Abstract
The field of Histone Acetyltransferases (HATs) has witnessed substantial progress over the last couple of decades, resulting in successful isolation of a variety of HATs from different organisms. These discoveries have led to the belief that HATs mostly have multiple subunits that perform diverse roles on their end. However, one HAT complex that is of prominence is the SAGA complex, which has been characterized as a multisubunit complex. Its role in chromatin modification is noteworthy and has been acknowledged in retrospect. The purpose of this document is to report on the findings from the lab experiments conducted on the subunit Spt7 of the SAGA coactivator complex. The lab involved largescale preparation of the yeast extracts, SDS-PAGE and Western Transfer, and tandem affinity purification (TAP) of the Spt7 subunit of SAGA complex. Tandem affinity purification (TAP) helped in purification to near homogeneity, which is a key requirement in identifying the components present in the various biological complexes.
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Introduction
In the year 1979, scientists were able to isolate the protein fractions with HAT activity from the brine shrimp larvae; almost a decade later, Hat1 and Gcn5 were isolated from the Saccharomyces cerevisiae, which was followed by the eventual isolation of the first multisubunit nuclear HAT known today as SAGA [1]. It wasn’t until 2004 that the layout of the SAGA complex was explored and highlighted by the Winston and Shultz Laboratories through electron microscopy structures [1]. SAGA can basically be characterized as a multiprotein complex possessing the histone activity of acetyltransferase or HAT [1]. It is primarily important because of its role in chromatin modification. It can be regarded as a coactivator complex encompassing the HAT activity of Gen5p, which performs key roles in gene activation through multistep pathways. It modifies chromatin by chemically altering the histones, which are the protein constituents of chromatin [2]. Since the process is often carried out by the involvement of several multisubunit complexes, the SAGA complex is often observed to be involved in the modification process.
The SAGA complex is unique and important because of its prominent attributes. To begin with, it is a highly conserved complex from yeast to human beings [3]. Secondly, it encompasses over 20 subunits with a size of 2 MDa, which is quite adequate. It also has a set of 15 putative chromatin-interacting domains, while the presence of several functional modules makes SAGA a highly important complex in this regard. Each of these chromatin-interacting domains hold their own respective significance. For instance, even though there are two proteins in the SAGA complex that have bromodomains, including Spt7 and Gcn5, the binding of the acetylated nucleosomes in vitro is exclusively carried out by the bromodomain of Gcn5 [3]. Furthermore, SAGA also offers a paradigm that could be used for numerous multi-subunit complexes that could modify histones. Although the presence of the Gcn5 subunit rendered it to be characterized as a histone acetyltransferase, SAGA is also known today for another important activity called histone deubiqutinase. It is further known for initiating interactions across various transcriptional activators along with the overall transcription mechanism in general. Not every microorganism has been studied in perspective of the SAGA complex as of yet. It has been studied in S. cerevisiae, Drosphila, and S. pombe, which reveals that this complex is also playing a part in the transcription elongation process, maintenance of the telomere, and protein stability regulation; nonetheless, these contentions are yet to be confirmed through multiple scientific studies. However, in order to gain a deeper comprehension of the functions of these distinct domains, they need to be studied within their respective protein complexes. It is through direct assistance of these modules and subunits that the SAGA complex is able to accomplish its functional roles [3]. Two of these modules hold the ability to perform enzymatic activities, whereas the others play a pivotal role in the mediation of SAGA interactions with proteins involved in control transcription. It is therefore widely argued that these modules work in close coordination in order to initiate transcription and the subsequent transition leading to the elongation of RNA [2]. This teamwork is necessary for the purpose of accurate enzymatic activity. This lab experiment involved a series of steps, after which, the findings on the subunit Spt7 of the SAGA coactivator complex will be reported. The lab involved largescale preparation of the yeast extracts, SDS-PAGE and Western Transfer, and tandem affinity purification (TAP) of the Spt7 subunit of SAGA complex. It also aimed at measuring the HAT activity associated with the chosen SAGA complex. For the purpose of activity detection, the reactions were analyzed through the Western blot with the help of an antibody that held the capability of recognizing histone H3 acetylated at lysine 18. As such, the HAT reaction master mix was prepared and listed in the order given in the lab instructions manual. The Tandem affinity purification (TAP) helped in purification to near homogeneity, which is a key requirement in identifying the components present in the various biological complexes.
Objectives
The current lab experiment comprised of multiple objectives that could only be achieved over a period of several days. To be specific, the objectives of this research study were as follows:
⦁ To detect the HAT activity per unit mass of the SAGA complex.
⦁ To measure the HAT activity specific to the SAGA complex by analyzing reactions y Western blot by virtue of the given antibody.
⦁ Largescale preparation of yeast extracts overnight in a liquid culture of YPD.
⦁ SDS-PAGE and Western Transfer.
Materials and Methods
Histone acetyltransferase assay (Part 1)
We prepared a HAT reaction master mix for 6×30 µL HAT reaction in a 1.5 mL tube. The master mix contained 1X HAT buffer 0.5 mM Acetyl-CoA, 20 µM histone H3 (1-20) peptide, 1 mM AEBSF, and 1 mM DTT, while water constituted the remaining volume of the master mix. To be specific, the master mix comprised of the following:
⦁ 5X HAT buffer
⦁ 250 mM Tris-HCl, pH 8.0
⦁ 25% glycerol
⦁ 0.5 mMEDTA
⦁ 250 mM KCl
⦁ 50 mM NaButyrate
⦁ 1 mM Acetyl-CoA
⦁ 300 µM H3 (aa 1-20) peptide
⦁ 50 mM AEBSF
⦁ 50 mM DTT
⦁ Water
The total stock concentrations were 28 µL. A total of 5 such tubes were set up while adding 1 µL of the Wild Type (WT) HAT complex to it. 1 µL CE.15 was further added to the tubes receiving 1 µL of HAT. This was followed by addition of 28 µL of the master mix to all the tubes that contained the HATs, and subsequent mixing by the help of fingers. The reactions were left to incubate for an interval of 30 minutes at 30oC in a water bath. The resultant samples were saved on ice and frozen for further analysis.
Histone acetyltransferase assay (Part 2)
The second part of the experiment involved SDS-PAGE and Western transfer, where we prepared 5 serial dilutions of the H3AcK18 peptide by adding concentrations as instructed. This was followed by the addition of 30µL of each dilution to 6 µL of 6X SDS sample buffer, which was later mixed to make 4 H3K18Ac peptide standards, namely 30, 15, 7.5 and 3.8 pmols. We subsequently prepared SDS-PAGE by removing gel from the package. A series of distinct steps. The top of the gel was rinsed around the comb with D1 water, while carefully pulling it out of the gel. This was followed by giving the previous week’s reaction a quick vortex, and heating the peptide gel samples, while centrifuging them at full speed for approximately 10 seconds. We then loaded 36 µL of each sample in the order shown in Table 1.
a. Prestained Protein Molecular Mass Markers
b. 30 pmol H3K18Ac peptide
c. 15 pmol H3K18Ac peptide
d. 7.5 pmol H3K18Ac peptide
e. 3.8 pmol H3K18Ac peptide
f. Wild-type SAGA (1 µL)
g. Wild-type SAGA (2 µL)
h. Mutant (1 µL)
i. Mutant (2 µL)
j. Gcn5Δ
Once loaded, a series of instructions provided in the lab manual were followed. Attention was given to not wring or bend the pads, as it would have substantially reduced their lifespan. It was ensured that the prestained protein ladder was not heated. The composition of the MES SDS Running buffer was as follows:
⦁ MES SDS Running Buffer
⦁ 50 mM MES
⦁ 50 mM Tris-base
⦁ 0.1% SDS
⦁ 1 mM EDTA
⦁ pH 7.3
⦁ Transfer buffer
⦁ 25 mM Tris Base
⦁ 190 mM glycine
⦁ 20% methanol
Large scale preparation of yeast extracts
The lab experiment also required largescale preparation of yeast extracts. The procedure lasted for a period of 4 days. Following the growth of the yeast and their cells, we used the glass beads and a bead beater to release the contents of the yeast cells. Once bead beating was completed, we performed high speed centrifugation at 3000g; cells were washed in 1 L of water, centrifuged, and transferred to a 50 mL conical tube and further centrifuged. This sedimented all the large cellular material that was insoluble. This mainly included chromatin and organelles, which gathered at the bottom of the tube. The supernatant was discarded and the cell pellet was frozen at -80oC.
Tandem Affinity Purification (TAP Purification)
Here, we purified the SAGA via TAP tag containing a protein A and calmodulin binding peptide (CBP) affinity tags, inserted on the C-terminus of the Spt7 subunit of the SAGA complex. We collected our beads through centrifugation, washed them with TEV protease and additional extraction buffer, and transferred them back to a tube containing TEV protease, while allowing the TEV cleavage reaction to take place overnight at 4oC. The samples were then frozen on the next day at -80oC. Once the purification procedure was complete, we ran sample from steps along the purification process along the sample we purified, while comparing it with a Wild-type (WT) sample. The procedure was completed over a period of 2 days.
On the 3rd day, we collected the proteins eluted by TEV protease in a tube with the help of the calmodulin binding solution CB.3 and froze it. We subsequently thawed and bound the samples to calmodulin-sepharose overnight. We used the CB.3 solution to wash the calmodulin beads in order to wash away the undesired protein contaminants that may have accumulated. We lowered the ion concentration by further washing the beads with CW.15, which reduced it 150 mM NACl. This was followed by subjecting the beads to a series of elutions in CE.15. The elute was later concentrated in a centrifugal ultraconcentrator with 10,000 kDa MWCO.
SDS-PAGE for Staining Gels
We used the SDS-PAGE method for visualizing the proteins in a sample through Sypro Red, which is a fluorescent dye that binds to the proteins while producing red light upon exposure to ultraviolet light. Specifically, we utilized the SDS-PAGE/Sypro Red staining method for analyzing the samples over the course of TAP purification, as elicited earlier. We loaded our final product alongside a purified preparation of the WT SAGA complex. This side-by-side comparison allowed in determining the purity of our preparation along with highlighting certain differences between the mutant and the wild-type complex.
Results
SDS-PAGE for Staining Gels
For this experiment, the final gel was photographed under U.
References
[1] Lee, K.K. and Workman, J.L., 2007. Histone acetyltransferase complexes: one size doesn’t fit all. Nature reviews Molecular cell biology, 8(4), p.284.
[2] Workman, J.L., 2016. It takes teamwork to modify chromatin. Science, 351(6274), pp.667-667.
[3] Hassan, A.H., Prochasson, P., Neely, K.E., Galasinski, S.C., Chandy, M., Carrozza, M.J. and Workman, J.L., 2002. Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes. Cell, 111(3), pp.369-379.
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