Sumoylation in Development and Differentiation.

Tissue morphogenesis is a fascinating aspect of both developmental biology and regeneration of certain adult organs, and timely control of cellular differentiation is a key to these processes. During development, events interrupting cellular differentiation and leading to organ failure are embryonic lethal; likewise, perturbation of differentiation in regenerating tissues leads to dysfunction and disease. At the molecular level, cellular differentiation is orchestrated by a well-coordinated cascade of transcription factors (TFs) and chromatin remodeling complexes that drive gene expression. Altering the localization, stability, or activity of these regulatory elements can affect the sequential organization of the gene expression program and result in failed or abnormal tissue development. An accumulating body of evidence shows that the sumoylation system is a critical modulator of these regulatory cascades. For example, inhibition of the sumoylation system during embryogenesis causes lethality and/or severe abnormalities from invertebrates to mammals. Mechanistically, it is now known that many of the TFs and components of chromatin remodeling complexes that are critical for development and differentiation are targets for SUMO modification, though the specific functional consequences of the modifications remain uncharacterized in many cases. This chapter will address several of the models systems that have been examined for the role of sumoylation in differentiation and development. Understanding the profound regulatory role of SUMO in different tissues should lead not only to a better understanding of developmental biology, stem cell linage control, and the mechanisms of cellular differentiation, but may also lead to the identification of new targets for drug therapy and/or therapeutic manipulation of damaged organs and tissues.

HPV E6 proteins target Ubc9, the SUMO conjugating enzyme.

The human papillomavirus oncogenic protein, E6, interacts with a number of cellular proteins, and for some targets, E6 directs their degradation through the ubiquitin-proteasome pathway. Post-translational modification with ubiquitin-like modifiers, such as SUMO, also influences protein activities, protein-protein interactions, and protein stability. We report that the high risk HPVE6 proteins reduce the intracellular quantity of the sole SUMO conjugation enzyme, Ubc9, concomitant with decreased host sumoylation. E6 did not significantly influence transcription of Ubc9, indicating that the effects were likely at the protein level. Consistent with typical E6-mediated proteasomal degradation, E6 bound to Ubc9 in vitro, and required E6AP for reduction of Ubc9 levels. Under stable E6 expression conditions in differentiating keratinocytes there was a decrease in Ubc9 and a loss of numerous sumoylated targets indicating a significant perturbation of the normal sumoylation profile. While E6 is known to inhibit PIASy, a SUMO ligase, our results suggest that HPV E6 also targets the Ubc9 protein to modulate host cell sumoylation, suggesting that the sumoylation system may be an important target during viral reproduction and possibly the subsequent development of cervical cancer.

Host cell sumoylation level influences papillomavirus E2 protein stability.

The stability of papillomavirus E2 proteins is regulated by proteasomal degradation, and regulation of degradation could contribute to the higher expression levels of E2 proteins observed in suprabasal layers of differentiated skin. We have recently shown that the E2 proteins are modified by sumoylation [Wu Y-C, Roark AA, Bian X-L, Wilson, VG (2008) Virol 378:329-338], and that sumoylation levels are up-regulated during keratinocyte differentiation [Deyrieux AF, Rosas-Acosta G, Ozbun MA, Wilson VG (2007) J Cell Sci 120:125-136]. These observations, coupled with the known ability of sumoylation to prevent proteasomal degradation of certain proteins, suggested that this modification might contribute to stabilizing E2 proteins in suprabasal keratinocytes. Conditions that increased overall sumoylation were found to increase the intracellular amounts of the HPV11, 16, and 18 E2 proteins. No effect of sumoylation was seen on E2 transcripts, and the increased levels of E2 proteins resulted from a greatly increased half-life for the E2 proteins. In vitro studies confirmed that sumoylation could block the proteasomal degradation of the 16E2 protein. Interestingly, this stabilization effect was indirect as it did not require sumoylation of 16E2 itself and must be acting through sumoylation of a cellular target(s). This sumoylation-dependent, indirect stabilization of E2 proteins is a novel process that may couple E2 levels to changes in the cellular environment. Specifically, our results suggest that the levels of papillomavirus E2 protein could be up-regulated in differentiating keratinocytes in response to the increased overall sumoylation that accompanies differentiation.

Sumoylation dynamics during keratinocyte differentiation.

SUMO modification regulates the activity of numerous transcription factors that have a direct role in cell-cycle progression, apoptosis, cellular proliferation, and development, but its role in differentiation processes is less clear. Keratinocyte differentiation requires the coordinated activation of a series of transcription factors, and as several crucial keratinocyte transcription factors are known to be SUMO substrates, we investigated the role of sumoylation in keratinocyte differentiation. In a human keratinocyte cell line model (HaCaT cells), Ca2+-induced differentiation led to the transient and coordinated transcriptional activation of the genes encoding crucial sumoylation system components, including SAE1, SAE2, Ubc9, SENP1, Miz-1 (PIASx beta), SUMO2 and SUMO3. The increased gene expression resulted in higher levels of the respective proteins and changes in the pattern of sumoylated substrate proteins during the differentiation process. Similarly to the HaCaT results, stratified human foreskin keratinocytes showed an upregulation of Ubc9 in the suprabasal layers. Abrogation of sumoylation by Gam1 expression severely disrupted normal HaCaT differentiation, consistent with an important role for sumoylation in the proper progression of this biological process.

In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line.

In vitro models to study the process of keratinocyte differentiation have been hindered by the stringent culture requirements and limitations imposed by the inherent properties of the cells. Primary keratinocytes only have a finite life span, while transformed cell lines exhibit many phenotypic features not found in normal cells. The spontaneously immortalized HaCaT cell line has been a widely employed keratinocyte model due to its ease of propagation and near normal phenotype, but protocols for differentiation and gene delivery into HaCaT cells vary widely in the literature. Here we report culture conditions for maintaining HaCaT cells in a basal-like state, for efficient differentiation of these cells, and for delivery of transgenes by transfection or adenoviral infection. This technological report will provide guidance to a large audience of scientists interested in investigating mechanisms of differentiation and skin morphogenesis.