Bogi Andersen

RXRβ: A coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements

The retinoic acid receptor (RAR) requires coregulators to bind effectively to response elements in target genes. A strategy of sequential screening of expression libraries with a retinoic acid response element and RAR identified a cDNA encoding a coregulator highly related to RXR alpha. This protein, termed RXR beta, forms heterodimers with RAR, preferentially increasing its DNA binding and transcriptional activity on promoters containing retinoic acid, but not thyroid hormone or vitamin D, response elements. Remarkably, RXR beta also heterodimerizes with the thyroid hormone and vitamin D receptors, increasing both DNA binding and transcriptional function on their respective response elements. RXR alpha also forms heterodimers with these receptors. These observations suggest that retinoid X receptors meet the criteria for biochemically characterized cellular coregulators and serve to selectively target the high affinity binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate DNA response elements.

Disruption of Paneth and goblet cell homeostasis and increased endoplasmic reticulum stress in Agr2−/− mice

Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that plays important roles in diverse processes in multiple cell lineages as a developmental regulator, survival factor and susceptibility gene for inflammatory bowel disease. Here, we show using germline and inducible Agr2-/- mice that Agr2 plays important roles in intestinal homeostasis. Agr2-/- intestine has decreased goblet cell Mucin 2, dramatic expansion of the Paneth cell compartment, abnormal Paneth cell localization, elevated endoplasmic reticulum (ER) stress, severe terminal ileitis and colitis. Cell culture experiments show that Agr2 expression is induced by ER stress, and that siRNA knockdown of Agr2 increases ER stress response. These studies implicate Agr2 in intestinal homeostasis and ER stress and suggest a role in the etiology of inflammatory bowel disease.

RLIM inhibits functional activity of LIM homeodomain transcription factors via recruitment of the histone deacetylase complex

LIM domains are required for both inhibitory effects on LIM homeodomain transcription factors and synergistic transcriptional activation events. The inhibitory actions of the LIM domain can often be overcome by the LIM co-regulator known as CLIM2, LDB1 and NLI (referred to hereafter as CLIM2; refs 2, 3, 4). The association of the CLIM cofactors with LIM domains does not, however, improve the DNA-binding ability of LIM homeodomain proteins, suggesting the action of a LIM-associated inhibitor factor. Here we present evidence that LIM domains are capable of binding a novel RING-H2 zinc-finger protein, Rlim (for RING finger LIM domain-binding protein), which acts as a negative co-regulator via the recruitment of the Sin3A/histone deacetylase corepressor complex. A corepressor function of RLIM is also suggested by in vivo studies of chick wing development. Overexpression of the gene Rnf12, encoding Rlim, results in phenotypes similar to those observed after inhibition of the LIM homeodomain factor LHX2, which is required for the formation of distal structures along the proximodistal axis, or by overexpression of dominant-negative CLIM1. We conclude that Rlim is a novel corepressor that recruits histone deacetylase-containing complexes to the LIM domain.

Brain and muscle Arnt-like protein-1 (BMAL1) controls circadian cell proliferation and susceptibility to UVB-induced DNA damage in the epidermis

The role of the circadian clock in skin and the identity of genes participating in its chronobiology remain largely unknown, leading us to define the circadian transcriptome of mouse skin at two different stages of the hair cycle, telogen and anagen. The circadian transcriptomes of telogen and anagen skin are largely distinct, with the former dominated by genes involved in cell proliferation and metabolism. The expression of many metabolic genes is antiphasic to cell cycle-related genes, the former peaking during the day and the latter at night. Consistently, accumulation of reactive oxygen species, a byproduct of oxidative phosphorylation, and S-phase are antiphasic to each other in telogen skin. Furthermore, the circadian variation in S-phase is controlled by BMAL1 intrinsic to keratinocytes, because keratinocyte-specific deletion of Bmal1 obliterates time-of-day–dependent synchronicity of cell division in the epidermis leading to a constitutively elevated cell proliferation. In agreement with higher cellular susceptibility to UV-induced DNA damage during S-phase, we found that mice are most sensitive to UVB-induced DNA damage in the epidermis at night. Because in the human epidermis maximum numbers of keratinocytes go through S-phase in the late afternoon, we speculate that in humans the circadian clock imposes regulation of epidermal cell proliferation so that skin is at a particularly vulnerable stage during times of maximum UV exposure, thus contributing to the high incidence of human skin cancers.

Dominant Mutations in GRHL3 Cause Van der Woude Syndrome and Disrupt Oral Periderm Development

Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6(+/-);Grhl3(+/-)) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.

Circadian clock genes contribute to the regulation of hair follicle cycling.

Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence (telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK–regulated genes are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle. Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin. While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes.

Mammary Morphogenesis and Regeneration Require the Inhibition of EMT at Terminal End Buds by Ovol2 Transcriptional Repressor

Epithelial cells possess remarkable plasticity, having the ability to become mesenchymal cells through alterations in adhesion and motility (epithelial-to-mesenchymal transition [EMT]). However, how epithelial plasticity is kept in check in epithelial cells during tissue development and regeneration remains to be fully understood. Here we show that restricting the EMT of mammary epithelial cells by transcription factor Ovol2 is required for proper morphogenesis and regeneration. Deletion of Ovol2 blocks mammary ductal morphogenesis, depletes stem and progenitor cell reservoirs, and leads epithelial cells to undergo EMT in vivo to become nonepithelial cell types. Ovol2 directly represses myriad EMT inducers, and its absence switches response to TGF-β from growth arrest to EMT. Furthermore, forced expression of the repressor isoform of Ovol2 is able to reprogram metastatic breast cancer cells from a mesenchymal to an epithelial state. Our findings underscore the critical importance of exquisitely regulating epithelial plasticity in development and cancer.

LMO4 can interact with Smad proteins and modulate transforming growth factor-beta signaling in epithelial cells

LIM-only protein 4 (LMO4) plays critical roles in mammalian development, and has been proposed to play roles in epithelial oncogenesis, including breast cancer. As LMO4 is highly expressed in the epithelial compartments at locations of active mesenchymal–epithelial interactions, we reasoned that LMO4 might act by modulating signaling pathways involved in mesenchymal–epithelial signaling. One such candidate signal is the transforming growth factor-β (TGFβ) cytokine pathway, which plays important roles both in development and cancer. We show here that the transcriptional response to TGFβ in epithelial cells is sensitive to LMO4 levels; both up- and downregulation of LMO4 can enhance TGFβ signaling as assessed by a TGFβ-responsive reporter gene. Furthermore, LMO4 can interact with the MH1 and linker domains of receptor-mediated Smad proteins, and associate with the endogenous TGFβ-responsive Plasminogen Activator Inhibitor-1 gene promoter in a TGFβ-dependent manner, suggesting that such interactions may mediate the effects of LMO4 on TGFβ signaling. When introduced into mammary epithelial cells, LMO4 potentiated the growth-inhibitory effects of TGFβ in those cells. These results define a new function for LMO4 as a coactivator in TGFβ signaling, and provide a potential novel mechanism for LMO4-mediated regulation in development and oncogenesis.

The epidermal differentiation‐associated Grainyhead gene Get1/Grhl3 also regulates urothelial differentiation

Skin and bladder epithelia form effective permeability barriers through the activation of distinct differentiation gene programs. Using a genome‐wide gene‐expression study, we identified transcriptional regulators whose expression correlates highly with that of differentiation markers in both the bladder and skin, including the Grainyhead factor Get1/Grhl3, which is already known to be important for epidermal barrier formation. In the bladder, Get1 is most highly expressed in the differentiated umbrella cells and its mutation in mice leads to a defective bladder epithelial barrier formation due to the failure of apical membrane specialization. Genes encoding components of the specialized urothelial membrane, the uroplakins, were downregulated in Get1−/− mice. At least one of these genes, uroplakin II, is a direct target of Get1. The urothelial‐specific activation of the uroplakin II gene is due to selective binding of Get1 to the uroplakin II promoter in urothelial cells, which is most likely regulated by histone modifications. These results show a crucial role for Get1 in urothelial differentiation and barrier formation.

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

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.