Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung

Abstract

Background Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes. Methods We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses. Results We identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4. Conclusion Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.

TBX2 controls a proproliferative gene expression program in melanoma

“…Of note, few peaks overlapped between our data sets and that previously published for mouse lungs (Supplemental Fig. S3C; Lüdtke et al 2021), possibly because the genomic distribution of Tbx2 in lungs and melanoma are different, or because immunoprecipitation with anti-Tbx2 antibody may be less specific compared with use of anti-HA antibody. Moreover, because the previous study did not generate replicate ChIPs, we were uncertain of the reproducibility of the Tbx2 binding sites identified.…”

“…Previous work to identify direct TBX2 target genes by ChIP-seq used an antibody against the endogenous protein. For example, using neuroblastoma cells, Decaesteker et al (2018) reported 557 TBX2 binding sites (q < 0.05) in total (41% intergenic; 30% lincRNAs; binding to consensus AGGTGTGA), while a more recent study of embryonic lungs (Lüdtke et al 2021) identified a total of 3062 Tbx2 peaks enriched >3.5-fold over the control, but only 177 were found within 5 kb of transcription start sites (TSSs), and in neither study were replicates used to robustly identify target sites. In contrast, by using high-affinity anti-HA antibody and HA-tagged endogenous Tbx2 in melanoma, we were able to identify up to 7500 binding sites (clone 9, R2) with a robust set of sites identified in common between multiple replicates, of which ∼38% were within 3 kb of the transcription start site.…”

“…Moreover, cooperating transcription factors whose binding is enhanced by interaction with Tbx2 could themselves mediate transcription activation, rather than Tbx2 itself. To date, in addition to the homeodomain transcription factor NKX2.5 (Habets et al 2002), TBX2 has been identified as interacting with HDAC1 (Vance et al 2005), RB1 (Vance et al 2010), a range of transcription factors such as HMGB2 and PBX1 identified by mass spectrometry (Lüdtke et al 2021), EGR1 (Redmond et al 2010), PML (Martin et al 2012), and the heterochromatin proteins CBX (Lüdtke et al 2021) andHP1α (Crawford et al 2019). Tbx2 also was reported to bind components of the NuRD chromatin remodeling complex (Lüdtke et al 2021).…”

“…To date, in addition to the homeodomain transcription factor NKX2.5 (Habets et al 2002), TBX2 has been identified as interacting with HDAC1 (Vance et al 2005), RB1 (Vance et al 2010), a range of transcription factors such as HMGB2 and PBX1 identified by mass spectrometry (Lüdtke et al 2021), EGR1 (Redmond et al 2010), PML (Martin et al 2012), and the heterochromatin proteins CBX (Lüdtke et al 2021) andHP1α (Crawford et al 2019). Tbx2 also was reported to bind components of the NuRD chromatin remodeling complex (Lüdtke et al 2021). Our mass spectrometry analysis identified many of these factors (e.g., HDAC1, HDAC2, CHD4, PBX1, and NKX2.5) but below our stringent 1% FDR statistical cutoff.…”

“…This is consistent with a previous study (Decaesteker et al 2018) significant bound sites (q < 0.05). Of these, only 107 could be converted to mouse genomic locations, none of which overlapped with those identified in a more recent ChIPseq data set from developing mouse lungs (Lüdtke et al 2021). These studies highlight the need for a robust and reproducible approach to Tbx2 ChIP.…”