Transcription factor 21 (TCF21), also known as pod-1, capsuling, or epicardin, is a protein that in humans is encoded by the TCF21 gene on chromosome 6. It is ubiquitously expressed in many tissues and cell types and highly significantly expressed in lung and placenta. TCF21 is crucial for the development of a number of cell types during embryogenesis of the heart, lung, kidney, and spleen. TCF21 is also deregulated in several types of cancers, and thus known to function as a tumor suppressor. The TCF21 gene also contains one of 27 SNPs associated with increased risk of coronary artery disease.
Discovery TCF21 was discovered in 1998 when search for novel cell-type-specific bHLH proteins expressed in human and mouse kidneys by performing a search of the expressed sequence tag (EST) databases. Because the transcript they found was highly expressed in visceral glomerular epithelial cells (podocytes), TCF21 was initialled named Pod-1. Comparison of Pod-1 with previously characterized bHLH proteins identified Pod-1 as a novel member of a subfamily of bHLH proteins with important roles in mesodermal development. The chromosomal location of Pod-1 in the mouse was then determined using an interspecific backcross panel along with genomic southern blot analysis to identify restriction fragment length polymorphisms (RFLPs) between inbred mouse strains. Analysis showed Pod-1 to map to a region of mouse chromosome 10 that is syntenic with human chromosome 6q23-q24.
The tissue distribution of Pod-1 was determined by hybridization of a human multiple tissue northern blot with a Pod-1 cDNA. A probe lacking the bHLH domain was used to minimize cross-reactivity along with high stringency hybridization and washing. Results showed that in humans and mice, Pod-1 was most highly expressed in the kidney, lung and heart, with selective expression at sites of epithelial-mesenchymal interaction in the kidney, lung, intestine and pancreas of developing mouse embryos. RNA in situ hybridization using 33P-labeled riboprobes was used to identify the cell types that expressed Pod-1 in the developing kidney and other tissues. This revealed Pod-1 expression in mesenchymal cells and podocytes, with expression coinciding with the onset of podocyte differentiation. It was found that expression of Pod-1 in embryonic kidney explants was inhibited through antisense oligonucleotides. This inhibition resulted in decreased mesenchymal cell condensation around the ureteric bud and a significant decrease in ureteric branching. Pod-1 was the first tissue-restricted bHLH protein to be identified in the developing kidney and tied to regulation of morphogenetic events.
In an effort to identify novel bHLH factors related to dHAND and eHAND (a novel subclass of cell type-restricted bHLH factors shown to play important roles in cardiac morphogenesis), they screened expressed sequence tag (EST) databases for sequences with homology to the bHLH regions of these factors. The novel bHLH protein they identified in their search was also Pod-1, but they used the name capsulin.
Whole-mount in situ hybridization of Capsulin transcripts were used to define sites of expression, which showed to be specific to mesodermal precursor cells that surround the epithelium of the developing gastrointestinal, genitourinary and respiratory systems during mouse embryogenesis. Expression patterns of capsulin mRNA in adult mouse tissues by Northern blot detected highest levels in the lungs, with lower levels in kidneys, heart and spleen. Capsulin transcripts were also found to mark the spiral septum of the heart and progenitor cells that give rise to the pericardium and coronary arteries.
Capsulin was translated in a rabbit reticulocyte lysate in the presence and absence of the widely expressed bHLH protein E12 and performed gel mobility shift assays with several E-box sequences as probes to test the protein's DNA binding activity. Capsulin alone failed to bind any of the sequences tested. However, in the presence of E12 plus capsulin, a DNA complex was generated with the probe that migrated faster than the E12 homodimeric complex alone, representing the binding of capsulin/E12 heterodimers. It was concluded that capsulin heterodimerizes with E12 and binds the specific E-box consensus sequence (CANNTG), though not activating transcription through this sequence on its own. Its restricted expression pattern and DNA binding activity identified Capsulin as a regulator of gene expression in specific subtypes of visceral mesodermal cells involved in organogenesis and in precursor cells that contribute to the pericardium, coronary arteries and regions of the heart.
Structure Gene The TCF21 gene resides on chromosome 6 at the band 6q23.2 and includes 3 exons. These three exons are associated with CpG islands CGI1, CGI2, and CGI3. DNA methylation analysis revealed hypermethylation at CGI1 and CGI3, but not CGI2 in samples from various cancer tissues. Luciferase reporter assays with constructs covering CGI3 sequences in sense and antisense orientation demonstrated that CGI3 harbors a promoter that directs the synthesis of a previously unknown long non-coding RNAs (lncRNAs) in antisense orientation to TCF21. This lncRNA has been named TARID (for TCF21 antisense RNA inducing demethylation).
Protein TCF21 is a member of the bHLH (basic helix-loop-helix) family of transcription factors. This protein is predicted to span 179 amino acid residues and contains a bHLH domain and an arginine-rich sequence that may facilitate DNA binding.
Function
TCF21 encodes a transcription factor of the basic helix-loop-helix (bHLH) family, which manages cell-fate specification, commitment and differentiation in various cell lineages during development. The TCF21 product is mesoderm-specific and expressed in embryonic epicardium, mesenchyme-derived tissues of lung, gut, gonad, and both mesenchymal and glomerular epithelial cells in the kidney.
TCF21 is essential for regulating properties of the mesenchyme that are critically important for several aspects of lung and kidney morphogenesis. TCF21 is also essential for cardiac fibroblast cell fate, as demonstrated by the failed development of cardiac fibroblasts in mice lacking TCF21. In the absence of TCF21, these fibroblast progenitor cells do not undergo epithelial-to-mesenchymal transition (EMT). While TCF21-expressing epicardial cells are initially multipotent, they become committed to the fibroblast lineage over time. Those TCF21-expressing cells that do not commit to the fibroblast lineage lose this expression and remain undifferentiated epicardial cells or coronary vascular smooth muscle cells. TCF21 is expressed in mesodermal cells in the proepicardial organ that give rise to coronary artery smooth muscle cells (SMC) and loss of TCF21 results in increased expression of smooth muscle markers by cells on the heart surface consistent with premature SMC differentiation. This suggests that early expression of TCF21 is important for expansion of the SMC compartment of the coronary circulation, with persistent TCF21 expression being required for cardiac fibroblast development.
TCF21 activation is directed by an antisense long non-coding RNA, TARID (TCF21 antisense RNA inducing demethylation). TARID activates TCF21 expression by inducing promoter demethylation and affects expression levels of target genes by functioning as epigenetic regulators of chromatin structure through interactions with histone modifiers, chromatin remodeling complexes, transcriptional regulators, and/or DNA methylation machinery.
Role in development Since the identification of Tcf21 significance in various cell lineages, further research has expanded understanding of the essential roles of this gene.
TCF21 is essential for regulating properties of the mesenchyme that are critically important for several aspects of lung and kidney morphogenesis. Null TCF21 mutant mice are born but die shortly after due to severely hypoplastic lungs and kidneys that lack alveoli and mature glomeruli. While TCF21 is exclusively expressed in the mesenchyme and podocytes, major defects are observed in adjacent epithelia of TCF21 mutant mice. In the kidney, TCF21 is required for conversion of condensing mesenchyme to epithelium of the nephron, branching morphogenesis and terminal differentiation of tubular epithelium. In the lung, TCF21 is required to correctly pattern the proximodistal axis of airway epithelium and for normal branching to occur.
TCF21 null mice also fail to form a spleen, as TCF21 acts after splenic specification to control morphogenetic expansion of the splenic anlage and in its absence, splenic precursor cells undergo apoptosis. Since this splenic phenotype resembles that of mice lacking the homeobox genes Hox11 and Bapx1, it is possible that TCF21, together with Hox11, and Bapx1 control a common essential early step in the developmental pathway for spleen organogenesis.
TCF21 is essential for cardiac fibroblast cell fate, as demonstrated by the failed development of cardiac fibroblasts in mice lacking TCF21. In the absence of TCF21, these fibroblast progenitor cells do not undergo epithelial-to-mesenchymal transition (EMT). While TCF21-expressing epicardial cells are initially multipotent, they become committed to the fibroblast lineage over time. Those TCF21-expressing cells that do not commit to the fibroblast lineage lose this expression and remain undifferentiated epicardial cells or coronary vascular smooth muscle cells. TCF21 is expressed in mesodermal cells in the proepicardial organ that give rise to coronary artery smooth muscle cells (SMC) and loss of TCF21 results in increased expression of smooth muscle markers by cells on the heart surface consistent with premature SMC differentiation. This suggests that early expression of TCF21 is important for expansion of the SMC compartment of the coronary circulation, with persistent TCF21 expression being required for cardiac fibroblast development.
Male TCF21 knockout mice, which die at birth due to respiratory failure, are reported to have feminized genitalia, implicating TCF21 in mouse gonadal development/differentiation. TCF21 transcriptionally represses steroidogenic factor 1 (Sf1), a gene expression regulator that mediates sexual differentiation and is involved in coordinating cell fate decisions in gonadal progenitors. Without TCF21, normal gonad development is disrupted as a result of ectopic expression of Sf1, which leads to abnormal committing of urogenital progenitor cells to steroidogenic cell fates. In the XY gonad, this disruption in organization contributes to changes in testicular structure and vasculature.
Clinical significance As a cancer suppressor
In humans, TCF21 has been identified as a candidate tumor suppressor gene and is frequently epigenetically silenced in various human cancers.