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