Characterization of Skn-1a/i POU Domain Factors and Linkage to Papillomavirus Gene Expression

Abstract

Tissue-restricted POU domain transcription factors, which bind octamer or octamer-like gene sequences, play roles in cellular differentiation and the development of several organs. We have previously identified a POU domain gene, Skn-1a/i, expressed primarily in epidermis, that encodes at least two products through alternative splicing. One of these, Skn-1a, acts as a transcriptional activator, and the other, Skn-1i, contains an inhibitory domain in the NH 2 terminus, which prevents DNA-binding in vitro and transcriptional activation in vivo. We now demonstrate that when Skn-1i is expressed in eukaryotic cells it can bind to an octamer site, suggesting that in vivo cellular factors modulate the activity of the inhibitory domain to permit DNA-binding. Yet the inhibitory domain does not allow transactivation by Skn-1i or by a heterologous transactivator containing this domain in cis. Furthermore, we demonstrate that Skn-1a, Tst-1, and Oct-1 are the major octamer-binding proteins in epidermis. Since Skn-1a is primarily expressed in suprabasal cells of the epidermis, we have tested its possible role in the regulation of epidermal papillomaviruses. In transient transfection assays, Skn-1a and Tst-1 can activate the long control region of the epidermis-specific human papillomavirus 1A (HPV-1A). Consistent with these in vivo transcription data, in vitro DNA binding studies identify three octamer-like sites, which are capable of binding Skn-1a, in the HPV-1A long control region. Mutations of all three octamer-like sites prevent transactivation by Skn-1a in transient transfection assays. Taken together, these results provide evidence that Skn-1a and Tst-1 may provide a molecular link between HPV gene expression and epidermal differentiation.During midgestation in mammals, the embryo is enclosed by a cellular bilayer composed of a basal layer of somatic ectoderm, which is covered by distinct epithelial cells referred to as periderm. While periderm is later shed, somatic ectoderm has several distinct fates in the mature organism. These fates include mammary epithelium, teeth, epidermis, and epidermal appendages such as hair, nails, and sweat glands (1). Epidermis, which forms relatively late in embryogenesis, is made of a single layer of proliferating basal keratinocytes and several layers of postmitotic suprabasal cells. The basal keratinocytes express a pair of keratins, K5 and K14, but, concomitant with departure from the basal layer, they exit the cell cycle, suppress expression of K5 and K14, and induce expression of another keratin pair, K1 and K10. As keratinocytes move outward to the surface of the skin, proteins required for formation of a cornified cell envelope are induced. These cells subsequently undergo programmed cell death to form the cornified layer of the epidermis. This differentiation process, first initiated during embryogenesis, continues throughout life with cell proliferation in the basal layer balanced by cell death and eventual shedding of the stratum corneum (2).Human papillomaviruses (HPVs) 1 ar...

Aberrant promoter methylation and silencing of the POU2F3 gene in cervical cancer

“…Thus, the cell layers above the basal layer support increased levels of viral DNA replication and synthesis of capsid proteins. Skn1a has been linked to the regulation of HPV transcription via the activation of E6/E7 promoters (Yukawa et al, 1996;Andersen et al, 1997;Kukimoto and Kanda, 2001). However, cellular transformation does not occur as a consequence of this productive infection because differentiated cells that express the oncoproteins have lost the ability to proliferate and are sloughed off.…”

“…hSkn-1a contributes to epidermal stratification by promoting keratinocyte proliferation and enhancing subsequent keratinocyte differentiation (Hildesheim et al, 2001). In addition, hSkn-1a can stimulate HPV transcription by activating E6/E7 promoters (Yukawa et al, 1996;Andersen et al, 1997;Kukimoto and Kanda, 2001).…”

The Differentiation-Specific Factor CDP/Cut Represses Transcription and Replication of Human Papillomaviruses through a Conserved Silencing Element

“…These three factors are each derived from multigene families, with the epithelial-cell specificity originating from the fact that different members of these gene families differ in function and are expressed in a cell-typespecific manner. While these three factors appear to bind the LCRs of all genital HPV types (44,50), another factor, skn-1, expressed exclusively in epithelial cells, has so far been found to activate only HPV-1 and HPV-18 gene expression (2,66). Consequently, regulation by skn-1 does not provide an explanation for the general phenomenon of epithelial-cell specificity of genital HPV enhancers.…”

Identification and characterization of Grainyhead‐like epithelial transactivator (GET‐1), a novel mammalian Grainyhead‐like factor

“…GST fusion proteins were expressed in Escherichia coli by using standard methods and stored on glutathione agarose beads at 4°C (Andersen et al, 1997a). 35 S-Labeled in vitro translated proteins were incubated with the GST proteins on a rotating wheel at 4°C for 30 min as previously described (Sugihara et al, 2001).…”