Histone Methylation In Gene Regulation

The methylation of histone is seen to play a key role in regulating gene expression. They repress or activate genes based on which residue of lysine is been methylated. For example, H3K27 or H3K9 (methylation of H3 at 27 or 9) act as repressive markers but the H3K4 is referred as an active mark (Lachner et al., 2003). The direct clue regarding functionality of histone methylation in case of the CT gene regulation was seen to be validated by the research that mouse ESCs which contain knockout of H3K9 methyltransferase enzymes, HLP or G9a results to create a marked increase in MAGE-4 expression (Tachibana et al., 2005; Tachibana et al., 2002). In another study, it is seen that the CT gene Prdm9 mediate the methylation of histone H3K4me3. Therefore, this process along with other factors leads to mark the chromatin to be involved in recombination (Hayashi et al., 2005). This informs that prdm9 which is CT gene has a functional role in modifying the histone.

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Heterochromatin and Euchromatin Structure: Heterochromatin is seen to have high density of nucleosome that results it to develop a closed form of chromatin. The Euchromatin is referred to open form of chromatin and it is been normally associated with the active transcription of gene. Thus, it can be seen that heterochromatin is related with the repression of transcription of gene. In case of each histone, it is seen that they have an amino tail in the terminal region which is able to be covalently changed through different epigenetic reactions (Russ et al., 2012).

Figure: Expression of TEX19 gene in human cancer shown through RT-PCR analysis

The TEX19 gene expression is presented through agarose gel in the cancer cell. The product size in the PCR of TEX19 is expected to be 344bp. The βACT gene expression is seen to be used for the positive control of cDNA samples synthesized from the cells of cancer. dH2O is been used as a form of negative control and the PCT product related to βACT gene is seen to have an expected size of 553bp.

Figure: Western blot analysis for detecting levels of protein TEX19 in various cancer cell line lysates.

The TEX19 is seen to be produced in the normal tissues of testis and it was detected by using Sheep polyclonal anti-TX19 antibody (R&D, #AF6319). The anti-GAPDH was seen to be used as a form of loading control. The provided samples are given by Abcam and Novus companies.

Figure: The use of siRNA to deplete TEX19 in the NTERA2

A The bar charts shown are presenting the TEX19 expression in NTERA2 cells followed by depletion through the process RT-PCR analysis. The NTERA2 cells are transinfected with the help of TEX19 siRNA molecules showing a decrease of the TEX19 mRNA when contrasted with the control. The p-value is mentioned through asterisks present in the bar graph. The GAPDH and βACT are used to normalize the data.

BThe western blot analysis has demonstrated the TEX19 protein levels in the NTERA2 followed by depletion. NTERA2 cells are seen to be transinfected with the TEX19 siRNA showing an important reduction in the protein level of TEX19 in comparison to the untreated cells and siRNA. The GADPH was seen to be used as a form of control for confirming equivalent loading in each well.

Figure: Transinfection of NTERA2 cells with the help of TEX19 siRNA

A The curve representing the cell count indicates the nature of proliferation of NTERA2 cells, cells treated with TEX19 siRNA and control siRNA for five days. The control siRNA and untreated cells are used in the form of control. The cells which are treated with TESX19 siRNA #7 are showing increased level of reduction of cell proliferation in comparison to controls. The error bars indicate the extent of standard deviation faced for the three replicates. The p-values are mentioned through the help of asterisks.

B Western blot analysis is showing protein level of TEX19 on cell proliferation of NTERA2. The findings reflect that the cells which are being experimented with TEX19 siRNA are showing a strong decrease in the protein level of TEX19 in comparison to the controls. The GAPDH was seen to be used in the form of control to load wells.

Western blot analysis of TEX19 sub-cellular localisation in SW480 cancer cell line

In the figure, the western blot analysis is representing cellular location related to TEX19 protein in the SW480 cell line by using a cytoplasmic fraction, a nuclear lysate fraction and a whole cell extract. The antibodies against Tubulin (Cytoplasmic) and Lamin B (Nuclear) are used in the experiment as a positive control for confirming fractionation efficiency as well as gel loading.

IF staining showing TEX19 protein localisation in SW480 cells

The image indicates IF staining for anti-α-Tubulin (SEGMA, T6074; 1:1000) and anti-TEX19 (Abcam, ab185507; 1:500) in the SW480 cells. (A) DNA is being stained with DAPI (blue) (B) Anti-α-Tubulin stained (green) (C) Anti-TEX19 stained (red) (D) Staining of DAPI, anti-α-Tubulin and anti-TEX19 (blue, green and red)

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The images present are being acquired through the Zeiss LSM 710 confocal microscope that has the objective lens of 20x.

The Histone Extraction Kit (ab113476) is referred to be a suitable thing for quickly isolating histone extracts from the tissues and cell samples from mammal and other species. The quantity of starting material may be low as 1mg of tissues or 105 cells. In order to get best results, the number of cells is required to be more than 106 or the amount of tissues is required to be more than 10 mg.

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