Nuclear LIM domains interact with a family of coregulators referred to as Clim͞Ldb͞Nli. Although one family member, Clim-2͞Ldb-1͞Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO͞Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix-loop-helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.The LIM motif, a cysteine-rich zinc-coordinating domain, originally was discovered adjacent to homeodomains in three transcription factors, lin-11, Isl-1, and mec-3 (1, 2). Several additional LIM homeodomain factors have been discovered, and these, as well as the original three members of this gene family, have been established to have critical functions in lineage specification and differentiation in diverse cellular systems (1, 2).Other nuclear LIM domain-containing factors have been identified, including the LIM-only (LMO) proteins, so named because they are composed almost entirely of two tandem LIM domains (1, 2). This subgroup of LIM proteins has three members: LMO-1 (RBTN1͞TTG1) and LMO-2 (RBTN2) were isolated at sites of chromosomal translocations in acute T-cell leukemia, and the third, LMO-3 (RBTN3) was isolated based on sequence similarity (3, 4). Both LMO-1 and LMO-2 have been shown to act as oncoproteins in lymphocytes (3). In addition to its role in human cancers, gene deletion of LMO-2 showed that this gene is essential for normal blood cell formation in mice (5, 6), and thus has roles in cellular determination and differentiation. Instead of directly binding DNA, LMO factors are thought to regulate gene transcription by associating with DNA-binding proteins. In addition to the nuclear LIM domain factors, LIM domains have been found on several cytoplasmic proteins, some of which also contain kinase domains (1).Prevailing evidence sup...

LMO-4 is an LIM-only factor that is highly expressed in many epithelial cells, including those of the epidermis and hair follicles. Because LMOs may function by interacting with DNA-binding proteins, we have used the yeast two-hybrid system to screen mouse skin libraries for LMO-4 -interacting DNA-binding proteins. In this screen, we isolated a novel LMO-4 -interacting factor highly related to the Drosophila gene Grainyhead. Grainyhead is epidermally expressed and carries out important functions in cuticular formation in the fly embryo. With the identification of this novel mammalian Grainyhead-like gene, referred to as Grainyhead-like epithelial transactivator 1 (GET-1), the known members of the mammalian Grainyhead-like gene family are extended to six, falling into two classes based on sequence homology. Of interest, the expression pattern of GET-1 is similar to that of Drosophila Grainyhead with highest expression in the somatic ectoderm/epidermis, but GET-1 is additionally expressed in epithelial cells of gastrointestinal, genitourinary, and respiratory tracks. The GET-1 protein localizes to the nucleus and binds to at least one Grainyhead DNA-binding site. The GET-1 DNA-binding domain maps to a region containing homology to the Drosophila Grainyhead DNA-binding domain. GET-1 homodimerizes in solution by means of a short C-terminally located domain that is homologous to other Grainyhead-like genes. A short domain located between amino acids 100 and 190, which bears no homology to known transactivation domains, is sufficient to confer transactivation to the heterologous GAL4 DNA-binding domain. In addition, GET-1 appears to contain repression domains consistent with the observation that Grainyhead and other mammalian Grainyhead-like genes can act both as activators and repressors. These data suggest that GET-1 is a transcriptional regulator that may perform important functions in epithelial tissues of mammals. Developmental Dynamics 226:604 -617, 2003.

domain factors and associated cofactors are important developmental regulators in pattern formation and organogenesis. In addition, overexpression of two LIM-only factors (LMOs) causes acute lymphocytic leukemia. The more recently discovered LMO factor LMO4 is highly expressed in proliferating epithelial cells, and frequently overexpressed in breast carcinoma. Here we show that while LMO4 is expressed throughout mammary gland development, it is dramatically upregulated in mammary epithelial cells during midpregnancy. The LMO coactivator Clim2/Ldb1/NLI showed a similar expression pattern, consistent with the idea that LMO4 and Clim2 act as a complex in mammary epithelial cells. In MCF-7 cells, LMO4 transcripts were upregulated by heregulin, an activator of ErbB receptors that are known to be important in mammary gland development and breast cancer. To test the hypothesis that LMO4 plays roles in mammary gland development, we created an engrailed-LMO4 fusion protein. This fusion protein maintains the ability to interact with Clim2, but acts as a dominant repressor of both basal and activated transcription when recruited to a DNA-regulatory region. When the engrailed-LMO4 fusion protein was expressed under control of the MMTV promoter in transgenic mice, both ductular development in virgin mice and alveolar development in pregnant mice were inhibited. These results suggest that LMO4 plays a role in promoting mammary gland development.

The genes encoding keratin 5 and 14 are highly expressed in the basal cell layer keratinocytes of the epidermis, but both genes are silenced when keratinocytes move into the suprabasal compartment. The POU homeodomain factors Skn-1a and Tst-1, which are expressed in epidermis, may play a role in the suprabasal repression of the keratin 5 and 14 genes because keratin 14 mRNA expression persists in suprabasal cells in Skn-1/ Tst-1 double knockout mice. In transfection experiments, both Skn-1a and Tst-1 repress the keratin 14 promoter, with the POU domain being sufficient for repression. The region of the keratin 14 gene sufficient and required for repression by Skn-1a is a 100-base pair sequence lacking POU-binding sites adjacent to the transcription start site. DNA-binding defective mutants of Skn-1a and Tst-1 are as effective at mediating repression as the wild type proteins, suggesting that proteinprotein interactions rather than direct DNA binding are important for repression. We also show that CREB-binding protein (CBP)/p300 co-activators are strong activators of keratin 14 gene expression, acting through sequences close to the keratin 14 promoter. Further, CBP interacts directly with the POU domain of Skn-1a, and increasing concentrations of CBP can overcome Skn-1a-mediated repression, suggesting that POU domain factors may repress keratin 14 gene expression by interfering with the activity of co-activators such as CBP/p300.The mammalian epidermis is a stratified squamous epithelial tissue characterized by differential expression of keratin genes in its different compartments. The basal cell layer, which is in contact with the basal lamina and contains proliferative cells, expresses keratin (K) 1 5 and 14 at high levels. In fact, these proteins constitute up to 30% of the protein content of keratinocytes in this layer (1). As keratinocytes move out of the basal cell layer into the suprabasal compartment, expression of the K5 and K14 genes is extinguished, whereas the K1 and K10 genes are activated (1). The cells moving progressively through the different layers of the suprabasal epidermis: stratum spinosum, granulosum and corneum, express other distinct keratinocyte markers, including loricrin and filaggrin, which are expressed in the most differentiated cells of the epidermis just prior to their death and the formation of the stratum corneum (2). Keratinocytes moving out of the basal cell layer into the suprabasal compartment also stop proliferating, suggesting a connection between differential keratin expression and the switch regulating proliferation/differentiation decisions in the epidermis (2, 3). Therefore, greater understanding of the molecular mechanisms regulating differential keratin expression may provide insight into the control of epidermal development and homeostasis.One of the genes highly suitable for such analysis is the K14 gene, whose regulation has been extensively studied both in vitro in cell culture models and in vivo in mice. Fuchs and co-workers (4 -7) have shown that ϳ2.2 kb of the...