TCF21

Transcription factor 21 (Tcf21) encodes TCF21, also referred to as Pod-1, Capsulin and/or Epicardin. TCF21 is a member of the class A basic helix-loop-helix (bHLH) family of transcription factors that manage cell-fate specification, commitment and differentiation in various cell lineages during development.[1] TCF21 is crucial for the development of a number of cell types during embryogenesis of the heart,[1] lung,[2] kidney,[2] and spleen.[3] TCF21 is also deregulated in several types of cancers, and thus known to function as a tumor suppressor.[4]

The third CGI of TCF21 contains a promoter that directs transcription of TARID (a lncRNA) in antisense orientation to TCF21. TARID then induces TET protein-dependent DNA demethylation, thus activating TCF21 transcription. This mechanism is regulated by the binding of TARID to the TCF21 promoter, which recruits GADD45A/TDG to direct BER for demethylation.

Discovery/Initial Research

Initial research on TCF21 was published in 1998. Susan E. Quaggin, Gregory B. Vanden Heuvel, and Peter Igarashi set out to identify novel cell-type-specific bHLH proteins expressed in human and mouse kidneys by performing a text search of the expressed sequence tag (EST) databases.[5] Because the transcript they found was highly expressed in visceral glomerular epithelial cells (podocytes), they termed the gene 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.[5] 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.[5]

In this same study, Quaggin et al. further examined the tissue distribution of Pod-1 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.[5] 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.[5] To begin examining the role of Pod-1 in kidney morphogenesis, 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. Together, Quaggin et al. found Pod-1 to be the first tissue-restricted bHLH protein to be identified in the developing kidney and tied to regulation of morphogenetic events.[5]

Two months later, in April 1998, Jianrong Lu, James A. Richardson, and Eric N. Olson published similar research. 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.[6] 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.[6] 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.[6]

Lu et al. translated Capsulin 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.[6] 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.[6] From this, Lu et al. 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.[6]

TCF21 and 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.[2] 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.[2]

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.[3] 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.[3]

TCF21 is essential for cardiac fibroblast cell fate, as demonstrated by the failed development of cardiac fibroblasts in mice lacking TCF21.[1] 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.[1] 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.[7]

Interestingly, 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.[8] 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.[8] 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.[8]

TCF21 as a Cancer Suppressor

TCF21 has been identified as a candidate tumor suppressor gene and is frequently epigenetically silenced in various human cancers.

In 2006, Laura T. Smith et al. applied restriction landmark genomic scanning (RLGS) along a region of recurrent loss of heterozygosity (LOH) at chromosome 6q23-q24 to profile DNA methylation. Smith et al. set out to investigate their hypothesis that abnormal promoter methylation could help pinpoint the location of a candidate tumor suppressor in regions of LOH. 6q23-q24 was the chosen chromosomal region due to frequently described LOH in human head and neck squamous cell carcinomas (HNSCC) and non-small-cell lung cancers (NSCLC) as well as in other tumor types, but with no identified tumor suppressor.[9] Hypermethylation was found to occur frequently in the same RLGS loci in HNSCC and NSCLC. Bisulfate sequencing further identified tumor-specific methylation of TCF21 when compared to normal controls. RNA samples were isolated from tumor tissues and analyzed to correlate the amount of TCF21 mRNA and DNA methylation in the samples. Overall, tumor samples with higher levels of CpG island hypermethylation had decreased TCF21 expression than normal controls.[9] Smith et al. went on to demonstrate that exogenous expression of TCF21 in cells with silenced endogenous TCF21 loci resulted in a reductions in tumor properties both in vitro and in vivo. Their researched showed that TCF21was indeed a previously unidentified tumor suppressor gene, often silenced by hypermethylation in cancer.[9]

TCF21 has also been linked to metastatic melanoma progression through the inhibition of the KISS1 metastasis-suppressor gene.[10] DNA methylation analysis in melanoma patient biopsies has demonstrated downregulation of TCF21 due to promoter hypermethylation, which also correlates with decreased survival in patients suffering from metastatic skin melanoma.[10] TCF21, together with E12 and TCF12, bind the KISS1 promoter, sustaining its activity. Without TCF21 to interact with the KISS1 promoter, KISS1 expression is lost. Melanoma cells overexpressing TCF21 have also been found to display reduced motility compared with vector-only control cells.[10]

Tobacco-induced lung cancer research has found TCF21 to be among the genes identified as highly methylated at both high and low concentrations of cigarette smoke condensate (CSC). In the presence genistein, one of the soy-derived bioactive isoflavones, methylation of TCF21 is significantly reduced.[11] Genistein has been known to affect tumorigenesis through epigenetic regulation, such as chromatin configuration and DNA methylation, activating other tumor suppressor genes that affect cancer cell survival.[12] These findings support the hypothesis that increasing hypermethylated tumor suppressor genes such as TCF21 is a potential chemopreventative pathway in tobacco-induced lung cancer.[11]

TCF21 may have also therapeutic potential for breast cancer treatment, as downregulation of TCF21 has been implicated in breast cancer tumorigenesis and proliferation.[4] TCF21 mRNA expression is very low in breast cancer cells compared with normal breast epithelial cells. This low expression is also associated with large tumor size and lymph node metastasis. Breast cancer tissues exhibit significantly downregulated expression of TCF21 mRNA and TCF21 mRNA overexpression has been found to inhibit cancerous cell proliferation.[4]

TCF21 Activation

In 2014, research by Arab et al. demonstrated that TCF21 activation was directed by an antisense long non-coding RNA that activates TCF21 expression by inducing promoter demethylation. The three exons that encode TCF21 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.[13] 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 lncRNA in antisense orientation to TCF21. Arab et al. named this lncRNA TARID (for TCF21 antisense RNA inducing demethylation). Long non-coding RNAs (lncRNAs) affect 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.[14] Along with CGI1 and CGI3 methylation, TCF21 and TARID were silent in lung, oral, and ovarian epithelium tumor samples, while normal tissues had unmethylated CGI1 and CGI3 with expression of TCF21 and TARID.[13]

Knockdown of TARID in human kidney cell lines led to decreased expression of TCF21 and an increase in DNA methylation around the TCF21 transcription start site. TARID splice variant transfections showed that sequences that promote transcriptional activation of TCF21 are contained within exon 2 of TARID.[13] Ectopic TARID also triggered demethylation of the TCF21 promoter in all cancer cell lines assayed. No significant change in TARID expression and promoter methylation was observed upon TCF21 siRNA knockdown, indicating that while TARID regulates TCF21, TCF21 does not regulate TARID.[13]

When TARID binds the TCF21 promoter, it recruits growth arrest and DNA-damage-inducible, alpha (GADD45A) and thymine-DNA glycosylase (TDG) to direct base excision repair (BER) for demethylation of cytosine residues.[13] GADD45A, a nuclear protein involved in maintenance of genomic stability, DNA repair and suppression of cell growth, has a key role in active DNA demethylation.[15] Knockdown of the GADD45A protein inhibits TARID-mediated demethylation of the TCF21 promoter and thus TCF21 expression in various tumor cell lines. GADD45A is known to recruit the BER machinery, and knockdown of TDG, an essential component of the BER pathway, also reduces TCF21 expression. Cells treated with BER inhibitors show inhibition of TCF21, and co-immunoprecipitation experiments confirm TDG-GADD45A associations.[13]

Knockdowns of ten-eleven translocation (TET) methylcytosine dioxygenase proteins TET1, TET2, and TET3 in tumor cells, either alone or in different combinations were conducted to examine whether TARID-mediated demethylation of the TCF21 promoter is also brought about by TET proteins.[13] Depletion of all three TET proteins was required to inhibit TARID-mediated demethylation and TCF21 expression. Quantitative hydroxymethylcytosine analysis of CpG residues revealed that TET proteins oxidized 5-methylcytosine to 5-hydroxymethylcytosine in the TCF21 promoter.[13] Together, these results reveal how TARID serves as a genomic address label for GADD45A-mediated demethylation and activation of TCF21.[13]

References

  1. 1 2 3 4 Acharya, A., Baek, S. T., Huang, G., Eskiocak, B., Goetsch, S., Sung, C. Y., … Tallquist, M. D. (2012). The bHLH transcription factor Tcf21 is required for lineage specific EMT of cardiac fibroblast progenitors. Development (Cambridge, England), 139(12), 2139–2149. http://doi.org/10.1242/dev.079970
  2. 1 2 3 4 Quaggin, S.E., Schwartz, L., Cui, S., Igarashi, P., Deimling, J., et al. (1999) The basic-helix loop-helix protein pod1 is critically important for kidney and lung organogenesis. Development 126: 5771–5783. PMID 10572052
  3. 1 2 3 Lu, J., Chang, P., Richardson, J. A., Gan, L., Weiler, H., & Olson, E. N. (2000). The basic helix loop–helix transcription factor capsulin controls spleen organogenesis. Proceedings of the National Academy of Sciences of the United States of America, 97(17), 9525–9530.
  4. 1 2 3 Wang, J., Gao, X., Wang, M., & Zhang, J. (2015). Clinicopathological significance and biological role of TCF21 mRNA in breast cancer. Tumour Biology: The Journal Of The International Society For Oncodevelopmental Biology And Medicine, 36(11), 8679-8683. http://dx.doi.org/10.1007/s13277-015-3476-1
  5. 1 2 3 4 5 6 Quaggin, S. E., Vanden Heuvel, G. B., Igarashi, P. (1998). Pod-1, a mesoderm specific basic helix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney. Mechanisms of Development, 71(1–2), 37-48. http://dx.doi.org/10.1016/S0925-4773(97)00201-3
  6. 1 2 3 4 5 6 Lu, J., Richardson, J. A., & Olson, E. N. (1998). Capsulin: a novel bHLH transcription factor expressed in epicardial progenitors and mesenchyme of visceral organs. Mechanisms of Development, 73 (1): 23-32.
  7. ↑ Sazonova, O., Zhao, Y., Nürnberg, S., Miller, C., Pjanic, M., Castano, V.G., et al. (2015). Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci. PLoS Genet 11(5): e1005202. doi:10.1371/journal.pgen.1005202
  8. 1 2 3 Cui, S., Ross, A., Stallings, N., Parker, K.L., Capel, B., Quaggin, S.E. (2014). Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout mice. Development, 131, 4095-4105. http://dx.doi.org/10.1242/dev.01266
  9. 1 2 3 Smith, L. T., Lin, M., Brena, R. M., Lang, J. C., Schuller, D. E., Otterson, G. A., …Plass, C. (2006). Epigenetic regulation of the tumor suppressor gene TCF21 on 6q23-q24 in lung and head and neck cancer. Proceedings of the National Academy of Sciences of the United States of America, 103(4), 982–987. http://doi.org/10.1073/pnas.0510171102
  10. 1 2 3 Arab, K., Smith, L. T., Gast, A., Weichenhan, D., Huang, J. P.-H., Claus, R., … Plass, C. (2011). Epigenetic deregulation of TCF21 inhibits metastasis suppressor KISS1 in metastatic melanoma. Carcinogenesis, 32(10), 1467–1473. http://doi.org/10.1093/carcin/bgr138
  11. 1 2 Lyn-Cook, L., Word, B., George, N., Lyn-Cook, B., Hammons, G. (2014). Effect of cigarette smoke condensate on gene promoter methylation in human lung cells. Tobacco Induced Diseases, 12:15. http://dx.doi.org/10.1186/1617-9625-12-15
  12. ↑ Zhang Y. and Chen H. (2011). Genistein, an epigenome modifier during cancer prevention. Epigenetics, 6:888–891. doi: 10.4161/epi.6.7.16315.
  13. 1 2 3 4 5 6 7 8 9 Arab, K., Park, Y. J., Lindroth, A. M., Schafer, A., Oakes, C., Weichenhan, D., Lukanova, A., Lundin, E., Risch, A., Meister, M., Dienemann, H., Dyckhoff, G., Herold-Mende, C., Grummt, I., Niehrs, C., Plass, C. (2014). Long noncoding RNA TARID directs demethylation and activation of the tumor suppressor TCF21 via GADD45A. Molecular Cell 55, 604-614. http://dx.doi.org/10.1016/j.molcel.2014.06.031
  14. ↑ Rinn, J. L., & Chang, H. Y. (2012). Genome regulation by long noncoding RNAs. Annual Review of Biochemistry, 81, 10.1146/annurev–biochem–051410–092902. http://doi.org/10.1146/annurev-biochem-051410-092902
  15. ↑ Barreto, G., Schäfer, A., Marhold, J., Stach, D., Swaminathan, S.K., Handa, V., Döderlein, G., Maltry, N., Wu, W., Lyko, F., and Niehrs, C. (2007).Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature, 445: 671–675.

Acharya, A., Baek, S. T., Huang, G., Eskiocak, B., Goetsch, S., Sung, C. Y., … Tallquist, M. D. (2012). The bHLH transcription factor Tcf21 is required for lineage specific EMT of cardiac fibroblast progenitors. Development (Cambridge, England), 139(12), 2139–2149. http://doi.org/10.1242/dev.079970

Arab, K., Smith, L. T., Gast, A., Weichenhan, D., Huang, J. P.-H., Claus, R., … Plass, C. (2011). Epigenetic deregulation of TCF21 inhibits metastasis suppressor KISS1 in metastatic melanoma. Carcinogenesis, 32(10), 1467–1473. http://doi.org/10.1093/carcin/bgr138

Arab, K., Park, Y. J., Lindroth, A. M., Schafer, A., Oakes, C., Weichenhan, D., Lukanova, A., Lundin, E., Risch, A., Meister, M., Dienemann, H., Dyckhoff, G., Herold-Mende, C., Grummt, I., Niehrs, C., Plass, C. (2014). Long noncoding RNA TARID directs demethylation and activation of the tumor suppressor TCF21 via GADD45A. Molecular Cell 55, 604-614. http://dx.doi.org/10.1016/j.molcel.2014.06.031

Barreto, G., Schäfer, A., Marhold, J., Stach, D., Swaminathan, S.K., Handa, V., Döderlein, G., Maltry, N., Wu, W., Lyko, F., and Niehrs, C. (2007).Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature, 445: 671–675.

Cui, S., Ross, A., Stallings, N., Parker, K.L., Capel, B., Quaggin, S.E. (2014). Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout mice. Development, 131, 4095-4105. http://dx.doi.org/10.1242/dev.01266

Lu, J., Chang, P., Richardson, J. A., Gan, L., Weiler, H., & Olson, E. N. (2000). The basic helix loop–helix transcription factor capsulin controls spleen organogenesis. Proceedings of the National Academy of Sciences of the United States of America, 97(17), 9525–9530.

Lu, J., Richardson, J. A., & Olson, E. N. (1998). Capsulin: a novel bHLH transcription factor expressed in epicardial progenitors and mesenchyme of visceral organs. Mechanisms of Development, 73 (1): 23-32.

Lyn-Cook, L., Word, B., George, N., Lyn-Cook, B., Hammons, G. (2014). Effect of cigarette smoke condensate on gene promoter methylation in human lung cells. Tobacco Induced Diseases, 12:15. http://dx.doi.org/10.1186/1617-9625-12-15

Rinn, J. L., & Chang, H. Y. (2012). Genome regulation by long noncoding RNAs. Annual Review of Biochemistry, 81, 10.1146/annurev–biochem–051410–092902. http://doi.org/10.1146/annurev-biochem-051410-092902

Quaggin, S.E., Schwartz, L., Cui, S., Igarashi, P., Deimling, J., et al. (1999) The basic-helix loop-helix protein pod1 is critically important for kidney and lung organogenesis. Development 126: 5771–5783. PMID 10572052

Quaggin, S. E., Vanden Heuvel, G. B., Igarashi, P. (1998). Pod-1, a mesoderm specific basic helix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney. Mechanisms of Development, 71(1–2), 37-48. http://dx.doi.org/10.1016/S0925-4773(97)00201-3

Sazonova, O., Zhao, Y., Nürnberg, S., Miller, C., Pjanic, M., Castano, V.G., et al. (2015). Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci. PLoS Genet 11(5): e1005202. doi:10.1371/journal.pgen.1005202

Smith, L. T., Lin, M., Brena, R. M., Lang, J. C., Schuller, D. E., Otterson, G. A., …Plass, C. (2006). Epigenetic regulation of the tumor suppressor gene TCF21 on 6q23-q24 in lung and head and neck cancer. Proceedings of the National Academy of Sciences of the United States of America, 103(4), 982–987. http://doi.org/10.1073/pnas.0510171102

Wang, J., Gao, X., Wang, M., & Zhang, J. (2015). Clinicopathological significance and biological role of TCF21 mRNA in breast cancer. Tumour Biology: The Journal Of The International Society For Oncodevelopmental Biology And Medicine, 36(11), 8679-8683. http://dx.doi.org/10.1007/s13277-015-3476-1

Zhang Y. and Chen H. (2011). Genistein, an epigenome modifier during cancer prevention. Epigenetics, 6:888–891. doi: 10.4161/epi.6.7.16315.


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