The Role of Sfp1 and NuA4 in Ribosome Regulation

Cells have developed complex systems to respond to changes in their environment. In budding yeast, one important player in this adaptation process is a protein called TORC1. This protein helps cells sense what's happening outside and make decisions about growth and energy use based on available nutrients and stress levels. One of the key tasks regulated by TORC1 is the creation of ribosomes, which are essential for making proteins.

#Ribosome Formation in Yeast

Yeast ribosomes are made up of 79 types of proteins, which are encoded by 139 different genes. The process of building ribosomes is energy-intensive and involves a lot of different regulatory elements, known as regulons. To avoid wasting energy, cells must carefully control the expression of ribosomal protein genes and their regulators. This requires various activating proteins that help manage when and how these genes are turned on.

Among the proteins involved, Sfp1 plays a critical role. Sfp1 is a kind of activator that helps manage the expression of Ribosomal Proteins and their regulators when nutrients fluctuate. Under normal growth conditions, TOR kinase activates Sfp1 by adding a phosphate group to it. This modification allows Sfp1 to move into the cell nucleus, where it teams up with other activating proteins to promote the expression of ribosomal genes. However, when nutrients run low or when cells are treated with rapamycin, a drug that blocks TORC1, Sfp1 moves to the cytoplasm, leading to reduced expression of ribosomal protein genes.

#Interaction Between Sfp1 and NuA4

While Sfp1 is known to interact with other proteins, it also works closely with a protein complex called NuA4. This complex contains several subunits that help modify the structure of DNA, making it easier for genes to be turned on. One way they do this is by adding chemical groups to histones, proteins that help package DNA in the cell.

NuA4 is particularly important because it contains the only essential histone acetyltransferase in yeast, known as Esa1. This protein is crucial for adding acetyl groups to certain histones, which is vital for regulating gene expression. Research has shown that NuA4 binds extensively to ribosomal protein gene promoters, and its association seems to be regulated by TORC1 signaling.

Given the roles of both Sfp1 and NuA4 in regulating ribosomal protein genes, it was important to understand how they might work together. To investigate this, researchers used various techniques in budding yeast to analyze their interactions.

#Experimental Evidence of Interaction

The researchers started by studying the physical association between Sfp1 and NuA4. They used a method called affinity purification to isolate Sfp1 and identify its interacting partners. They found that a crucial component of NuA4, known as Tra1, was present in the samples where Sfp1 was purified. This confirmed that Sfp1 interacts physically with the NuA4 complex.

To dig deeper, they also looked for specific NuA4 subunits like Eaf1 and Arp4 in the Sfp1 samples, which further confirmed the interaction. Even when the TORC1 pathway was inhibited, the Sfp1-NuA4 interaction persisted, indicating that their connection is robust and independent of nutrient conditions.

Next, they used a method called GST pulldown to assess if the interaction could happen directly between Sfp1 and NuA4. They demonstrated that when they mixed Sfp1 with NuA4, they could pull down the activity of NuA4, suggesting a direct interaction.

#Sfp1's Role in NuA4 Function

After confirming that Sfp1 and NuA4 interact, the researchers wanted to find out how this relationship affects gene expression. They looked into whether Sfp1 impacts the recruitment of the NuA4 complex to the promoters of ribosomal protein genes. To minimize the effects of Sfp1 deletion on growth, they employed a technique called anchor-away to rapidly remove Sfp1 from the nucleus.

Though Sfp1-depleted cells showed growth defects, the researchers found that NuA4 binding to ribosomal gene promoters remained unchanged. Even when they used cells that lacked Sfp1 altogether, they observed that NuA4 was still present at these promoters. This suggested that Sfp1 does not play a direct role in recruiting NuA4 to the gene promoters.

#Effects of Sfp1 Depletion on Histone Acetylation

While Sfp1 does not affect the binding of NuA4 to promoters, the researchers next wanted to see if Sfp1 plays a role in NuA4’s function. They measured the levels of acetylation on two key histones, H3 and H4, to find out if Sfp1 influences the activity of NuA4. They discovered that the depletion of Sfp1 led to a significant decrease in the acetylation of both histones at the promoters of ribosomal protein genes. This suggests that while NuA4 could still bind to these regions, its ability to modify histones was impaired in the absence of Sfp1.

Interestingly, they also noticed that the level of Htz1, a variant of histone H2A, increased at ribosomal gene promoters when Sfp1 was depleted. This observation suggests that the presence of Sfp1 promotes the acetylation of Htz1 as well, which may be crucial for regulating when genes are turned on.

#NuA4's Influence on Sfp1 Binding

Given that Sfp1 does not affect the binding of NuA4 but can modulate its function, the researchers investigated whether NuA4 might influence Sfp1's ability to bind to ribosomal protein gene promoters. Using the anchor-away technique again, they quickly depleted the Esa1 subunit of NuA4. The depletion of Esa1 reduced acetylation levels at ribosomal gene promoters and affected Sfp1 binding.

When they analyzed Sfp1 binding across different categories of ribosomal protein genes, they found that its recruitment was significantly reduced in the main categories of ribosomal protein genes except for a specific subset. This indicates that NuA4 is necessary for optimal Sfp1 recruitment to these genes.

#Acetylation of Sfp1

Aside from histones, NuA4 can also modify non-histone proteins, including Sfp1 itself. The researchers identified that Sfp1 gets acetylated at two specific lysine residues, K655 and K657. To further understand the importance of these acetylation sites, they created two mutants of Sfp1: one that cannot be acetylated and another that mimics acetylation.

When the researchers examined the acetylation levels of Sfp1, they found that mutations at K655 and K657 did not completely eliminate Sfp1 acetylation, indicating that other sites may also be targeted. Interestingly, they noticed that the acetyl-mimic mutant showed increased sensitivity to stress and nutrient deprivation, meaning that it might mislead the cell's signaling about nutrient availability.

#Impact of Acetylation on Gene Expression

Next, they wanted to see how the acetylation of Sfp1 influences its function as a transcriptional activator. They compared gene expression levels of certain ribosomal protein and ribosome biogenesis genes between the wild-type Sfp1 and the mutant strains. The mutant strain that mimics acetylation showed a significant increase in the expression of ribosome biogenesis genes, while the expression of ribosomal protein genes remained steady.

Wide-scale RNA sequencing confirmed that specific clusters of genes related to ribosome biogenesis were upregulated in the acetyl-mimic mutant. This indicates that the NuA4-dependent acetylation of Sfp1 significantly affects its ability to regulate gene expression.

#Effects of Carbon Sources on Gene Expression

The researchers found that the type of carbon source available to yeast cells impacted the expression of ribosomal protein and ribosome biogenesis genes. They conducted experiments in which yeast were grown in different carbon sources, noting that when cells were shifted from a rich carbon source to a less favorable one, the expression of ribosomal protein genes decreased.

When examining the response of Sfp1 mutants to these shifts, they observed that the acetyl-mimic mutant had a surprising decline in the expression of ribosomal biogenesis genes under nutrient-limited conditions. This suggests that Sfp1's regulatory mechanisms might differ based on the availability of nutrients in the environment.

#Glucose Pulse Experiments

To better understand how cells sense changes in nutrient availability, the researchers designed an experiment where they shifted cells from a poor carbon source to a rich one. They wanted to see how quickly and effectively cells could adjust their transcription in response to the nutrient shift.

Upon introducing glucose after a period in raffinose, they found that both wild-type and mutant strains showed an increase in the expression of ribosomal protein and ribosome biogenesis genes. The results indicated different regulatory mechanisms for ribosomal protein genes compared to ribosome biogenesis genes in response to changing nutrient conditions.

#Conclusion

In summary, this research sheds light on how Sfp1 interacts with the NuA4 complex to regulate ribosomal protein and ribosome biogenesis gene expression. Sfp1's presence is essential for the local histone modification necessary for proper gene expression, and while it does not influence the binding of NuA4, it is critical for NuA4’s acetyltransferase activity.

Moreover, the study highlights the importance of Sfp1 acetylation in eliciting proper responses to nutrient changes, with different regulatory mechanisms at play for ribosomal protein and ribosome biogenesis genes. This work reveals a complex relationship between transcription factors and coactivators that governs how cells adapt to their environments and ensure proper growth and metabolism. The findings have implications for broader discussions about gene regulation and cellular responses, showing that how proteins interact and are modified can significantly influence cellular functions.