Author response: NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets (2015)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease. https://doi.org/10.7554/eLife.06942.001 eLife digest Many genes working within large gene networks influence the development of heart muscle cells in humans and other animals. The activity of these genes is controlled in part by proteins called transcription factors, which bind to DNA and act as molecular switches. One transcription factor that is particularly important for the development of heart muscle cells is called NKX2-5. Mice lacking NKX2-5 have abnormal hearts and many humans who are born with congenital heart disease carry mutations in the gene that encodes this protein. Many of these mutations alter a section of the protein called the homeodomain, and therefore interfere with the ability of NKX2-5 to bind to DNA or associate with other important cardiac proteins called cofactors. However, it is not clear how such mutations alter the behaviour of NKX2-5 across all of its targets. Bouveret et al. have now used a technique called ‘DNA adenine methyltransferase identification’ to study how NKX2-5 interacts with other proteins and DNA. The experiments found that, as expected, the mutant NKX2-5 proteins were unable to associate with many of the usual gene and protein targets of normal NKX2-5. However, the mutant proteins were still able to bind to some of their usual targets, plus many other targets that the normal NKX2-5 protein was not able to bind to. A particular NKX2-5 mutant protein that the experiments analysed was missing the entire homeodomain, yet it was still able to associate with the normal NKX2-5 protein and bind to cofactors that help NKX2-5 to find its usual targets. This finding led Bouveret et al. to discover the role of a section of the NKX2-5 protein called the tyrosine-rich domain, which in the absence of the homeodomain can direct interactions of NKX2-5 with itself and its cofactors. Bouveret et al.'s findings suggest that protein cofactors of NKX2-5 help mutant NKX2-5 proteins retain some of their normal activities, but also direct the mutant proteins to abnormal gene targets, which could contribute to congenital heart disease. The next steps are to carry out experiments in animals to confirm these findings, and to understand the activities of mutant NKX2-5 and other mutant transcription factors across the whole genome. This could lead to new therapeutic approaches to treat congenital heart disease and other conditions. https://doi.org/10.7554/eLife.06942.002 Introduction The mammalian heart is a highly modified muscular vessel whose lineage programmes are governed by conserved gene regulatory networks (GRNs) (Davidson and Erwin, 2006). The cardiomyocyte GRN is controlled by lineage-restricted transcription factors (TFs), which interact to form a recursively wired sub-network termed the cardiac ‘kernel’ (Davidson and Erwin, 2006). Kernel TFs, such as NKX2-5, GATA4, TBX5/20, and serum response factor (SRF), show regionally restricted expression and act as selector proteins that help define developmental and organ-specific territories. A well-studied kernel TF is NKX2-5, a member of the NK-2 homeodomain (HD) factor subclass that plays an early role in development of hearts and heart-like organs of diverse species (Elliott et al., 2010). Loss of Nkx2-5 in mice leads to arrested heart morphogenesis due to blocked progenitor growth, defective chamber and conduction system development, and a deranged GRN (Prall et al., 2007). In humans, NKX2-5 is one of the most commonly mutated single genes in congenital heart disease (CHD), with dominant alleles causing atrial septal defect and atrioventricular conduction block most commonly, and a host of more severe defects at lower penetrance (Elliott et al., 2010). Many CHD-causing NKX2-5 mutations are located within the conserved HD (Figure 1A), which serves as both a sequence-specific DNA-binding domain and protein-binding interface for interactions with other kernel TFs (Elliott et al., 2010). It is widely assumed that CHD is caused by haploinsufficiency and an inability of mutant proteins to recognise target genes. However, we know little about disease causation at the genome level, and in fact, most CHD mutations lie outside of the HD (Figure 1A), often in conserved domains with largely unknown functions. Figure 1 with 9 supplements see all Download asset Open asset 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). https://doi.org/10.7554/eLife.06942.003 Here, we take a functional genomics approach to understanding the mechanism of NKX2-5 CHD at the chromatin level. We applied the technique of DNA adenine methyltransferase identification (DamID) (Vogel et al., 2006) to identify target genes of NKX2-5 wild type and NKX2-5 mutant proteins mimicking those found in patients with CHD. While binding of severe NKX2-5 mutants to targets was compromised, they nonetheless associated with hundreds of targets, including many normally bound by NKX2-5 wild type, and could regulate a subset of these in cellular assays. We demonstrate that severe NKX2-5 mutant proteins retained an ability to interact with NKX2-5 wild type and other cardiac TFs via a novel tyrosine-rich protein:protein interface in NKX2-5 that lies outside of the HD. NKX2-5 mutant proteins also bound hundreds of ‘off-targets’ not bound by NKX2-5 wild type, via altered DNA-recognition or via interactions with previously unrecognised cofactors. These cofactors, which include members of the ETS family, are embedded in the cardiac GRN, indicating a broad role for ubiquitous TFs in cardiac network logic and mechanism of CHD. Results DamID identification of NKX2-5 WT targets DamID is a sensitive enzymatic method developed to identify protein:DNA interactions (van Steensel and Henikoff, 2000; van Steensel et al., 2001). It is complementary to chromatin immunoprecipitation (ChIP) but avoids artifacts associated with chromatin crosslinking and poor-quality ChIP antibodies (Waldminghaus and Skarstad, 2010; Teytelman et al., 2013). DamID involves creation of a fusion between a chromatin-binding protein of interest and Escherichia coli DNA adenine methyltransferase (Dam). When expressed, Dam fusions bind to DNA, whereby Dam locally methylates adenine within its target sites (5′GATC3′). Target DNA can then be released using DpnI, a nuclease specific for the methylated Dam site, and analysed on arrays (Vogel et al., 2006). Here, we used DamID in HL-1 cells, which resemble atrial cardiomyocytes, to profile NKX2-5 WT and mutants that mimic those found in CHD (Claycomb et al., 1998). Mouse Nkx2-5 cDNAs were cloned into a lentiviral Dam vector carrying heat shock and ponasterone A-inducible promoters (Vogel et al., 2006). Only N-terminal Dam fusions (Dam-NKX2-5) were analysed because C-terminal fusions were sterically compromised (data not shown). Ponasterone A induction after viral transduction of HEK Ecr-293 cells confirmed nuclear localisation and the correct size of fusions (Figure 1—figure supplement 1). Transduction of HL-1 cells was efficient (∼97%; Figure 1—figure supplement 2). DamID fusions were expressed from the uninduced heat shock protein 68 promoter at very low levels (undetectable by western blotting), which was sufficient for Dam enzymatic activity on chromatin (Figure 1—figure supplement 3) but avoided skewing of the network. DamID is dependent on the density of DpnI sites (5′GATC3′), which occur on average every 260 bp in the mouse genome, as in the fly genome (van Steensel and Henikoff, 2000). After DpnI digestion of virus-transduced HL-1 cell DNA, PCR-amplification lead to fragments of ∼200-2000 bp (Figure 1—figure supplement 3). We determined that of all perfect NKX2-5 high-affinity binding sites (NKE; 5′AAGTG3′) in the mouse genome, 90% had a DpnI site within 1 kb upstream and downstream (median of ∼289 bp), suggesting that DamID captures the vast majority of NKX2-5 direct targets. Methylated genomic fragments were hybridised to Affymetrix Mouse promoter microarrays in biological triplicate. These microarrays represent 7.3% of the mouse genome, and we estimate from published NKX2-5 ChIPseq data that they would capture 20–30% of all NKX2-5 peaks (He et al., 2011; van den Boogaard et al., 2012). DamID peaks were determined using CisGenome after subtraction of signal obtained from cells expressing Dam alone (Ji et al., 2008) (see Supplementary file 1, and UCSC genome browser http://genome.ucsc.edu/cgi-bin/hgTracks?db=mm9&type=bed&hgt.customText=ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE44nnn/GSE44902/suppl/GSE44902_DamID.bed.gz [Kent et al., 2002]). For NKX2-5 WT, we identified 1524 peaks, which displayed low-false discovery rates (<0.08; Figure 1—figure supplement 4). ChIP-PCR confirmed NKX2-5 occupancy over NKX2-5 DamID peaks for 10 out of 11 targets tested, while DamID-negative regions were not occupied (Figure 1—figure supplement 5). De novo motif discovery using Weeder or Trawler (Pavesi and Pesole, 2006; Haudry et al., 2010) revealed that the top 3 over-represented motifs were 5′AAGTG3′, identical to the NKE; 5′GATAA3′, identical to the GATA-binding motif; and 5′TGCCAA3′, similar to the binding motif of Nuclear Factor 1 (NF1) (Figure 1B). After counting of their exact sequences, the NKE was present in 79% of NKX2-5 peaks, with half of these bearing over 2 and up to 12 NKEs. The GATA- and NF1-binding motifs were present in 59% and 36% of peaks, respectively (Figure 1C). These results suggest that the majority of NKX2-5 WT targets are directly bound via the NKE and also support the combinatorial action of NKX2-5 with GATA factors on many targets (Durocher et al., 1997; He et al., 2011). NKX2-5 did not bind directly to the NF1-like site in vitro (Figure 1—figure supplement 6), suggesting that NKX2-5 could act in combination with NF1 in HL-1 cells. Using a yeast two-hybrid (Y2H) assay, we showed that NKX2-5 interacted with NF1-B1 and NF1-B3 but not NF1-A or NF1-X (Figure 1D), as for the related family members NKX2-1 and NF1 in lung (Bachurski et al., 2003). This suggests that NF1-B1/3 regulate a subset of target genes in combination with NKX2-5 in cardiomyocytes. Using GREAT (McLean et al., 2010), NKX2-5 peaks were assigned to 1490 unique genes, including previously identified NKX2-5 target genes (Figure 1—figure supplement 7). NKX2-5 also bound its own promoter, potentially reflecting auto-regulation (Prall et al., 2007). Gene Ontology (GO) analysis of DamID NKX2-5 targets showed that the most enriched biological processes were heart development and muscle contraction (Supplementary file 1), attesting to the specificity of DamID. The most represented words amongst the top 50 GO terms, were muscle, development, regulation, cardiac, and cell. Other enriched processes included cytoskeleton organisation and related terms, and metabolic GO terms such as regulation of cellular carbohydrate catabolic process, regulation of glycolysis, and regulation of generation of precursor metabolites and energy, indicating that NKX2-5 exerts high-level network control over metabolic processes. In silico analysis using the BioGPS repository of 91 tissue transcriptomes revealed that the median expression level of NKX2-5 target genes was highest in adult hearts (Figure 1E). Transcriptome data for HL-1 cells (Mace et al., 2009) showed that NKX2-5 targets were strongly skewed towards higher expression values compared to genes randomly selected from the array, although some genes were expressed at lower levels (Figure 1F). We note from immunofluorescence studies that the most intense NKX2-5 staining co-localised in speckle-like nuclear foci with active histone marks H3K9Ac and H3K4me3, but not with repressive mark H3K27me3 (Figure 1—figure supplement 8). Probing the mechanisms of CHD using DamID Having established a robust NKX2-5 WT target list, we set out to probe the mechanisms of CHD by identifying the genome-wide targets of NKX2-5 mutant proteins. We first constructed N-terminal Dam fusions for the human CHD mutation NKX2-5Y191C (Benson et al., 1999), which disrupts tyrosine 54 of the HD, necessary for the unique binding site specificity of the NK-2 homeoprotein sub-class to the NKE (Figure 2A). In vitro, NKX2-5Y191C dimerises normally with co-factors but shows 80-fold reduced binding to the NKE (Kasahara et al., 2000; Kasahara and Benson, 2004). We also created NKX2-5∆HD, in which the HD was deleted and replaced by a glycine-linker. We anticipated that NKX2-5∆HD would be functionally dead, since the HD has been shown to serve as both DNA-binding domain and interface for homo- and hetero-dimerisation (Elliott et al., 2010). However, we were interested in testing whether the presence of other conserved domains in NKX2-5 and often mutated in NKX2-5-related CHD (Figure 2A) could confer any functionality to the NKX2-5 mutants. Both mutants were stable (Figure 1—figure supplements 1, 6) and, as anticipated, did not bind efficiently to the NKE of the known target Nppa (natriuretic peptide precursor A) (Durocher et al., 1996) in vitro (Figure 1—figure supplement 6). Figure 2 with 3 supplements see all Download asset Open asset NKX2-5 mutants bind to hundreds of targets in HL-1 cells. (A) Structure of NKX2-5Y191C and NKX2-5∆HD mutant proteins (‘G’ indicates a Glycine linker). (B) Overlapping binding peaks between NKX2-5 WT and mutants. Proportional Venn diagrams show peaks unique to WT NKX2-5 (A sets); common to WT and mutant proteins (B sets); unique to mutant proteins (C sets). (C) Representative autocorrelation function (ACF) curves and fits for NKX2-5 WT and mutant proteins measured by photoactivatable fluorescence correlation spectroscopy (paFCS) in HL-1 cells. (D) Top over-represented motifs discovered de novo using Weeder in A-, B- and C-target sets of NKX2-5 WT and mutant comparisons. https://doi.org/10.7554/eLife.06942.013 Surprisingly, in HL-1 cells, both NKX2-5Y191C and NKX2-5∆HD associated with a large number of loci (1149 and 792 peaks, respectively; Figure 2B; Supplementary file 1) with low false discovery rates (Figure 1—figure supplement 4). Even though NKX2-5Y191C cannot bind DNA, the most over-represented motifs were 5′AAGTGT3′ (NKE), 5′GATAA3′ (GATA), and 5′TGCCAA3′ (NF1-like), exactly as for NKX2-5 WT fusion (Supplementary file 1). NKX2-5Y191C peaks also showed over-representation of the motif 5′TAATC3′, which is similar to the binding sites of many non-NK-2 class HD proteins, including HOX proteins, as well as that of NK-2 proteins that lack HD tyrosine 54, such as NKX1-2 (Berger et al., 2008). NK-2 class proteins which do carry Y54 in their HDs, including NKX2-5, also bind to this HOX-like site, albeit with a 10-fold-reduced affinity compared to that of the NKE (Chen and Schwartz, 1995). For NKX2-5∆HD peaks, there were many over-represented motifs, although none resembled known cardiac TF-binding sites (Supplementary file 1). The ability of NKX2-5Y191C and NKX2-5∆HD to bind a large number of targets was unexpected. Hence we sought to confirm using an independent approach that these mutants could interact with chromatin in vivo. We employed photoactivatable fluorescence correlation spectroscopy (paFCS) (Kaur et al., 2013), measuring the diffusion dynamics of NKX2-5 WT and mutants within individual HL-1 nuclei (Figure 2—figure supplement 1A). Expression vectors encoding NKX2-5 proteins fused to photoactivatable GFP (paGFP) were introduced by transfection. As for other TFs, NKX2-5 WT showed a biphasic behaviour, with a freely diffusing fraction similar to paGFP (adjusted for molecular mass) and a slower, chromatin-interacting fraction. NKX2-5 WT displayed slower diffusion (dwell time τDslow = 121 ms) than that mutants (NKX2-5Y191C: τDslow = 64 ms; NKX2-5ΔHD: τDslow = 11 ms), which diffused more slowly than paGFP alone (Figure 2C, Figure 2—figure supplement 1B-D). This confirmed that NKX2-5Y191C and NKX2-5ΔHD physically interact with chromatin (Kaur et al., 2013) and suggests that a gradation in chromatin tethering correlates with the apparent severity of the NKX2-5 mutation (WT>Y191C>ΔHD>paGFP). NKX2-5 mutant targets overlap partially with NKX2-5 WT targets We compared NKX2-5 WT and mutant peaks and adopted a simple nomenclature (A, B, and C) for peak subsets, where A is bound by NKX2-5 WT only, B is bound by both WT and a mutant, and C is bound only by a mutant (Figure 2B). In both comparisons, the A sets predominated (81% and 53% of NKX2-5 WT peaks in the NKX2-5 WT/∆HD and NKX2-5 WT/Y191C comparisons, respectively), and their most enriched motif determined de novo was the NKE (Figure 2D), demonstrating that, as anticipated, NKX2-5 mutants failed to bind to most WT targets, including Nppa. Therefore, the HD mutations result in a severe loss of function. However, a high proportion of mutant peaks overlapped with NKX2-5 WT peaks (B sets; 36% of 792 NKX2-5∆HD peaks and 63% of 1149 NKX2-5Y191C peaks). To investigate the A- and B-set logic further, we used Clover (Frith et al., 2004) to calculate the enrichment of known TF motifs present in TRANSFAC (BIOBASE) and JASPAR databases (Bryne et al., 2008) in A and B sets (Figure 2—figure supplement 2). The NKE was enriched in both A and B that A and B sets direct NKX2-5 targets. A- and also enrichment for many known TF motifs, for those for cardiac kernel TFs GATA4, and were only enriched in the B-set of the NKX2-5 WT/∆HD both mutants can bind a subset of direct WT targets crippled DNA This via with NKX2-5 WT its cofactors (see and et al., for NKX2-5∆HD, which lacks a DNA-binding domain, data suggest that a protein:protein interface within NKX2-5 that is from the HD can the mutant protein to NKX2-5 WT targets. a GO level, the A- and were also (Figure 2—figure supplement Supplementary file 1). For the the showed enrichment for GO terms and the B-set was enriched in related to heart For the WT/∆HD the overlap in GO terms between the and B-set was also very with the enriched in terms related to cellular such as organisation and and the B-set enriched in related to heart and vessel This suggests that NKX2-5 mutants associate with genes in cardiac related processes. of NKX2-5 mutants on the DamID and data an between NKX2-5 mutants and we the functionality of NKX2-5 mutants in of DNA binding 3 as a B set gene bound by NKX2-5 WT, and for analysis (Figure encodes a factor that in heart progenitor cells and is as and levels of NKX2-5 et al., 2006). Using we confirmed data that is in Nkx2-5 mouse mutant similar to other cardiac progenitor genes (Prall et al., a known directly NKX2-5 is (Figure supplement 1). The downstream cloned into a vector in and into HEK cells, transcription was a by (Figure supplement 1), which DamID also bound to this (Figure and NKX2-5 WT, and both the and activity (Figure suggesting that NKX2-5 mutants retain some WT functionality as of Figure 3 with 2 supplements see all Download asset Open asset NKX2-5 mutant proteins retained partial functionality. (A) UCSC DamID transcription factor in HL-1 cells with of DNA binding 3 (B) activity in HEK cells with NKX2-5 and serum response factor was in the were to = not or alone The of vector is in (C) of and for the generation of cell for expression of NKX2-5 WT and mutants. was in by induction of on (D) of in NKX2-5 cell 5). The of independent experiments is shown. of (D) and cells (E) at of A- and B-set target genes in NKX2-5 WT or cell at in three independent experiments Results were to expression in uninduced cells. were using a To functionality of NKX2-5 mutants in a developmental we mouse cells in which expression of NKX2-5 WT or mutants could be by of a ubiquitous promoter et al., 2011). induction of NKX2-5 WT, and proteins were in most or all cells with at 1 NKX2-5 in a of cells within (Figure levels of NKX2-5 WT and mutant and protein were (Figure supplement 2). NKX2-5 WT strongly of at (Figure and of and cells at (Figure of cardiac by NKX2-5 in cells is with its early role as a of cardiac induction and heart gene expression (Prall et al., and as an of of cardiac to a cardiomyocyte et al., 2010). was nuclear in only of cells at due to of the nuclear localisation signal within the Figure supplement and had on cardiac or cell (Figure and was not NKX2-5Y191C was nuclear and, while on or cells in three independent showed a towards of cardiomyocyte at = suggesting that it also retained some repressive activity in this assay. To into the we therefore measured in gene expression in the cell at in three independent testing a of A and B-set genes from the The majority of both A and B set genes were by NKX2-5 WT that DamID NKX2-5 WT developmental targets. B-set genes, most were by NKX2-5 WT, with the of by NKX2-5 WT at early of cardiac lineage (Prall et al., 2007). A subset of A and B set genes was also by NKX2-5Y191C Figure in early in the NKX2-5Y191C with regulation by in the as for NKX2-5 As and were by both NKX2-5 WT and while Nppa and were by These data show that while NKX2-5Y191C its ability to bind and regulate most normal NKX2-5 targets, it nonetheless some of the regulatory of NKX2-5 interface for NKX2-5 Our results that the NKE is enriched in both suggest that NKX2-5 mutants can bind targets by with NKX2-5 While to NKX2-5 WT has been for NKX2-5Y191C (Kasahara and Benson, it was for NKX2-5ΔHD because the HD has been previously as for NKX2-5 (Elliott et al., 2010). NKX2-5∆HD was largely in cells (Figure which lack NKX2-5. This is a of the of the NKX2-5 nuclear localisation signal located in the of the HD (Kasahara and However, NKX2-5∆HD nuclear with NKX2-5 WT, which itself was nuclear alone (Figure GATA4, and which have been to bind NKX2-5 WT via the HD also nuclear of NKX2-5∆HD (Figure supplement 1). In HL-1 cells, which NKX2-5 and its cardiac cofactors, NKX2-5∆HD was nuclear although some protein was present in the (Figure These data demonstrate that NKX2-5 WT and its cofactors are able to carry NKX2-5∆HD into the via an unknown protein:protein interaction not the HD. Figure with 3 supplements see all Download asset Open asset The YRD is for interaction between NKX2-5 and (A) localisation of NKX2-5ΔHD and NKX2-5 cells were with NKX2-5ΔHD only NKX2-5 WT only or both and the cellular and nuclear respectively. (B) localisation of NKX2-5 WT and NKX2-5ΔHD in HL-1 cells. (C) NKX2-5 homo- and hetero-dimerisation measured by in HEK cells. and represent the and C-terminal respectively. Data are represented as of the activity was using an (D) NKX2-5 homo- and hetero-dimerisation measured by and represent the and is represented as were using a between and its is as for for for values are (E) Representative using up used in (F) Yeast-two-hybrid assay. were fused to Gal4-activation and DNA-binding