Staged assembly of histone gene expression machinery at subnuclear foci in the abbreviated cell cycle of human embryonic stem cells

Abstract

Human embryonic stem (hES) cells have an abbreviated G(1) phase of the cell cycle. How cells expedite G(1) events that are required for the initiation of S phase has not been resolved. One key regulatory pathway that controls G(1)/S-phase transition is the cyclin E/CDK2-dependent activation of the coactivator protein nuclear protein, ataxia-telangiectasia locus/histone nuclear factor-P (p220(NPAT)/HiNF-P) complex that induces histone gene transcription. In this study, we use the subnuclear organization of factors controlling histone gene expression to define mechanistic differences in the G(1) phase of hES and somatic cells using in situ immunofluorescence microscopy and fluorescence in situ hybridization (FISH). We show that histone gene expression is supported by the staged assembly and modification of a unique subnuclear structure that coordinates initiation and processing of transcripts originating from histone gene loci. Our results demonstrate that regulatory complexes that mediate transcriptional initiation (e.g., p220(NPAT)) and 3'-end processing (e.g., Lsm10, Lsm11, and SLBP) of histone gene transcripts colocalize at histone gene loci in dedicated subnuclear foci (histone locus bodies) that are distinct from Cajal bodies. Although appearance of CDK2-phosphorylated p220(NPAT) in these domains occurs at the time of S-phase entry, histone locus bodies are formed approximately 1 to 2 h before S phase in embryonic cells but 6 h before S phase in somatic cells. These temporal differences in the formation of histone locus bodies suggest that the G(1) phase of the cell cycle in hES cells is abbreviated in part by contraction of late G(1).

Fig. 1.

The p220^NPAT foci are associated with histone gene loci in H9 hES cells and normal diploid WI-38 fibroblasts. (A) IF microscopy images were obtained using antibodies against p220^NPAT (green), phospho-T1270-p220^NPAT (green), or coilin (blue), and a DIG-labeled FISH probe adjacent to the histone gene cluster on 6p22 (red). The arrows in the bottom row, left column, indicate the position of coilin foci. The Insets in the lower right of each panel in the bottom row indicate colocalization between p220^NPAT/colin/6p. DAPI staining (blue) is used to visualize the nucleus (top 2 rows). There are typically 2 or 4 p220^NPAT foci, depending on the cell cycle stage, that are consistently in proximity to histone gene clusters. In 50–60% of cells, coilin foci (Cajal bodies) overlap with at least one p220^NPAT foci. (B) IF images as in A, using antibodies against p220^NPAT (green) and Ki-67 (blue), a marker for cell cycle position, and the 6p22 FISH probe. Cell cycle stages are indicated at the lower left of each panel.

Fig. 2.

Colocalization of factors mediating histone gene transcriptional initiation and 3′-end transcript processing of histone mRNA. IF microscopy images were obtained for H9 hES cells (Left column) and normal diploid WI-38 cells (Right column) using antibodies against p220^NPAT (green) and factors that process or interact with histone transcripts (Lsm10, Lsm11, SLBP, or 3′ hExo; red). SLBP interacts with the 3′ hairpin in histone mRNA; the protein only partially colocalizes with p220^NPAT foci. Foci of 3′ hExo show no colocalization with p220^NPAT foci (green, row 4) and complete overlap with PML/ND10 bodies (green, row 5) in both hES cells and somatic WI-38 cells. The percentages in the lower left of the panels represent positive cells for colocalization of respective factors in each cell type.

Fig. 3.

Association of p220^NPAT and 3′-end processing factors with histone gene loci during the hES cell cycle. Mitotically synchronized hES cells at various cell cycle stages were monitored by IF microscopy for association of Lsm10 or SLBP (red) with p220^NPAT (green; rows 2 and 3), and spatial linkage of the histone gene cluster at 6p22 (red) with p220^NPAT or coilin (green; rows 4 and 5). Ki-67 (green) staining (row 1) was done to establish cell cycle position, and DAPI staining (all rows; blue) was used to visualize the nucleus.

Fig. 4.

Colocalization of 3′-end processing factors SLBP and Lsm10 with p220^NPAT during the cell cycle in somatic cells. Synchronized WI-38 fibroblasts were examined at various cell cycle stages by IF microscopy for spatial interactions between Lsm10 or SLBP (red) with p220^NPAT (green). Ki-67 (green) staining (Top row) was done to establish cell cycle position, and DAPI staining (all rows; blue) was used to visualize the nucleus. The percentages in the lower left of the panels represent cells positive for Ki-67 (Top row) and SLBP (Bottom row). The images in row 1 were taken at 40× magnification.