A complex array of DNA-binding proteins required for pairing-sensitive silencing by a polycomb group response element from the Drosophila engrailed gene

Regulatory DNA from the Drosophila gene engrailed causes silencing of a linked reporter gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transgene and has been called "pairing-sensitive silencing." The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), we tested the activities of each of these engrailed fragments in a construct designed to detect PRE activity in embryos. Both fragments were found to behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment showed additional silencing capabilities. Using the mini-white reporter gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggested that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function.     

Regulation of the Drosophila engrailed gene by Polycomb repressor complex 2

Suppressor-of-zeste-12 (Su(z)12) is a core component of the Polycomb repressive complex 2 (PRC2), which has a methyltransferase activity directed towards lysine residues of histone 3. Mutations in Polycomb group (PcG) genes cause de-repression of homeotic genes and subsequent homeotic transformations. Another target for Polycomb silencing is the engrailed gene, which encodes a key regulator of segmentation in the early Drosophila embryo. In close proximity to the en gene is a Polycomb Response Element, but whether en is regulated by Su(z)12 is not known. In this report, we show that en is not de-repressed in Su(z)12 or Enhancer-of-zeste mutant clones in the anterior compartment of wing discs. Instead, we find that en expression is down-regulated in the posterior portion of wing discs, indicating that the PRC2 complex acts as an activator of en. Our results indicate that this is due to secondary effects, probably caused by ectopic expression of Ubx and Abd-B.     

Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila.

The stable maintenance of expression patterns of homeotic genes depends on the function of a number of negative trans-regulators, termed the Polycomb (Pc) group of genes. We have examined the pattern of expression of the Drosophila segment polarity gene, engrailed (en), in embryos mutant for several different members of the Pc group. Here we report that embryos mutant for two or more Pc group genes show strong ectopic en expression, while only weak derepression of en occurs in embryos mutant for a single Pc group gene. This derepression is independent of two known activators of en expression: en itself and wingless. Additionally, in contrast to the strong ectopic expression of homeotic genes observed in extra sex combs- (esc-) mutant embryos, the en expression pattern is nearly normal in esc- embryos. This suggests that the esc gene product functions in a pathway independent of the other genes in the group. The data indicate that the same group of genes is required for stable restriction of en expression to a striped pattern and for the restriction of expression of homeotic genes along the anterior-posterior axis, and support a global role for the Pc group genes in stable repression of activity of developmental selector genes.     

GAGA facilitates binding of Pleiohomeotic to a chromatinized Polycomb response element

Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Here, using DNaseI footprinting analysis, we provide a high-resolution map of sites for the sequence- specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. We found that while GAGA could efficiently bind to a chromatinized PRE, PHO alone was incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, was strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins.     

Coordinate regulation of bovine prion protein gene promoter activity by two Sp1 binding site polymorphisms

Relationships between insertion/deletion (Ins/Del) polymorphisms of the bovine prion protein gene (PRNP) promoter and bovine spongiform encephalopathy (BSE) susceptibility have been reported. Our previous study has shown that polymorphisms of −6C → T included in the specific protein 1 (Sp1) site in the 5′-flanking region of bovine PRNP influence the promoter activity of bovine PRNP. The present study shows that 12 and 23 bp Ins/Del polymorphisms in the upstream region and an additional polymorphism (−47C → A) in the Sp1 binding site coordinately affect the promoter activity. Reporter gene assays demonstrated that the bovine PRNP promoter containing −47A and 23 bp Del/12 bp Ins or 23 bp Ins/12 bp Ins showed lower promoter activity compared with other haplotypes (23 bp Del/12 bp Ins or 23 bp Ins/12 bp Del with −47C) or the wild-type haplotype (23 bp Del/12 bp Del with −47C). Furthermore, gel shift assays showed that the binding activity of Sp1 to the PRNP promoter was influenced by both polymorphisms with corresponding effects on the promoter activity. The coordinate regulation of the bovine PRNP promoter suggests the two Sp1 binding site polymorphisms control Sp1 binding to the PRNP promoter and its activity.     

A novel human polycomb binding site acts as a functional polycomb response element in Drosophila

Polycomb group (PcG) proteins are key chromatin regulators implicated in multiple processes including embryonic development, tissue homeostasis, genomic imprinting, X-chromosome inactivation, and germ cell differentiation. The PcG proteins recognize target genomic loci through cis DNA sequences known as Polycomb Response Elements (PREs), which are well characterized in Drosophila. However, mammalian PREs have been elusive until two groups reported putative mammalian PREs recently. Consistent with the existence of mammalian PREs, here we report the identification and characterization of a potential PRE from human T cells. The putative human PRE has enriched binding of PcG proteins, and such binding is dependent on a key PcG component SUZ12. We demonstrate that the putative human PRE carries both genetic and molecular features of Drosophila PRE in transgenic flies, implying that not only the trans PcG proteins but also certain features of the cis PREs are conserved between mammals and Drosophila.