Arabidopsis TCP20 links regulation of growth and cell division control pathways -- Li et al. 102 (36): 12978 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Li et al. 10.1073/pnas.0504039102.

Supporting Information

Files in this Data Supplement:

Supporting Text
Supporting Table 1
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9

Supporting Figure 7

Fig. 7.
The GCCCR element is occupied in vivo in cells competent to proliferate. DNA was isolated from Arabidopsis suspension cells or tissues treated with dimethyl sulfate (DMS) (lanes 2, 3, 5, 7, and 9) or isolated from untreated material and exposed to DMS in vitro after purification (lanes 1, 4, 6, 8). Sequences protected from DMS modification were amplified by ligation-mediated PCR (1), and visualized by autoradiography. The sequences protected in vivo are highlighted by thick black bars to the left of the autoradiograph and the corresponding bases are boxed in the DNA sequence of the CYCB1;1 promoter between –143 and –225. Lanes 1 and 2: in vitro or in vivo treated DNA isolated from suspension cells, respectively; lane 3: in vivo treated DNA isolated from suspension cells grown without auxin for 12 h; lanes 4 and 5: in vitro or in vivo treated DNA isolated from young leaves, respectively; lanes 6 and 7: in vitro or in vivo treated DNA isolated from suspension cells, respectively; lanes 8 and 9: in vitro or in vivo treated DNA isolated from senescing leaves, respectively.

1. Garrity, P. A. & Wold, B. J. (1992) Proc. Natl. Acad. Sci. USA 89, 1021-1025.

Supporting Figure 8

Fig. 8.
Functional analysis of Arabidopsis genes with four copies of the GCCCR element. The frequency of each functional term in the list of genes containing GCCCR elements was compared to the frequency in the whole genome, and a P value of overrepresentation was calculated by using the hypergeometric distribution (R.A.G., L. Lejay, and G. M. Coruzzi, unpublished data).

Supporting Figure 9

Fig. 9.
p33^TCP20 binds to GCCCR elements in the CYCB1;1 promoter. Radiolabeled oligonucleotides with wild-type GCCCR sequences were used for in vitro binding reactions with purified recombinant p33^TCP20. Lane 1, GCCCR probe alone; lanes 2-4, addition of increasing amounts of p33^TCP20. Addition of increasing amounts of recombinant p33^TCP20 leads to the appearance of two bands with reduced mobility. Formation of the lower-mobility band is concentration-dependent, which likely reflects the ability of TCP proteins to dimerize (1). Lanes 5 and 6, competition with increasing amounts of un-labeled oligonucleotide with wild-type GCCCR motif. Radiolabeled probe is marked with an asterisk, bands with lower mobility than the free label due to binding of p33^TCP20 to probe are marked with arrows.

1. Kosugi, S. & Ohashi, Y. (2002) Plant J. 30, 337-348.

Table 1. Relative root growth rates of Col-0 and CYCB1;1::CYCB1;1-transformed lines

	Plant line
	Col-0	11D	12F	17E	26A
Relative root growth rate	100	120	122	119	124
n	76	43	77	48	58

Root growth rates were assessed with seedlings grown on vertical plates.

Supporting Text
Oligonucleotides Used for Constructs or EMSA.

GCCCR-1: 5´-CAGGCCCGTAACAAGCCCACGCAGGCCCGTACACAGCCCGCGTAGGCCCAT-3´; mutGCCCR (5´-CAGTAATGTAACAATAATACGCAGTAATGTACACATAATGCGTAGTAATAT-3´). MSA: 5´-CCGTTGGGGTTCAATTTATTTTCCCAACGGTCCCTTCATAATTTATTTATACCGTTGGG-3´.

Oligonucleotides Used for Priming Reverse Transcription Reactions and Quantitative PCR.

Priming: b-ATPase: 5´-AAACCTGTAAGTCCAACACG-3´, cyclin-GUS (CGU): 5´-GCTCCATCACTTCCTGATTA-3´, and NtCYM: 5´-GCAACTTTGCACAACCCAG-3´.

PCR: b-ATPase: 5´-GGTTCTCCTATCACCGTTCCTG-3´, and 5´-AGCACAGTTTTCCCCACACC-3´; CGU: 5´-GTGGAGGATAATCTCAAAAAACCTG-3´, and 5´-ACAGTTTTCGCGATCCAGAC-3´; NtCYM: 5´-TTCACAGCCTGAGATAACTGCA-3´, and 5´-CACACAAAGTCATTCACC TCAG-3´.

Oligonucleotides Used for in Vivo Footprinting.

Set 1: 1a739TP1: 5´-TCTCTAAACCGTACTAATTCTAATG-3´; 512ATP2new: 5´-CTAATTCTAATGCAAACCGAACCAAAATC-3´; 512ATP3new 5´-CTAATTCTAATGCAAACCGAACCAAAATCAATTTTCATTTC-3´. Set 4: 512BP1new: 5´-AGATCTTTCTCTTACCGTGGATT-3´; 1a1241BP2: 5´-TCTTACCCGTGGATTGTTGAGGAACAAT-3´; 1234BP3: 5´-CGTGGATTGTTGAGGAACAATCGAACGAGAAG-3´. Adaptor and linkers: Long linker: 5´-CACCCGGGAGATCTGAATTC-3´; short linker: 5´-GAATTCAGATC-3´.

Oligonucleotides Used for Modification of Ribosomal Protein (RP) Gene Promoters.

RPS27aB: (only upper strand shown for internal primers mut1-2,4; mutated bases are underlined)

wt-5': 5´-ATGGGCCATTAAGCAGCGAA-3´, mut-5': 5'ATATTTCATTAAGCAGCGAA-3´, wt/mut-3': 5´-CAGCTAGCACACTGATTCTCACCAT-3´, mut-1: 5´-CTTTTAACTACATAAAATATTTTTAATACC-3´, mut-2: 5´-TTACAAATATTTCTAATTATGTATCAATAATTACACTAGT TA-3´; mut-4: 5´-GTATCAATAATTACACTAGTTATGTATTTATAATATTCTTAGTTATAC-3´.

RPL24B: (only upper strand shown for internal primers mut1-2; mutated bases are underlined)

wt/mut-5': 5´-GGTCCATTGACAAGTTTCCTCTA-3´, wt/mut-3': 5´-CTGAAACGAGCTAGCTCCGTCC-3´, mut-1: 5'-CAAAAATATATCACATAATAAAATTATCACACTATTTGTGATCACACTATTTAG-3´, mut-2: 5´-CTATTTGTGATCACACTATTTAGTCACATTAAAGTGTCACACAGAATAATATATATAG-3´.^.

Oligonucleotides Used for Chromatin Immunoprecipitation.

Negative controls:

chr1-neg-1: 5´-ATCACTCGACACAGCATTCG-3´, chr1-neg-2: 5´-ACGAAAGAAGCCGAATCAGA-3´; chr4-neg-1: 5´-TTGGATCTTCATCCGGAAAG-3´, chr4-neg-2: 5´-GGAGACTGCACCCAGGTA TC-3´;

Gene-specific sets:

At4g37490 (CycB1;1): CycB1;1-1: 5´-CCATGTAAACCAAACAGAGATCA-3´, CycB1;1-2: 5´-TAAATGAACCCCAACGGTCT-3´; At2g29570 (PCNA2): PCNA2-1: 5´-TCCTTCCTCAATGATTTCTGG-3´, PCNA2-2: 5´-GCCTGTGTGTGACGATGAAT-3´; At3g53020 (RPL24B): RP24-1: 5´-GGTCCATTGACAAGTTTCCTCTA-3´, RP24-2: 5'CCCAATGCCATTTTCGTCTA-3´; At1g23290 (RPS15aD): RP15-1: 5´-GGGCGAGACTTTAGGGTTTA-3´, RP15-2: 5´-CAGCTAGGGTTTTCGGAGA-3´; At3G46040 (RPS27aB): RP27-1: 5'-TGAACGGCTAAGCATCTCTC-3´, RP27-2: 5´-TTCGCTTTTATCGCTTTCAC-3´; At5g03545 (At4): At4-1: 5´-GGATGGCCCCAAACACAAG-3´, At4-2: 5´-TAAACCGGAAACAAAGTAAACACG-3´.