Np95 is implicated in pericentromeric heterochromatin replication and in major satellite silencing

Abstract

Heterochromatin plays an important role in transcriptional repression, for the correct segregation of chromosomes and in the maintenance of genome stability. Pericentric heterochromatin (PH) replication and formation have been proposed to occur in the pericentric heterochromatin duplication body (pHDB). A central question is how the underacetylated state of heterochromatic histone H4 tail is established and controlled, because it is a key event during PH replication and is essential to maintain the compacted and silenced state of these regions. Np95 is a cell cycle regulated and is a nuclear histone-binding protein that also recruits HDAC-1 to target promoters. It is essential for S phase and for embryonic formation and is implicated in chromosome stability. Here we show that Np95 is part of the pHDB, and its functional ablation causes a strong reduction in PH replication. Depletion of Np95 also causes a hyperacetylation of lysines 8, 12, and 16 of heterochromatin histone H4 and an increase of pericentromeric major satellite transcription, whose RNAs are key players for heterochromatin formation. We propose that Np95 is a new relevant protein involved in heterochromatin replication and formation.

Figure 1.

During mid-S, Np95 targets pericentric heterochromatin and is part of pHDB. (A) Distribution of Np95 during S-phase. NIH-3T3 cells were synchronized in G0. At different time after release, cells were pulse-labeled with BrdU and then stained with anti-Np95 together with either anti-BrdU or anti-pH3 (specific for histone H3 phosphorylated at serine 10). Nuclear counterstaining was visualized with DAPI. (B) Np95 is part of pHDB. NIH-3T3 cells in mid-S phase were pulse-labeled with BrdU and then stained with anti-Np95 together with anti-BrdU. Nuclear counterstaining was visualized with DAPI. Representative pictures are shown. The insets correspond to magnifications of the areas indicated. On the right is a schematic representation of the pHDB of the inset. (C) NIH-3T3 cells in mid-S phase were stained as above and then analyzed by confocal microscopy. Representative pictures are shown. The insets correspond to magnifications of the areas indicated. The histogram shows the local intensity distribution (diagonal white lines through the images) of Np95 in green, BrdU in red, and DAPI in blue.

Figure 2.

Loss of Np95 leads to slow S-phase progression. (A) Whole-cell extracts of NIH-3T3 transfected with the RNAi oligo anti-Np95 (Np95 RNAi) or with the control oligo (ctrl RNAi) were analyzed by Western blot using antibody anti-Np95. An antibody anti-lamin B was used as a loading control. (B) Cell proliferation assay. NIH-3T3 cells were transfected with the RNAi oligo anti-Np95 (RNAi Np95) or with the control oligo (RNAi ctrl). Twenty-four hours after the second transfection, the cells were serum-starved for another 36 h to synchronize them in G0. At the indicated times after release, cells were harvested and counted with a Burker chamber. Each timing point corresponds to the average of three experiments. (C) Np95 depleted and control NIH-3T3 cells were pulsed for 10 min or 24 h with 10 μM BrdU and stained with antibodies anti-Np95 together with anti-BrdU. Nuclear counterstaining was visualized with DAPI. Representative pictures are shown. The plot indicates the percentage of cells positive for anti-BrdU. (D) NIH-3T3 cells treated as above were pulsed for 10 min with 10 μM BrdU and then stained with anti-Np95 together with anti-PCNA. Nuclear counterstaining was visualized with DAPI. Representative pictures are shown. The plot indicates the percentage of cells positive for anti-PCNA. Quantitative assessment in C and D was performed on three independent experiments, in which at least 250 cells per experiment were counted.

Figure 3.

Np95 depletion interferes with PH replication. NIH-3T3 cells were transfected with oligo against Np95 (Np95 RNAi) and a control (Ctrl RNAi). Twenty-four hours after treatment, the cells were pulsed for 10 min with 10 μM BrdU and then fixed onto glass slides and immunostained with anti-Np95 together with anti-BrdU. Representative pictures are shown. Arrows indicate the cells are replicating. The plot indicates percentage of cells in early (dark, bar), mid- (gray, bar), and late (white, bar) S phase. Quantitative assessment was performed on three independent experiments, in which at least 250 cells per experiment were counted.

Figure 4.

Reduction of Np95 leads to specific enrichment of H4 acetylated at Lys 8, 12, and 16 at the periphery of heterochromatic dense DAPI dots. (A) Np95-depleted (Np95 RNAi) and control cells (Ctrl RNAi) were stained with anti-Np95, together with either anti-H2A-K5, -H4-K8, -H4-K12, and -H4-K16. Nuclear counterstaining was visualized with DAPI. Representative pictures are shown. The plots represent the percentage of cells that show H2A-AcK5, H4-AcK8, H4-AcK12, and H4-AcK16 acetylation on the periphery of heterochromatic dense DAPI dots. Quantitative assessment was performed on three independent experiments, in which at least 250 cells per experiment were counted. (B) Lysates of whole cells treated as above were analyzed by immunoblot with the indicate antibodies.

Figure 5.

Confocal analyses of distribution histone H4 acetylated at Lys 8 and 16 in Np95-depleted cells. Np95-depleted cells (Np95 RNAi) were stained with anti-Np95 antibody, together with antibodies against either acetylated H2A-K5, H4-K8, and H4-K16 and then analyzed by confocal microscopy. Nuclear counterstaining was visualized with DAPI. Representative pictures are shown (right panel). The histogram shows the local (diagonal white lines through the images) intensity distribution of Np95 in green, H4 acetylated in red, and DAPI in blue (left panel).

Figure 6.

Np95 depletion increases the transcription expression of major satellites. Top, schematic representation of major (pericentric heterochromatin) and minor satellite (centromeric heterochromatin) repeats on a mitotic mouse chromosome. Bottom, relative RNA levels for major satellites, minor satellites, and GAPDH was determined by RT-PCR analyses on cDNA derived from total RNA obtained from Np95-depleted (Np95 RNAi) and control cells (Ctrl RNAi). The results of 18 cycles of amplification are shown. The negative digital image of agarose gel analysis is shown.