Spps, a Drosophila Sp1/KLF family member, binds to PREs and is required for PRE activity late in development

Abstract

The Polycomb group of proteins (PcG) is important for transcriptional repression and silencing in all higher eukaryotes. In Drosophila, PcG proteins are recruited to the DNA by Polycomb-group response elements (PREs), regulatory sequences whose activity depends on the binding of many different sequence-specific DNA-binding proteins. We previously showed that a binding site for the Sp1/KLF family of zinc-finger proteins is required for PRE activity. Here, we report that the Sp1/KLF family member Spps binds specifically to Ubx and engrailed PREs, and that Spps binds to polytene chromosomes in a pattern virtually identical to that of the PcG protein, Psc. A deletion of the Spps gene causes lethality late in development and a loss in pairing-sensitive silencing, an activity associated with PREs. Finally, the Spps mutation enhances the phenotype of pho mutants. We suggest that Spps may work with, or in parallel to, Pho to recruit PcG protein complexes to PREs.

Fig. 1.

Amino acid sequence comparison between Drosophila Spps and human Sp3. The boxed amino acids are the btd box, a domain present in Sp-subfamily members (Suske et al., 2005). The double-headed arrows indicate the extent of the three zinc-finger domains. Amino acids in bold are conserved between the two proteins. Underlined amino acids have similar properties.

Fig. 2.

Spps is a ubiquitous nuclear protein. (A) Anterior half of a stage 13 embryo (anterior leftwards, dorsal upwards), showing ubiquitous staining of Spps antigen. (B) Ventral view of abdominal segments of a stage 16 embryo expressing Spps via an enGal4 driver that causes Spps to be expressed in stripes and in specific cells of the CNS. This shows that the Spps antibody can detect Spps. (C,D) Embryos derived from Spps germline clones, stained with Spps antibody (C) and DAPI (D). The lower embryo received a wild-type Spps gene from the father, whereas the upper embryo received a mutant Spps1 chromosome from the father and thus has no Spps antigen. The absence of Spps staining in the upper embryo (broken line in C) shows the specificity of the Spps antibody for Spps. Both embryos are stage 15. The anterior end of the lower embryo and the lateral posterior end of the upper embryo are shown.

Fig. 3.

Immunostaining of polytene chromosomes. (A) Green and red panels show the binding of antibodies against Psc and Spps respectively. The third panel shows the merged images. Virtually all of the bands are coincident. Boxed regions are magnified in the merged figure. (B) Green and red panels show the binding of antibodies against Psc and PHO proteins respectively. The third panel shows the merged images. The green asterisks represent some of the positions where Psc is bound at a higher incidence than PHO based on the comparable intensities of other bands nearby. The red asterisks represent positions where PHO is bound at a higher incidence than Psc when compared with the intensities of nearby bands.

Fig. 4.

Spps colocalizes with Pho, Ph and Pc on en and Ubx PREs in S2 cells and larvae. (A) Schematic of the en transcription start and the upstream 2.4 kb containing the identified PREs and the fragments amplified in the PCR after ChIP. The diagram is not to scale. (B) PCR of fragments after ChIP with antibody shown on the left. The numbers above the lanes indicate the fragment that was amplified in the PCR as shown in A. RpII₁₄₀ primers were included as a negative control, as PcG proteins do not bind to this DNA. A DNA control and a sample incubated with pre-bleed from the rabbit used to generate the Spps antibody are also shown. (C) Schematic of Ubx DNA fragments used to analyze ChIP products over PRE_D. (D) PCR of fragments after ChIP with antibody shown on the left and the same controls as in B. (E,F) qPCR showing that Pho and Spps bind to the en and Ubx PREs in larvae.

Fig. 5.

Pairing-sensitive silencing is alleviated in Spps¹ homozygotes. (A,B) Eye from a fly homozygous for a mini-white construct without a PRE in either a heterozygous or homozygous Spps¹ background, showing that Spps¹ does not alter the eye color in the absence of a PRE. (C,D) Eye from a fly homozygous for a PRE-mini-white construct in either a heterozygous or homozygous Spps¹ background, showing that Spps alleviates pairing-sensitive silencing. This PRE-mini-white construct contains en-181 bp PRE in pCaSpeR [insert 8-10C on chromosome 2R (Kassis, 1994)]. (E,F) Eyes from flies with the insertion of another en-181 bp PRE pCaSpeR construct (4-8-3-1B, inserted on chromosome 2L, unpublished line). (G,H) Eyes from a line of flies with an insertion of a construct that has Ubx-PRE_D cloned in pCaSpeR (line K2-1, inserted on the X chromosome).