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. 2015 Jul 6;4:e06942. doi: 10.7554/eLife.06942

Figure 1. DNA adenine methyltransferase identification (DamID) identifies a robust set of NKX2-5 targets in HL-1 cardiomyocytes.

(A) Structure of the human NKX2-5 protein (TN, tinman domain; NK2SD, NK-2 specific domain; YRD, tyrosine-rich domain). Bars and arrows indicate missense and termination mutations associated with congenital heart disease (CHD), respectively. (B) Top over-represented motifs discovered de novo in NKX2-5 peaks using Trawler or Weeder. NKX2-5, GATA, and Nuclear Factor 1 (NF1) binding motifs deposited in TRANSFAC are shown. (C) Distribution of NKX2-5, GATA, and NF1 binding sequences in NKX2-5 peaks. (D) Yeast-two-hybrid assay. NKX2-5 and NF1 proteins were fused to Gal4-activation and DNA-binding domains, respectively. Positive signs (+) show interaction as growth on selective medium from three independent experiments. (E) Normalized median expression of NKX2-5-target genes in 91 murine cell types (data collected from BioGPS). Tissues with the highest median expressions are shown in colour, including heart (red). (F) Expression of NKX2-5 target genes and random genes in HL-1 cells. Data collected from (Mace et al., 2009).

DOI: http://dx.doi.org/10.7554/eLife.06942.003

Figure 1.

Figure 1—figure supplement 1. DamID validation in HL-1 cardiomyocytes.

Figure 1—figure supplement 1.

(A) Expression of V5-tagged DNA adenine methyltransferase (Dam) and NKX2-5-Dam in HEK Ecr-293 cells in presence of 10 mM Ponasterone A detected by immunofluorescence microscopy using anti-V5 antibodies. (B) Expression of Dam alone (‘D’) and Dam/NKX2-5 fusion proteins (WT, ∆HD, and Y191C) in HEK Ecr-293 cells in absence (−) or presence (+) of 5 mM Ponasterone A detected by western blotting using anti-NKX2-5 antibodies.
DOI: http://dx.doi.org/10.7554/eLife.06942.004

Figure 1—figure supplement 2. Expression of PGK-GFP control 24 hr post-transduction of HL-1 cells transduced with lentivirus (LV).

Figure 1—figure supplement 2.

DOI: http://dx.doi.org/10.7554/eLife.06942.005

Figure 1—figure supplement 3. PCR-amplified methylated fragments of HL-1 genomic DNA 40 hr post-transduction with Dam alone and Dam-NKX2-5.

Figure 1—figure supplement 3.

Controls are ‘not transduced’ cells, or cells transduced with empty lentivirus (‘LV’), or with Dam alone and amplified with ‘no ligase’ or ‘no DpnI’.
DOI: http://dx.doi.org/10.7554/eLife.06942.006

Figure 1—figure supplement 4. False discovery rate of Dam/TF fusion protein binding peaks as determined using CisGenome/TileMapv2 with moving average ≥ 3.5.

Figure 1—figure supplement 4.

DOI: http://dx.doi.org/10.7554/eLife.06942.007

Figure 1—figure supplement 5. Chromatin immunoprecipitation (ChIP)-PCR validation of NKX2-5 WT target peakes determined by DamID.

Figure 1—figure supplement 5.

DOI: http://dx.doi.org/10.7554/eLife.06942.008

Figure 1—figure supplement 6. NKX2-5 binds the NKE but not the NF1-like motif.

Figure 1—figure supplement 6.

(A) Cell-free expression of NKX2-5 WT, NKX2-5Y191C, and NKX2-5∆HD proteins revealed by western-blotting using anti-V5 antibodies. (B) Electrophoretic mobility shift assay using NKX2-5 and DIG-labelled NKE (lanes 1–7) and NF-1 oligos (lanes 8–9). Controls include no protein (lane 1), supershifts after addition of anti-V5 (lane 3) or anti-NKX2-5 (lane 4) antibodies and competition with unlabelled oligos (lane 5). See ‘Material and methods’ for details.
DOI: http://dx.doi.org/10.7554/eLife.06942.009

Figure 1—figure supplement 7. Identification of NKX2-5, SRF, and ELK1/4 target genes in HL-1 cells.

Figure 1—figure supplement 7.

UCSC Genome Browser screen shots showing DamID TF association in HL-1 cells with known NKX2-5 target genes (Myocd [Ueyama et al., 2003], Actc1 [Chen and Schwartz, 1997], and Gata4 [Riazi et al., 2009]).
DOI: http://dx.doi.org/10.7554/eLife.06942.010

Figure 1—figure supplement 8. Nuclear localisation of NKX2-5 and histone modifications in HL-1 cardiomyocyte nuclei.

Figure 1—figure supplement 8.

Scale bars represent 10 mm.
DOI: http://dx.doi.org/10.7554/eLife.06942.011

Figure 1—figure supplement 9. Proportional Venn diagram showing the overlapping binding peaks between NKX2-5 determined by DamID (this study) or ChIP-seq (He et al., 2011 ) and (van den Boogaard et al., 2012).

Figure 1—figure supplement 9.

Only peaks that fall in the regions covered by the Affymetrix mouse promoter microarrays are represented.
DOI: http://dx.doi.org/10.7554/eLife.06942.012