Satb2 regulates proliferation and nuclear integrity of pre-osteoblasts

Abstract

Special AT-rich sequence binding protein 2 (Satb2) is a matrix attachment region (MAR) binding protein. Satb2 impacts skeletal development by regulating gene transcription required for osteogenic differentiation. Although its role as a high-order transcription factor is well supported, other roles for Satb2 in skeletal development remain unclear. In particular, the impact of dosage sensitivity (heterozygous mutations) and variance on phenotypic severity is still not well understood. To further investigate molecular and cellular mechanisms of Satb2-mediated skeletal defects, we used the CRISPR/Cas9 system to generate Satb2 mutations in MC3T3-E1 cells. Our data suggest that, in addition to its role in differentiation, Satb2 regulates progenitor proliferation. We also find that mutations in Satb2 cause chromatin defects including nuclear blebbing and donut-shaped nuclei. These defects may contribute to a slight increase in apoptosis in mutant cells, but apoptosis is insufficient to explain the proliferation defects. Satb2 expression exhibits population-level variation and is most highly expressed from late G1 to late G2. Based on these data, we hypothesize that Satb2 may regulate proliferation through two separate mechanisms. First, Satb2 may regulate the expression of genes necessary for cell cycle progression in pre-osteoblasts. Second, similar to other MAR-binding proteins, Satb2 may participate in DNA replication. We also hypothesize that variation in the severity or penetrance of Satb2-mediated proliferation defects is due to stochastic variation in Satb2 binding to DNA, which may be buffered in some genetic backgrounds. Further elucidation of the role of Satb2 in proliferation has potential impacts on our understanding of both skeletal defects and cancer.

Keywords: Chromatin defects; MAR-binding protein; Osteoblast differentiation; Satb2.

Figure 1:. Overview of CRISPR strategy and outcomes

A) Diagram of Satb2 protein with the location of the Cas9 cut site, qPCR primer sets, and the binding region of the antibody used in this study highlighted. B) Colony cell lines used in this study are described. The colony number, Satb2 genotype, cut site DNA sequence and translation, as well as protein product produced are listed.

Figure 2:. Mutations in Satb2 reduce protein levels

Confocal maximum intensity projections showing representative Satb2 protein levels in undifferentiated (T0) cells of colonies used in this study. Note the presence of Satb2 protein in C4, indicating the production of a mutant protein, as well as the absence of protein in C8. DNA in green; Satb2 in pink. Asterisks highlight chromatin bridges. All scale bars represent 10 μm.

Figure 3:. Mutations in Satb2 reduce pre-osteoblast proliferation rates

A) Growth curves for undifferentiated (T0) cells. Data represent the means of three replicate experiments, with 95% confidence intervals shown. Asterisks indicate significance at p<0.0001. B) Representative flow cytometry detection of Annexin V (x-axis) and propidium iodide (y-axis) staining in WT (left) and C4 (right) cells. Yellow rectangles indicate live cells, orange boxes indicate early apoptotic cells, and red boxes indicate late apoptotic cells. C) Quantification of flow cytometry for live cells (green bars), early apoptotic cells (orange bars), and late apoptotic cells (magenta bars) for all colonies is shown. Data represent the means of two replicates with standard deviation.

Figure 4:. Mutations in Satb2 cause aberrant nuclear morphology.

Confocal images of Satb2 mutant osteoblasts showing representative nuclear aberrations. A,B) C9 cells with a chromatin bridge. Lamin B immunostaining highlights nuclear folds being pulled into bridge (arrow in B). C) Large cell with small hole shown relative to other normal-sized cells. D) Chromatin herniation from nucleus. E, F) Nuclear blebbing and donut-shaped nuclei in C9 (E) and C4 (F) cells. Note the presence of nuclear folds in F (arrows). DNA in green; alpha-tubulin in teal; lamin in pink (Lamin A in C and E; Lamin B in A, B, and F). Images in A, B, C, and D are maximum intensity projections. Images in E and F are single confocal sections. Panel F includes a z-section at the top. All scale bars represent 10 μm.

Figure 5:. Over-expression of Satb2 in mutant cells reduces the frequency of abnormal nuclei.

Confocal images of C4 cells transfected with a Satb2-GFP over-expression plasmid. A) DNA stained with Hoechst. Asterisks highlight abnormal nuclei. B) Satb2 (magenta) is detected in nuclei of transfected cells. C) Overlay showing GFP-positive transfected cells. D) Pie-chart showing distribution of different types of nuclear aberrations observed in GFP-negative and GFP-positive C4 cells. Scale bar represents 10 μm.

Figure 6:. Mutations in Satb2 reduce, but don’t inhibit, osteogenic differentiation.

A) In vitro differentiation assay timeline. B) Overview scans of wells (left) and 10x magnification of Alizarin Red staining on WT, C19, C9, and C8 cells after 21 days (T21) in differentiation. Data are shown for seeding densities of 50,000 cells, 100,000 cells, and 200,000 cells. Note that clusters start to form when mutant cells are plated at higher density (compare asterisk in C19 at 200K to WT at 50K). C) Expression of Opn (pink bars), Ocn (purple bars), Bsp (green bars), and Osx (yellow bars) is shown relative to WT at T21. Data are the means of three experiments with standard deviation. Standard deviation for Osx in C8 at T28 is 5.01.

Figure 7:. Reduction in Satb2 increases gene expression variance

Upper panels show t-distributed Stochastic Neighbor Embedding (tSNE) plots of single cell gene expression in wild-type (WT; blue squares) and colony 9 (C9; pink triangles) cells at A) T0 (initial dimensions= 80, perplexity=15, iterations 4000, error was 0.528 at the 4000th iteration) and B) T14 (initial dimensions= 80, perplexity=10, iterations 3000, error was 0.524 at the 3000th iteration). Lower panels show plots of principal components 1 and 2 from Principal Components Analysis (PCA) on single cell gene expression in WT (blue squares) and C9 (pink triangles) cells at C) T0 and D) T14.

Figure 8:. Model of Satb2-mediated variation in molecular and cellular outcomes

Satb2 in known to regulate gene expression through high-order chromatin organization, including looping of chromatin to associate long-distance regulatory elements with promotors into “transcription factories” (1). Satb2 also associates with other transcription factors, including Runx2, to more directly regulate gene expression (2). A) In WT cells, Satb2-mediated gene regulation in a population is coordinated. B) When Satb2 is reduced, it is predicted to fail to bind to some of its DNA and/or protein partners randomly. Thus, cell to cell variation in gene expression will exist in a population, contributing to molecular and cellular variation in severity of phenotypes.