Volume 1, Issue 1, Pages 58-67 (January 2008) The Clock Protein CCA1 and the bZIP Transcription Factor HY5 Physically Interact to Regulate Gene Expression in Arabidopsis  Andronis Christos , Barak Simon , Knowles Stephen M. , Sugano Shoji , Tobin Elaine M.   Molecular Plant  Volume 1, Issue 1, Pages 58-67 (January 2008) DOI: 10.1093/mp/ssm005 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Recombinant HY5 Binds to the A2 Fragment of the Lhcb1*3 Promoter and its Binding is Dependent on the G-Box Element. (A) Schematic diagram of the –221 to +7 region of the Lhcb1*1 promoter and the –160 to –73 region (A2 fragment) of the Lhcb1*3 promoter. Numbers indicate distance from transcription start. ACGT core sequences (G-box elements) are highlighted in blue and CCA1-binding sites (CBS) are highlighted in green. A site that deviates from the consensus sequence by one residue is labeled in brackets. The MutG fragment contains mutations in the G-box element, whereas the m1 fragment contains mutations in and adjacent to the CBSs. Base substitutions are shown as lower-case letters. Dashes represent bases identical to those of the A2 fragment. (B) EMSA of recombinant HY5-binding to the A2 (lanes 1–3) or the MutG (lanes 4–6) fragment of the Lhcb1*3 promoter. HY5 protein was produced in rabbit reticulocyte lysate (RRL); lanes 1, 4: free probe; lanes 2, 5: RRL minus HY5; lanes 3, 6: HY5. Molecular Plant 2008 1, 58-67DOI: (10.1093/mp/ssm005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Whole-Plant Nuclear Extracts Contain a HY5-Binding Activity Specific to the G-box Element of the Lhcb1*3 Promoter. Nuclear extracts were incubated with either the A2 (lanes 1–4) or MutG probe (lanes 5–7) in an EMSA. Lanes 1, 5: free probe; lanes 2, 6: wild-type extract; lanes 3, 7: HY5-OX extract; lane 4: HY5-OX extract plus HY5 antibody. Arrow indicates position of HY5-OX-binding activity. Broken arrow indicates supershifted HY5-OX-binding activity. Asterisks indicate unspecific bands. Molecular Plant 2008 1, 58-67DOI: (10.1093/mp/ssm005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 HY5 and CCA1 Interact In Vitro and in Yeast. (A) Autoradiograph of SDS–PAGE analysis showing in vitro interaction between 35S-labeled HY5 or GBF4 and GST–CCA1. Lanes 1 and 4 represent 10% of the total amount of 35S-labeled protein used in the binding reaction. Lanes 2 and 5 show the amount of protein retained by GST, whereas lanes 3 and 6 show the amount of HY5 or GBF4 protein retained by GST–CCA1, respectively. (B) Interaction of CCA1 and HY5 in yeast. Each panel shows triplicate patches of yeast expressing CCA1-BD (left column) or SNF1-BD (negative control; right column), transformed with HY5-AD (top row) or AD alone (bottom row). Blue coloration results from accumulated β-galactosidase activity after incubation with the chromogenic substrate, X-Gal. AD, GAL4 transcription-activation domain; BD, GAL4 DNA-binding domain; n.a., not applicable. Molecular Plant 2008 1, 58-67DOI: (10.1093/mp/ssm005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 CCA1 and CKB3 Alter the DNA-Binding Activity of HY5. (A) Recombinant CCA1 and HY5 alter each other's binding to the Lhcb1*3 promoter. EMSA using the A2 (lanes 1–4), MutG (lanes 5–8) and m1 (lanes 9–11) fragments of the Lhcb1*3 promoter as probes. Lanes 1, 5: free probe; lanes 2, 6, 10: 10 ng HY5; lanes 3, 7, 9: 0.5 ng CCA1; lanes 4, 8, 11: 0.5 ng CCA1 plus 10 ng HY5. The CCA1–HY5 DNA-binding complex is indicated by an asterisk. CCA1 and HY5 were expressed as GST-fusion proteins in E. coli. The GST tag was removed after protein purification (see Materials and Methods). (B) CK2 β3 (CKB3) subunit enhances the DNA-binding activity of HY5 on the Lhcb1*3 promoter. EMSA using the A2 fragment as a probe. Lane 1: free probe; lane 2: 0.5 ng CCA1; lanes 3, 5: 10 ng HY5; lane 4: 10 ng HY5 plus 10 ng CKB3; Lane 6: 10 ng HY5 plus 50 ng BSA. Molecular Plant 2008 1, 58-67DOI: (10.1093/mp/ssm005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 CCA1 Increases the Binding of HY5 to the Lhcb1*1 Promoter in a Yeast One-Hybrid Assay. (A) and (B) Quantification of β-galactosidase activity in a yeast strain harboring the LacZ reporter gene fused to the Lhcb1*1 promoter (–221 to +7 relative to transcription start) and co-expressing GAL4 transcription–activation domain (AD) fusion proteins. The results from three independent experiments were averaged and are shown ±SEM. CCA1-AD, CCA1 fused to the AD; HY5-AD, HY5 fused to the AD; CCA1, CCA1 lacking the AD. Molecular Plant 2008 1, 58-67DOI: (10.1093/mp/ssm005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Lhcb1*1 but not CAT3 Circadian mRNA Oscillations Show a Shorter Period in hy5 Plants. Wild-type (WT) and hy5 plants were grown for 12 d in a 12 h light:12 h dark photoperiod before being transferred to continuous light. Tissue was collected every 4 h during the interval between 40 and 108 h in continuous light (LL) and the resulting RNA was subjected to northern analysis. (A) Lhcb1*1 transcript levels in wild-type and hy5 plants. A representative autoradiogram along with quantification (based on UBQ10 transcript levels) from one of three independent experiments is shown. (B) CAT3 transcript levels in wild-type and hy5 plants. A representative autoradiogram along with quantification (based on UBQ10 transcript levels) from one of two independent experiments is shown. Because the overall CAT3 expression level in hy5 was substantially higher than in wild-type plants, relative RNA levels were normalized to the highest value in each line. Wild-type: blue squares; hy5: red triangles. Alternating white and gray bars within the chart represent subjective light:dark photoperiods, respectively. (C) Northern analysis of UBQ10 transcript levels in wild-type and hy5 plants, used for the quantification of (A) and (B). Molecular Plant 2008 1, 58-67DOI: (10.1093/mp/ssm005) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions