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Product Upgrade | TF-centered Y1H (Reverse Yeast One-Hybrid) Service Newly Upgraded
Release time:
2025-01-14
Reverse Yeast One-Hybrid (TF-centered Y1H) is a molecular biology technique used to screen DNA sequences that interact with a specific protein. Unlike the traditional Yeast One-Hybrid (Y1H), which uses known DNA sequences as baits, the TF-centered Y1H uses a known protein or protein fragment as the bait, while the prey library consists of DNA sequences instead of proteins.
Thus, the standard Yeast One-Hybrid is primarily used to identify proteins, such as transcription factors, that bind to specific DNA sequences and to investigate the interaction between transcription factors and DNA cis-elements. In contrast, TF-centered Y1H is used to identify new DNA response elements or enhancers.
Specifically, TF-centered Y1H works by constructing a random motif library, where each motif consists of seven nucleotides. A gene encoding a transcription factor (usually on a pGADT7 vector) serves as the bait, and the library of random motifs is screened to identify those interacting with the bait transcription factor. The identified motifs are then used to infer potential target genes that may be regulated by the transcription factor.
TF-centered Y1H technology enables accurate, fast, and efficient identification of elements recognized by transcription factors, providing broad applications in the study of protein-DNA interactions.
Case Analysis
Case 1
Next, we will explore the application of reverse yeast one-hybrid through a paper (Top-tier journal, IF=6.5).
Research indicates that TaCAMTA4 may play a role in the negative regulation of wheat resistance to leaf rust fungus. Using calcium-binding transcriptional activator 4 (TaCAMTA4) as a bait, reverse yeast one-hybrid (TF-centered-Y1H) screening was performed. Among the 48 positive yeast clones obtained, 29 were sequenced and confirmed to interact. These clones carried plasmids containing four different DNA motifs, which were further confirmed by Y1H as the cis-elements recognized by TaCAMTA4.
The promoter sequences within the -2000 to -1 bp range upstream of the translation initiation site (ATG) in the wheat genome were analyzed. It was found that 43, 163, 188, and 355 genes contained motifs 1, 2, 3, and 4, respectively. The expression patterns of these genes were regulated positively or negatively by TaCAMTA4, suggesting that TaCAMTA4 may directly or indirectly influence the synthesis of these transcripts.
Ten genes related to plant immunity were selected, which exhibited different expression patterns in wheat when interacting with the Pt affinity and non-affinity groups. Based on the positive correlation between the transcription levels of TaCAMTA4 and TaCAT1 (catalase 1), along with gene silencing results, it is inferred that TaCAT1 is a target gene directly activated by TaCAMTA4.
Case 2
(Top-tier journal, IF=11.4)
In Asian white birch, BplMYB46 regulates the expression of target genes by binding to motifs such as MYBCORE and AC-box, thereby enhancing plant stress tolerance and promoting the biosynthesis of secondary cell walls.
Reverse yeast one-hybrid (TF-centered-Y1H) screening was performed using BplMYB46 as the bait. Five positive clones were identified, and among them, three showed high binding affinity to BplMYB46. The clones were sequenced, and the inserted DNA sequences were analyzed using the Plant DNA Cis-Regulatory Elements Database (PLACE) and the Plant Cis-Acting Regulatory Elements Database (PLANTCARE). Sequence 1 was found to contain the E-box “CAAATG.”Other sequences did not match any known motifs, representing previously unidentified new candidate DNA motifs for MYB binding.
By performing yeast one-hybrid, the new core motif sequences were identified. It was found that the third DNA base (T) on the left boundary and the fifth DNA base (C) on the right boundary of motif 2 were critical for BplMYB46 binding. Thus, its core sequence was identified as “TGTCGC,” and it was named the TC-box. Similarly, the third DNA base (C) on the right boundary of motif 3 was essential for BplMYB46 binding. The core sequence of motif 3 was identified as “AGTAGTTC,” and it was named the GT-box.
From Case 2, it was found that by using PLACE and PlantCARE to analyze the inserted DNA sequences of the positive clones, the identified motifs could be classified into conserved motifs (those reported in previous studies) and unknown motifs. The conserved motifs were annotated based on past research, laying the foundation for future studies on interactions between transcription factors, kinases, disease resistance genes, and others. For the unknown motifs, their core sequences were validated through yeast one-hybrid and subsequently named. These motifs were then analyzed in relation to the transcription factors and genes containing these unknown motifs, thus gaining insights into the regulatory networks and mechanisms of the transcription factor.
Pronetbio Supports Transcription Factor Research
Based on common issues encountered during transcription factor research, Pronetbio has upgraded the reverse yeast one-hybrid (TF-centered-Y1H) technology to assist in transcription factor studies.
Upgrade Details
Using the New PLACE (Plant DNA Cis-acting Regulatory Element Database, upgraded version), all motifs are derived from previously published reports. It only covers vascular plants. In addition to the originally reported motifs, variations of these motifs in other genes or later-reported plant species have also been compiled.
The PLACE database also includes a brief description of each motif and relevant literature with PubMed ID numbers. The sequences obtained from the reverse yeast one-hybrid screening are analyzed, and previously published motif sequences (referred to as conserved motifs) are identified based on the database analysis, while the remaining sequences are classified as unknown motifs.
Based on the research results of conserved motifs in the New PLACE database, potential target genes are identified and categorized according to research areas such as transcription factors, kinases, and disease resistance genes, helping to predict the interaction partners of the bait transcription factors and guiding transcription factor research.
For unknown motif sequences, the boundaries of the motif are determined step by step from small to large, identifying the core region, which helps in researching new transcription factor interaction pathways.
Technical Advantages
1.Rapid identification of various cis-acting elements bound by a specific transcription factor.
2.When identifying various elements bound by transcription factors, the false positive rate is much lower than that of Gene-centered Y1H.
3.After the upgrade, more meaningful small-scale data are delivered, making it easier to choose appropriate validation and research targets for subsequent studies.
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