Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction

Abstract

BackgroundThe molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized.ResultsTo identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation. We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription.ConclusionsOur results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3853-9) contains supplementary material, which is available to authorized users.

A prion-like protein regulator of seed germination undergoes hydration-dependent phase separation

“…An entry vector containing the YFP gene was donated by Dr. Zhiyong Wang (Carnegie Institution for Science, USA) and another one, G18395, containing FLOE1's coding sequence was obtained from ABRC. The two genes were then transferred from the entry vector into the binary vector pB7HFC3_0 (Bossi et al, 2017) using Gateway cloning (Life Technologies), to create the vectors p35S:YFP-FLAG and p35S:FLOE1-FLAG. Transgenes for tobacco experiments: Arabidopsis genes: The coding sequences of FLOE1's isoforms, FLOE1.1 and FLOE1.2, were amplified by Phusion (Thermo Fisher Scientific) PCR from the entry vector G18395 using priFLOE1.1-FWD/REV and priFLOE1.2-FWD/REV (Table S3) and then BP recombined into pDONR221 (Thermo Fisher Scientific) to generate pDONR221-FLOE1.1 and pDONR221-FLOE1.2.…”

Toll/interleukin-1 Receptor Domain-Only Protein of Arabidopsis Thaliana AtTX14 2IR, Produced By Alternative Splicing, Can Induce Defense Responses

“…Sequence alignment of the C-terminal part of AtTX14 Full and related proteins, a transcriptional regulator CHIQ1 and ten CHIQLs, revealed that the C-terminal half of AtTX14 Full contains a truncated DUF641 domain (Bossi et al 2017) (Fig. S2).…”

LPCAT1 controls phosphate homeostasis in a zinc-dependent manner

“…We next assessed the effect of these motif sequence changes on the activity of the LPCAT1 promoter using a quantitative in planta transactivation assay ( Bossi et al, 2017 ). In this assay, we co-transformed tobacco leaves with an effector construct (35S::bZIP23 or 35S::YFP) with a reporter construct, containing either the LPCAT1 Col-0 native promoter (with GTGTC GA A motif), the LPCAT1 Col-0 mutated promoter (with GTGTC AC A motif), or the promoter of the zinc transporter ZIP4 promoter (as positive control) fused to a β-glucuronidase ( GUS )-encoding reporter gene ( Figure 3C ).…”

“…35S promoter. The 35S::C-YFP construct was provided by Dr. Seung Y. Rhee ( Bossi et al, 2017 ). Each construct was transformed into Agrobacterium.…”

“…Three days after infiltration, leaves were collected, and the infiltrated areas in each leaf were excised and pooled into one sample. The GUS extraction and the GUS enzymatic activity measurements was performed as described by Bossi et al, 2017 . The relative GUS enzymatic activity was determined by comparing the effect of bZIP23 TF and C-YFP protein on each promoter.…”