Topic Next step: deciding which genes to clone Problem = correlating enzymes with genes Who matches the pH? Who localizes where? Which isoform if alternatively spliced? Clone several, using one known to work to find orthologs Use sequence to design primers to clone cDNA

Issues All genes that have been cloned and expressed have acidic pH optima: but Trinchant found bacteroid with pH 8 opt Oxalate oxidase belongs to the cupin superfamily functionally diverse proteins that have a highly conserved pattern of two histidine-containing motifs: the cupin signature

Cupins functionally diverse proteins that have a highly conserved pattern of two histidine-containing motifs: the cupin signature Proposal: you each clone one safe and one risky oxalate ox Safe = known Oxalate oxidase or decarboxylase Risky = based on homology

Cupins functionally diverse proteins that have a highly conserved pattern of two histidine-containing motifs: the cupin signature Proposal: you each clone one safe and one risky oxalate ox Safe = known Oxalate oxidase or decarboxylase Wheat oxalate oxidase GF-2.8: P15290http:// / / Barley oxalate oxidase: CAA74595 Rice oxalate oxidase: Os03g or Os03g Ceriporiopsis subvermispora oxalate oxidase: AJ B. subtilis oxalate decarboxylase: O34714 White rot oxalate decarboxylase: AF200683

Risky = based on homology, eg to rice OXO1 SORBIDRAFT_01g [Sorghum bicolor] Sequence ID: ref|XP_ | Length: 225ref|XP_ | Expect Identities Positives Gaps 1e /216(79%)196/216(90%)0/216(0%) LOC precursor [Zea mays] Sequence ID: ref|NP_ | Length: 225 ref|NP_ | Expect Identities Positives Gaps 3e /228(76%) 203/228(89%) 4/228(1%) oxalate oxidase GF-2.8-like [Brachypodium distachyon] Sequence ID: ref|XP_ | Length: 226ref|XP_ | Expect Identities Positives Gaps 4e /222(76%) 191/222(86%) 4/222(1%)

Risky = based on homology, eg to Bacillus subtilis oxdC as query Accession: NP_ oxalate decarboxylase oxdC [Neurospora crassa OR74A] Sequence ID: ref|XP_ |Length: 455 Expect Identities Positives Gaps 5e /356(59%) 269/356(75%) 2/356(0%) Oxalate decarboxylase [Mesorhizobium sp. LNHC209A00] Sequence ID: ref|WP_ |Length: 377 Expect Identities Positives Gaps 8e /367(57%) 259/367(70%) 4/367(1%) hypothetical protein BJ6T_62210 [Bradyrhizobium japonicum] Sequence ID: ref|YP_ |Length: 357 Expect Identities Positives Gaps 3e /355(69%) 283/355(79%) 1/355(0%)

Risky = based on homology, eg to Agrobacterium oxalate decarboxylase Accession: NP_ OxdD [Rhizobium sp. IRBG74] Sequence ID: ref|YP_ |Length: 415 Expect Identities Positives Gaps /415(97%) 406/415(97%) 0/415(0%) cupin [Rhizobium lupini] Sequence ID: ref|WP_ |Length: 415 Expect Identities Positives Gaps /415(97%) 407/415(98%) 0/415(0%) Oxalate decarboxylase [Bradyrhizobium sp.] Sequence ID: gb|EJZ |Length: 406 Expect Identities Positives Gaps /403(66%) 320/403(79%) 2/403(0%) oxalate decarboxylase [Bradyrhizobium japonicum USDA 6] Sequence ID: ref|YP_ |Length: 415 Expect Identities Positives Gaps /413(64%) 321/413(77%) 3/413(0%)

Risky = based on homology, eg to Agrobacterium oxalate decarboxylase Accession: NP_ uncharacterized protein LOC [Alligator sinensis] XP_ [XM_ ] Length = 341 Expect = 5e-12, Identities = 67/277 (24%), Positives = 119/277 (42%), Gaps = 28/277 (10%) uncharacterized protein C18orf54 [Bos taurus] XP_ [XM_ ] Length = 453 Expect = 7e-12,Identities = 78/312 (25%), Positives = 132/312 (42%), Gaps = 36/312 (11%)

How to proceed? Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

Using the genome Bisulfite sequencing to detect C methylation ChIP-chip or ChIP-seq to detect chromatin modifications: 17 mods are associated with active genes in CD-4 T cells

Using the Genome Acetylation, egH3K9Ac, is associated with active genes Phosphorylation shows condensation Ubiquitination of H2A and H2B shows repression Methylation is more complex: H3K36me3 = on H3K27me3 = off H3K4me1 = off H3K4me2 = primed H3K4me3 = on

Histone code Modifications tend to group together: genes with H3K4me3 also have H3K9ac Cytosine methylation is also associated with repressed genes

Generating the histone code Histone acetyltransferases add acetic acid

Generating the histone code Histone acetyltransferases add acetic acid Many HAT proteins: mutants are very sick!

Generating the histone code Histone acetyltransferases add acetic acid Many HAT proteins: mutants are very sick! HATs are part of many complexes

Generating the histone code Bromodomains specifically bind acetylated lysines

Generating the histone code Bromodomains specifically bind acetylated lysines Found in transcriptional activators & general TFs

Generating the histone code acetylated lysines Deacetylases “reset” by removing the acetate

Generating the histone code acetylated lysines Deacetylases “reset” by removing the acetate Deacetylase mutants are sick!

Generating the histone code Deacetylases “reset” by removing the acetate Deacetylase mutants are sick! Many drugs are histone deacetylase inhibitors

Generating the histone code Deacetylases “reset” by removing the acetate Deacetylase mutants are sick! Many drugs are histone deacetylase inhibitors SAHA = suberanilohydroxamic acid = vorinostat Merck calls it Zolinza, treats cutaneous T cell lymphomacutaneous T cell lymphoma

Generating the histone code Deacetylases “reset” by removing the acetate Deacetylase mutants are sick! Many drugs are histone deacetylase inhibitors SAHA = suberanilohydroxamic acid = vorinostat Merck calls it Zolinza, treats cutaneous T cell lymphomacutaneous T cell lymphoma Binds HDAC active site & chelates Zn 2+

Generating the histone code When coupled SAHA to PIPS (pyrrole-imidazole Polyamides) got gene- specific DNA binding & gene activation

Generating the histone code Other drugs are activators of histone deacetylases Resveratrol increases Sirtuin 1 expression and activity, possibly by enhancing its binding to Lamin A