Pits, a protein interacting with Ttk69 and Sin3A, has links to histone deacetylation

Abstract

Histone deacetylation plays an important role in transcriptional repression. Previous results showed that the genetic interaction between ttk and rpd3, which encodes a class I histone deacetylase, is required for tll repression. This study investigated the molecular mechanism by which Ttk69 recruits Rpd3. Using yeast two-hybrid screening and datamining, one novel protein was found that weakly interacts with Ttk69 and Sin3A, designated as Protein interacting with Ttk69 and Sin3A (Pits). Pits protein expressed in the early stages of embryos and bound to the region of the tor response element in vivo. Expanded tll expression patterns were observed in embryos lacking maternal pits activity and the expansion was not widened by reducing either maternal ttk or sin3A activity. However, in embryos with simultaneously reduced maternal pits and sin3A activities or maternal pits, sin3A and ttk activities, the proportions of the embryos with expanded tll expression were significantly increased. These results indicate that all three gene activities are involved in tll repression. Level of histone H3 acetylation in the tll proximal region was found to be elevated in embryo with reduced these three gene activities. In conclusion, Ttk69 causes the histone deacetylation-mediated repression of tll via the interaction of Pits and Sin3A.

Figure 1. CG11138 interacts with both Ttk69 and Sin3A.

(a) The shaded and black boxes indicate amino acid residues that are identical among Drosophila species and insects, respectively. The dotted box in Sin3A represents the domain that interacts with HDACs. For CG11138 and Ttk69, full-length (FL) and three protein fragments, N, M or C, as indicated by bars below the diagram. The four highly conserved domains in Sin3A, PAH1 to PAH4, as previously described, were expressed in bacteria. (b,c) Crude extracts containing the GST fusion proteins shown at the top of the panels, were mixed with crude extracts containing one of the S-tag fusion proteins, except for the full-length of Ttk69, which was His-tagged. The proteins were pulled down by glutathione agarose and detected by western blotting using either anti-S tag or anti-His tag antibody. Protein samples, consisting of 10% of the sample used for the pull-down assay, were used as the controls and are designated as Input. (d) Ttk69 and Sin3A in a cross-linked protein complex immunoprecipitated by an anti-Pits antibody. The co-IPed protein complexes were separated in SDS agarose-polyacrylamide gels and proteins in the complexes were detected by western blotting with an anti-Ttk69 antibody. The membrane was then stripped to allow detection of Sin3A.

Figure 2. Pits distribution patterns in Drosophila embryo.

A preabsorbed anti-Pits antibody was used to detect Pits distribution in Drosophila embryos by immunostaining. Embryos at stage 3 (a), stage 4 (b,i,i’), stage 5 (c,j,j’), stage 6 (d), stage 9 (e), stage 10 (f) and stage 13 (g,h) are shown. The anterior is displayed to the left. Except for panel h, which shows a ventral view, all panels show a sagittal view of the embryo. (i,j) To reveal whether Pits is in the nucleus, wild-type embryo was stained with the pre-absorbed anti-Pits (green) and anti-LamC (red) antibodies, and observed using confocal microscopy. Chromosome was labeled with Hoechst 33342 (blue). Punctate staining of Pits was detected in early stage 4 (i,i’) and 5 embryos (j,j’). Dashed yellow circles in panels i’ and j’ represent regions of embryonic nuclei to clearly show Pits nuclear localization. Scale bars are 5 μm.

Figure 3. Pits and Ttk69 co-localize to the vicinity of the tor-RE in vivo.

At the top of panel a, a diagram shows the relative positions of the transcription unit of tll, the tor-RE and a set of primers that is represented by arrows. Embryos were collected from parents of Oregon-R/w¹¹¹⁸ (wt) or pits⁹⁴ (pits) (a) or ttk^1e11 da-GAL4/+ da-GAL4 females crossed with either UAS-GFP (GFP) or UAS-ttk69-HA (ttk-HA) males (b) every 2 hours. Embryos aged for 30 min (a) or 60 min (b) were used in ChIP experiments with anti-Pits (“+”; 6 μg) or anti-HA tag antibody (“+”; 3 μg). Chromatin samples, consisting of 5% of the sample used for ChIP, were used as “input” controls. Mock control, “−”, used the same experimental procedure without antibody. The primer set, tor, was used to reveal whether Pits or Ttk69-HA exists in the vicinity of the tor-RE. Rp is a set of primers to detect the RPII140 gene that serves as a PCR negative control. The PCR products were separated in a 4% agarose gel, followed by ethidium bromide fluorography.

Figure 4. Structure and alleles of the pits gene.

(a) Exons are represented by boxes. Three subregions of Pits, N, M and C as shown in Fig. 1a, are shown by grid, solid and dotted rectangles. The pits gene encodes three putative transcripts, represented by RC, RD and RE. Transcripts C and E are the result of alternative splicing inside exon 2. Furthermore, the 3’ untranslated regions in these three transcripts are different. Using the imprecise P-element excision method and a P-element line, EP1313 inserted 38 bp downstream of the putative transcription initiation site for the transcripts, three pits deletions, 64, 94 and 240, were obtained. The arrows indicate the positions and direction of the primers used for screening the pits deletions. The range of each deletion, from position +39 to +1621, is indicated by brackets. The dashed bracket indicates that the 5′ end of the deletion has not been determined. (b) Pits levels in w¹¹¹⁸, pits⁹⁴, pits²⁴⁰ and pits⁶⁴ embryos from 0 to 4 hours were assessed by western blotting with an anti-Pits antibody. The membrane was then stripped to allow detection of β-Tubulin (β-Tub), which served as the loading control. M represents the protein size marker.

Figure 5. Maternal pits activity is important for tll repression.

The tll expression patterns in embryos from w¹¹¹⁸ (a–d), a cross of pits⁹⁴ with w¹¹¹⁸ (pits/+; e–h) and a cross of pits⁹⁴/Df(X)BSC624 with pit⁹⁴ (pits/Df(X); i–l) were determined by in situ hybridization using digoxigenin-labeled antisense tll RNA as the probe. The embryonic stages are indicated at the top of the panels. Embryos are arranged in a sagittal view, with the anterior towards the left.

Figure 6. The genetic interactions of pits with sin3A and ttk are important for tll repression.

To test the maternal effect of the gene activities, embryos from females pits⁹⁴ (a,b), pits⁹⁴; sin3A^ex4/+ (c,d), pits⁹⁴; ttk^1e11/+ (g,h), sin3A^ex4/+; ttk^1e11/+ (k,l) and pits⁹⁴; sin3A^ex4/+; ttk^1e11 (o,p) crossed with w¹¹¹⁸ males were collected to determine tll expression patterns. To test the maternal and zygotic effect of the genes’ activities, females and males that had the same genotypes, as shown at the left, were mated (e,i,m,q). Due to undetectable GFP protein expressed by the ubi-GFP transgene in the balancer chromosome in early embryonic stages, genotypes of embryos homozygous for sin3A or ttk could not be determined. Therefore, RNAi was used. Females carrying the da-GAL4 transgene were mated with RNAi lines to knock down at least two gene activities simultaneously (f,j,n,r). The tll expression patterns in the embryos were revealed by in situ hybridization with digoxigenin-labeled antisense tll RNA. Embryos are arranged in a sagittal view, with the anterior towards the left. (s) A bar graph presents percentages of embryos with expanded tll expression patterns. “M” in brackets represents embryos only with reduced maternal gene activities. Percentage indicates proportion of embryos with expanded tll expression patterns over the total number of stage-4 (solid bars) or stage-5 (open bars) embryos. The left and right numbers beneath each genotype are the total numbers of stage-4 and stage-5 embryos. Df(X) represents Df(X)BSC624. Fisher’s exact test was used to determine statistical significance of proportion of embryos with the expanded tll expression from pits (M) mothers or Df(X)/pits crossed with pits males against those with further reduction of one or two more gene activities (*: increase, #: decrease, p < 0.001).

Figure 7. Simultaneous reduction of pits, sin3A and ttk activity increases the level of histone acetylation in the tll proximal region.

Embryos were collected from da-GAL4 females crossed with w¹¹¹⁸ males (da-GAL4/+) or pits⁹⁴/+; sin3A^ex4/+; ttk^1e11 da-GAL4/daGAL4 females crossed with males carrying multiple transgenes to knock down pits, sin3A and ttk69 mRNA (pits; sin; ttk). Embryos from these crosses were used to determine the level of histone acetylation in region adjacent to the tor-RE by ChIP with an anti-acetyl histone H3 specific antibody (Merck Millipore). A cluster of Pho binding sites, called 5X Pho that is 173 bp upstream the tor-RE, is at the 5′ end of a putative PRE. Arrows indicate a set of primers used for real-time PCR. Detection of act-5C served as an endogenous control. C_T values of act were used to normalize tll, designated as ΔC_T. Relative amounts are represented by −ΔΔC_T where the ΔC_T value for the pits; sin; ttk embryos are subtracted from that of the control embryos. Significance difference was determined by Student’s t-test (*p < 0.05).

Figure 8. A model of Pits associating with Rpd3 and Mi-2/NuRD complexes with and without stimulation of the active tor pathway.

Previous results showed that GAF, Hsf and Ttk69 form a protein complex that binds to the tor-RE (grid rectangle). tll expression is attenuated in the middle of embryo where tor is inactive. Here, Pits serves as a mediator to recruit the Rpd3/Sin3A complex. In addition, based on information from the literature, GAF likely recruits the Mi-2/NuRD complex (indicated by shaded diagrams). Both Rpd3 and NuRD complexes associate with Ttk69 and GAF to inhibit histone acetylation (Ac) in the tll locus. At both poles of Drosophila embryo, Erk activated by the active tor pathway phosphorylates Ttk69, Sin3A and Hsf, indicated by asterisks. The phosphorylated Hsf becomes an activator, whereas the phosphorylated Ttk69 is released from the protein complex, leading to the disruption of the association of the Rpd3 and NuRD complex with the tor-RE, and also converts to an activator that binds to TC5. Previous work has shown that Sin3A is phosphorylated by Erk and converts it to an activator. Therefore, phosphorylated Sin3A may associate with Mi-2/MBD-like proteins to activate tll expression.