Skip to main content
. 2014 Jun 23;29(6):740–749. doi: 10.1016/j.devcel.2014.05.001

Figure 3.

Figure 3

H4K20 Monomethylation Occurs at the CENP-A Nucleosomes

(A) Chromatin was completely digested with MNase, and the mononucleosome fraction was isolated from HeLa or DT40 cells expressing CENP-A-FLAG (input). Immunoprecipitation (IP) experiments were performed with anti-human CENP-A (for HeLa) or FLAG (for DT40) antibodies, and the CENP-A nucleosomes were recovered. Western blot analysis was carried out for the CENP-A nucleosomes with two kinds of anti-H4K20me1 (15F11 or 22G3) and -panH4 antibodies. Silver-stained gel image is also shown.

(B) Gel electrophoresis of DNAs extracted from the mononucleosome fractions used in (A).

(C) IP experiments were performed with anti-H4K20me1 antibodies in the mononucleosome fraction from DT40 cells expressing CENP-A- FLAG, and western blot analysis was carried out with anti-H4K20me1 and -CENP-A antibodies.

(D) An experimental design to prepare CENP-A nucleosome (B) and histone H3 nucleosome (UB) fractions in centromere region. Chromatin from HeLa or DT40 cells expressing CENP-A-FLAG was partially digested with MNase and performed IP with anti-human CENP-A (for HeLa) or FLAG (for DT40) antibodies. The precipitated fraction was completely digested with MNase again. The beads unbound supernatant fraction (UB) was expected to contain H3 nucleosome around CENP-A nucleosomes, and beads bound fraction (B) was expected to contain CENP-A mononucleosomes.

(E) DNAs were recovered from UB or B fractions, and Southern blot analysis was performed with a centromeric DNA probe (CenZ). DNAs from both fractions were hybridized the CenZ probe.

(F) Western blot analysis for UB or B fractions prepared from DT40 or HeLa cells with anti-H4K20me1, -pan H4, -CENP-A, and -H3 antibodies. In the UB fraction, H3, but not CENP-A, was detected, and CENP-A, but not H3, was detected in the B fraction. H4K20me1 was highly enriched in CENP-A-containing chromatin from both DT40 and HeLa cells.

(G) Purification of the cytoplasmic CENP-A-H4 complex before deposition to nucleosome from DT40 cells expressing CENP-A-FLAG. Western blot analysis for the CENP-A-H4 complex with anti-H4K20me1 and -panH4 antibodies was performed. Silver-stained gel image is also shown.

(H) Western blot analysis for the cytoplasmic CENP-A-H4 complex before deposition to nucleosome from HeLa cells with anti-H4K20me1, -panH4, and -CENP-A antibodies.