User:Nkuldell/SAGA swap: Difference between revisions
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==Idea== | ==Idea== | ||
===Justification=== | ===Justification=== | ||
The genetic and epigenetic requirements for gene expression will need to be considered if logic circuits and genetic programs are to be reliably written for eukaryotic cells. Since chromatin dynamics are an integral part of eukaryotic gene regulation, a better understanding of chromatin modifying complexes will be necessary if DNA sequences are to preform, i.e. express, in a predictable way. Ideally a common chromatin remodelling complex could be described that would perform seemlessly in any (or less ambitiously, several) hosts/operating system. Is there a generic complex that could plug into multiple eukaryotic hosts and properly regulate histone acetylation/deacetylation as well as nucleosome positioning over transcribed regions? As a first step in designing such a common chromatin remodeller, the SAGA subunits of S. cerevisiae can be replaced with the S. pombe homologs. Shoot, if Apple can find a way to run a MS operating system, smart yeast should make this SAGA swap possible. | The genetic and epigenetic requirements for gene expression will need to be considered if logic circuits and genetic programs are to be reliably written for eukaryotic cells. Since chromatin dynamics are an integral part of eukaryotic gene regulation (see as recent example [http://science.slashdot.org/article.pl?sid=06/07/25/1438229][http://www.nytimes.com/2006/07/25/science/25dna.html?ex=1311480000&en=34d8e6ced8d42f47&ei=5089&partner=rssyahoo&emc=rss,]) a better understanding of chromatin modifying complexes will be necessary if DNA sequences are to preform, i.e. express, in a predictable way. Ideally a common chromatin remodelling complex could be described that would perform seemlessly in any (or less ambitiously, several) hosts/operating system. Is there a generic complex that could plug into multiple eukaryotic hosts and properly regulate histone acetylation/deacetylation as well as nucleosome positioning over transcribed regions? As a first step in designing such a common chromatin remodeller, the SAGA subunits of S. cerevisiae can be replaced with the S. pombe homologs. Shoot, if Apple can find a way to run a MS operating system, smart yeast should make this SAGA swap possible. | ||
H.s. to S.c. swap of SGF73 at [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed]] | |||
Other yeast to S.c. swap of SPT3 at | |||
[[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=9559549&query_hl=1&itool=pubmed_docsum]] | |||
[[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=12072450&query_hl=1&itool=pubmed_docsum]] | |||
===Proposal Details=== | ===Proposal Details=== | ||
Standard yeast techniques can be used to replace the S. cerevisiae subunits with the homologs from S. pombe and effects on gene expression can be assessed. Specifically it should be possible to integrate a URA3 marker at a gene encoding a SAGA-subunit and then replace that marker wtih the S. pombe gene by transformation and seletion on FOA. The pombe genes would be amplified from a cDNA library, the source of which is still to be determined. Expression of each replaced subunit can be followed by Western if Abs are available or with epitope tags (less desirable), SAGA integrity can be functionally and biochemically assessed, cell-wide effects on gene expression can be followed by array, mutant phenotypes (Spt, drug sens etc...) Single gene replacements of multisubunit are often unsuccessful but activity can be restored with multiple replacements of interacting subunits (check Stan Fields STE12 and MCM1?) | Standard yeast techniques can be used to replace the S. cerevisiae subunits with the homologs from S. pombe and effects on gene expression can be assessed. Specifically it should be possible to integrate a URA3 marker at a gene encoding a SAGA-subunit and then replace that marker wtih the S. pombe gene by transformation and seletion on FOA. The pombe genes would be amplified from a cDNA library, the source of which is still to be determined. Expression of each replaced subunit can be followed by Western if Abs are available or with epitope tags (less desirable), SAGA integrity can be functionally and biochemically assessed, cell-wide effects on gene expression can be followed by array, mutant phenotypes (Spt, drug sens etc...) Single gene replacements of multisubunit are often unsuccessful but activity can be restored with multiple replacements of interacting subunits (check Stan Fields STE12 and MCM1?) | ||
| Line 44: | Line 48: | ||
*Spt7 50aa deletion leads to loss of Spt8 from SAGA | *Spt7 50aa deletion leads to loss of Spt8 from SAGA | ||
*Spt8 still associated with Spt7 in ada1, spt20 deletions | *Spt8 still associated with Spt7 in ada1, spt20 deletions | ||
*dstI, sgf73 synthetic lethal (Krogan in Mol Cell 2003 12(6):1565) | |||
===S. cerevisiae vs S. pombe SAGA subunits=== | ===S. cerevisiae vs S. pombe SAGA subunits=== | ||
<font color = red> check codon bias Sc vs Sp </font color> | <font color = red> check codon bias Sc vs Sp </font color> [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=7992504&query_hl=24&itool=pubmed_docsum] | ||
Codon Usage Comparison using GeneArt tool called Graphical Codon Usage Analyzer [http://gcua.schoedl.de/sequential.php] | |||
===S. cerevisiae vs human SAGA subunits=== | ===S. cerevisiae vs human SAGA subunits=== | ||
Latest revision as of 10:01, 23 August 2006
Idea
Justification
The genetic and epigenetic requirements for gene expression will need to be considered if logic circuits and genetic programs are to be reliably written for eukaryotic cells. Since chromatin dynamics are an integral part of eukaryotic gene regulation (see as recent example [1][2]) a better understanding of chromatin modifying complexes will be necessary if DNA sequences are to preform, i.e. express, in a predictable way. Ideally a common chromatin remodelling complex could be described that would perform seemlessly in any (or less ambitiously, several) hosts/operating system. Is there a generic complex that could plug into multiple eukaryotic hosts and properly regulate histone acetylation/deacetylation as well as nucleosome positioning over transcribed regions? As a first step in designing such a common chromatin remodeller, the SAGA subunits of S. cerevisiae can be replaced with the S. pombe homologs. Shoot, if Apple can find a way to run a MS operating system, smart yeast should make this SAGA swap possible.
H.s. to S.c. swap of SGF73 at [[3]] Other yeast to S.c. swap of SPT3 at [[4]] [[5]]
Proposal Details
Standard yeast techniques can be used to replace the S. cerevisiae subunits with the homologs from S. pombe and effects on gene expression can be assessed. Specifically it should be possible to integrate a URA3 marker at a gene encoding a SAGA-subunit and then replace that marker wtih the S. pombe gene by transformation and seletion on FOA. The pombe genes would be amplified from a cDNA library, the source of which is still to be determined. Expression of each replaced subunit can be followed by Western if Abs are available or with epitope tags (less desirable), SAGA integrity can be functionally and biochemically assessed, cell-wide effects on gene expression can be followed by array, mutant phenotypes (Spt, drug sens etc...) Single gene replacements of multisubunit are often unsuccessful but activity can be restored with multiple replacements of interacting subunits (check Stan Fields STE12 and MCM1?)
Four distinct classes of genes make up the multisubunit SAGA complex in S. cerevisiae
1. The Ada proteins
- Ada1, Ada2, Ada3, Gcn5, Ada5
2. The Spt proteins
- Spt3, Spt7, Spt8, Spt20
3. The TAF proteins
- TAF5, TAF6, TAF9, TAF10, TAF12
4. The Tra1 protein
- essential gene
- target of gene specific activators
Can generic versions of these subcomplexes be described and then used to intelligently specify the chromatin packaging needed to execute a genetic program? Success would provide an existance proof for a re-usable genetic module capable of appropriately remodelling chromatin in N>1 host.
Fact tables
S. cerevisiae SAGA chemistry
summarized from TiBS 05 review File:Macintosh HD-Users-nkuldell-Desktop-SAGA swap-SAGAunveiled TiBS05.pdf
- GCN5 has HAT activity
- Ada2, Ada3 regulate GCN5
- Ada5 is = Spt20
- SAGA structural integrity depends on Ada1, Spt7, Spt20, and TAF12
- interaction with TBP requires Spt8 and Ada3
- gene specific transcriptional activators Gcn4, VP16 and Gal4 target Tra1
- histone fold pairs: TAF6 and TAF9, TAF10 and Spt7, TAF12 and Ada1
- SAGA variants such as SALSA, SLIK without Spt8 and truncation of Spt7/ of questionable functional significance
- Other SAGA associated subunits that don't fall into one of 4 catagories above but that purify with SAGA: Sgf73, Sgf29, Sgf11, Ubp8 and Sus1 (in S. cerevisiae)which correspond to SPCC126.04c, SPBC1921.07c, SPA(C)57A10.14, SPAC13A11.04c, SPBC6B1.12c (in pombe).
summarized from Dom/Mark/Fred's talks, Winston retreat 06.07.06
- Structural role: Spt7, Ada1, Spt20
- Histone fold pairs: Taf6 and Taf9, Taf10 and Spt7, Taf12 and Ada1
- H3, H2B acetylation: Gcn5, Ada2, Ada3
- H2B deubiq: Ubp8, perhaps Sgf11
- TBP recruitment: Spt3, Spt8
- Intn w/ activators: Tra1
- Unknown: Sgf73 (though mutations in this subunit confer PAU defects) and Sgf29
Genetic interactions:
- Spt3 still in SAGA in spt20 deletion strain
- Spt3 not in SAGA in ada1 deletion or spt7 deletion strain (region withing 873-1125 req'd)
- Spt7 50aa deletion leads to loss of Spt8 from SAGA
- Spt8 still associated with Spt7 in ada1, spt20 deletions
- dstI, sgf73 synthetic lethal (Krogan in Mol Cell 2003 12(6):1565)
S. cerevisiae vs S. pombe SAGA subunits
check codon bias Sc vs Sp [6] Codon Usage Comparison using GeneArt tool called Graphical Codon Usage Analyzer [7]
S. cerevisiae vs human SAGA subunits
1. structures
- EM of human TFTC in File:Macintosh HD-Users-nkuldell-Desktop-TFTCvsSAGA Sci99.pdf
- EM of ySAGA in File:Macintosh HD-Users-nkuldell-Desktop-SAGA swap-SAGAstruct MolCell05.pdf
2. common subunits
| subunit | cerevisiae | gene size, chromosome, null p-type | human | gene size, chromosome, null p-type | BLAST comparison info |
|---|---|---|---|---|---|
| Spt7 | [[8]] | 3.999 kb, Chr. II, viable | |||
| TAF6 (aka TAF60) | [[9]] | 1.551 kb, Chr. VII, inviable | hTAF80 |
3. human specific
TFTC-subunits(from Wu in Mol Cell 04)
- PAF65-beta
- Taf2
- Taf4
TBP Comparisons
| subunit | cerevisiae | pombe | human | other |
|---|---|---|---|---|
| TBP | SPT15,BTFI [[10]] | SPTFIID [[11]] | TATA binding factor [[12]] | C. albicans [[13]] |
