Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development.

The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1+/+ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1+/+ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.

Correction to: Similarities and differences in tissue distribution of DLK1 and DLK2 during E16.5 mouse embryogenesis.

In the original publication of the article, some symbols in Figure 3 were not correctly aligned with the image.

Similarities and differences in tissue distribution of DLK1 and DLK2 during E16.5 mouse embryogenesis.

DLK1 and DLK2 are transmembrane proteins belonging to the EGF-like repeat-containing family that function as non-canonical NOTCH inhibitory ligands. DLK1 is usually downregulated after embryo development and its distribution in some adult and embryonic tissues has been described. However, the expression and role of DLK2 in embryo and adult tissues remains unclear. To better understand the relevance of both proteins during embryo development, we analyzed the expression pattern of DLK1 and DLK2 in 16.5-day-old mouse embryos (E16.5) and evaluated the possible relationship between these two proteins in embryo tissues and cell types. We found that DLK1 and DLK2 proteins exhibited a broad distribution pattern, which was detected in developing mouse organs from each of the three germ layers: ectoderm (brain, salivary glands), mesoderm (skeletal muscle, vertebral column, kidney, cartilage), and endoderm (thymus, lung, pancreas, intestine, liver). The expression pattern of DLK1 and DLK2 indicates that both proteins could play a synergistic role during cell differentiation. This study provides additional information for understanding temporal and site-specific effects of DLK1 and DLK2 during embryo morphogenesis and cell differentiation.

Reduced salivary gland size and increased presence of epithelial progenitor cells in DLK1-deficient mice.

DLK1 (PREF1, pG2, or FA1) is a transmembrane and secreted protein containing epidermal growth factor-like repeats. Dlk1 expression is abundant in many tissues during embryonic and fetal development and is believed to play an important role in the regulation of tissue differentiation and fetal growth. After birth, Dlk1 expression is abolished in most tissues but is possibly reactivated to regulate stem cell activation and responses to injury. We have recently reported that DLK1 regulates many aspects of salivary gland organogenesis. Here, we have extended our studies of the salivary gland phenotype of Dlk1 knock-out mice. We have observed that salivary glands are smaller and weigh significantly less in both Dlk1 knock-out males and females compared with gender and age-matched wild-type mice and regardless of the natural sexual dimorphism in rodent salivary glands. This reduced size correlates with a reduced capacity of Dlk1-deficient mice to secrete saliva after stimulation with pilocarpine. However, histological and ultrastructural analyses of both adult and developing salivary gland tissues have revealed no defects in Dlk1 ((-/-)) mice, indicating that genetic compensation accounts for the relatively mild salivary phenotype in these animals. Finally, despite their lack of severe anomalies, we have found that salivary glands from Dlk1-deficient mice present a higher amount of CK14-positive epithelial progenitors at various developmental stages, suggesting a role for DLK1 in the regulation of salivary epithelial stem cell balance.

Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression.

Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes.

Elevated expression of Ets2 or distinct portions of Ets2 can reverse Ras-mediated cellular transformation.

Ets transcription factors are important downstream targets of oncogenic Ras. The transcriptional activity of several Ets family members is regulated by Ras, and interfering with Ets-dependent transcription by expression of just the Ets2 DNA binding domain can inhibit or reverse Ras-mediated cellular transformation. To better understand the role of Ets proteins in Ras transformation, we have now analyzed the effects of stably expressing a variety of Ets2 constructs in Ras-transformed NIH3T3 (DT) cells. Expression of only the Ets2 transactivation domains, which also inhibits Ras or Neu/ErbB-2-mediated activation of Ets-dependent transcription, strongly inhibited anchorage-independent growth, but did not revert the transformed DT cell morphology. Unexpectedly, high expression of full-length Ets2, a transcriptional activator, broadly reversed the transformed properties of DT cells, including anchorage-independent growth, transformed morphology, and tumorigenicity, but did not impair attached cell growth. Increasing full-length Ets2 transcriptional activity by fusing it to the VP16 transactivation domain enhanced its ability to reverse DT cell transformation. Mutational analysis revealed that the mitogen-activated protein kinase phosphorylation site required for Ras-mediated activation, Ets2(T72), was not essential for Ets2 reversion activity. The distinct reversion activities of the highly expressed Ets2 transactivation domains or full-length Ets2, along with the specific reversion activity by Ets2 constructs that either inhibit or activate Ets-dependent transcription, suggests multiple roles for Ets factors in cellular transformation. These results indicate that several distinct approaches for modulating Ets activity may be useful for intervention in human cancers.

Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1.

Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Ras and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of deltaRaf-1:ER, a conditionally active form of oncogenic human Raf-1. Promoter activation by deltaRaf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp -974 and -988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following deltaRaf-1:ER activation. Cotransfection of Ets-2 and p44 mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to deltaRaf-1:ER. Potentiated activation required both p44 MAP kinase catalytic activity and threonine 72 in the Pointed domain of Ets-2. Biochemical assays demonstrated the ability of the p42 and p44 MAP kinases to phosphorylate Ets-2 on threonine 72. Importantly, in intact cells, the kinetics of phosphorylation of Ets-2 on this residue closely mirror the activation of the p42 and p44 MAP kinases and the observed onset of HB-EGF gene transcription following deltaRaf-1:ER activation. These data firmly establish Ets-2 as a direct target of the Raf-MEK-MAP kinase signaling pathway and strongly implicate Ets-2 in the regulation of HB-EGF gene expression.

The Saccharomyces cerevisiae RIB4 gene codes for 6,7-dimethyl-8-ribityllumazine synthase involved in riboflavin biosynthesis. Molecular characterization of the gene and purification of the encoded protein.

6,7-Dimethyl-8-ribityllumazine, the immediate biosynthetic precursor of riboflavin, is synthesized by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate. The gene coding for 6,7-dimethyl-8-ribityllumazine synthase in Saccharomyces cerevisiae (RIB4) has been cloned by functional complementation of a mutant accumulating 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which can grow on riboflavin- or diacetyl- but not on 3,4-dihydroxy-2-butanone-supplemented media. Gene disruption of the chromosomal copy of RIB4 led to riboflavin auxotrophy and loss of enzyme activity. Nucleotide sequencing revealed a 169-base pair open reading frame encoding a 18.6-kDa protein. Hybridization analysis indicated that RIB4 is a single copy gene located on the left arm of chromosome XV. Overexpression of the RIB4 coding sequence in yeast cells under the control of the strong TEF1 promoter allowed ready purification of 6,7-dimethyl-8-ribityllumazine synthase to apparent homogeneity by a simple procedure. Initial structural characterization of 6,7-dimethyl-8-ribityllumazine synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a pentamer of identical 16.8-kDa subunits. The derived amino acid sequence of RIB4 shows extensive homology to the sequences of the beta subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes.

Riboflavin biosynthesis in Saccharomyces cerevisiae. Cloning, characterization, and expression of the RIB5 gene encoding riboflavin synthase.

Saccharomyces cerevisiae has a monofunctional riboflavin synthase that catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine. We have isolated the gene encoding this enzyme from a yeast genomic library by functional complementation of a mutant, rib5-10, lacking riboflavin synthase activity. Deletion of the chromosomal copy of RIB5 led to riboflavin auxotrophy and loss of enzyme activity. Intragenic complementation between point and deletion mutant alleles suggested that the encoded protein (Rib5p) assembles into a multimeric complex and predicted the existence of a discrete functional domain located at the N terminus. Nucleotide sequencing revealed a 714-base pair open reading frame encoding a 25-kDa protein. Rib5p was purified to apparent homogeneity by a simple procedure. The specific activity of the enzyme was enriched 8500-fold. The N-terminal sequence of the purified enzyme was identical to the sequence predicted from the nucleotide sequence of the RIB5 gene. Initial structural characterization of riboflavin synthase by gel filtration chromatography and both nondenaturing pore limit and SDS-polyacrylamide gel electrophoresis showed that the enzyme forms a trimer of identical 25-kDa subunits. The derived amino acid sequence of RIB5 shows extensive homology to the sequences of the alpha subunits of riboflavin synthase from Bacillus subtilis and other prokaryotes. In addition, the sequence also shows internal homology between the N-terminal and the C-terminal halves of the protein. Taken together, these results suggest that the Rib5p subunit contains two structurally related (substrate-binding) but catalytically different (acceptor and donator) domains.

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.