User:Nkuldell/SAGA swap

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.

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).

S. cerevisiae vs S. pombe SAGA subunits

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) [[1]]	1.467 kb, Chr. XVI, viable	Nucleotide comparison identifies clone spc05247 Protein comparison identifies SPBC887.18c [[2]]	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 [[3]] Protein comparison Identities = 72/271 (26%), Positives = 123/271 (45%), Gaps = 26/271 (9%), E = 6.3e-15 [[4]]
	Ada2 (aka SWI8) [[5]]	1.305 kb, Chr. IV, viable	SPCC24B10.08c [[6]]	1.314 kb, Chr. III, hypothetical protein NP_588011.1	Identities = 384/645 (59%), E=1e-24 [[7]]
	Ada3 (aka NGG1, SWI7) [[8]]	2.109 kb, Chr. IV, viable	Nucleotide comparison identifies cosmid c16H5 [[9]] Protein comparison identifies SPBC28F2.10c [[10]]	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 [[11]] Protein comparison Identities = 53/153 (34%), Positives = 84/153 (54%), Gaps = 20/153 (13%), E=1.6e-28[[12]]
	Gcn5 (aka ADA4, SWI9) [[13]]	1.32 kb, Chr. VII, viable	SPAC1952.05 [[14]]	1.365 kb,Chr. I, hypothetical protein NM_001020236	Identities = 718/1103 (65%)E= 4.7e-79 [[15]]
	Ada5 (aka SPT20) [[16]]	1.815 kb, Chr. XV, viable	Sequence Comparison identifies SPBC21D10.09c [[17]] Protein Comparison identifies SPAC4D7.10c [[18]]	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 [[19]] Protein Comparison Identities = 15/43 (34%), Positives = 27/43 (62%), E= 0.0002 [[20]]

Spt subunits	cerevisiae	gene size, chromosome, null p-type	pombe	gene size, chromosome, null p-type	BLAST comparison info
	Spt3 [[21]]	1.014 kb, Chr. IV, viable	SPCC61.02 [[22]]	0.924 kb, Chr. III, hypothetical protein NP_588193.1	Identities = 194/297 (65%) E=1.6e-36 [[23]]
	Spt7 (aka GIT2) [[24]]	3.999 kb, Chr. II, viable	SPBC25H2.11c [[25]]	2.940 kb, Chr. II , hypothetical protein NP_596356.1	Identities = 570/979 (58%) E =4.3e-28 [[26]]
	Spt8 [[27]]	1.809 kb, Chr. XII, viable	SPBC14C8.17c [[28]]	1.581 kb, Chr. II, hypothetical protein NP_595920.1	Identities = 135/230 (58%) E= 0.053 [[29]]
	Spt20 (aka Ada5) [[30]]	same as Ada5	Sequence Comparison identifies SPBC21D10.09c [[31]] Protein Comparison identifies SPAC4D7.10c [[32]]	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 [[33]] Protein Comparison Identities = 15/43 (34%), Positives = 27/43 (62%), E= 0.0002 [[34]]

TAF subunits	cerevisiae	gene size, chromosome, null p-type	pombe	gene size, chromosome, null p-type	BLAST comparison info
	TAF5 (aka TAF90) [[35]]	2.397 kb, Chr. II, inviable	SPCC5E4.03c (aka spTAF72)
	TAF6 (aka TAF60) [[36]]	1.551 kb, Chr. VII, inviable	SPCC16C4.18c (aka spTAF50)	inviable	39% identical File:Macintosh HD-Users-nkuldell-Desktop-ScSpTAFs NAR02.pdf
	TAF9 (aka TAF17) [[37]]	0.474 kb, Chr. XIII, inviable	SPAC12G12.05c
	TAF10 (aka TAF23, TAF25) [[38]]	0.621 kb, Chr. IV, inviable	SPBC21H7.02
	TAF12 (aka TAF61, TAF68) [[39]]	1.620 kb, Chr. IV, inviable	Nucleotide comparison identifies cosmid clone 1465-1759 [[40]] 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 [[41]] Protein comparison Identities = 76/209 (36%), Positives = 117/209 (56%), Gaps = 11/209 (5%), E= 2.2e-37 [[42]]

Tra1 subunit	cerevisiae	gene size, chromosome, null p-type	pombe	gene size, chromosome, null p-type	BLAST comparison info
	Tra1 [[43]]	11.235 kb, Chr. VIII, inviable	SPBP16F5.03c [[44]]	Chr. II, hypothetical protein	GeneDB [[45]]

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	[[46]]	3.999 kb, Chr. II, viable
TAF6 (aka TAF60)	[[47]]	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 [[48]]	SPTFIID [[49]]	TATA binding factor [[50]]	C. albicans [[51]]