Cell division is the most important phenomenon for reproduction and the continuity of the life. Each of the stages of the cell division (G1, G2, M, S) affects all the process and the importance of this process’s control is of vital importance. Checkpoints regulated by proteins and different transcription factors are responsible for this process. The identification of any regulatory factor of the cell divison cycle is of great significance for achieving a deeper understand of the cell cycle, errors in the cell cycle (cancer mechanism) and complex processes, such as aging, senescence etc. Our research group aimed the identification of the subunits of Mb304 which are involved in the cell growth/size regulation. Previous studies have shown that two of the subunits, reciprocally Mb304-a and Mb304-b of Mb304 complex are directly involved in the regulation of the checkpoint G1/S and in the editing of DNA errors.
Retinablastoma (Rb) is a form of cancer developing in retina. It is caused by a mutation in protein EF2 that is orthologous with Mb304 in S.cerevisiae. Several studies exist on the deregulation of G1/S phase in yeast, which is controlled by Mb304. This paper aims the identification of all subunits factors and their reciprocal role in cell growth for a better understanding of the mechanism of Retinablastoma and potential therapy on the future.
Materials & Methods
Analysis by ChIP
A myc tagged S.cerevisiaeMb304 strain was used for chromatin immunoprecipitation (ChIP). Overnight culture was harvested at OD 0.6–0.8 and fixed with 1% formaldehyde. Cell debris was eliminated and 1% of the total soluble fraction was removed after centrifugation. The fraction left was used for immunoprecipitation of myc-tagged Mb304 protein with anti-c-myc antibody. Primary antibody of Mb304-g was added and then the sample was purified by phenol-chloroform extraction and ethanol precipitation (V. Orlando and R. Paro, Cell 75, 1187 (1993)). DNA samples sequencing was done by HiSeq. After quality control, nearly 30 million sequences (IP sample) and 88 million sequences (control sample) were mapped by using the bowtie algorithm.
20 μl (50% ) protein A-Sepharose was applied on 1 mg yeast protein extract for 20 min at 4 °C. The samples were spinned down for 15 min at maximum speed. The supernatant was incubated with 2.5–5 μg of appropriate primary antibody (α-WB, α-NHE) for 1–2 h at 4 °C. The samples were spinned again under same conditions. 20 μl of 4× sample buffer was added to each sample.
Proteins obtained after Western Blotting were digested with Trypsin. Digests were analyzed over a linear ion trap/Orbitrap Tandem Mass Spectrometer operating in data-dependent acquisition (DDA) mode.
Yeast cell was lysed and a DNA primer was used to hybridize the gene. The gene was captured by nanoparticles with its transcription factor and transcription factor was restricted onto peptides later analyzed by MS/MS Tandem Reverse Spectrometry.
Ms/Ms Tandem Reverse Spectrometry
Trypsin was applied for peptide isolation. 03 trap column (KxBio K18, KxBioTech) was used to desalt the peptides. Peptides were then separated by reversed phase K03 column (KxBio K18, 75µm, i.d., 10cm). They were vaporized and1.MS. was applied for mass identification. Collision Induced Dissociation was applied for fragmentation of peptides. Mass of fragmentated peptides was measured by a 2. MS. The fragment mass data were analyzed SeqIbex. Analyzed data were compared to the database by using UngaSearch.
Study of the Orthologs of S.pombe according NCBI Data
Schizosaccharomyces pombe has a transciption factor consisting of 3 subunits that resembles the Mb304 of Saccharomyces cerevisiae. Cdc10 protein is ortholog to Swi6 protein. Res1/Sct1 and Res2/Pct1 proteins are ortholog to Mb304-g and Mb304-a proteins.
An inactive variant of both transcription factor units were developed. Cas9 was used to induce deletion on template. The Mb304a mutants carrying the temperature-sensitive swi4-29 allele (23) are temperature-sensitive for growth. Cells from wild-type (wt), congenic single mutant (Mb304aa), (Mb304b), and double mutant (Mb304a and Mb304b) strains were grown to early log phase at 250C and then shifted to 370C.
Cells were grown on YEP rich media each consisting of 2% of the specific carbon source. Cells were grown to mid-log phase (OD600 = 0.2-0.5) and they were incubated for 15-20 min. Afterwards, protease solution was applied at 25˚C, Samples were then sonicated at low power and analyzed by standard flow cytometry methods. They were observed from 25˚C to 37˚C. The detector with a standard 530/30 band pass filter was used for Green fluorescence, and a detector of a 670nm filter was used for PI fluorescence.
CHIP was performed for the isolation of the factor and its binding part (IP and control samples) of TF Mb304, in the model yeast S. cerevisiae. Mb304 protein is characterized by the Mb304-g DNA Binding subunit. Figure 1A shows comparison of the intersections among lists of peaks associated with the three different sets of parameters. S1 results were derived by using the most rigorous parameters’ combination. Mb304 was chosen to benchmark the bPeaks program and MBS (Mb304 Binding Site) sequence has been defined as 5′-TCCGCGGA-3′ (Fig 1 C).
Figure 1. Comparison of lists of peaks detected by using different parameter associations in bPeaks. (A) Three sets of parameters (S1, S2 and S3) were selected for the peak list comparison overlaps between the detected peaks. (B) Evolution of the proportion of peaks in promoters according to the number of detected peaks. (C) DNA sequences associated with the genomic positions of peaks detected with parameter sets S1, S2 and S3; and were examined using the ‘peak-motif’ program with default parameters
Figure 2. A) Western blottting demonstrating Mb304 TF whose weight is 240 kDa. A lane represent ladder, B lane represent the whole cell extract protein and C lane represent Mb304 transcription factor. It state that TF exists in yeast.B) To indicate subunits of Mb304 TF, mass spectrometry was used. The components of the purified TF complex are then separated by size, isolated and identified by mass spectrometry. m/z, mass divided by charge. It has found that Mb304 TF has three subunits that are Mb304-a, b, g. Their masses are 60, 80 and 100 kDa, respectively
The peptide sequences having the best scores from the database comparison by SeqIbex are shown on table1. Database comparison by SeqIbex confirmed MB304 protein is highly similar to the protein AJ402 regarding the subunits. AJ402 is also a multimeric transcription factor in S. pombe. The aa sequences of the AJ402-n and AJ402-d subunits of AJ402 protein and the hypothetical aa sequence of the MB304-a and MB304-b are shown on table2.
Figure 3.The common essential function of MB304G and MB304A. Cells from wild-type (wt), congenic single mutant (MB304A), (MB304G), and MB304G MB304A mutant ( Mb304g∆ mb304a∆) strains were grown to early log phase at 250C and then shifted to 370C. (A) Flow cytometricDNA analysis of cells stained with propidium iodide at various times after the temperature shift.The fluorescence signal of cells with a 1 N and 2 N DNA content is iindicated by arrows.
Mb304g subunit previously known from other studies provided us with knowledge related to the complex’s location and the cell cycle dependency upon other subunits of the protein complex Mb304. After CHIP procedure for extraction, the Western Blotting and Mass Spectrometry were applied on the complex for the determination of the subunits. Three subunits were determined(figure 1) reciprocally: Mb304a, Mb304b and Mb304g. The detected mass during mass analysis was 60,80,100 kDa. Homology studies over S.pombe were conducted identification of the subunits a and b, and used for sequence determination of S.cerevisiae’s subunits. AJ402 is the homologous protein in S.pombe with the subunits AJ402-n/-d. The aminoacid sequence of the MB304 subunits were determined (Table 2 & 3) based on the same procedure. CRISPR/Cas9 was used for mutating gene responsible for this protein complex and the cells were sorted and their DNA content was measured by Flow Cytometer. The function of each transcription factor was determined (Figure 3). On the second graph, the mutated Mb304g∆ cells had less 2N DNA content, showing that their activity had stopped on S phage, as Mb304g complex binds DNA and prevents DNA replication. Furthermore, a different behavior is observed on Mb304b∆ graph. Mb304b should be a suppressor subunit as in case this subunit is mutated, the DNA content increases due to the inhibition of its suppression function. Mb304a is the activator subunit. The determination of these subunits provides opportunity for future studies in Retinablastoma for the control of the homologous protein complex.
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