SNF5 is an essential executor of epigenetic regulation during differentiation

Abstract

Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation.

Figure 1. OCT4 target genes show distinctive nucleosome occupancy patterns that underlie the potential for gene expression.

(A) Genome-wide studies were performed in human embryonic stem cells (H1) using ENCODE and GEO data (wgEncodeHudsonalphaMethylSeqRegionsRep1H1hesc for DNA methylation, GSM518373 for OCT4 ChIP-Seq and wgEncodeUwDnaseSeqPeaksRep1H1es for DNaseI). The data comprised 100 bp windows of OCT4 binding regions (29740 sites), DNA methylated regions (43659 sites) and DNaseI hypersensitive regions (123778 sites). (B and E) H1 and NCCIT cells were exposed to 10 uM RA for the indicated days. The expression levels of OCT4, NANOG, PAX6 and NEUROG1 were determined by quantitative PCR (normalized to PCNA). Quantitative PCR data represent the average of three biological experiments (the mean +SEM) (C, D, F and G) Nucleosome occupancy at the PAX6 and NEUROG1 promoters was analyzed by NOMe-seq during differentiation of H1 and NCCIT cells. Blue circles represent GpC sites of the DNA (unfilled blue circles represent GpC sites which are inaccessible to GpC methyltransferase, teal-filled circles represent cytosines accessible to GpC methyltransferase). Pink bars represent regions of inaccessibility large enough to accommodate a nucleosome (around 150 bp). The data is representative of three biological experiments.

Figure 2. SNF5 is recruited to OCT4-activated and -repressed genes with distinctive chromatin landscape during differentiation.

(A–E) Chromatin from NCCIT cells was immunoprecipitated with anti-OCT4 (A), anti-EZH2 (B), anti-SNF5 (C), anti-BRM (D), anti-BRG1 (E) or anti-H3 antibodies and their binding at the DNA regulatory regions of OCT4 target genes were analyzed by quantitative PCR. Quantitative PCR data represent the average of three biological experiments (the mean +SEM). A Mann-Whitney test was performed and the increase in recruitment of SNF5, BRM and BRG1 at OCT4 target genes during differentiation as found to be statistically significant with p-values of 0.013 (SNF5), 0.012 (BRM) and 0.029 (BRG1). (F) The protein level of EZH2, SNF5, BRG1, BRM and loading control ACTIN were subsequently analyzed by western blot. The data is representative of three biological experiments.

Figure 3. Knockdown of SNF5 enhances a stem cell like state and blocks differentiation.

(A) SNF5, OCT4, EZH2, and loading control histone H3 were analyzed by western blot, 72 h post-transfection with SNF5 siRNA in NCCIT cells. (B and C) To get the nucleosome footprint, we have performed at least three biological replicates of NOMe-seq at 72 h post-transfection with SNF5 siRNA in NCCIT and selected ∼10 sequences in an unbiased manner to represented in the figures. (D) Stably infected SNF5 knockdown NCCIT cells were selected for 21 days with antibiotics and SNF5, OCT4, EZH2, and loading control histone H3 were subsequently analyzed by western blot. G401 cells were used for a SNF5 knockdown control. (E) At the same time point, cell morphology micrographs (200X) were taken. The data is representative of three biological experiments. (F) Apoptosis of SNF5 knockdown NCCIT cells after RA treatment was determined by flow cytometric analysis. The X axis indicates Annexin V and the Y axis indicates Propidium iodide (PI). The data are representative of three biological experiments (the mean +SEM).

Figure 4. Overexpression of SNF5 disturbs epigenetic regulation and enhances differentiation.

(A–D) Exogenous SNF5 was overexpressed in NCCIT cells and 72 h later, SNF5, OCT4, EZH2, and loading control histone H3 were analyzed by western blot (A). After exogenous SNF5 transfection, glycerol density centrifugation assay was performed. Fractions of 0.5 ml of the 10 ml 10∼30% glycerol gradient were collected and subjected to western bolt analysis for various BAF complex subunits (B). NOMe-seq was performed indicated OCT4 target regions (C and D). The data is representative of three biological experiments. (E) After overexpression, SNF5 binding at the DNA regulatory regions of OCT4 target genes were analyzed by quantitative PCR.

Figure 5. Overexpression of SNF5 alters SNF5 binding distribution, especially to OCT4 target genes.

(A) Percentage distribution of ChIP-seq binding regions for SNF5 in control and overexpression state. (B) The binding plots show the localization of SNF5 bound sites relative to OCT4 bound sites. SNF5 bound sites (y axis) are displayed within a 5 kb window centered on the OCT4 bound site. Intensity at position 0 indicates that site overlap. (C) Venn diagram showing overlapping of OCT4 and SNF5 (the number of OCT4 only binding genes; 3412, the number of SNF5 only binding genes; 7185, and the number of both binding genes; 1862) bound genes after overexpression of SNF5 based on ChIP-seq data in NCCIT cells.

Figure 6. SNF5 controls the balance between pluripotency and differentiation.

(A and B) 2D matrix and heat plots depicting gene expression changes in SNF5 knockdown/SNF5 overexpression and RA 7 d treated NCCIT cells. Axes indicate degree of fold change, from the middle of axis. The numbers indicate the median fold change of genes in each column or row. The intensity of each square represents the number of genes that fall in that square. (C) Fold change of SNF5 target genes (over two fold changes in opposite direction) among previously defined ES signature genes .

Figure 7. SNF5 is a key executor of epigenetic regulation in pluripotency and differentiation.

(A and B) Differentiation signals cause recruitment of SNF5 to both OCT4 activated and repressed target genes with distinctive roles (closing or generating NDRs) dependent on cellular context. Changes in exogenous SNF5 levels disrupt the balance between pluripotent and differentiated states. (Refer to Discussion for a detailed explanation).