User:Nkuldell/SAGA swap: Difference between revisions

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H.s. to S.c. swap of SGF73 at [[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed]]
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?)  

Revision as of 04:27, 15 July 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, 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 [[1]] Other yeast to S.c. swap of SPT3 at [[2]] [[3]]

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 [4] Codon Usage Comparison using GeneArt tool called Graphical Codon Usage Analyzer [5]

Ada subunits cerevisiae gene size,chromosome,null p-type pombe gene size,chromosome,null p-type BLAST comparison info
Ada1 (aka HFI1, SUP110, SRM12, GAN1) [[6]] 1.467 kb/489 aa,
Chr. XVI, viable 		Nucleotide comparison identifies clone spc05247
Protein comparison identifies SPBC887.18c [[7]] 		Nucleotide comparison
0.167 kb, late log phase mRNA
Protein comparison
339 aa, Chr. II, hyptothetical protein NM_001022412.1 		Nucleotide comparison
 Identities = 74/114 (64%), E= 0.94 [[8]]
Protein comparison
 Identities = 72/271 (26%), Positives = 123/271 (45%), Gaps = 26/271 (9%), E = 6.3e-15 [[9]]
Ada2 (aka SWI8) [[10]] 1.305 kb/434aa,
Chr. IV, viable 		SPCC24B10.08c [[11]] 1.314 kb/437aa, Chr. III, hypothetical protein NP_588011.1 Identities = 384/645 (59%), E=1e-24 [[12]]
Ada3 (aka NGG1, SWI7) [[13]] 2.109 kb/702aa,
Chr. IV, viable 		Nucleotide comparison identifies cosmid c16H5 [[14]]
Protein comparison identifies SPBC28F2.10c [[15]] 		Nucleotide comparison
Chr. II
Protein comparison
551 aa, Chr. II, Spt-Ada-Gcn5-Acetyltransferase NP_595671 		Nucleotide comparison
 Identities = 342/619 (55%), E= 0.001 [[16]]
Protein comparison
 Identities = 53/153 (34%), Positives = 84/153 (54%), Gaps = 20/153 (13%), E=1.6e-28[[17]]
Gcn5 (aka ADA4, SWI9) [[18]] 1.32 kb/439aa,
Chr. VII, viable 		SPAC1952.05 [[19]] 1.365 kb/454aa,Chr. I, hypothetical protein NM_001020236 Identities = 718/1103 (65%)E= 4.7e-79 [[20]]
Ada5 (aka SPT20) [[21]] 1.815 kb/604aa,
Chr. XV, viable 		Sequence Comparison identifies SPBC21D10.09c [[22]]
Protein Comparison
identifies SPAC4D7.10c [[23]] 		Sequence Comparison 4.833 kb, Chr. II, hypothetical protein NP_596004.1
Protein Comparison
473 aa, Chr. I, hypothetical protein NP_594963 		Sequence Comparison
Identities = 270/488 (55%),
E = 0.99 [[24]]
Protein Comparison
Identities = 15/43 (34%), Positives = 27/43 (62%),
E= 0.0002 [[25]]
Spt subunits cerevisiae gene size, chromosome, null p-type pombe gene size, chromosome, null p-type BLAST comparison info
Spt3 [[26]] 1.014 kb/337aa,
Chr. IV, viable 		SPCC61.02 [[27]] 0.924 kb/307aa, Chr. III, hypothetical protein NP_588193.1 Identities = 194/297 (65%)
E=1.6e-36 [[28]]
Spt7 (aka GIT2) [[29]] 3.999 kb/1332aa,
Chr. II, viable 		SPBC25H2.11c [[30]] 2.940 kb/979aa, Chr. II , hypothetical protein NP_596356.1 Identities = 570/979 (58%)
E =4.3e-28 [[31]]
Spt8 [[32]] 1.809 kb/602aa,
Chr. XII, viable 		SPBC14C8.17c [[33]] 1.581 kb/526aa, Chr. II, hypothetical protein NP_595920.1 Identities = 135/230 (58%)
E= 0.053 [[34]]
Spt20 (aka Ada5) [[35]] same as Ada5
1.815 kb/604aa, Chr. XV, viable 		Sequence Comparison identifies SPBC21D10.09c [[36]]
Protein Comparison
identifies SPAC4D7.10c [[37]] 		Sequence Comparison 4.833 kb, Chr. II, hypothetical protein NP_596004.1
Protein Comparison
473 aa, Chr. I, hypothetical protein NP_594963 		Sequence Comparison
Identities = 270/488 (55%),
E = 0.99 [[38]]
Protein Comparison
Identities = 15/43 (34%), Positives = 27/43 (62%),
E= 0.0002 [[39]]
TAF subunits cerevisiae gene size, chromosome, null p-type pombe gene size, chromosome, null p-type BLAST comparison info
TAF5 (aka TAF90) [[40]] 2.397 kb/798aa, Chr. II, inviable SPCC5E4.03c (aka spTAF72) 643aa
TAF6 (aka TAF60) [[41]] 1.551 kb/516aa, Chr. VII, inviable SPCC16C4.18c (aka spTAF50) inviable 452aa 39% identical File:Macintosh HD-Users-nkuldell-Desktop-ScSpTAFs NAR02.pdf
TAF9 (aka TAF17) [[42]] 0.474 kb/157aa, Chr. XIII, inviable SPAC12G12.05c 163aa
TAF10 (aka TAF23, TAF25) [[43]] 0.621 kb/206aa, Chr. IV, inviable SPBC21H7.02 215aa
TAF12 (aka TAF61, TAF68) [[44]] 1.620 kb/539aa, Chr. IV, inviable Nucleotide comparison identifies cosmid clone 1465-1759 [[45]]
Protein comparison identifies SPAC15A10.02 		Nucleotide comparison Chr. III
Protein comparison 450 aa, Chr. I, hypothetical protein NM_001019712.1 		Nucleotide comparison Identities = 187/313 (59%) E = 1 [[46]]
Protein comparison Identities = 76/209 (36%), Positives = 117/209 (56%), Gaps = 11/209 (5%), E= 2.2e-37 [[47]]
Tra1 subunit cerevisiae gene size, chromosome, null p-type pombe gene size, chromosome, null p-type BLAST comparison info
Tra1 [[48]] 11.235 kb/3744aa, Chr. VIII, inviable SPBP16F5.03c [[49]] 3655aa, Chr. II, hypothetical protein GeneDB [[50]]
other subunits cerevisiae gene size, chromosome, null p-type pombe gene size, chromosome, null p-type BLAST comparison info
Sgf73 [[51]] 1.974 kb/657aa, Chr. VII , viable Protein comparison identifies SPCC126.04c [[52]] Protein comparison 344aa, Chr. III, hypothetical protein NP_588447 Protein comparison identifies best stretch as Identities = 48/100 (48%), Positives = 60/100 (60%), Gaps = 1/100 (1%) E=1.7e-26 File:Sc Sp Sgf73.pdf
Sgf29 [[53]] 0.779 kb/259aa, Chr. III, viable Protein comparison identifies SPBC1921.07c [[54]] Protein comparison
244 aa, Chr. II, hypothetical protein NP_596000 		Protein comparison Identities = 70/125 (56%), Positives = 85/125 (68%), Gaps = 5/125 (4%)E=3.1e-31 File:Sp Sc Sgf29p .pdf
Sgf11 [[55]] 0.3 kb/99aa, Chr.XVI, viable Protein comparison identifies SPA(C)57A10.14 [[56]] Protein comparison
117 aa, Chr. I, hypothetical protein NP_001018231 		Protein comparison Identities = 20/51 (39%), Positives = 27/51 (52%), Gaps = 3/51 (5%)E=3.6e-05
Ubp8 [[57]] 1.416 kb/471aa, Chr. XIII, viable Protein comparison identifies SPAC13A11.04c [[58]] Protein comparison
449 aa, Chr. I, hypothetical protein NP_592992 		Protein comparison Identities = 115/364 (31%), Positives = 177/364 (48%), Gaps = 50/364 (13%)E=1.8e-44
Sus1 [[59]] gene with intron, Chr. II, viable Protein comparison identifies SPBC6B1.12c [[60]] Protein comparison
108 aa, Chr. II, hypothetical protein NP_001018822 		Protein comparison
Identities = 24/86 (27%), Positives = 47/86 (54%) E=1.6e-09

S. cerevisiae vs human SAGA subunits

1. structures

2. common subunits

subunit cerevisiae gene size, chromosome, null p-type human gene size, chromosome, null p-type BLAST comparison info
Spt7 [[61]] 3.999 kb, Chr. II, viable
TAF6 (aka TAF60) [[62]] 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 [[63]] SPTFIID [[64]] TATA binding factor [[65]] C. albicans [[66]]
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