Hello everyone, this week's article is "Dual-role transcription factors stabilize intermediate expression levels" published in Cell. The article was published by Yinqing Li's research group at the School of Pharmacy at Tsinghua University and Pilong Li's research group at the School of Life Sciences, Tsinghua University. Realize single-cell multiomics of epigeneomics, transcriptome, and signalome, and comprehensively characterize the changes in molecular, cell, and intercellular interactions at both temporal and spatial scales. Pilong Li's team focuses on the universal regulatory mechanism and law of phase separation, the biological significance of phase separation in the fields of autophagy, transcriptional regulation, epigenetics, and the development and application of phase separation-based technology.
summary
Precise control of gene expression levels is essential for the proper functioning of cellular functions. However, the mechanism by which gene expression levels, especially intermediate levels, are defined and strictly maintained remains unclear. Through a series of newly developed sequencing, imaging, and functional detection techniques, the authors have uncovered a class of transcription factors with dual effects of activation and inhibition, which are known as condensate-forming horizontally regulated dual-acting transcription factors. They achieve stable intermediate expression levels by decreasing high expression levels and increasing low expression levels. These dual-acting transcription factors selectively separate core transcription units by exerting activation and repressor functions directly through the condensate-forming domain. Associated clinical mutations impair the selective and dual-acting transcription factor activity of condensates and are associated with a variety of developmental disorders. These findings not only answer a fundamental question in the field of expression regulation, but also demonstrate the potential of level-modulating double-acting transcription factors as powerful effectors to achieve control of expression levels.
Image summary
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The regulatory mechanisms of gene expression in eukaryotes are critical to cell function. Aberrant fluctuations in gene expression levels have been associated with a variety of human diseases, but the precise regulatory mechanisms are not fully understood. The availability, binding accessibility, and residence time of transcription factors (TFs) are correlated with transcription intensity, but traditional models have failed to fully explain the robustness of gene regulation. The polyvalent dynamic interaction between TF and intrinsically disordered regions (IDRs) plays an important role in transcriptional regulation, but its effects on the genome have not been systematically studied.
Synthetic transcriptional activators are used as a platform to study gene regulation, but their activation effects are significantly different among different gene loci and affected by chromatin background. The authors have developed a set of tools through the application of which a special class of transcription factors have been discovered, which are able to activate or inhibit genes at a certain expression threshold through selective interactions of condensate-forming domains, enabling precise control of gene expression levels. This discovery not only reveals a novel mechanism of gene expression regulation, but also provides new tools and methods for bioengineering, which is helpful for understanding the role of chromatin-associated condensates in transcriptional regulation.
outcome
Methods for the detection of chromatin-binding condensates
The authors have developed a new method called Exploratory Sequencing of Chromatin-Binding Aggregates (ACC-seq) for genome-wide detection of chromatin-bound condensates. This technique generates a pattern of DNA accessibility that reflects the presence of condensates by altering the accessibility of the Tn5 transposase to specific DNA regions (Figures 1A and 1B). In combination with ATAC-seq, these patterns can be used to identify the occupancy of condensates. To verify the effectiveness of ACC-seq, the authors utilized a fusion protein of SOX2 and FUS IDR, which is capable of forming condensates and binding to specific DNA motifs. Experimental results showed that in the natural state, these condensates were accessible to Tn5 transposase, and after chemical fixation, the condensates became inaccessible, but after 1,6-hexanediol pretreatment, the condensates were destroyed and DNA accessibility was restored. In addition, this detected "deep U-shaped" pattern is specific and is observed only on condensate-bound DNA, not on bare DNA or condenser-bound DNA that is not associated with agglomerate formation. The robustness of ACC-seq has been validated under a variety of experimental conditions, demonstrating that it is a reliable method for the detection of chromatin-bound condensates.
The authors conducted an in-depth study of the effects of 1,6-hexanediol on non-DNA-binding proteins, especially the condensates formed by MED1 (IDR), by ACC-seq technology. It was found that although MED1 (IDR) condensates were sensitive to 1,6-hexanediol, they did not hinder DNA accessibility under fixed conditions and did not produce a self-release pattern. This may be because the fixatives used in ACC-seq require cross-linked DNA and direct binders within the condensate to produce the self-release pattern. Experiments have also found that DNA lacking the SOX2 binding motif does not exhibit a self-release pattern.
Applying ACC-seq on mouse embryonic stem cells (mESCs), the authors found that the accessibility intensity profile under different conditions was highly reproducible between technical and biological replicates. By grouping the genomic regions by the k-means algorithm, the authors found large clusters that exhibited a "deep U-shaped" hexaphic pattern, similar to the condensate occupancy observed in the FUS(IDR)-SOX2 condensate, i.e., the addition of 1,6-hexanediol could release the target DNA prior to fixation (Figures 1C and 1D). Therefore, these areas are referred to as self-released accessible areas. In addition, clusters with less variation under ACC-seq conditions were also observed, known as consistent accessible regions.
Figure 1. Methods for the detection of chromatin-binding condensates
Moderately regulated medium-level transcription within the region accessible to release
The authors conducted an in-depth study of the relationship between chromatin-binding condensates and transcriptional activity using ACC-seq (Figure 2A). The study found that the consistently accessible region had higher DNA accessibility, while the released accessible region had lower DNA accessibility (Figure 2B). Nonetheless, gene expression levels in the released accessible regions ranged between the consistently accessible and inaccessible regions, showing moderate levels of transcriptional activity (Figure 2C). This was further confirmed by the authors through RNA-seq and GRO-seq data analysis.
At the functional level, consistently accessible regions are primarily associated with housekeeping processes, while released accessible regions are closely associated with developmental and lineage-specific functions (Figure 2D). The authors ruled out the possibility that low expression levels themselves led to enrichment of developmental functions through expression level grouping and enrichment analysis, indicating that genes in the released accessible regions were specific during development.
In addition, the authors used single-cell RNA-seq data to explore the expression dynamics of genes in the released accessible region during stem cell differentiation. The results showed that these genes were expressed at low levels in stem cells but were rapidly upregulated during differentiation (Figure 2E), and that these genes were rich in transcription factors and key differentiation regulators (Figure 2F). This finding suggests that genes that have released accessible regions may be in a state of readiness to respond quickly during differentiation (Figure 2G).
Although the bivalent gene model is an important mechanism for regulating gene expression, the authors found that the released accessible regions were not enriched for bivalent markers, and the accessibility and expression levels of these regions differed significantly from those of the bivalent regions (Fig. 2H). These suggests that gene expression in the released reachable region may be controlled by other regulatory mechanisms, which may coexist with traditional bivalent models and participate in the fine regulation of gene expression.
Figure 2. Hexagonal release access to highly regulated transcription at the intermediate level of the region
Enrichment in different interactions of TFs in the hexagonal release accessible region
The authors propose a new view that the pattern of condensation formed within the region accessible to self-release may indicate the presence of specific transcription factors that regulate transcription at intermediate levels. By analyzing transcription factors in mouse embryonic stem cells, the authors identified a panel of transcription factors that are site-rich in regions accessible for release, particularly those containing large intrinsically disordered regions that promote dynamic intermolecular interactions (Figure 3A). These transcription factors, such as ETV5, FUBP1, PITX2, and ZIC3, not only form nuclear spots in living cells, but are also implicated in dynamic chromatin interactions.
Using live-cell imaging techniques, the authors observed that the plaques formed by transcription factors such as ETV5 had a spherical structure that was able to fuse and recover after photobleaching, suggesting that they were involved in dynamic intermolecular interactions (Figure 3B). In addition, these transcription factor spots occupy unique nuclear domains that differ from known regions of chromatin repression and activation (Figures 3C and 3D). Using an in vitro recombinant protein system, the authors found that ETV5 was able to interact with Pol II (CTD) (Figure 3E), but its intensity was weaker than that of MED1 (IDR) and other strong activators (Figure 3G).
To further explore the function of these transcription factors, the authors developed a dCas9-based seed system capable of nucleating agglomerate factors at specific genomic locations, and verified that transcription factors such as ETV5 are capable of forming nuclear domains that selectively restrict other factors through domain/probe analysis (Fig. 3H). These transcription factors not only have affinity for homotype probes, but are also selectively limited to heterotype probes such as MED1 (IDR).
Therefore, the authors suggest that these TFs be referred to as condensed double-acting TFs.
Figure 3. Enrichment in the hexagonal release of the different interactions of the double-acting TF in the accessible region
Coagulation into double-acting TF regulates stable intermediate transcription
The authors' research shows that specific condensate-forming double-acting transcription factors (TFs) have a unique ability to regulate gene expression, enabling simultaneous activation and inhibition at moderate levels. These transcription factors are localized at specific locations on the genome by dCas9-6xMCP technology, enabling down-regulation of expression levels of high-expressing genes and up-regulation of expression levels of silent genes to moderate levels (Figure 4A). This regulatory mechanism differs from traditional activators or inhibitors in that they exhibit a switching effect based on a threshold of expression levels, rather than relying on a co-inhibitory pathway (Figure 4B).
In addition, these double-acting TFs are functionally distinct from traditional weak activators or weak inhibitors. They have been shown to significantly activate reporter genes and, when lined, to highly expressed endogenous genes, do not inhibit these genes, but slightly enhance their expression (Figures 4C and 4D).
The authors also explored the potential function of double-acting TFs to form condensates, suggesting that condensates may serve as a noise buffering mechanism to reduce sensitivity to external changes by limiting concentration fluctuations within condensers (Fig. 4E). Through mathematical models and experimental verification, the authors found that when the double-acting TF concentration reached the condensate formation threshold, the transcriptional output quickly reached a stable level, and the transcription level remained relatively stable even when the TF concentration increased further. This shows higher stability compared to traditional weakly activator-driven reporter output. Taken together, these results position the dual-acting TF of agglutination formation as a unique class of expression level-regulating TFs.
Figure 4. Coagulation into double-acting TF regulates transcription at intermediate levels
The authors' work challenges the conventional wisdom of transcription factors (TFs) as weak regulators, especially for double-acting TFs capable of forming condensates. Experimentally, the authors found that these double-acting TFs, unlike traditional weak activators or weak inhibitors (Figure 5A), were able to significantly activate the reporter gene in the plasmid-based reporter system, rather than the expected weak activation or weak inhibitory effect (Figure 5B). These double-acting TFs exhibit unique regulatory properties, not only transcriptional regulation at moderate expression levels, but also higher stability.
In addition, the authors propose that condensate formation may provide a noise buffering mechanism for double-acting TFs, reducing their susceptibility to external changes (Figure 5C). Using mathematical models and experimental validation, the authors found that when the concentration of double-acting TFs reached a certain threshold and the reporter gene was activated, the expression level of the reporter gene remained relatively stable even when the concentration of TFs increased substantially, in contrast to the weakly activator-driven system (Figure 5D).
The authors' research reveals novel roles of dual-acting TFs in transcriptional regulation, which not only have the dual functions of activation and repression, but also may provide stability of transcriptional regulation through the formation of condensates, which make them play an important role in the regulation of gene expression in eukaryotes.
Figure 5. Condensation into double-acting TFs regulates stable transcription at the intermediate level
Dual functionality is encoded in the IDR domain
The authors' research provides an in-depth analysis of the effects of intrinsically disordered regions (IDRs) of transcription factors (TFs) on their dual transcriptional regulatory functions. By constructing double-acting TF mutants that are deletion of IDRs, the authors found that IDRs are essential for the activation and inhibition of these TFs. In particular, in FUBP1 and ZIC3, two TFs containing two IDR domains, mutants containing only the intact IDR domain were able to exhibit a dual effect, while mutants containing only a single IDR domain were significantly less functional. These results reveal the core role of IDRs in double-acting TFs, and suggest that the differences in their intermolecular interactions are the key to realizing the complex regulatory function of TFs.
Further, the authors explored the functional sequence units in the ETV5 IDR and found that it contains scattered activation and suppression units. Despite the deletion of the sequence fragment containing the activation unit, the ETV5 mutant retains the ability to activate the reporter gene and exhibits significant transcriptional stability. This suggests that the dual effect of double-acting TFs is not solely determined by the combination of these sequence units, but may involve more complex molecular mechanisms (Figs. 6B-6D).
Figure 6. Coding of double effects and differential interactions in the IDR domain
Through a series of experiments, the authors' research revealed the important role of sequence characteristics of transcription factors (TFs) in their condensate formation ability and dual regulatory functions. Through mutational analysis, the authors found that mutations that affect the ability of ETV5 condensate formation, such as the removal of the prion domain (PLD), attenuate its activation on silencing genes and repression on highly expressed genes (Figures 6B-6D). In addition, by attaching a maltose-binding protein (MBP) tag to ETV5, the authors found that the ability to form and double regulate condensates was diminished at low concentrations, but these functions were retained at high concentrations, further confirming the importance of condensate formation sequences for regulatory potential (Figure 6E).
The authors also explored the role of charge modes in double-acting TFs, and by introducing patterned charge blocks in FUBP1, ZIC3, PITX2, and ETV5, they found that this alteration affected the interaction and assignment of TFs in a sequence-context-dependent manner. These results suggest that the overall charge pattern and the IDR sequence unit work together to influence the function of double-acting TFs (Figure 6F).
Finally, the authors investigated double-acting TF variants associated with cardiac malformations and tumors, and found that small deletions or nonsense mutations in the IDR domain of these variants led to selective alterations in the nuclear domain and decreased regulatory function (Fig. 7A-C). These clinical variants exhibit varying degrees of reduced activity, the extent of which correlates with the severity of the associated developmental disorder.
In summary, the authors' study highlights the need for a comprehensive analysis of the IDR domain of double-acting TFs to understand their role in transcriptional regulation. These findings not only provide new insights into the molecular mechanisms of transcriptional regulation, but also provide possible new directions for the molecular pathology of related diseases.
Figure 7. Double-acting TF regulates stable developmental functions through the condensate forming domain
The condensation forms a dual-acting TF that stably regulates developmental functions
By analyzing the expression and localization of bifunctional transcription factors (TFs) in mouse embryonic stem cells, the authors revealed specific distribution patterns of these factors in the nucleus. Using ChIP-seq technology, the chromatin-binding properties of these TFs were investigated and they were found to be spatially separated from known transcriptional regulators and heterochromatin markers (Figs. 7D, 7E). In particular, the localization of ETV5, a bifunctional TF, on chromatin is significantly different from that of other factors, suggesting that it may play a unique role in regulating gene expression (Figs. 7F-7H). In addition, other bifunctional TFs with condensate formation ability also exhibited a similar distribution pattern, suggesting that such TFs may play an important role in cell development and gene regulation.
By regulating the levels of bifunctional transcription factors (TFs) in mouse embryonic stem cells, the authors revealed the important role of these factors in the maintenance and differentiation of stem cells. Through RNA sequencing technology, it was found that overexpression of these TFs caused only minor transcriptomic changes, in which the up-regulated genes were mainly related to stem cell maintenance, while the down-regulated genes were associated with developmental processes, suggesting that bifunctional TFs may help maintain the homeostatic state of stem cells. Conversely, knockdown of these TFs by shRNA technology resulted in a wide range of transcriptional changes associated with multiple differentiation processes, most of which manifested as upregulation of gene expression (Figure 7I).
Further single-cell RNA sequencing analysis showed that the downregulation of bifunctional TFs led to the deinhibition of their target genes, and the expression of these target genes was negatively correlated with the expression level of TFs. In particular, target genes with higher TF motif densities showed stronger correlations, and these genes were expressed at stable levels with low variability when TFs levels were normal, while variability increased when TFs levels were reduced. These findings suggest that bifunctional TFs have an important impact on the differentiation and development of stem cells by precisely regulating the expression of target genes, emphasizing their critical role in cell fate determination.
discuss
The authors' research reveals that precise regulation of gene expression levels is essential for normal cellular function, with transcription factors (TFs) playing a key role. Traditionally, TFs function primarily by activating or inhibiting gene expression, and some bifunctional TFs are able to respond to changes in the cellular environment by recruiting different cofactors. However, the mechanism by which stable control of gene expression can be achieved at moderate levels has been unclear. This study is the first to identify bifunctional TFs that can form condensates and regulate gene expression levels. These TFs differ from typical TFs, effector units, and bifunctional regulators in that they are able to switch between the two modes of action in a threshold-dependent manner to modulate moderate-level expression without relying on traditional co-repressors or activators. Most notably, these condensate-forming bifunctional TFs are able to maintain stable expression levels and are robust to a wide range of dose variations. This finding adds a powerful dimension to the regulatory mechanism (see Figure 7J). These TFs may operate in a combinatorial manner, modulating expression levels in the moderate range, providing direction for further investigation.
Author: DBK
Review: FY
Original link: https://www.sciencedirect.com/science/article/pii/S0092867424003143?via%3Dihub