Supplemental information
ARC 32 kDa
MQREEKQLEASLDHLLSQVADLKNSLGSFICKLENEYGRLTWPSVLDSFALLSGQLNTLNKVLKH
EKTPLFRNQVIIPLVLSPDRDEDLMRQTEGRVPVFSHEVVPDHLRTKPDPEVEEQEKQLTTDAARIG
ADAAQKQIQSLNKMCSNLLEKISKEERESESGGLRPNKQTFNPTDTNALVAAVAFGKGLSNWRPS
GSSGPGQAGQPGAGTILAGTSGLPQVQMAGAPSQQQPMLSGVQMAQAGQPGKMPSGIKTNIKSA
SMHPYQR*
Peptides obtained by microsequencing of the ARC32 protein are underlined
Compilation of translated sequences from the following ESTs: R60110; T86784; AA037068; AA147608;
AA554428; AA662369
ARC 36 kDa
MAASSSGEKEKERLGGGLGVAGGNSTRERLLSALEDLEVLSRELIEMLAISRNQKLLQAGEENQVL
ELLIHRDGEFQELMKLALNQGKIHHEMQVLEKEVEKRDSDIQQLQKQLKEAEQILATAVYQAKEK
LKSIEKARKGAISSEEIIKYAHRISASNAVCAPLTWVPGDPRRPYPTDLEMRSGLLGQMNNPSTNGV
NGHLPGDALAAGRLPDVLAPQYPWQSNDMSMNMLPPNHSSDFLLEPPGHNKENEDDVEIMSTDS
SSSSSESD*
Peptides obtained by microsequencing of the ARC36 protein are underlined
Compilation of translated sequences from the following ESTs: R17240; AA400109; D81336; AA042832;
W03615; AA453188
ARC 42 kDa
MLWSGCRRFGARLGCLPGGLRVLVQTGHRSLTSCIDPSMGLNEEQKEFQKVAFDFAAREMAPNM
AEWDQKELFPVDVMRKAAQLGFGGVYIQTDVGGSGLSRLDTSVIFEALVTGCTSTTAYISIHNMC
AWMIDSFGNEEQRHKFCPPLCTMEKFASYCLTEPGSGSDAASLLTSAKKQGDHYILNGSKAFISGA
GESDIYVVMCRTGGPGPKGISCIVVEKGTPGLSFGKKEKKVGWNSQPTRAVIFEDCAVPVANRIGS
EGQGFLIAVRGLNGGRINIASCSLGAAHASVILTRDHLNVRKQFGEPLAVTSTCNSHWLIWQQGW
WPRGLMVRNAAVALQEERKDAVALCSMAKLFATDECFAICNQALQMHGGYGYLKDYAVQQYV
RDSRVHQILEGSNEVMRILISRSLLQE*
Peptides obtained by microsequencing of the ARC42 protein are underlined
Compilation of translated sequences from the following ESTs: AA309011; H23157; AA663798;
AA282272; AA741056; AA775859
The ARC42 protein exhibits significant sequence homology to human medium-chain acylCoA
dehydrogenase. The functional significance of this homology and whether ARC42 is a bona fide ARC
component or just happens to co-purify with the ARC complex is under investigation.
ARC 92 kDa
...KTEQWPQKLIMQLIPQQLLTTLGPLFRNSRMVQFHFTNKDLESLKGLYRIMGNGFAGCVHFPHT
APCEV RVLMLLYSSKKKIFMGLIPYDQSGF...
Peptides obtained by microsequencing of the ARC92 protein are underlined
Translated sequence from the following human EST: R17150
The peptides KPKPASVDANTK and KLLAWSGVLEWQ match the AA617203 mouse EST. The
peptides KAAPPALLEPLQ and KQPVPLPPAAPSG match the AA589362 mouse EST.
ARC 240 kDa
…DEHGSAKNVSFNPAKISSNFSSIIAEKLRCNTLPDTGRRKPQVNQKDNFWLVTARSQSAINTWFT
DLAGTKPLTQLAKKVPIFSKKEEVFGYLAKYTVPVMRAAWLIKMTCAYYAAISETKVKKRHVDPF
MEWTQIITKYLWEQLQKMAEYYRPGPAGSGGCGSTIGPLPHDVEVAIRQWDYTEKLAMFMFQDG
MLDRHEFLTWVLECFEKIRPGEDELLKLLLPLLLRYSGEFVQSAYLSRRLAYFCTRRLALQLDGVS
SHSSHVISAQSTSTLPTTPAPQPPTSSTPSTPFSDLLMCPQHRPLVFGLSCILQTILLCCPSALVWHYS
LTDSRIKTGSPLDHLPIAPSNLPMPEGNSAFTQQVRAKLREIEQQIKERGQAVEVRWSFDKCQEATA
GFTIGRVLHTLEVLDSHSFERSDFSNSLDSLCNRIFGLGPSKDGHEISSDDDAVVSLLCEWAVSCKR
SGRHRAMVVAKLLEKRQAEIEAERCGESEAADEKGSIASGSLSAPSAPIFQDVLLQFLDTQAPMLT
DPRSESERVEFFNLVLLFCELIRHDVFSHNMYTCTLISRGDLAFGAPGPRPPSPFDDPADDPEHKEA
EGSSSSKLEDPGLSESMDIDPSSSVLFEDMEKPDFSLFSPTMPCEGKGSPSPEKPDVEKEVKPPPKEK
IEGTLGVLYDQPRHVQYATHFPIPQEESCSHECNQRLVVLFGVGKQRDDARHAIKKITKDILKVLN
RKGTAETDQLAPIVPLNPGDLTFLGGEDGQKRRRNRPEAFPTAEDIFAKFQHLSHYDQHQVTAQVS
RNVLEQITSFALGMSYHLPLVQHVQFIFDLMEYSLSISGLIDFAIQLLNELSVVEAELLLKSSDLVGS
YTTSLCLCIVAVLRHYHACLILNQDQMAQVFEGLCGVVKHGMNRSDGSSAERCILAYLYDLYTSC
SHLKNKFGELFSDFCSKVKNTIYCNVEPSESNMRWAPEFMIDTLENPAAHTFTYTGLGKSLSENPA
NRYSFVCNALMHVCVGHHDPDRVNDIAILCAELTGYCKSLSAEWLGVLKALCCSSNNGTCGFND
LLCNVDVSDLSFHDSLATFVAILIARQCLLLEDLIRCAAIPSLLNAACSEQDSEPGARLTCRILLHLF
KTPQLNPCQSDGNKPTVGIRSSCDRHLLAASQNRIVDGAVFAVLKAVFVLGDAELKGSGFTVTGG
TEELPEEEGGGGSGGRRQGGRNISVETASLDVYAKYVLRSICQQEWVGERCLKSLCEDSNDLQDP
VLSSAQAQRLMQLICYPHRLLDNEDGENPQRQRIKRILQNLDQWTMRQSSLELQLMIKQTPNNEM
NSLLENIAKATIEVFQRSAETGSSSGSTASNMPSSSKTKPVLSSLERSGVWLVAPLIAKLPTSVQGH
VLKAAGEELEKGQHLGSSSRKERDRQKQKSMSLLSQQPFLSLVLTCLKGQDEQREGLLTSLYSQV
HQIVNNWRDDQYLDDCKPKQLMHEALKLRLNLVGGMFDTVQRSTQQTTEWAMLLLEIIISGTVD
MQSNNELFTTVLDMLSVLINGTLAADMSSISQGSMEENKRAYMNLAKKLQKELGERQSDSLEKV
RQLLPLPKQTRDVITCEPQGSLIDTKGNKIAGFDSIFKKEGLQVSTKQKISPWDLFEGLKPSAPLSW
GWFGTVRVDRRVARGEEQQRLLLYHTHLRPRPRAYYLEPLPLPPEDEEPPAPTLLEPEKKAPEPPK
TDKPGAAPPSTEERKKKSTKGKKRSQPATKTEDYGMGPGRSGPYGVTVPPDLLHHPNPGSITHLN
YRQGSIGLYTQNQPLPAGGPRVDPYRPVRLPMQKLPTRPTYPGVLPTTMTGVMGLEPSSYKTSVY
RQQQPAVPQGQRLRQQLQQSQGMLGQSSVHQMTPSSSYGLQTSQGYTPYVSHVGLQQHTGPAGT
MVPPSYSSQPYQSTHPSTNPTLVDPTRHLQQRPSGYVHQQAPTYGHGLTSTQRFSHQTLQQTPMIS
TMTPMSAQGVQAGVRSTAILPEQQQQQQQQQQQQQQQQQQQQQQQQQQYHIRQQQQQQILRQQ
QQQQQQQQQQQQQQQQQQQQQQQQHQQQQQQQAAPPQPQPQSQPQFQRQGLQQTQQQQQTAA
LVRQLQQQLSNTQPQPSTNIFGRY*
Peptides obtained by microsequencing of the ARC240 protein are underlined
Identified as KIAA0192, Genbank accession #: D83783
ARC 250 kDa
…CCNDCYLALEHGRQFMDNMSGGKVDEALVKSSCLHPWSKRNDVSMQCSQDILRMLLSLQPVL
QDAIQKKRTVRPWGVQGPLTWQQFHKMAGRGSYGTDESPEPLPIPTFLLGYDYDYLVLSPFALPY
WERLMLEPYGSQRDIAYVVLCPENEALLNGAKSFFRDLTAIYESCRLGQHRPVSRLLTDGIMRVGS
TASKKLSEKLVAEWFSQAADGNNEAFSKLKLYAQVCRYDLGPYLASLPLDSSLLSQPNLVAPTSQ
SLITPPQMTNTGNANTPSATLASAASSTMTVTSGVAISTSVATANSTLTTASTSSSSSSNLNSGVSSN
KLPSFPPFGSMNSNAAGSMSTQANTVQSGQLGGQQTSALQTAGISGESSSLPTQPHPDVSESTMDR
DKVGIPTDGDSHAVTYPPAIVVYIIDPFTYENTDESTNSSSVWTLGLLRCFLEMVQTLPPHIKSTVSV
QIIPCQYLLQPVKHEDREIYPQHLKSLAFSAFTQCRRPLPTSTNVKTLTGFGPGLAMETALRSPDRPE
CIRLYAPPFILAPVKDKQTELGETFGEAGQKYNVLFVGYCLSHDQRWILASCTDLYGELLETCIINI
DVPNRARRKKSSARKFGLQKLWEWCLGLVQMSSLPWRVVIGRLGRIGHGELKDWSCLLSRRNLQ
SLSKRLKDMCRMCGISAADSPSILSACLVAMEPQGSFVIMPDSVSTGSVFGRSTTLNMQTSQLNTP
QDTSCTHILVFPTSASVQVASATYTTENLDLAFNPNNDGADGMGIFDLLDTGDDLDPDIINILPASP
TGSPVHSPGSHYPHGGDAGKGQSTDRLLSTEPHEEVPNILQQPLALGYFVSTAKAGPLPDWFWSA
CPQAQYQCPLFLKASLHLHVPSVQSDELLHSKHSHPLDSNQTSDVLRFVLEQYNALSWLTCDPAT
QDRRSCLPIHFVVLNQLYNFIMNML*
Peptides obtained by microsequencing of the ARC250 protein are underlined
Identified as KIAA0593, Genbank accession #: AB011165
The following ARC subunits exhibit significant homology to C. elegans sequences (Gene name and
accession number noted) deposited in the Genbank data base:
ARC250: F07H5.7; Z68314
ARC240: F47A4.4; Z49888
ARC150: C38C10.5; Z19153
ARC130: F39B2.4B; Z92834
ARC105: Y113B8 (potential, sequence not completed)
ARC77: Y113G7 (potential, sequence not completed)
ARC42: U29377; K05F1.1
ARC36: U29380; ZK546.13
ARC34: AL032653; Y54E5B.3
ARC33: U78081; ceMed6
ARC32: Y62F5 (potential, sequence not completed)
For ARC 205, ARC100, ARC 92, and ARC70 there are no obvious homologous sequences.
Supplemental figures:
Figure legends and methods
Supplemental Figure 1.
Glycerol gradient purification of the ARC complex. a, Lane 1 shows ARC directly
purified from HeLa NE using GST-SREBP activation domain affinity chromatography.
Lanes 2-11 show the glycerol gradient fractions (fraction numbers indicated below the
panel) from the sedimentation of ARC affinity-purified using the SREBP activation
domain. The majority of ARC polypeptides co-sediment as one distinct large complex
(fractions 12-16) with an estimated molecular weight of 2 MDa when compared with size
standards separated using identical conditions. Most of CBP/p300 present in the ARC
preparation appears to migrate as monomer (fractions 6-8), suggesting that much of the
CBP/p300 bound to GST-SREBP is "free", with only a small portion co-purifying with
ARC. The molecular weights of ARC subunits are indicated to the left of the panel.
Migration of size standards are indicated at the top of the panel. b, In vitro transcription
experiments demonstrate that the glycerol gradient-purified complex harbors cofactor
activity. Lanes 1-4 show the transactivation by Sp1 and SREBP-1a in the absence of the
ARC complex. Lanes 5-8 show the strong effect on SREBP-1a/Sp1-activated
transcription at the LDLR chromatin template by the addition of affinity-purified ARC.
Lanes 9-12 show the stimulation by the glycerol gradient-purified ARC complex. The
weaker enhancement observed with the glycerol gradient fraction is due to the lower
concentration of ARC complex added as compared with the SREBP affinity-purified
ARC.
Methods: Hela NE (2 ml) was incubated with 50 µl of GST-SREBP-1a beads. After 3 hrs
nutating at 4˚C, the beads were washed extensively with 0.5 M KCl HEG+0.1% NP40,
followed by one wash with 0.1 M KCl HEG+0.1% NP40, and elution using 2 x 50 µl 10
mM Glutathione in 0.1 M KCl HEG+0.1% NP40 for 1 hr nutating at 4˚C. The eluted
material (100 µl) was applied to a 2 ml glycerol gradient (15-40%) in 0.1 M KCl HEG
and spun for 7 hrs at 4˚C at 50,000 rpm in a TL100 rotor (Beckman). Fractions (100 µl)
were collected from the top of the gradient, then analyzed by SDS-PAGE/silver staining
and transcription. Transcription reactions were performed by combining SREBP-1a (5
nM) and Sp1 (2 nM) with or without affinity-purified ARC or glycerol gradient-purified
ARC (0.125-0.5 nM) and chromatin template (0.5 nM) for 30 min. at 27˚C followed by
addition of general transcription factors and NTPs to initiate transcription.
Supplemental Figure 2.
S-500 gel filtration and GST-SREBP-1a(1-50) affinity purification of the ARC complex.
Lane 1 shows ARC directly purified from HeLa NE using GST-SREBP activation
domain affinity chromatography. Lanes 2-13 show the SREBP affinity-purified fractions
from the S-500 gel filtration column. The ARC polypeptides apparently purify as one
large complex with an estimated molecular weight of 2 MDa when compared with size
standards separated using identical conditions. As the results from the glycerol gradient
show, the majority of CBP/p300 appears to migrate as a monomer or in a small complex
distinct from ARC when separated by sizing chromatography. Additionally, CBP/p300
can apparently interact with the activation domain of SREBP-1a independent of the ARC
complex. The molecular weights of ARC subunits are indicated to the left of the panel.
Migration of size standards are indicated at the top of the panel.
Methods: Hela NE (5 ml) was applied to a 325 ml S-500 (Pharmacia) gel filtration
column pre-equilibrated in 0.4 M KCl HEG+0.05% NP40 and 10 ml fractions were
collected. Every other fraction was diluted to 20 ml with HEG (no KCl) and 50 µl of
GST-SREBP-1a beads. After 3 hrs nutating at 4˚C, the beads were washed extensively
with 0.5 M KCl HEG+0.1% NP40 and then analyzed by SDS-PAGE and silver staining.
Supplemental Figure 3.
Anti-CBP and anti-p300 immunoblotting analyses of ARC affinity-purified using
different activation domains. Both CBP and p300 are recruited by the three different
activation domains, although the immobilized VP16 activation domain binds
considerably less CBP/p300, than the SREBP-1a and p65 activation domains. The upper
panel shows immunoblotting using affinity-purified antibody directed against CBP, while
the lower panel shows the immunoblotting using affinity-purified antibody directed
against p300. Lane 1 shows 0.5 % of the input HeLa nuclear extract. Lane 2: 10 % of
ARC affinity-purified and eluted from the VP16 activation domain-coupled resin. Lane 3:
10 % of ARC affinity-purified and eluted from the SREBP-1a activation domain-coupled
resin. Lane 4: 10 % of ARC affinity-purified and eluted from the NF-B p65 activation
domain-coupled resin.
Methods: Hela NE (1 ml) was incubated with 30 µl of GST-SREBP-1a beads. After 3 hrs
nutating at 4˚C, the beads were washed extensively with 0.5 M KCl HEG+0.1% NP40,
followed by one wash with 0.1 M KCl HEG+0.1% NP40, and elution using 2 x 30 µl 10
mM Glutathione in 0.1 M KCl HEG+0.1% NP40 for 1 hr nutating at 4˚C. The eluted
material was analyzed using 7 % SDS-PAGE followed by transfer to nitrocellulose
membranes. After blocking with 5% milk in PBS+0.1% Tween 20, immunoblotting was
performed by standard methods and visualized by ECL (antibodies: CBP A-22, p300 N15; Santa Cruz Biotechnology).
Supplemental Figure 4.
ARC exerts little effect on NF-B and Sp1 activation with naked HIV LTR template.
Transcription reactions were performed by combining NF-B (5 nM) (lanes 2, 4, 6, 8)
and Sp1 (2 nM) (lanes 3, 4, 7, 8) with affinity-purified ARC (0.5 nM) (lanes 1-4) or
without (lanes 5-8) and naked HIV LTR template (0.5 nM) for 30 min. at 270C followed
by addition of general transcription factors and NTPs to initiate transcription. ARC was
purified from HeLa NE by SREBP-1a activation domain affinity-chromatography as
described above.
Supplemental Figure 5.
The same pattern of polypeptides is observed when ARC complex affinity-purified using
SREBP-1a (lane 1), VP16 (lane 2), and p65 (lane 3) activation domains was
immunoprecipitated with antibody against ARC105/TIG-1.
Methods: ARC was purified using GST-SREBP-1a, GST-VP16, and GST-p65A beads as
described under Supplemental Figure 3. Two hundred µl of Glutathione-eluted ARC
complex was incubated with 15 µl protein A/G beads pre-bound with 4 µl of anti-
ARC105/TIG-1 antiserum for 4 hrs nutating at 4oC. After extensive washing, specifically
bound material was analyzed by SDS-PAGE and silver staining.