condensation, nuclear kinesis, and assembly of spindle apparatus. The aberrations in the nuclear division cycle are correlated with defects in the formation of centric/centromeric heterochromatin and point to a failure in the assembly of functional centromeres.

Actins↗
PubMed2005

Nanos downregulates transcription and modulates CTD phosphorylation in the soma of early Drosophila embryos.

nanos (nos) specifies posterior development in the Drosophila embryo by repressing the translation of maternal hb mRNA. In addition to this somatic function, nos is required in the germline progenitors, the pole cells, to establish transcriptional quiescence. We have previously reported that nos is required to keep the Sex-lethal establishment promoter, Sxl-Pe, off in the germline of both sexes. We show here that nos also functions to repress Sxl-Pe activity in the surrounding soma. Sxl-Pe is inappropriately activated in the soma of male embryos from nos mothers, while Sxl-Pe can be repressed in female embryos by ectopic Nos protein. nos appears to play a global role in repressing transcription in the soma as the effects of nos on promoter activity are correlated with changes in the phosphorylation status of the carboxy terminal domain (CTD) repeats of the large RNA polymerase II subunit. Finally, we present evidence indicating that the suppression of transcription in the soma by Nos protein is important for normal embryonic development.

3' Untranslated Regions↗
PubMed2005

The Drosophila fragile X protein functions as a negative regulator in the orb autoregulatory pathway.

Translational regulation of maternal mRNAs in distinct temporal and spatial patterns underlies many key decisions in developing eggs and embryos. In Drosophila, Orb is responsible for mediating the translational activation of mRNAs localized within the developing oocyte. Orb is a germline-specific RNA binding protein and is one of the founding members of the CPEB family of translational regulators. Here we show that Orb associates with the Drosophila Fragile X Mental Retardation (dFMR1) protein as part of a ribonucleoprotein complex that controls the localized translation of mRNAs in developing egg chambers. One of the key orb regulatory targets is orb mRNA, and this autoregulatory activity is critical for ensuring that Orb protein is expressed at high levels in the oocyte. We show that dFMR1 functions as a negative regulator in the orb autoregulatory circuit, downregulating orb mRNA translation.

Animals↗
PubMed2005

HMGCoA reductase potentiates hedgehog signaling in Drosophila melanogaster.

Drosophila HMGCoA reductase (hmgcr) catalyzes the biosynthesis of a mevalonate precursor for isoprenoids and has been implicated in the production of a signal by the somatic gonadal precursor cells (SGPs) that attracts migrating germ cells. Here, we show that hmgcr functions in the hedgehog (hh) signaling pathway. When hmgcr activity is reduced, high levels of Hh accumulate in hh-expressing cells in each parasegment, while the adjacent "Hh-receiving" cells cannot sustain wg expression and fail to relocalize the Smoothened (Smo) receptor. Conversely, ectopic Hmgcr upregulates Hh signaling when it is produced in hh-expressing cells, but has no effect when produced in the receiving cells. These findings suggest that Hmgcr might orchestrate germ cell migration by promoting the release and/or transport of Hh from the SGPs. Consistent with this model, there are substantial germ cell migration defects in trans combinations between hmgcr and mutations in different components of the hh pathway.

Animals↗
PubMed2004

Characterization of the grappa gene, the Drosophila histone H3 lysine 79 methyltransferase.

We have identified a novel gene named grappa (gpp) that is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development.

Animals↗
PubMed2004

Developmental modulation of Fab-7 boundary function.

The Fab-7 boundary functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains in the Drosophila Bithorax Complex from early embryogenesis through to the adult stage. Although Fab-7 is required only for the proper development of a single posterior parasegment, it is active in all tissues and stages of development that have been examined. In this respect, Fab-7 resembles conventional constitutive boundaries in flies and other eukaryotes that act through ubiquitous cis-elements and trans-acting factors. Surprisingly, however, we find that the constitutive activity of Fab-7 is generated by combining sub-elements with developmentally restricted boundary function. We provide in vivo evidence that the Fab-7 boundary contains separable regions that function at different stages of development. These findings suggest that the units (domains) of genetic regulation that boundaries delimit can expand or contract by switching insulator function off or on in a temporally regulated fashion.

Animals↗
PubMed2004

Overlapping mechanisms function to establish transcriptional quiescence in the embryonic Drosophila germline.

In Drosophila melanogaster, the germline precursor cells, i.e. pole cells, are formed at the posterior of the embryo. As observed for newly formed germ cells in many other eukaryotes, the pole cells are distinguished from the soma by their transcriptional quiescence. To learn more about the mechanisms involved in establishing quiescence, we ectopically expressed a potent transcriptional activator, Bicoid (Bcd), in pole cells. We find that Bcd overrides the machinery that downregulates transcription, and activates not only its target gene hunchback but also the normally female specific Sex-lethal promoter, Sxl-Pe, in the pole cells of both sexes. Unexpectedly, the terminal pathway gene torso-like is required for Bcd-dependent transcription. However, terminal signaling is known to be attenuated in pole cells, and this raises the question of how this is accomplished. We present evidence indicating that polar granule component (pgc) is required to downregulate terminal signaling in early pole cells. Consistently, pole cells compromised for pgc function exhibit elevated levels of activated MAP kinase and premature transcription of the target gene tailless (tll). Furthermore, pgc is required to establish a repressive chromatin architecture in pole cells.

3' Untranslated Regions↗
PubMed2004

The enhancer-blocking activity of the Fab-7 boundary from the Drosophila bithorax complex requires GAGA-factor-binding sites.

In the work reported here we have analyzed the role of the GAGA factor [encoded by the Trithorax-like (Trl) gene] in the enhancer-blocking activity of Frontabdominal-7 (Fab-7), a domain boundary element from the Drosophila melanogaster bithorax complex (BX-C). One of the three nuclease hypersensitive sites in the Fab-7 boundary, HS1, contains multiple consensus-binding sequences for the GAGA factor, a protein known to be involved in the formation and/or maintenance of nucleosome-free regions of chromatin. GAGA protein has been shown to localize to the Fab-7 boundary in vivo, and we show that it recognizes sequences from HS1 in vitro. Using two different transgene assays we demonstrate that GAGA-factor-binding sites are necessary but not sufficient for full Fab-7 enhancer-blocking activity. We show that distinct GAGA sites are required for different enhancer-blocking activities at different stages of development. We also show that the enhancer-blocking activity of the endogenous Fab-7 boundary is sensitive to mutations in the gene encoding the GAGA factor Trithorax-like.

Animals↗
PubMed2004

The Drosophila GAGA factor is required for dosage compensation in males and for the formation of the male-specific-lethal complex chromatin entry site at 12DE.

Drosophila melanogaster males have one X chromosome, while females have two. To compensate for the resulting disparity in X-linked gene expression between the two sexes, most genes from the male X chromosome are hyperactivated by a special dosage compensation system. Dosage compensation is achieved by a complex of at least six proteins and two noncoding RNAs that specifically associate with the male X. A central question is how the X chromosome is recognized. According to a current model, complexes initially assemble at approximately 35 chromatin entry sites on the X and then spread bidirectionally along the chromosome where they occupy hundreds of sites. Here, we report that mutations in Trithorax-like (Trl) lead to the loss of a single chromatin entry site on the X, male lethality, and mislocalization of dosage compensation complexes.

Animals↗
PubMed2003

Protein:protein interactions and the pairing of boundary elements in vivo.

Although it is now well-established that boundary elements/insulators function to subdivide eukaryotic chromosomes into autonomous regulatory domains, the underlying mechanisms remain elusive. One idea is that boundaries act as barriers, preventing the processive spreading of "active" or "silenced" chromatin between domains. Another is that the partitioning into autonomous functional units is a consequence of an underlying structural subdivision of the chromosome into higher order "looped" domains. In this view, boundaries are thought to delimit structural domains by interacting with each other or with some other nuclear structure. The studies reported here provide support for the looped domain model. We show that the Drosophila scs and scs' boundary proteins, Zw5 and BEAF, respectively, interact with each other in vitro and in vivo. Moreover, consistent with idea that this protein:protein interaction might facilitate pairing of boundary elements, we find that that scs and scs' are in close proximity to each other in Drosophila nuclei.

Animals↗
PubMed2003

Functional conservation of the sex-lethal sex determining promoter, Sxl-Pe, in Drosophila virilis.

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.

5' Untranslated Regions↗
PubMed2003

Masters change, slaves remain.

Sex determination offers an opportunity to address many classic questions of developmental biology. In addition, because sex determination evolves rapidly, it offers an opportunity to investigate the evolution of genetic hierarchies. Sex determination in Drosophila melanogaster is controlled by the master regulatory gene, Sex lethal (Sxl). DmSxl controls the alternative splicing of a downstream gene, transformer (tra), which acts with tra2 to control alternative splicing of doublesex (dsx). DmSxl also controls its own splicing, creating an autoregulatory feedback loop that ensures expression of Sxl in females, but not males. A recent paper has shown that in the dipteran Ceratitis capitata later (downstream) steps in the regulatory hierarchy are conserved, while earlier (upstream) steps are not. Cctra is regulated by alternative splicing and apparently controls the alternative splicing of Ccdsx. However, Cctra is not regulated by CcSxl. Instead it appears to autoregulate in a manner similar to the autoregulation seen with DmSxl.

Animals↗
PubMed2002

First seduction, then transfiguration.

Members of the fibroblast growth factor (FGF) family contribute to various cellular processes. Recent reports elucidate the involvement of FGFs as chemoattractants that influence diverse cellular events such as imaginal patterning in Drosophila and gastrulation in amniotes. These studies underscore the enticing activity of FGFs and raise interesting questions regarding subsequent transformations in cellular identity.

Animals↗