The authors have declared that no competing interests exist.
Conceived and designed the experiments: AS VLB LAP. Performed the experiments: AS VLB SF SG. Analyzed the data: AS VLB SF SG LAP. Wrote the paper: AS VLB LAP.
The role of the Sin3A transcriptional corepressor in regulating the cell cycle is established in various metazoans. Little is known, however, about the signaling pathways that trigger or are triggered by Sin3A function. To discover genes that work in similar or opposing pathways to
Histone acetylation levels are maintained by the opposing activities of histone lysine acetyltransferases (KATs) and histone deacetylases (HDACs). Modulation of acetylation levels that affect regulation of gene expression has been shown to be an important process during
(A, B) Images of wild type (
Sin3A has been implicated in the regulation of signaling that directs developmental pathways.
Function |
CG Number |
Gene Symbol |
Transcription | CG12809 |
|
CG2702 |
|
|
CG10488 |
|
|
CG10704 |
|
|
CG10390 |
|
|
CG43662 |
|
|
CG9461 |
|
|
CG7467 |
|
|
CG4107 |
|
|
CG3909 |
|
|
CG5358 |
|
|
CG1070 |
|
|
CG11033 |
|
|
Signaling | CG5974 |
|
CG11848 |
|
|
CG31110 |
|
|
CG12876 |
|
|
CG7910 |
|
|
Cell Proliferation and Division | CG2669 |
|
CG10061 |
|
|
CG6875 |
|
|
CG5814 |
|
|
GTPase activity | CG1081 |
|
CG1250 |
|
|
Apoptosis | CG10233 |
|
DNA repair | CG10018 |
|
Translation | CG2957 |
|
Larval Development | CG2723 |
|
Metabolism | CG5804 |
|
CG1152 |
|
|
CG1939 |
|
|
Proteolysis | CG11951 |
|
Chitin binding | CG32024 |
|
Unknown | CG32023 |
|
CG10053 |
|
|
CG11993 |
|
|
CG12347 |
|
|
CG14463 |
|
Functional process information was obtained from the listing for the individual gene on FlyBase
Flies carrying mutations or having reduced expression of these genes suppressed the
Sin3A also plays an important role in regulating the cell cycle. In
Although Sin3A and histone acetylation have been implicated in various developmental processes, the specific pathways regulated by the Sin3A HDAC complex during development are unknown. Genetic screens in
Images of representative wings from progeny of
CG Number | Gene Symbol | SIN3 KD I |
SIN3 KD II |
||
RNAi | LOF | RNAi | LOF | ||
CG11848 |
|
55±6 |
28±11 |
55±10 |
35±5 |
24±9 | 18±3 | ||||
CG6193 |
|
30±12 | n.t. | 17±9 | n.t. |
CG1451 |
|
n.t. | 4±1 | n.t. | 6±3 |
CG7926 |
|
11±6 | n.t. | 3±1 | n.t. |
CG3352 |
|
0 |
n.t. | 0 |
n.t. |
CG8384 |
|
25±19 | n.t. | 40±3 | n.t. |
CG34403 |
|
48±6 | n.t. | 52±17 | n.t. |
CG10225 |
|
53±8 | n.t. | 45±6 | n.t. |
CG2621 |
|
0 |
7±2 | 0 |
8±2 |
CG11895 |
|
7±8 | n.t. | 10±1 | n.t. |
CG13345 |
|
0 |
n.t. | 0 |
n.t. |
SIN3 KD I and II/
The percentage of straight winged flies in the progeny of the cross that are knocked down for
Flies had a wing phenotype that was neither straight nor curved.
n.t., not tested.
Generation of constitutive wing imaginal disc
Images of representative wings from progeny of
qRT-PCR analysis of the mRNAs of the indicated genes. mRNA from control
Wing images (63X) and adult fly images (30X) were taken with an Olympus DP72 camera coupled to an Olympus SZX16 microscope.
Total RNA was extracted from wing discs isolated from wandering third instar larvae using the RNeasy mini kit (Qiagen). cDNA was generated from total RNA using the ImProm-II Reverse Transcription System (Promega) with random hexamers. The cDNA was used as template in a quantitative real-time PCR (qPCR) assay. The analysis was performed using ABsolute SYBR Green ROX master mix (Fisher Scientific) and carried out in a Stratagene Mx3005P real-time thermocycler. Primers used for analysis are given in
CG Number | Gene Symbol | Cell Cycle Phase | SIN3 KD I |
SIN3 KD II |
||
RNAi | LOF | RNAi | LOF | |||
CG4654 |
|
G1/S |
0 |
n.t. | 0 |
n.t. |
CG7405 |
|
G1/S |
35±9 | n.t. | 38±4 | n.t. |
CG7413 |
|
G1/S |
0 | n.t. | 0 | n.t. |
CG10498 |
|
G1/S |
6 |
2±1 | 0 |
3±1 |
CG3510 |
|
G2/M |
11±1 | n.t. | 15±3 | n.t. |
CG5940 |
|
G2/M |
0 |
n.t. | 0 |
n.t. |
CG6759 |
|
M |
0 |
26±1 | 0 |
28±1 |
20±1 | 23±3 | |||||
CG10308 |
|
M |
23±1 | n.t. | 44±9 | n.t. |
CG5814 |
|
M |
11±4 | 2±2 | 13±2 | 8±3 |
SIN3 KD I and II/
The percentage of straight winged flies in the progeny of the cross that are knocked down for
Flies had a wing phenotype that was neither straight nor curved.
The double knockdown resulted in a partial lethal phenotype.
n.t., not tested.
Reduction of Sin3A protein levels by RNAi knockdown in cells of the wing imaginal disc results in a curved wing phenotype in the adult fly (
Images of representative wings from progeny of
In this first phase (phase I) of the screen we found 21 out of a total of 148 deletions tested that suppressed the penetrance of the curved wing phenotype to varying degrees (
In phase II of the screen we attempted to narrow down the cytogenetic intervals that interact with
Inspection of the deletions identified in phase II allowed us to generate a list of hundreds of genes that when reduced in expression could potentially modify the curved wing phenotype. To identify individual genes that interact with
CG Number | Gene Symbol | Mediator Module | SIN3 KD I |
SIN3 KD II |
||
RNAi | LOF | RNAi | LOF | |||
CG10572 |
|
Kinase | 79±9 |
n.t. | 89±6 |
n.t. |
CG7281 |
|
Kinase | 0 |
n.t. | 0 |
n.t. |
CG8491 |
|
Kinase | 0 |
53±30 | 0 |
34±16 |
CG9936 |
|
Kinase | n.t. | 82±5 | n.t. | 87±5 |
CG7957 |
|
Head | n.t. | 0 | n.t. | 0 |
CG18267 |
|
Head | n.t. | 2±2 | n.t. | 0 |
CG5057 |
|
Middle | n.t. | 0 | n.t. | 0 |
CG7162 |
|
Middle-Tail Junction | n.t. | 54±4 | n.t. | 49±3 |
CG3695 |
|
Tail | n.t. | 0 | n.t. | 0 |
CG7999 |
|
Tail | n.t. | 4±3 | n.t. | 3±1 |
SIN3 KD I and II/
The percentage of straight winged flies in the progeny of the cross that are knocked down for
Flies had a wing phenotype that was neither straight nor curved.
n.t., not tested.
We observed three classes of phenotypes in the double knockdown flies. These phenotypes included curved wings similar to the
Results of phase III allow us to group the genes into four distinct categories. In the first are genes whose knockdown in the wing had no phenotype on their own but when combined with knockdown of
In summary, in phase III of our unbiased screen, we identified 38 genes that suppressed the
Genes that showed a suppression of the
Images of representative wings from progeny of
Images of wings from progeny of
As expected, mutant alleles in number of genes that function in a variety of processes involved in transcription and regulation of gene expression were found to suppress the curved wing phenotype. These processes included gene specific regulation by DNA binding factors (
The DNA binding factors that suppress the
MIA is one isoform of TAF6, a component found in TFIID, the general transcription factor
In addition to histone modification, a second major enzymatic activity to affect chromatin and regulate transcription is that which is carried out by the ATP-dependent nucleosome remodeling complexes. It has long been appreciated that histone modifiers and remodeling complexes work in concert to affect transcriptional outcomes
The
Interestingly, multiple genes, including
KDM2 is a histone demethylase that targets histone H3K36 dimethylation in S2 cells and H3K4 trimethylation in
In addition to the above genes whose mutation suppresses the
Through this screen three additional genes were identified as having a role in wing development and affecting the
The Wnt pathway has been implicated in cell division in the wing disc and development of the wing
Using the QueryBuilder tool on FlyBase
When expression of two of the negative Wnt regulators,
Knockdown of
Negative regulators of the Wnt pathway interfere with signal transduction at various stages of the pathway ultimately resulting in downregulation of Wnt responsive genes
Previous work on
To extend the analysis of whether
Reduction by RNAi of three genes
Finally, while knockdown of
Taken together, these results suggest the following. First, all of these cell cycle genes are important for some process of wing development. Wing phenotypes result from reduced expression of the gene, or the reduced expression suppresses the wing phenotype brought about by knockdown of
Multiple CDKs are expressed in a cell, some of which are directly involved in regulating the cell cycle while others may have an indirect role. Some have also been implicated in regulating transcription. One such CDK is
While Mediator is essential for the majority of RNA polymerase II dependent transcription, individual genes depend on RNA polymerase II association with distinct Mediator modules and subunits
One possible explanation for the interaction of
We also tested other Mediator components for their ability to interact with
Seven RNAi fly stocks yielded an abnormal wing phenotype in the
Knockdown of
The other four genes that had an altered wing phenotype that was the same in the single knockdown and when combined with reduced
The role of the fourth gene,
A final gene that falls into the category of a fly yielding the same phenotype when mutated individually or when in combination with
Sin3A has been implicated in development in various organisms including
(DOCX)
(XLSX)
We thank Wassim Gharib, Youssef Moussa, Blake Walker and Sana Choudhry for their assistance with scoring flies for the genetic screen. We thank the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing transgenic RNAi fly stocks and/or plasmid vectors used in this study.