Apoptosis and Histones

H2A.X and H2B

The Role of Histone Modifications in Apoptosis and DNA Damage

Many key scientific discoveries have been made that link specific histone modifications with important biological phenomena. In particular, several histone modifications have been discovered that are useful biomarkers of apoptosis and DNA damage. Reagents and kits specific for these modifications are of great value to any researcher studying these processes.

H2A.X Phosphorylation Western Blot

Western blot using anti-phospho-Histone H2A.X (Ser 139), clone JBW301 (Millipore cat. no. 05-636) antibody at 1:2000 dilution to detect accumulation of g-H2A.X in Jurkat cells treated with 1 µg/mL Staurosporine to induce apoptosis. Cells were harvested after the number of hours indicated above each lane.

Apoptosis is defined as a growth-limiting regulatory mechanism by which cells can trigger their own demise due to advanced age in response to extracellular signals or as a result of irreparable cellular or DNA damage. Many key regulators of apoptosis are critical to cancer development, as the function of one or more apoptotic inducers (e.g., p16, p53, BRCA1) is lost in most types of cancer. The loss of these key growth checkpoint proteins allow cells to become immortalized by escaping important regulatory growth restrictions. Additionally, some genes that normally function to block premature apoptosis are hyperactivated in specific cancers (Bcl2), thus preventing cells from terminating their growth. Apoptosis is also an important developmental mechanism, such as preventing the overgrowth of particular neuronal cell lineages in the developing brain.

H2A.X Phosphorylation During Apoptosis

Immunofluorescence of HeLa cells using Anti-phospho-Histone H2A.X (Ser139) clone JBW301 (Millipore cat. no. 05-636).
Cells were treated with 1 ìg/mL Staurosporine for two hours to induce DNA damage and apoptosis. Left panel, Anti-phospho-H2A.X (Ser139), FITC conjugate ( Millipore cat. no.16-202A). Middle panel, DNA stained with DAPI. Right panel, phase contrast image.

H2A.X Phosphorylation as a Biomarker for DNA Damage and Apoptosis

Many agents, such as ionizing radiation and DNAbinding drugs, cause damage to nuclear DNA – the most severe type being double-strand breakage (DSB). DSBs must be repaired quickly and with high fidelity because they can lead to rearrangements in the genome through recombination. Specific protein exist within the nucleus for just this purpose. The process of apoptosis occurs in successive stages, each with characteristic changes in cell morphology. Along with these changes, specific post-translational modifications occur on the histones. The histone variant H2A.X is rapidly phosphorylated at serine 139 by the ATM kinase in response to even a few double strand DNA breaks. The accumulation of high levels of H2A.X phosphorylation resulting from severe DNA damage is a very early and accurate indicator of apoptosis. Interestingly, H2A.X phosphorylation also occurs when chromosomal DNA is cleaved intentionally, such as during meiotic recombination between sister chromatids and immunoglobulin gene VDJ recombination. The detection of H2A.X phosphorylation is illustrated in the “H2A.X Phosphorylation During Apopto” images.

References

Rogakou, E. P. et al., J Biol Chem 1998; 273:5858–5868.
Rogakou, E. P. et al., J Cell Biol 1999; 146: 905–916.
Talasz, H. et al., Cell Death Differ 2002; 9:27–39.
Rogakou, E. P. et al., J Biol Chem 2000; 275: 9390–9395.
Paull, T. T. et al., Curr Biol 2000; 10:886–895.

Anti-phospho-Histone H2B and DAPI Staining

Detection of Histone H2B phosphorylated at serine 14 with Anti-phospho-Histone H2B (Ser14) (Millipore cat. no. 07-191) by immunofluorescence in HL-60 cells stimulated to undergo apoptosis by treatment with VP-16. Top panel, Anti-phospho- Histone H2B (Ser14) (Millipore cat. no. 07-191) staining. Bottom panel, DAPI staining. Note the distinctive morphology of the nuclear DNA as it breaks down, concomitant with the detection of H2B serine 14 phosphorylation (arrows). Courtesy David Allis, The Rockefeller University.

H2B Phosphorylation as a Biomarker of Irreversible Commitment to Apoptosis

Another modification, phosphorylation of serine 14 on histone H2B, occurs at the stage in apoptosis when the condensed nuclear DNA is cleaved by caspase- associated DNase enzymes. It is a hallmark of a cell irrevocably committed to the apoptotic pathway. H2A.X phosphorylation is also observed during this stage as a result of the DNase-induced DNA breaks. A third phosphorylation event linked to apoptosis, at serine 32 of histone H2B, has also been reported. Millipore’s upstate antibodies that recognize these modifications, by themselves or as part of kits, are extremely useful in monitoring DNA damage and apoptosis.

Reference

Cheung, W. et al., Cell 2003;113: 507–517

HL-60 cells were cultured with 20 ìg/mL of etoposide for 18 hours. Cells were aliquoted at 3 X 105 per well into a 96-well round-bottom microtiter plate, permeabilized with a buffer containing Tween®-20 and stained with 20 ìg/mL of Anti-phospho-Histone H2B (Ser14), clone MC603 (Millipore cat. no. 05-751)(filled histogram) or 20 ìg/mL of Normal Rabbit IgG (Millipore cat. no. 12-370) (open histogram) followed by a goat anti-rabbit IgG (H+L) phycoerythrin conjugate. Data was acquired and analyzed using a Guava® PCA-96 System.

	H2A.X Phosphorylation Assay, FACS
Log phase Jurkat cells were treated with Staurosporine (1 ìg/mL) for the indicated time. Histone H2A.X (Ser139) phosphorylation was detected using the H2A.X Phosphorylation Assay Kit for Flow Cytometry (Millipore cat. no. 17-344), and the cells were analyzed on a Becton-Dickinson FACSCalibur™ flow cytometer

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	H2A.X Phosphorylation Western Blot
Western blot using anti-phospho-Histone H2A.X (Ser 139), clone JBW301 (Millipore cat. no. 05-636) antibody at 1:2000 dilution to detect accumulation of g-H2A.X in Jurkat cells treated with 1 µg/mL Staurosporine to induce apoptosis. Cells were harvested after the number of hours indicated above each lane.

	H2A.X Phosphorylation During Apoptosis
Immunofluorescence of HeLa cells using Anti-phospho-Histone H2A.X (Ser139) clone JBW301 (Millipore cat. no. 05-636). Cells were treated with 1 ìg/mL Staurosporine for two hours to induce DNA damage and apoptosis. Left panel, Anti-phospho-H2A.X (Ser139), FITC conjugate ( Millipore cat. no.16-202A). Middle panel, DNA stained with DAPI. Right panel, phase contrast image.