Here we show that abo encodes an evolutionary conserved chromosomal protein that localizes exclusively to the histone gene cluster and binds to the regulatory regions of such genes. We also show a significant increase of histone transcripts in eggs of abo mutant mothers and a partial rescue of the abo maternal-effect defect by deficiencies of the histone gene cluster. On the basis of these results, we suggest that the Abo protein functions specifically as a negative regulator of histone transcription and propose a molecular model to account for the ability of heterochromatin to partially rescue the abo maternal-effect defect.
Our model proposes that increased doses of specific heterochromatic regions titrate out abnormally high levels of histones present in embryos from mutant abo mothers and that a balanced pool of histones is critical for normal embryogenesis in Drosophila.
The maternal effect gene, abnormal oocyte abo , of Drosophila melanogaster encodes a specific negative regulator of histones. T1 - The maternal effect gene, abnormal oocyte abo , of Drosophila melanogaster encodes a specific negative regulator of histones. The active promoter is marked by dimethylation and trimethylation of Lys 4. For the repressed promoter, we observed high levels of H3 methyl Lys 9 and H3 methyl Lys 27, particularly of the trimethylated isoforms.
These findings are confirmed by the results from the abd-A region Fig 1C , in which the repressed promoter is marked by H3 trimethyl Lys 9 and H3 trimethyl Lys 27 in both cell lines. The presence of both modifications was independently confirmed using different antibodies specific for the same modifications Table 1 and supplementary information online.
A bp part of it, containing a nuclease hypersensitive region HS3 , has been isolated as a minimal PRE Mishra et al , , and references therein. As for the promoter regions, the repressed state can be correlated with the presence of Pc and E z and is marked by trimethylation of Lys 9 and Lys 27 of H3 Fig 2B. We observed a main peak of Pc binding for the primer pair p9, which contains the minimal PRE. Interestingly, no significant upmethylation of Lys 4 in the PRE is observed when Abd-B is active, indicating that Lys 4 methylation is specifically linked to transcriptional activity in promoter regions or genes.
Taken together, our data on iab-7 -PRE are consistent with the activity of its cis -regulatory target, the Abd-B gene. A Schematic representation of the iab-7 region and the position of the three primer pairs used for X-ChIP.
The different antisera used for the IPs are indicated above the gels. After four rounds of treatment with Pc -dsRNA, a spliced, polyadenylated abd-A transcript is detected. The Abd-A protein is produced after knockdown, although it is detected only in a subset of cells Fig 3B.
After reactivation of abd-A , we see patterns that resemble those observed for the active Abd-B gene in S3 cells Fig 1B —that is, high levels of Pol II, Tbp and the elongation factors and low occupancy for Pc as expected, as this protein was knocked down and E z. Thus, the artificial derepression of the abd-A promoter is a slow process that requires the cells to go through repeated rounds of replication.
We suggest that this happens, as it is necessary to dilute out, through cell division, PcG components and the repressive methylation marks near the promoter by histone replacement as suggested by Wang et al , Similarly, H3 Lys 9 and Lys 27 trimethylation will be lost, as the maintaining action of E z is missing.
In parallel or subsequently, the methylation of H3 Lys 4 possibly by the activity of the two trxG HMTs Ash1 and Trx and the recruitment of remodelling activities such as the Brahma protein would enable functional initiation and transcriptional elongation Beisel et al , In addition, we observed a mild upregulation of ph and Psc expression after Pc knockdown see supplementary information online , which could compensate for the loss of Pc and further slow down the process of abd-A reactivation.
As an input control, a primer pair specific for transcripts of the RP gene was included in the analysis. Amplified products using a primer pair specific for abd-A are shown on the left of each gel, and those using a primer pair specific for RP are shown on the right. The expressions of PcG proteins in control and knocked down cells are shown below the reactivation of Abd-A in the knocked down cells. The expression of Abd-A in control cells and the staining patterns of the secondary antibodies used alone are shown at the bottom.
In a last set of experiments, we also knocked down Ph and Psc. Psc is virtually absent after four rounds of dsRNA treatment, whereas the level of Ph is strongly reduced Fig 3C and supplementary information online. After knockdown of Ph, as for Pc, a spliced, polyadenylated abd-A transcript is present, which is detected only at very low levels after Psc knockdown Fig 3A , lane This fits with the different efficiency of the Pc and ph knockdown.
Chromatin was prepared from various dsRNA-treated cells and used for immunoprecipitation as described before Fig 4. Again, we observed a weak upregulation of Psc and Pc expression after ph knockdown supplementary information online , which could result in a mild compensation for the reduction of Ph levels by overexpression of other PcG components, weakening the effect of the ph knockdown on abd-A reactivation.
Reduction of Psc leads to little change, as expected, as the Psc knockdown did not result in the expression of Abd-A. Also in imaginal discs, no reactivation of target genes has been observed in clones mutant for Psc Beuchle et al , Thus, the functions of the Psc and Su z 2 proteins are at least partly redundant. Our results are in contrast with published immunohistochemical data, which solely identify H3 methyl Lys 27 as the mark for PcG-repressed regions Fischle et al , This discrepancy may be due to the resolution of the X-ChIP method as compared with immunohistochemistry and the specificity of the single antibody used in that study.
We therefore suggest that PcG-repressed regions, core promoters and PREs are marked by a specific pair of histone H3 modifications, trimethyl Lys 9 and trimethyl Lys Wang et al suggest a model of hierarchical binding of PcG proteins, where E z , recruited to PREs by the PcG protein Pho Pleiohomeotic , creates a binding substrate for Pc-containing complexes in the PRE and by the formation of a loop, also in promoter regions.
Then, PRC1 recruitment leads to the establishment of the repression of the target. Our data suggest that E z has an important role also in maintaining repression. Both the loss of E z and the loss of PRC1 components lead to the derepression of PcG target genes, as well as to the loss of repressive methylation marks Breiling et al , ; Cao et al , ; Wang et al , ; this report. The presence of both proteins or protein complexes is necessary to maintain the repressed state of HOX genes.
Thus, E z has an important function in maintenance, in particular in keeping the PcGspecific methylation marks. This suggests that the necessary interaction of E z with the target promoter region during maintenance depends also on PRC1, in particular Pc.
This interdependence may provide the flexibility necessary to adapt maintained transcription patterns to new developmental situations. Culture cell growth. S2 and S3 cells were grown in Schneider's Drosophila medium Gibco supplemented with This strain of S2 cells does not express the Abd-B protein Fig 1 , in contrast to a strain used previously Breiling et al , Psc and Ph antibodies were described previously Breiling et al , Paro, J.
Kadonaga, F. Winston, R. Link to publication in Scopus. Link to citation list in Scopus. Fingerprint Dive into the research topics of 'Binding of Trithorax and Polycomb proteins to the bithorax complex: Dynamic changes during early Drosophila embryogenesis'.
Together they form a unique fingerprint. View full fingerprint. EMBO Journal , 17 17 , EMBO Journal. Orlando, Valerio ; Jane, Esther P.
0コメント