A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.

The interdependence of mammary-specific super-enhancers and their native promoters facilitates gene activation during pregnancy

Lineage-specific genetic programs rely on cell-restricted super-enhancers, which are platforms for high-density transcription factor occupation. It is not known whether super-enhancers synergize specifically with their native promoters or provide autonomous and independent regulatory platforms. Here, we investigated the ability of the mammary Wap super-enhancer to activate the promoter of the juxtaposed and ubiquitously expressed Tbrg4 gene in the mouse mammary gland. The Wap super-enhancer was fused, alone or in combination with the Wap promoter, to the Tbrg4 gene. While the super-enhancer increased the expression of the Tbrg4 promoter five-fold, the combination of the super-enhancer and promoter resulted in 80-fold gene upregulation, demonstrating lineage-specific promoter–enhancer synergy. Employing ChIP-seq profiling to determine transcription factor binding and identify activating histone marks, we uncovered a chromatin platform that enables the high-level expression of the native promoter–enhancer but not the heterologous promoter. Taken together, our data reveal that lineage-specific enhancer–promoter synergy is critical for mammary gene regulation during pregnancy and lactation.

RelB/NF-κB2 Regulates Corticotropin-Releasing Hormone in the Human Placenta

Placental CRH may be part of a clock that governs the length of human gestation. The mechanism underlying differential regulation of CRH in the human placenta is poorly understood. We report here that constitutively activated RelB/nuclear factor-κB2 (NF-κB)-2 (p100/p52) acts as an endogenous stimulatory signal to regulate CRH by binding to an NF-κB enhancer of CRH gene promoter in the human placenta. Nuclear staining of NF-κB2 and RelB in villous syncytiotrophoblasts and cytotrophoblasts was coupled with cytoplasmic CRH in syncytial knots of cytotrophoblasts. Chromatin immunoprecipitation identified that CRH gene associated with both RelB and NF-κB2 (p52). Dexamethasone increased synthesis and nuclear translocation of RelB and NF-κB2 (p52) and their association with the CRH gene. In contrast, progesterone, a down-regulator of placental CRH, repressed NF-κB2 (p100) processing, nuclear translocation of RelB and NF-κB2 (p52), and their association with the CRH gene. Luciferase reporter assay determined that the NF-κB enhancer of CRH was sufficient to regulate transcriptional activity of a heterologous promoter in primary cytotrophoblasts. RNA interference-mediated repression of RelB or NF-κB2 resulted in significant inhibition of CRH at both transcriptional and translational levels and prevented the dexamethasone-mediated up-regulation of CRH transcription and translation. These results suggest that the noncanonical NF-κB pathway regulates CRH production in the human placenta and is responsible for the positive regulation of CRH by glucocorticoids.

The 26-Amino Acid ß-Motif of the Pit-1ß Transcription Factor Is a Dominant and Independent Repressor Domain

The POU-homeodomain transcription factor Pit-1 governs the pituitary cell-specific expression of Pit-1, GH, prolactin (PRL), and TSHß genes. Alternative splicing generates Pit-1ß, which contains a 26-amino acid ß-domain inserted at amino acid 48, in the middle of the Pit-1 transcription activation domain (TAD). Pit-1ß represses GH, PRL, and TSHß promoters in a pituitary-specific manner, because Pit-1ß activates these same promoters in HeLa nonpituitary cells. Here we comprehensively analyze the role of ß-domain sequence, position, and context, to elucidate the mechanism of ß-dependent repression. Repositioning the ß-motif to the Pit-1 amino terminus, hinge, linker, and carboxyl terminus did not affect its ability to repress basal rat (r) PRL promoter activity in GH4 pituitary cells, but all lost the ability to repress Ras-induced rPRL promoter activity. To determine whether ß-domain repression is independent of Pit-1 protein and DNA binding sites, we generated Gal4-Pit-1TAD, Gal4-Pit-1ßTAD, and Gal4-ß-domain fusions and demonstrated that the ß-motif is sufficient to actively repress VP16-mediated transcription of a heterologous promoter. Moreover, ß-domain point mutants had the same effect whether fused to Gal4 or within the context of intact Pit-1ß. Surprisingly, Gal4-ß repression lost histone deacetylase sensitivity and pituitary specificity. Taken together, these results reveal that the ß-motif is a context-independent, modular, transferable, and dominant repressor domain, yet the ß-domain repressor activity within Pit-1ß contains cell type, promoter, and Pit-1 protein context dependence.

Deletion of Many Yeast Introns Reveals a Minority of Genes that Require Splicing for Function

Splicing regulates gene expression and contributes to proteomic diversity in higher eukaryotes. However, in yeast only 283 of the 6000 genes contain introns and their impact on cell function is not clear. To assess the contribution of introns to cell function, we initiated large-scale intron deletions in yeast with the ultimate goal of creating an intron-free model eukaryote. We show that about one-third of yeast introns are not essential for growth. Only three intron deletions caused severe growth defects, but normal growth was restored in all cases by expressing the intronless mRNA from a heterologous promoter. Twenty percent of the intron deletions caused minor phenotypes under different growth conditions. Strikingly, the combined deletion of all introns from the 15 cytoskeleton-related genes did not affect growth or strain fitness. Together, our results show that although the presence of introns may optimize gene expression and provide benefit under stress, a majority of introns could be removed with minor consequences on growth under laboratory conditions, supporting the view that many introns could be phased out of Saccharomyces cerevisiae without blocking cell growth.

c-Myc and ChREBP regulate glucose-mediated expression of the L-type pyruvate kinase gene in INS-1-derived 832/13 cells

Increased glucose flux generates metabolic signals that control transcriptional programs through poorly understood mechanisms. Previously, we demonstrated a necessity in hepatocytes for c-Myc in the regulation of a prototypical glucose-responsive gene, L-type pyruvate kinase (L-PK) (Collier JJ, Doan TT, Daniels MC, Schurr JR, Kolls JK, Scott DK. J Biol Chem 278: 6588–6595, 2003). Pancreatic β-cells have many features in common with hepatocytes with respect to glucose-regulated gene expression, and in the present study we determined whether c-Myc was required for the L-PK glucose response in insulin-secreting (INS-1)-derived 832/13 cells. Glucose increased c-Myc abundance and association with its heterodimer partner, Max. Manipulations that prevented the formation of a functional c-Myc/Max heterodimer reduced the expression of the L-PK gene. In addition, glucose augmented the binding of carbohydrate response element binding protein (ChREBP), c-Myc, and Max to the promoter of the L-PK gene in situ. The transactivation of ChREBP, but not of c-Myc, was dependent on high glucose concentrations in the contexts of either the L-PK promoter or a heterologous promoter. The glucose-mediated transactivation of ChREBP was independent of mutations that alter phosphorylation sites thought to regulate the cellular location of ChREBP. We conclude that maximal glucose-induced expression of the L-PK gene in INS-1-derived 832/13 cells involves increased c-Myc abundance, recruitment of c-Myc, Max, and ChREBP to the promoter, and a glucose-stimulated increase in ChREBP transactivation.

σE Regulates and Is Regulated by a Small RNA in Escherichia coli

ABSTRACT RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA, encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on σE. The sequence upstream of the +1 of rybB contains a consensus σE promoter. The activity of rybB-lacZ was increased in cells lacking the RseA anti-sigma factor and when σE was overproduced from a heterologous promoter. The activity of rybB-lacZ and the detection of RybB were totally abolished in an rpoE-null strain. In vitro, σE efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increased expression of both rybB-lacZ and rpoE-lacZ fusions, consistent with negative regulation of the σE response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate σE-dependent promoters in an RseA-independent fashion.

Metabolic Regulation of IMD2 Transcription and an Unusual DNA Element That Generates Short Transcripts

ABSTRACT Transcriptional regulation of IMD2 in yeast (Saccharomyces cerevisiae) is governed by the concentration of intracellular guanine nucleotide pools. The mechanism by which pool size is measured and transduced to the transcriptional apparatus is unknown. Here we show that DNA sequences surrounding the IMD2 initiation site constitute a repressive element (RE) involved in guanine regulation that contains a novel transcription-blocking activity. When this regulatory region is placed downstream of a heterologous promoter, short poly(A)+ transcripts are generated. The element is orientation dependent, and sequences within the normally transcribed and nontranscribed regions of the element are required for its activity. The promoter-proximal short RNAs are unstable and serve as substrates for the nuclear exosome. These findings support a model in which intergenic short transcripts emanating from upstream of the IMD2 promoter are terminated by a polyadenylation/terminator-like signal embedded within the IMD2 transcription start site.