Table 1.
PCR primers used for gene expression analyses.
Table 2.
PCR primers used for ChIP assays.
Fig 1.
In vivo and in vitro expression of Pax2 and LSD1 in otic progenitors.
(A-A”) Mouse E10.5 otic vesicle stained with Pax2 (green) and LSD1 (red) antibodies. (B-B”) Mouse embryonic otic vesicle at E9.5 stained with Pax2 (green) and TUJ1 (red) antibodies. Nuclei were counter stained using DAPI (blue). (C-C”) N33 cells were cultured under proliferative conditions and stained with Pax2 (green) and LSD1 (red) antibodies. Scale bars, 50 μm.
Fig 2.
Pax2 interacts with LSD1 and is associated with the NuRD complex.
(A, B) Physical interaction between endogenously expressed Pax2 and LSD1 proteins in N33 cells. Nuclear cell lysates were prepared from undifferentiated N33 cells and immunoprecipitations with either anti-LSD1 or anti-Pax2 antibody were performed. (C, D) Pax2 and LSD1 immunoprecipitated complexes were probed against various members of the NuRD complex as indicated. Immunoprecipitated complexes were resolved by SDS-PAGE followed by immunoblot analysis using indicated antibodies. Rabbit IgG was used as a negative control for non-specific immunoprecipitation. Cell lysates before co-immunoprecipitation were used as input controls.
Fig 3.
Generation and characterization of PRS4-EGFP N33 (PE) stable cells.
(A) A linearized PRS4-EGFP construct was transfected into N33 cells. G418 antibiotic at 500 μg/ml was used as a negative selection for stable clones. The upper panel indicates cells after 24 hours of transfection. As indicated in the lower panel, almost all non-transformed cells died by day 9, but cells with stable incorporation of the PRS4-EGFP cassette (PE) survived. (B) FACS sorting of PE positive and negative cells based upon GFP expression. N33 cells were used as a negative control for gate setup during separation of PE negative cells. The lower panel in the figure shows histogram representation of the upper panel. (C) FACS sorted PE positive (PE + ve) and PE negative (PE—ve) cells were separately grown and analyzed for GFP expression by fluorescence microscopy. (D) GFP expression was determined by Western blot analysis using GFP antibodies. Upper panel in the Western blot shows the absence of GFP expression in the PE—ve cells. β-ACTIN protein was used as a loading control in the lower panel. (E) PCR analysis was performed to confirm the presence of the PE cassette integration in DNA isolated from PE negative cells using PRS4 sequence specific primers (upper panel). PCR primers designed in the promoter region of Gapdh were used as an equal loading control for PCR products (lower panel). Chromatin isolated from normal N33 or PE positive cells were used as negative and positive controls, respectively. Scale bars, 100 μm.
Fig 4.
PAX2-LSD1/NuRD complexes are recruited on the Pax2 binding elements.
ChIP experiments were performed on PE negative cells using a variety of antibodies as indicated in each graph. Primer pairs flanking to the Pax2 response element were used for real-time quantitative PCR and relative amounts of PCR products are expressed as a percent of input chromatin. Equal concentrations of rabbit or mouse IgG were used as negative controls for respective ChIP experiments. All values are expressed as the mean of 3 replicates; error bars indicate one standard deviation. Statistically significant differences are indicated (*P<0.05).
Fig 5.
Blockade of LSD1/NuRD activity derepresses Egfp expression in PE negative cells.
(A) PE negative cells were treated with DMSO (control) or 0.25 mM TCP for 48 hours and DIC and fluorescence images were captured. (B) PE negative cells were treated with DMSO (control) or TCP (48 hours) to inhibit LSD1. ChIP analyses were performed using H3K4 me1, me2 or me3 methyl marks specific antibodies as indicated. Rabbit IgG was used as a negative control. The immunoprecipitated chromatin was quantified by quantitative PCR using primers flanking to the Pax2 response element. The relative amounts of PCR products are expressed as a percent of input chromatin. All values are expressed as the mean of 3 replicates; error bars are one standard deviation. Statistically significant differences are indicated (*P<0.05). (C) PE negative cells were treated with DMSO (control), TSA, and TCP or alternatively differentiated by temperature induction for 48 hours followed by Western blot analysis of GFP protein expression. β-ACTIN was used as a protein loading control. (D) Schematic figure showing how GFP expression is activated in PE N33 cells upon differentiation.
Fig 6.
PAX2/LSD1/NuRD complex suppresses neuronal differentiation of N33 cells.
(A, B) N33 cells were cultured under proliferative conditions. Cells were washed once the following day with PBS and treated with DMSO (control) or TSA, LSD1-C12 for 48 hours in the absence of gamma interferon. Quantitative real-time RT-PCR analysis was used to examine changes in the mRNA expression of the proneural markers NeuroD and Ngn1, the migrating neuroblast marker Phox2b and the inner ear sensory neural markers Tlx3 and Brn3a. Expression levels were normalized to those of the housekeeping gene L27. All values are expressed as the mean of 3 replicates; error bars indicate one standard deviation. Statistically significant differences are indicated (*P<0.05). (C) N33 cells were cultured and treated under similar conditions to those described in A and B. Cells were washed once with PBS and fixed in 4% PFA at room temperature. Immunostaining was performed for analysis of NeuroD protein expression. Nuclei were counter stained using DAPI (blue). Scale bar, 100 μm. (D) N33 cells were treated with DMSO control or TSA for 48 or 72 hours. Western blot analysis was performed to confirm a significant up-regulation of NeuroD. β-ACTIN was used as a loading control. Histone 3 Acetyl mark specific (Ac-H3) antibody was used to show an increase in acetylation, as a positive control to TSA treatment.
Fig 7.
A model of LSD1-mediated epigenetic gene regulation in otic progenitors.
Schematic illustration of the association of LSD1 with Pax2 and the NuRD repressor complex and recruitment of this complex onto Pax2 responsive genes leading to transcriptional repression in otic progenitors prior to differentiation. Inhibition of LSD activity results in an increase in H3K4 methylation, thereby triggering transcriptional activation of Pax2 downstream target genes. Activation of prosensory genes leads to neurosensory fate specification and differentiation in the inner ear.