Transcriptional Regulation Of Mouse Nestin Gene In The Developing Central Nervous System

Intermediate filament proteins are one of the major components of cytoskeletonand their expression and organization are rigidly regulated during development ofmany tissues. Nestin, a member of type VI intermediate filament protein, is expressedin the progenitor cells during neurogenesis and myogenesis. We first characterized 5′flanking promoter in the mouse embryonic carcinoma (EC) P19 cells. Primerextension showed that mouse nestin mRNA was transcribed from at least threedifferent initiation sites. Two closely-spaced Sp1-binding sites in the minimalpromoter were critical for promoter activity, and Sp1 and Sp3 proteins specificallybound to the Sp1 sites. Furthermore, overexpression of dominant-negative Sp1 or Sp3mutants significantly inhibits promoter activity. These results suggest the key role ofgeneral transcription factors Sp1 and Sp3 in the basal expression of nestin gene.The second intron of nestin gene possesses the central nervous system (CNS)specific enhancer. However, the molecular mechanism of expressional regulation ofnestin gene in the neural development remained unclear. P19 cells can be induced byretinoic acid (RA) to differentiate into neural cells. In this process, the expression ofnestin was upregulated by the neural enhancer in the second intron of mouse nestingene. Using in ovo electroporation of chick embryos and transgenic mice, we showedthat 3′ 320-bp fragment in the second intron of mouse nestin gene is sufficient to drivereporter expression in the developing neural tube of chick and mouse embryos, andthe POU factor-binding site in this 320-bp mini-enhancer is necessary for reporterexpression. The neural enhancer might regulate nestin expression through recruitmentof Class V POU factor Oct4, Class II POU factor Oct1, and Class III POU factorsBrn1, Brn2 to bind to the same POU factor-binding site in P19EC cells and P19neural progenitor cells, respectively. Our findings indicate a role for the same POUbinding site in nestin regulation in both P19EC cells and P19 neural progenitor cells.We also characterized the first intron of mouse nestin gene during skeletalmuscle differentiation of mouse myoblast C2C12 cells. The first intron might functionas skeletal muscle-specific enhancer in that it contains several E-boxes and is highlyinducible by bHLH transcription factor MyoD, and by muscle cell differentiation. Themuscle enhancer could direct the repeort gene expression to myotome, as shown by inovo chick electroporation.