Homeobox genes, which are found in all eukaryotic organisms, encode transcriptional regulators involved in cell-type differentiation and development. Several homeobox genes encoding homeodomain proteins that bind and activate the insulin gene promoter have been described. In an attempt to identify additional beta-cell homeodomain proteins, we designed primers based on the sequences of beta-cell homeobox genes cdx3 and lmxl and the Drosophia homeodomain protein Antennapedia and used these primers to amplffy inserts by PCR from an insulinoma cDNA library. The resulting amplification products include sequences encoding 10 distinct homeodomain proteins; 3 of these proteins have not been described previously. In addition, an insert was obtained encoding a splice variant of engrafled-2, a homeodomain protein previously identified in the central nervous system. Northern analysis revealed a distinct pattern of expression for each homeobox gene. Interestingly, the PCR-derived clones do not represent a complete sampling of the beta-cell library because no inserts encoding cdx3 or lmxl protein were obtained. Beta cells probably express additional homeobox genes. The abundance and diversity of homeodomain proteins found in beta cells illustrate the remarkable complexity and redundancy of the machinery controlling beta-cell development and differentiation.RIPE3B element (16) and the P1 element (8) [also called CT1 (9)] lie on either side of the IEB1 element. The A/T elements and the E boxes function synergistically: none of the elements can function in isolation, but combination of an E box and an A/T element results in dramatic activation of transcription (11,16,19). A number of complexes from beta-cell nuclei bind to the A/T elements (6, 8-11, 16, 19 Based on evidence that the A/T-binding proteins cloned so far do not account for all the beta-cell nuclear complexes that bind the A/T elements (H.O. and M.S.G., unpublished data), we assumed that beta cells contained additional homeodomain proteins. We attempted to identify additional beta-cell homeodomain proteins by PCR. We report here the PCR amplification and cDNA cloning of several additional betacell homeodomain proteins. ¶ The beta cells in the pancreatic islets of Langerhans are distinguished by their ability to produce insulin. The cellspecific expression of insulin derives, at least in part, from regulation of the insulin gene promoter by a unique set of nuclear proteins that bind to the promoter and activate it in beta cells. Mapping of transcriptionally active sequences within the insulin promoter has identified several important sequence elements within the rat insulin I (1, 2), rat insulin II (3, 4), and human insulin promoters (5) and has led to the recognition of beta-cell nuclear protein complexes that bind these elements (4-11) (for reviews, see refs. 12 and 13).Many of the critical sequence elements of the insulin promoter fall into two groups: the E box elements and the (A+T)-rich elements. The proximal human and rat insulin I promoters contain two...

LIM-homeodomain proteins direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-homeodomain protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) protein E47/Pan-1 to activate the insulin promoter in transfected fibroblasts. In this study, we show that two proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.1 and Lmx1.2. We have used yeast genetic systems to delineate the functional domains of the Lmx1 proteins and to characterize the physical interactions between Lmx1 proteins and E47/Pan-1 that produce synergistic transcriptional activation. The LIM domains of the Lmx1 proteins, and particularly the second LIM domain, mediate both specific physical interactions and transcriptional synergy with E47/Pan-1. The LIM domains of the LIM-homeodomain protein Isl-1, which cannot mediate transcriptional synergy with E47/Pan-1, do not interact with E47/Pan-1. In vitro studies demonstrate that the Lmx1.1 LIM2 domain interacts specifically with the bHLH domain of E47/Pan-1. These studies provide the basis for a model of the assembly of LIM-homeodomain-containing complexes on DNA elements that direct cell-type-restricted transcription in differentiated tissues. Development of differentiated cells depends on the differential expression of transcriptional activators (23).Moreover, cooperative effects of several activators with restricted expression patterns determine the subset of genes turned on in a given cell type (4,18,25,33). Determination of the molecular basis of cooperation between activators that leads to synergy in promoting the transcription of cell-type-restricted genes will help define the molecular basis for the existence of differentiated cells.LIM-homeodomain (LIM-HD) proteins are a major class of transcriptional activators that cooperate with other activators to direct cellular differentiation. LIM-HD proteins contain a DNA-binding homeodomain and two N-terminal, zinc-binding LIM domains. The LIM-HD protein Lmx1.2 specifies a dorsal cell fate during vertebrate limb development (28, 32). Lim1 is a LIM-HD protein found in the organizer region of vertebrate embryos that is essential for formation of head structures (30). The mec-3 and lin-11 LIM-HD proteins are required for the asymmetric division of precursor cells in Caenorhabditis elegans (12,39). Other LIM-HD proteins, including Isl-1, mec-3, and apterous, have been found to be crucial components of nervous system development in different species (22,27,39).The LIM domains in LIM-HD proteins and several nonhomeodomain-containing proteins are often found in pairs (and sometimes clusters of three) at the N or C termini of proteins, and they have been found to mediate both intramolecular and intermolecular interactions (6). LIM domains in the LIM-only protein cysteine-rich protein (CRP) appear to mediate both dimerization and the interaction of CRP with another LIM-only protein, zyxin (10, 29...

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