Regulation of chromatin structure by site-specific histone H3 methyltransferases

Regulation of chromatin structure by site-specific histone H3 methyltransferases. associated with DNA degradation during apoptosis. and and analysis of apoptotic signal transduction15,16,17. We previously found that histone H2B was phosphorylated during the induction of apoptosis6. In this report, we examined the regulatory mechanism of the apoptosis-specific S14 H2B phosphorylation in relation to internucleosomal DNA degradation and deacetylation of the K15 lysine. RESULTS Glucocorticoids induce apoptosis in thymocytes The induction of thymocyte apoptosis by dexamethasone was initially analyzed by flow cytometry (Fig. 1A). Thymocytes treated with 1 M dexamethasone for 4 hrs contain a population of cells showing reduced forward scatter light but increased side scatter light. This occurrence of a shrunken, granular population of cells is characteristic of programmed cell death (Fig. 1A, d). In contrast, this cell population develops only to a small extent (2.9% vs. 26.6% in Dex treated) in the non-glucocorticoid treated control cells incubated at 37 C and is not detected in freshly isolated cells or cells incubated on ice for 4 hrs. This 2.9% of cells seen in our control sample likely reflects spontaneous apoptosis of these cells in culture (Fig. 1A, c). Ultrastructural analysis of thymocytes by electron microscopy shows distinct areas of euchromatin and heterochromatin (light and dark staining, respectively) in the nuclei of control Sodium Aescinate thymocytes (Fig. 1B, left) in contrast to apoptotic cells where nuclear components appear degraded and showed homogeneous dark staining (Fig. 1B, right). Analysis of DNA by agarose gel electrophoresis (Fig. 1C) shows the characteristic pattern of internucleosomal DNA cleavage Sodium Aescinate in nuclei from apoptotic thymocytes. After 4 hrs incubation of thymocytes with dexamethasone, only lower molecular weight DNA was observed in the soluble chromatin fraction (Fig. 1C, lane 4). Together these observations indicate that glucocorticoid treatment for 4 hrs is sufficient to induce classic apoptotic DNA fragments in rat thymocytes. Open in a separate window Open in a separate window Open in a separate window FIG. 1 Dexamethasone induces apoptosis in thymocytes(A) Rat primary thymocytes induced to Sodium Aescinate undergo apoptosis by glucocorticoid treatment. Thymocytes were incubated with 1M dexamethasone in RPMI1640 medium for 4 hrs at 37C, and analyzed by flow cytometry. X-axis, forward scatter indicating cell size. Y-axis, side scatter indicating cell granularity. Apoptotic cells show a decrease in cell size (forward scatter light) with a concomitant increase in granularity (side scatter light). Dexamethasone treatment increases the percentage of apoptotic cells (box). a. freshly isolated thymocytes, Sodium Aescinate b. untreated thymocytes incubated on ice for 4 hrs, c. untreated thymocytes incubated at 37C for 4 hrs, d. dexamethasone treated thymocytes at 37C for 4 hrs. (B) Electron microscopic observation of apoptotic thymocytes. Thymocytes treated with dexamethasone were fixed as described in the Methods. Con, Control thymocytes. Dex, Dexamethasone. Nuclear materials of apoptotic cells are degraded. Magnification: 9, 900 . (C) Analysis of DNA fragments from whole or soluble chromatin. Thymocytes treated with dexamethasone were harvested after 4 hrs. The DNA (1 g) was run on 1.8% agarose gel and stained with ethidium bromide. Lane 1: Freshly isolated DNA from nuclei, lane 2: Control nuclei incubated 4 hrs, lane 3: nuclei from 1 M dexamethasone treated nuclei for 4 hrs, lane 4: DNA from soluble chromatin from nuclei of the dexamethasone treated cells. Marker DNA; 100 bp DNA ladder. Note: DNA of soluble chromatin (lane Sodium Aescinate 4) does not have HMW DNA as indicated by *. Phosphorylated histone H2B at S14 is abundant in the soluble chromatin Degraded DNA fragments with their associated histones are separable from whole genomic DNA based on their solubility in nonionic detergent. Soluble chromatin from thymocytes exposed to 4 hrs of 1 1 M dexamethasone was analyzed by SDS gel electrophoresis (Fig. 2A). The relative amount of histones H1, H2A, H2B, H3 and H4 present in the soluble chromatin fraction increased in a time dependent manner upon incubation with dexamethasone, indicating the release of histones from chromatin during apoptosis. In the control cells incubated for 6 h without dexamethasone, only small amounts of histones were also released which likely reflects spontaneous apoptosis of a small percentage of our Mouse monoclonal to Epha10 cultured cells18. Open in a separate window Open in a separate window FIG..