The Regulation of rRNA Gene Transcription during Directed Differentiation of Human Embryonic Stem Cells.

It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ~50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor, UBTF, from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency, hESCs were treated with the Pol I inhibitor, CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers, reduces the expression of pluripotency markers, and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene, and corresponding reduction in transcription, represent early regulatory events during the directed differentiation of pluripotent stem cells

The reduction in rRNA gene synthesis is concomitant with dissociation of UBTF from the promoter, and precedes heterochromatin formation.

<p>(A) UBTF ChIP-seq from either untreated, or ACTIVIN A-treated H9 ESCs for 6 hours was aligned to a consensus unit of the human rRNA gene, and visualized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157276#pone.0157276.g002" target="_blank">Fig 2A</a>. (B) UBTF ChIP-QPCR across the indicated regions of the human rRNA gene. At each locus, the percent input of UBTF (and IgG) is shown. The percent input from each trial was then normalized to the percent input at <i>GAPDH</i>. The geometric mean is presented plus or minus the 95% confidence interval. Asterisks are used to indicate statistical significance (*** p < 0.001, * p < 0.05). The location of each primer set within the consensus rRNA gene is indicated beneath the schematic from (A) (5’-ETS = 5’-external transcribed spacer; NTS = non-transcribed spacer). (C) A Western blot of UBTF, Fibrillarin (FBL), and GAPDH in untreated and ACTIVIN A-treated H9 ESCs at 6 hours. (D) ChIP-seq of H3K27me3 (H1 ESCs: SRR212453; 48 hours ACTIVIN A treatment: SRR212471) was aligned to the consensus rRNA gene as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157276#pone.0157276.g002" target="_blank">Fig 2A</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157276#pone.0157276.ref037" target="_blank">37</a>]. (E) A ChIP-QPCR of H3K27me3 across the indicated regions of the rRNA gene in untreated and ACTIVIN A-treated H9 ESCs for 2 hours was calculated and presented as described in (B). ChIP-QPCR of H3K9me3 (F), and H4K20me3 (G) was carried out and presented as described in (B), but was done at 48 hours and normalized to <i>PER1</i>.</p

ACTIVIN A–induced differentiation coincides with rapid downregulation of rRNA synthesis.

<p>(A) The alignment of GRO-seq reads from GSE41009 to a consensus unit of the rRNA gene visualized using the Integrative Genomics Viewer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157276#pone.0157276.ref058" target="_blank">58</a>]. The scale is set from 0 to 2000 reads per million. A schematic diagram of the rRNA gene is shown below the reads. The thin vertical line after the 28S region indicates the transcriptional termination site. (B) The metabolic labeling of total RNA from H9 ESCs either untreated or grown in ACTIVIN A (50 ng/ml) for the indicated length of time. Cells were first grown in phosphate-free media and then ³²P was added (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157276#sec008" target="_blank">Methods</a>). A two hour time lag exists between the onset of ACTIVIN A treatment and the introduction of ³²P. Total RNA was harvested and electrophoresed on a 1% agarose-formaldehyde denaturing gel. The RNA was transferred to a Zeta-Probe membrane and imaged using a phosphoimager. An identical gel was simultaneously stained with SYBR gold to visualize total RNA (shown below). The rRNA species is labeled to the left of the image. (C) The newly synthesized, radiolabeled, rRNA was quantified using the Typhoon phosphoimager, and then normalized to total RNA using the SYBR gold stain and imageJ software. The ratio of newly synthesized RNA to total RNA (28S rRNA) was then plotted after 6 hours of ACTIVIN A treatment (Student’s t-test p = 0.026, N = 4). A control experiment was carried out in an identical manner using HEK293T cells (gray bars). The y-axis represents counts per minute per pixel (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157276#sec008" target="_blank">Methods</a>). (D) Two representative gels, as described in (B), are lined up below their corresponding quantitation in (C).</p

Direct inhibition of Pol I by CX-5461 induces loss of pluripotency and differentiation.

<p>(A) The metabolic labeling of H9 ESCs with ³²P was carried out for 6 hours, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157276#pone.0157276.g002" target="_blank">Fig 2B</a>, but with the addition of a range of concentrations of CX-5461. The dose of CX-5461 is shown on the x-axis and the ratio of c.p.m (newly synthesized rRNA) to total 28S rRNA was calculated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157276#sec008" target="_blank">Methods</a>) and plotted on the y-axis. (B) A phase-contrast image of CX-5461-treated H9 ESCs at 48 hours is shown. (C) A Venn diagram indicating the overlap between significantly upregulated genes after 48 hours of either ACTIVIN A or CX-5461 treatment. A list of the top PANTHER DB GO terms from the common genes is shown below. The full list is available in the Supporting Information. (D) The same analysis as described in (C) was performed for the downregulated genes. (E-H) The fold change in gene expression after 6 or 48 hours of CX-5461 treatment is shown for the same panel of genes from endoderm (E), mesoderm (F), ectoderm (G), and pluripotency-markers (H) that were depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157276#pone.0157276.g001" target="_blank">Fig 1</a>, alongside the fold change in gene expression after 48 hours of ACTIVIN A treatment (* p-adjusted < 0.1, Benjamini/Hochberg correction).</p