MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

Thyroid gland organogenesis results in an organ the shape, size, and position of which are largely conserved among adult individuals of the same species, thus suggesting that genetic factors must be involved in controlling these parameters. In humans, the organogenesis of the thyroid gland is often disturbed, leading to a variety of conditions, such as agenesis, ectopy, and hypoplasia, which are collectively called thyroid dysgenesis (TD). The molecular mechanisms leading to TD are largely unknown. Studies in murine models and in a few patients with dysgenesis revealed that mutations in regulatory genes expressed in the developing thyroid are responsible for this condition, thus showing that TD can be a genetic and inheritable disease. These studies open the way to a novel working hypothesis on the molecular and genetic basis of this frequent human condition and render the thyroid an important model in the understanding of molecular mechanisms regulating the size, shape, and position of organs.

We used the lung epithelial cell-specific surfactant protein B (SPB) gene promoter as a model with which to investigate mechanisms involved in transcriptional control of lung-specific genes. In a previous study, we showed that the SPB promoter specifically activated expression of a linked reporter gene in the continuous H441 lung cell line and that H441 nuclear proteins specifically protected a region of this promoter from bp -111 to -73. In this study, we further show that this region is a complex binding site for thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3 (HNF-3). Whereas TTF-1 bound two highly degenerate and closely spaced sites, HNF-3 proteins bound a TGT3 motif (TGTTTGT) that is also found in several liver-specific gene regulatory regions, where it appears to be a weak affinity site for HNF-3. Point mutations of these binding sites eliminated factor binding and resulted in significant decreases in transfected SPB promoter activity. In addition, we developed a cotransfection assay and showed that a family of lung-specific gene promoters that included the SPB, SPC, SPA, and Clara cell secretory protein (CCSP) gene promoters were specifically activated by cotransfected TTF-1. We conclude that TTF-1 and HNF-3 are major activators of lung-specific genes and propose that these factors are involved in a general mechanism of lung-specific gene transcription. Importantly, these data also show that common factors are involved in organ-specific gene expression along the mammalian foregut axis.The lung forms as an endodermal bud from the anteromedian foregut wall. Mesenchymal tissue interacts with this bud, inducing a process of branching morphogenesis that establishes a highly branched network of epithelially lined airways. Cellular differentiation in the lung is complex, and several morphologically distinct cell types comprise the airway epithelium. By definition, the onset of cellular differentiation is signalled by expression of differentiated gene products; hence, one fruitful approach to understanding mechanisms of cellular differentiation in mammals has been to identify factors that control expression of genes that define the cellular phenotype (14,22,34,41,71 (513) 559-7868. sue-specific genes are often sufficient to target expression of a reporter gene to the appropriate tissue in vivo (36). Extensive study has shown that DNA-binding proteins specifically interact with these sequences to stimulate gene transcription (37,46,50). One model suggests that the mechanism by which these proteins act depends on their restricted cellular distribution and interaction with only one type of tissue-specific gene family. The control of skeletal muscle cell differentiation by the MyoD family of transcription factor proteins supports this model. The expression of these proteins is restricted to the skeletal muscle cell lineage, and ectopic expression in vitro or in vivo is sufficient to convert most cell types to skeletal muscle-like cells (22,49,73,74). This property of myogenic conversion d...

The cDNA for TTF‐1, a thyroid nuclear factor that binds to the promoter of thyroid specific genes, has been cloned. The protein encoded by the cDNA shows binding properties indistinguishable from those of TTF‐1 present in nuclear extracts of differentiated rat thyroid cells. The DNA binding domain of TTF‐1 is a novel mammalian homeodomain that shows considerable sequence homology to the Drosophila NK‐2 homeodomain. TTF‐1 mRNA and corresponding binding activity are detected in thyroid and lung. The chromosomal localization of the TTF‐1 gene has been determined in humans and mice and corresponds to chromosomes 14 and 12, respectively, demonstrating that the TTF‐1 gene is not located within previously described clusters of homeobox‐containing genes.

Permanent congenital hypothyroidism (CH) is a common disease that occurs in 1 of 3,000-4,000 newborns. Except in rare cases due to hypothalamic or pituitary defects, CH is characterized by elevated levels of thyroid-stimulating hormone (TSH) resulting from reduced thyroid function. When thyroid hormone therapy is not initiated within the first two months of life, CH can cause severe neurological, mental and motor damage. In 80-85% of cases, CH is associated with and presumably is a consequence of thyroid dysgenesis (TD). In these cases, the thyroid gland can be absent (agenesis, 35-40%), ectopically located (30-45%) and/or severely reduced in size (hypoplasia, 5%). Familial cases of TD are rare, even though ectopic or absent thyroid has been occasionally observed in siblings. The pathogenesis of TD is still largely unknown. Although a genetic component has been suggested, mutations in the gene encoding the receptor for the thyroid-stimulating hormone (TSHR) have been identified in only two cases of TD with hypoplasia. We report mutations in the coding region of PAX8 in two sporadic patients and one familial case of TD. All three point mutations are located in the paired domain of PAX8 and result in severe reduction of the DNA-binding activity of this transcription factor. These genetic alterations implicate PAX8 in the pathogenesis of TD and in normal thyroid development.

The Pax-8 gene, a member of the murine family of paired box-containing genes (Par genes), is expressed in adult thyroid and in cultured thyroid cell lines. The Pax-8 protein binds, through its paired domain, to the promoters of thyroglobulin and thyroperoxidase, genes that are exclusively expressed in the thyroid. In both promoters, the binding site of Pax-8 overlaps with that of TTF-l1, a homeodomain-containing protein involved in the activation of thyroid-specific transcription. Pax-8 activates transcription from cotransfected thyroperoxidase and thyroglobulin promoters, indicating that it may be involved in the establishment, control, or maintenance of the thyroid-differentiated phenotype. Thus, the promoters of thyroglobulin and thyroperoxidase represent the first identified natural targets for transcriptional activation by a paired domain-containing protein.

A rat thyroglobulin promoter fragment, capable of directing thyroid‐specific transcription, binds at least three different factors, TTF‐1, TTF‐2 and UFA, which are all present in nuclear extracts of the differentiated rat thyroid cell line FRTL‐5. TTF‐1 and TTF‐2 are FRTL‐5 specific, as demonstrated by their absence in nuclear extracts prepared from cell lines that do not express any thyroid‐differentiated function, while UFA is present in all cell lines tested. TTF‐1 has been extensively purified. It binds to the rat thyroglobulin promoter at three different sites which share sequence homology. Mutations in two of the three sites decrease both binding of TTF‐1 in vitro and promoter function in vivo. This suggests that the tissue‐specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.

The gene encoding the Na/I symporter (NIS) is expressed at high levels only in thyroid follicular cells, where its expression is regulated by the thyroid-stimulating hormone via the second messenger, cyclic AMP (cAMP). In this study, we demonstrate the presence of an enhancer that is located between nucleotides ؊2264 and ؊2495 in the 5-flanking region of the NIS gene and that recapitulates the most relevant aspects of NIS regulation. When fused to either its own or a heterologous promoter, the NIS upstream enhancer, which we call NUE, stimulates transcription in a thyroid-specific and cAMP-dependent manner. The activity of NUE depends on the four most relevant sites, identified by mutational analysis. The thyroid-specific transcription factor Pax8 binds at two of these sites. Mutations that interfere with Pax8 binding also decrease transcriptional activity of the NUE. Furthermore, expression of Pax8 in nonthyroid cells results in transcriptional activation of NUE, strongly suggesting that the paired-domain protein Pax8 plays an important role in NUE activity. The NUE responds to cAMP in both protein kinase A-dependent and -independent manners, indicating that this enhancer could represent a novel type of cAMP responsive element. Such a cAMP response requires Pax8 but also depends on the integrity of a cAMP responsive element (CRE)-like sequence, thus suggesting a functional interaction between Pax8 and factors binding at the CRE-like site.Cell type-specific gene transcription is often dependent on a set of transcription factors whose combination is unique to that cell type. Three transcription factors, TTF-1, TTF-2, and Pax8, have been implicated in such a control in the case of thyroidspecific transcription of the thyroglobulin and thyroperoxidase genes (13). TTF-1 is an homeodomain (HD)-containing protein, present in the developing thyroid, lung, and diencephalon (26). TTF-2, a forkhead protein, has been detected in the endoderm of the developing foregut, including the thyroid anlage, and in the anterior pituitary (41), while the paireddomain (PD) factor Pax8 is present in both the thyroid and kidney (35). The unique combination of these factors in the thyroid follicular cells strongly suggests that their interaction plays an important role in inducing a specific pattern of gene expression in these cells. Cyclic AMP (cAMP), whose intracellular level is elevated by the thyroid-stimulating hormone (TSH), is an important modulator of gene expression in thyroid cells (1,2,14,18,20,22,34,37). Nevertheless, direct roles for the thyroid-restricted factors TTF-1, TTF-2, and Pax8 in mediating the cAMP effects in thyroid cells have not yet been demonstrated. Interestingly, well-known mediators of transcriptional regulation by cAMP, such as those acting through the cAMP responsive element (CRE) sequence (5) have been proposed to be involved only in the regulation of TSH receptor gene expression (22), but no conclusive evidence on their roles in the control exerted by TSH cAMP or on other thyroidspecific genes has been pr...