BHLH1a localizes in the nucleus, and its overexpression alters rhythmic diel gene expression. bHLH1a localizes in the nucleus, and its overexpression alters.

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bHLH1a localizes in the nucleus, and its overexpression alters rhythmic diel gene expression. bHLH1a localizes in the nucleus, and its overexpression alters rhythmic diel gene expression. (A) Confocal fluorescence microscopy of P. tricornutum cells expressing the bHLH1a-YFP protein under the control of the Lhcf2p promoter. (Scale bar: 15 µm.) (B) Immunoblot analysis of bHLH1a-HA protein at ZT7 and ZT12 in 16L:8D entrained WT and OE lines, using the anti-HA and anti-D2 (loading control) antibodies. (C) qRT-PCR analysis of bHLH1a, bHLH1b, and bHLH3 in WT and OE1 cultures sampled in L:D every 3 h over 24 h (mean ± SEM, n = 2). (D) qRT-PCR analysis of the same genes at ZT7 and ZT12 in WT and OE1, OE2, and OE3 lines (mean ± SEM, n = 3). Inset in the bHLH3 graph magnifies the ZT7 time point. (E) nCounter analysis of the bHLH1a, bHLH1b, and bHLH3 over 24 h of continuous dark (D:D) in WT and OE1 lines (mean ± SEM, n = 3). (F) qRT-PCR analysis of the same genes at DD7 and DD12 in WT and OE1, OE2, and OE3 lines (mean ± SEM, n = 3). bHLH1a-total includes endogenous bHLH1a and bHLH1a-HA transgene transcripts; for all of the other genes, we refer to endogenous transcripts. The expression value of each gene is relative to its maximum expression. *P < 0.05, **P < 0.01, ***P < 0.001, t test. Rossella Annunziata et al. PNAS doi:10.1073/pnas.1819660116 ©2019 by National Academy of Sciences