Pygo2 regulates histone gene expression and H3 K56 acetylation in human mammary epithelial cells

Abstract

Histone gene expression is tightly controlled during cell cycle. The epigenetic mechanisms underlying this regulation remain to be fully elucidated. Pygopus 2 (Pygo2) is a context-dependent co-activator of Wnt/beta-catenin signaling and a chromatin effector that participates in histone modification. In this study, we show that Pygo2 is required for the optimal expression of multiple classes of histone genes in cultured human mammary epithelial cells. Using chromatin immunoprecipitation assay, we demonstrate that Pygo2 directly occupies the promoters of multiple histone genes and enhances the acetylation of lysine 56 in histone H3 (H3K56Ac), previously shown to facilitate yeast histone gene transcription, at these promoters. Moreover, we report reduced global levels of H3K56Ac in Pygo2-depleted cells that occur in a cell cycle-independent manner. Together, our data uncover a novel regulator of mammalian histone gene expression that may act in part via modifying H3K56Ac.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the UCI Genomics High Throughput Facility (GHTF) and Sue and Bill Gross Stem Cell Research Center Core Facility for expert service. We also thank Julie Wells for technique assistance. This work was supported by NIH Grant R01-GM083089 (to X.D.). B.G. was supported by a California Breast Cancer Research Program (CBCRP) Postdoctoral Fellowship (14FB-0129). K.W. was supported by a US Department of Defense Breast Cancer Research Program (DOD BCRP) Postdoctoral Fellowship (W81XWH-10-1-0383).

Figures and Tables

Figure 1 Altered histone gene expression in Pygo2-knockdown MCF10A cells. (A) Protein gel blot analysis showing effective depletion of Pygo2 protein at 24 h after siRNA treatment. (B) Pie diagram showing microarray data on histone gene expression upon Pygo2 knockdown. (C) RT-qPCR analysis of select histone genes. Shown are average values from two independent experiments with standard deviations. (D) Flow cytometry analysis of DNA content (propidium iodide, or PI) and DNA synthesis (BrdU) in siRNA-treated cells (24 h after treatment). Shown are profiles from a representative experiment as well as average values from three independent experiments with standard deviations. *p = 0.17 using two-tailed t-tests assuming equal variance.

Figure 2 Histone gene expression change precedes cell cycle arrest in Pygo2-depleted MCF10A cells. (A) Schematic diagram showing the design of siRNA/shRNAs for human Pygo2. (B) Protein gel blot analysis of MCF10A whole-cell lysates showing knockdown of Pygo2 at 48 and 72 h post-infection. Signals are quantified by ImageJ 1.43u and normalized as indicated. (C) Flow cytometry analysis of cell cycle progression of infected cells as in (B). Shown are profiles from a representative experiment as well as average values from three independent experiments with standard deviations. (D) RT-qPCR analysis of select histone genes after knockdown of Pygo2. Similar results are obtained from two independent experiments. Expression levels are quantified and normalized against GAPDH control.

Figure 3 Pygo2 occupancy and H3K56 acetylation at select histone promoters. (A) Immunofluorescence analysis of Pygo2 (green) and NPAT-associated HLBs (red) 24 h after siRNA treatment. White arrows indicate Pygo2-depleted cells. Cell nuclei are stained blue with DAPI. Scale bar: 10 µm. Quantification of HLBs in control (n = 103) and Pygo2-knockdown (n = 91) cells is shown on the right. Error bars indicate standard deviations of two independent experiments. (B) ChIP analysis of Pygo2 occupancy on select histone gene promoters. GAPDH promoter was used as a negative control. (C–F) ChIP analysis of H3K56Ac (C and D) and H3K9/K14Ac (E and F) at select histone promoters after treatment with the indicated shRNAs. (D and F) are quantifications of ChIP experiments in (C and E), respectively. Error bars are standard deviations of two independent experiments.

Figure 4 Reduced global H3K56Ac in Pygo2-knockdown cells. (A) Protein gel blot analysis of cellular H3K56Ac levels in shRNA-treated cells. MCF10A cells were infected with the indicated shRNA-expressing lentivirus for 3 d. Signals are quantified and normalized as indicated. (B) Immunofluorescence analysis of H3K56Ac in siRNA-treated cells (24 h). (C) RT/semi-quantitative PCR analysis of known H3K56Ac-regulating proteins 3 d after shRNA-viral infection. (D) Protein gel blot analysis of H3K56Ac in cells infected with lentiviruses that express the indicated shRNAs plus either lentiviruses that express a RNAi-resistant form of Pygo2 or vector control. Signals are quantified and normalized as indicated. (E) Flow cytometry analysis of H3K56Ac during cell cycle progression. Shown at the top are representative profiles of H3K56Ac/DNA content (PI) from siRNA-treated MCF10A cells (24 h). Bottom, altered H3K56Ac levels at the indicated cell cycle stages. (F) Quantification of results from (E). Error bars are standard deviations of three independent experiments. p-values are calculated using two-tailed t-tests assuming equal variance.