Feedback Regulation of DYT1 by Interactions with Downstream bHLH Factors Promotes DYT1 Nuclear Localization and Anther Development

Abstract

Transcriptional regulation is one of the most important mechanisms controlling development and cellular functions in plants and animals. The Arabidopsis thaliana bHLH transcription factor (TF) DYSFUNCTIONL TAPETUM1 (DYT1) is required for normal male fertility and anther development and activates the expression of the bHLH010/bHLH089/bHLH091 genes. Here, we showed that DYT1 is localized to both the cytoplasm and nucleus at anther stage 5 but specifically to the nucleus at anther stage 6 and onward. The bHLH010/bHLH089/bHLH091 proteins have strong nuclear localization signals, interact with DYT1, and facilitate the nuclear localization of DYT1. We further found that the conserved C-terminal BIF domain of DYT1 is required for its dimerization, nuclear localization, transcriptional activation activity, and function in anther development. Interestingly, when the BIF domain of DYT1 was replaced with that of bHLH010, the DYT1(N)-bHLH010(BIF) chimeric protein shows nuclear-preferential localization at anther stage 5 but could not fully rescue the dyt1-3 phenotype, suggesting that the normal spatio-temporal subcellular localization of DYT1 is important for DYT1 function and/or that the BIF domains from different bHLH members might be functionally distinct. Our results support an important positive feedback regulatory mechanism whereby downstream TFs increase the function of an upstream TF by enhancing its nucleus localization through the BIF domain.

Figure 1.

The Subcellular Localization of DYT1 in Arabidopsis Anthers and Various DYT1 Dimers in Tobacco Leaves. (A) The subcellular localization of DYT1-EYFP in Arabidopsis tapetal cells at anther stage 4-7. Images show longitudinal sections of anthers. (B) and (C) The enlarged tapetal cells at early anther stage 5 (Sanders et al., 1999) (E-stage 5; [B]) and anther stage 6 (C). Green shows the EYFP signals from the DYT1-EYFP fusion proteins, magenta shows the DAPI-stained nuclei, and white shows the merged signals from YFP and DAPI. (D) The subcellular localization of DYT1 homodimer and different heterodimers. (E) The subcellular localization of the three DYT1-interaction proteins: bHLH010, bHLH089, and bHLH091. (F) The localization of DYT1-EYFP in the control or bHLH010/089/091 coexpression background. Yellow shows the signal from YFP, blue indicates the DAPI-stained nuclei, and white represents the merged signals from YFP and DAPI. Bar = 25 μm in (A), 3 μm in (B) and (C), and 20 μm in (D) to (F).

Figure 2.

Y2H and EMSA Analyses to Determine the Motif and Amino Acids Critical for DYT1 DNA Occupation and Dimerization. (A) Schematic diagram to show the length and domains in full-length DYT1 and DYT1 truncations. Green and blue boxes represent β-sheets and α-helices, respectively. (B) EMSA of binding of various truncated and mutant DYT1 forms to the G-box in the MS1 promoter region (ProMS1). DYT1 with mutations in the bHLH basic region (mDYT1^R40AR41A) was used as a negative control. (C) DNA sequence of probes used in the EMSA. ProMS1, positive probe; m-ProMS1, competition probe with mutation. The core G-box sequence is underlined, and the mutated nucleotide is indicated in red. (D) Relative reporter activity (LUC/REN) of DYT1 and DYT1^{△ BIF} on the MS1 promoter. Empty vector was used as the control. The black asterisks indicate a statistically significant difference from the control, and the red asterisk indicates a significant difference between full-length DYT1 and DYT1^△BIF (P < 0.01, t test). Error bars indicate sd of the biological replicates, n ≥ 3. (E) Interactions between truncated DYT1 and DYT1, bHLH010, bHLH089, bHLH091, and AMS. Blue indicates positive protein-protein interactions. (F) Interactions between DYT1, bHLH010, bHLH089, bHLH091, and AMS with DYT1 carrying point mutations. Expression of DYT1 and DYT1 mutant proteins in the Y2H system is shown in Supplemental Figure 6. (G) The predicted 3D structure of DYT1^BIF homodimer. Four amino acids (i.e., F139, L141, I143, and I144) are shown in red. (H) Schematic of full-length DYT1 showing the location of F139, L141, I143, and I144 in the BIF domain. The amino acid sequence of the black line-labeled part was shown above, and the red fonts indicated F139, L141, I143, and I144.

Figure 3.

Deletion and Mutations in the BIF Domain Disturbing the Protein Interactions Also Affect the Nuclear Localizations. (A) and (B) The subcellular localization of mDYT1^F139DL141D and mDYT1^I143DI144D alone or when coexpressed with bHLH010/089/091. Yellow shows the signal from YFP, blue indicates the DAPI-stained nucleus, and white represents the merged signals from YFP and DAPI. Bars = 20 μm. (C) to (F) The subcellular localization of DYT1-EYFP (Line-A1/dyt1-3), DYT1^△BIF-EYFP (Line-C1/WT), mDYT1^F139DL141D-EYFP (Line-D1/WT), and mDYT1^I143DI144D-EYFP (Line-E1/WT) in Arabidopsis tapetal cells at anther stage 6 and stage 7. Green indicates EYFP signals, and magenta shows the DAPI-stained cell nucleus. Bars = 3 μm.

Figure 4.

Phenotypic Analyses of Plant Growth, Flowers, and Anthers of the Wild-Type, dyt1-3, Line-A1/dyt1-3, Line-B1/dyt1-3, Line-C1/dyt1-3, Line-D1/dyt1-3, Line-E1/dyt1-3, and Line-B1/WT Plants. Bar = 3 cm for plants (in the first panel), 500 μm for flowers (in the second panel), and 20 μm for anthers (in the third panel).

Figure 5.

Real-Time PCR Analysis Showing the Relative Expression Levels of Detected DYT1-Downstream Genes in the Wild-Type, dyt1-3, and Different Transgenic Plant Inflorescences. DYT1 (A), MYB35 (B), MS1 (C), MYB99 (D), MYB103/MYB80 (E), and AMS (F). EF1α serves as the internal control. Error bars indicate sd of three technical repeats.

Figure 6.

The BIF Domain Is Involved in the Transcriptional Specificity of Different DYT1 Heterodimers. (A) and (B) Transcriptional activation of MS1 and MYB35 by dimers of DYT1 and bHLH proteins. (C) Activation of MYB35 by dimers of mutant DYT1 proteins and bHLH089. Transiently expressing blank vector and reporter serve as the control. The y axis shows relative reporter activity (LUC/REN). Error bars indicate sd of at least three biological repeats. (D) EMSA using DYT1 and bHLH089 alone or in combination. The first lane shows the interaction between DYT1 and the E-box sequence of ProMS1, serving as the positive control. Comp, competition probe; ++, double the amount of protein as +. (E) Proposed model in which the DYT1-bHLH089 heterodimer binds to the CATGTG E-box of MYB35 promoter and activates the transcription of MYB35. (F) DYT1 and bHLH089 regulate the expression of MYB35 through both feed-forward and positive feedback regulatory loops. DYT1 somehow activates the expression of bHLH089 and interacts with bHLH089 products and together they promote the expression of MYB35, thus forming a feed-forward loop. The downstream bHLH089 interacts with cytosolic DYT1 and increases DYT1 translocation to the nucleus; thus, bHLH089 positively regulates the activity of DYT1. Orange line indicates protein interaction, green arrows show transcriptional activation, and the magenta arrow implies a positive feedback regulation.

Figure 7.

The BIF Domain Contributes to the Conditional Regulation of DYT1 Nuclear Distribution. (A) The subcellular localization of DYT1^ΔBIF, bHLH010^ΔBIF_, and bHLH089^ΔBIF. (B) The subcellular localization of the BIF domain from DYT1 (DYT1^BIF), bHLH010 (bHLH010^BIF), and bHLH089 (bHLH089^BIF). (C) Schematic maps of DYT1, bHLH010, bHLH089, and the DYT1 chimeric proteins, which combined the N-terminal region of DYT1 (DYT1^N) with the BIF domain of bHLH010 (DYT1^N-bHLH010^BIF) or bHLH089 (DYT1^N-bHLH089^BIF). The gray box and the black box together show the bHLH domain of DYT1 (DYT1^bHLH), the light-purple box and light blue box together show bHLH010^bHLH, and the light-green box and green box together show bHLH089^bHLH. The light-gray box indicates DYT1^BIF, the dark-blue box indicates bHLH010^BIF, and the dark-green box indicates bHLH089^BIF. The first white box in each protein represents amino acids before the bHLH domains, and the second white box in each protein is for amino acids between the bHLH domain and BIF domain. (D) The subcellular localization of DYT1 and the two DYT1 chimeric proteins DYT1^N-bHLH010^BIF and DYT1^N-bHLH089^BIF. Bar = 20 μm. (E) and (F) The subcellular distribution of DYT1-EYFP and DYT1^N- bHLH010^BIF-EYFP in Arabidopsis tapetal cells at anther stage 5. Bar = 3 μm. (G) Statistical analysis of transgenic lines showed percentages of normal (blue) and abnormal (red) fertility in the dyt1-3, DYT1/dyt1-3, DYT1^N-bHLH010^BIF/dyt1-3, and DYT1^N-bHLH089^BIF/dyt1-3 transgenic plants. The blue boxes represent transgenic lines with normal fertility, and the red boxes indicate plants with reduced fertility. (H) Phenotypic analysis of anthers of the DYT1^N-bHLH010^BIF/dyt1-3 and DYT1^N-bHLH089^BIF/dyt1-3 transgenic lines. Bar = 20 μm.

Figure 8.

Proposed Working Model of DYT1-bHLH010/089/091 Transcriptional Regulatory Loops. (A) DYT1 expression is initiated at early anther stage 5, and DYT1 monomers and homodimers do not localize to the nucleus. Some unknown factors (in gray and black) could interact with DYT1 and help some DYT1 translocate into the nucleus, and DYT1 then directly or indirectly activates the expression of bHLH010/089/091. bHLH transcripts are translated into proteins, which then interact with DYT1 to form heterodimers. (B) The bHLH010/bHLH089/bHLH091 proteins interact with DYT1 and facilitate DYT1 nuclear localization, and DYT1-bHLH089 heterodimers bind to the E-box of MYB35 and promote MYB35 expression. Green indicates DYT1; yellow, bHLH010; magenta, bHLH089; and blue, bHLH091. Black lines and arrows show transcription; blue arrows with dotted lines show direct or indirect activation; the magenta arrow, the yellow arrow, and the blue arrow indicate the protein translocation from the cytoplasm to the nucleus; ovals in different colors all indicate BIF domains, and the two interacting bHLH motifs are shown as two coiled helices.