Epigenetic Control

A. 1. Epigenetic Control of Stem Cell Pluripotency

The term “epigenetics” has first been introduced by Conrad Waddington (1942) to define the phenotypic execution of developmental programs. Epigenetics now broadly describes the additional information superimposed on the genome that contributes to the heritable establishment and maintenance of transcriptional states and cellular identity (Bernstein et al. 2007, Ptashne 2007, Rajasekhar and Begemann 2007). Vertebrates employ two types of epigenetic modulation of gene expression: (i) DNA methylation and (ii) hyperdynamic changes in chromatin structure. Changes in chromatin structure greatly alter ES cell circuitry and germ layer differentiation. Our lab is studying the role of a chromatin-associated pluripotency factor, CP27, in ES cell pluripotency and differentiation.

cp27 cloning, sequencing and characterization. In 1995, five years prior to the closure of the Human Genome Project, we cloned and sequenced a number of novel genes in our laboratory. One of them, we first termed it MOMO (after a Dallas restaurant in Deep Ellum) and later cp27 (after Craniofacial Protein with the Molecular Weight of 27 kDa), turned out to be of particular interest in craniofacial development and was pursued thereafter. Following cloning and sequencing, we entered cp27 into the genbank database (EBI database entry # Y08219 [1996]) and characterized the gene (Diekwisch et al. 1999).

CP27 function. In our subsequent experiments, the cp27 gene exhibited a number of powerful effects. When CP27 gene products were blocked in organ culture, teeth were completely destroyed. When we specifically enhanced CP27 activity, teeth grew to almost twice their original size (Diekwisch and Luan 2002). CP27 was also expressed in significant locations at the beginning of tooth formation and during the development of other craniofacial organs (Diekwisch et al. 2002). Currently we are trying to understand aspects of cp27 function in tooth development. We are also studying how the cp27 gene is regulated. Already now we have learned that cp27 is an important and unique novel gene in the signaling networks involved in early craniofacial development.

Figure 1: Mouse embryonic stem cells. The nuclei are labeled yellow/red for CP27 while the cytoskeleton is labeled in green.

Figure 2: cp27 chromosome localization. Genetic analysis links the cp27 gene to the long arm of chromosome 16. The exact region is 16q22.2-22.3, an area associated with a number of genetic diseases.

Figure 3

Figure 4

Figure 3-4: CP27 function modulation in organ culture. Each panel represents four experiments related to CP27 function modulation. A CP27 specific peptide enhances tooth organ growth (upper right figure in each panel) in comparison to a control tooth organ (upper left in each panel), while addition of an antibody blocking CP27 causes the tooth organ to disintegrate (lower right figure in each panel). A control experiment with another antibody has no effect (lower left in each panel). Figure 2illustrates the whole tooth organs, Figure 3 represents individual sections, and Figure 4 features apoptotic cells highlighted by the TUNEL procedure. Note the intensely red colored apoptotic cells in the tooth organ following CP27 function inhibition. Reprinted from: Diekwisch and Luan (2002). CP27 function is necessary for cell survival and differentiation during tooth morphogenesis in organ culture. Gene 287, 141-147. Copyright(2002), with permission from Elsevier Science.

Selected Publications

Ito, Y., Zhang, Y., Dangaria, S., Luan, X., and Diekwisch, T.G.H. (2011). NF-Y and USF1 transcription factor binding to CCAAT box and E-box elements activates the CP27 promoter. Gene in press.

Luan, X., Ito, Y., Zhang, Y., and Diekwisch, T.G.H. (2010). Characterization of the mouse CP27 promoter and NF-Y mediated gene regulation. Gene 460, 8-19.

Diekwisch, T.G.H., Luan, X., and McIntosh, J.E. (2002). CP27 localization in the dental lamina basement membrane and in the stellate reticulum of developing teeth. J. Histochem. Cytochem 50, 583-585.

Luan, X., and Diekwisch, T.G.H. (2002). CP27 affects viability, proliferation, attachment, and gene expression in embryonic fibroblasts. Cell Proliferation 35, 207-219.

Diekwisch, T.G.H. and Luan, X. (2002). CP27 function is necessary for cell survival and differentiation during tooth morphogenesis in organ culture. Gene 287, 141-147.

Diekwisch, T.G.H., Marches, F., Williams, A., and Luan, X. (1999). Cloning, gene expression, and characterization of CP27, a novel gene in mouse embryogenesis. Gene 235, 19-30.