Neighborhood regulation by lncRNA promoters, transcription, and splicing

Mammalian genomes are pervasively transcribed to produce thousands of spliced long noncoding RNAs (lncRNAs), whose functions remain poorly understood. Because recent evidence has implicated several specific lncRNA loci in the local regulation of gene expression, we sought to determine whether such local regulation is a property of many lncRNA loci. We used genetic manipulations to dissect 12 genomic loci that produce lncRNAs and found that 5 of these loci influence the expression of a neighboring gene in cis. Surprisingly, however, none of these effects required the specific lncRNA transcripts themselves and instead involved general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Interestingly, such effects are not limited to lncRNA loci: we found similar effects on local gene expression at 4 of 6 protein-coding loci. These results demonstrate that 'crosstalk' among neighboring genes is a prevalent phenomenon that can involve multiple mechanisms and cis regulatory signals, including a novel role for RNA splicing. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia

Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia

Cigarette Smoke Exposure Alters mSin3a and Mi-2α/β Expression; implications in the control of pro-inflammatory gene transcription and glucocorticoid function

<p>Abstract</p> <p>Background</p> <p>The key co-repressor complex components HDAC-2, Mi-2α/β and mSin3a are all critical to the regulation of gene transcription. HDAC-2 function is impaired by oxidative stress in a PI3Kδ dependant manner which may be involved in the chronic glucocorticoid insensitive inflammation in the lungs of COPD patients. However, the impact of cigarette smoke exposure on the expression of mSin3a and Mi2α/β and their role in glucocorticoid responsiveness is unknown.</p> <p>Methods</p> <p>Wild type, PI3Kγ knock-out (PI3Kγ^-/-) and PI3K kinase dead knock-in (PI3Kδ^D910/A910) transgenic mice were exposed to cigarette smoke for 3 days and the expression levels of the co-repressor complex components HDAC-2, mSin3a, Mi-2α and Mi-2β and HDAC-2 activity in the lungs were assessed.</p> <p>Results</p> <p>Cigarette smoke exposure impaired glucocorticoid function and reduced HDAC-2 activity which was protected in the PI3Kδ^D910/A910mice. Both mSin3a and Mi-2α protein expression was reduced in smoke-exposed mice. Budesonide alone protected mSin3a protein expression with no additional effect seen with abrogation of PI3Kγ/δ activity, however Mi-2α, but not Mi-2β, expression was protected in both PI3Kδ^D910/A910and PI3Kγ^-/-budesonide-treated smoke-exposed mice. The restoration of glucocorticoid function coincided with the protection of both HDAC activity and mSin3a and Mi-2α protein expression.</p> <p>Conclusions</p> <p>Cigarette smoke exposure induced glucocorticoid insensitivity and alters co-repressor activity and expression which is prevented by blockade of PI3K signaling with glucocorticoid treatment. Inhibition of PI3Kδ signalling in combination with glucocorticoid treatment may therefore provide a therapeutic strategy for restoring oxidant-induced glucocortiocid unresponsiveness.</p

The Chromosomal High-Affinity Binding Sites for the Drosophila Dosage Compensation Complex

Dosage compensation in male Drosophila relies on the X chromosome–specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called “high-affinity sites” (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features

On the Interaction of Clostridium perfringens Enterotoxin with Claudins

Clostridium perfringens causes one of the most common foodborne illnesses, which is largely mediated by the Clostridium perfringens enterotoxin (CPE). The toxin consists of two functional domains. The N-terminal region mediates the cytotoxic effect through pore formation in the plasma membrane of the mammalian host cell. The C-terminal region (cCPE) binds to the second extracellular loop of a subset of claudins. Claudin-3 and claudin-4 have been shown to be receptors for CPE with very high affinity. The toxin binds with weak affinity to claudin-1 and -2 but contribution of these weak binding claudins to CPE-mediated disease is questionable. cCPE is not cytotoxic, however, it is a potent modulator of tight junctions. This review describes recent progress in the molecular characterization of the cCPE-claudin interaction using mutagenesis, in vitro binding assays and permeation studies. The results promote the development of recombinant cCPE-proteins and CPE-based peptidomimetics to modulate tight junctions for improved drug delivery or to treat tumors overexpressing claudins

Stem Cell Factor SALL4 Represses the Transcriptions of PTEN and SALL1 through an Epigenetic Repressor Complex

Background The embryonic stem cell (ESC) factor, SALL4, plays an essential role in both development and leukemogenesis. It is a unique gene that is involved in self-renewal in ESC and leukemic stem cell (LSC).Methodology/Principal Findings To understand the mechanism(s) of SALL4 function(s), we sought to identify SALL4-associated proteins by tandem mass spectrometry. Components of a transcription repressor Mi-2/Nucleosome Remodeling and Deacetylase (NuRD) complex were found in the SALL4-immunocomplexes with histone deacetylase (HDAC) activity in ESCs with endogenous SALL4 expression and 293T cells overexpressing SALL4. The SALL4-mediated transcriptional regulation was tested on two potential target genes: PTEN and SALL1. Both genes were confirmed as SALL4 downstream targets by chromatin-immunoprecipitation, and their expression levels, when tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR), were decreased in 293T cells overexpressing SALL4. Moreover, SALL4 binding sites at the promoter regions of PTEN and SALL1 were co-occupied by NuRD components, suggesting that SALL4 represses the transcriptions of PTEN and SALL1 through its interactions with the Mi-2/NuRD complex. The in vivo repressive effect(s) of SALL4 were evaluated in SALL4 transgenic mice, where decreased expressions of PTEN and SALL1 were associated with myeloid leukemia and cystic kidneys, respectively.Conclusions/Significance In summary, we are the first to demonstrate that stem cell protein SALL4 represses its target genes, PTEN and SALL1, through the epigenetic repressor Mi-2/NuRD complex. Our novel finding provides insight into the mechanism(s) of SALL4 functions in kidney development and leukemogenesis

Faced with inequality: chicken do not have a general dosage compensation of sex-linked genes

<p>Abstract</p> <p>Background</p> <p>The contrasting dose of sex chromosomes in males and females potentially introduces a large-scale imbalance in levels of gene expression between sexes, and between sex chromosomes and autosomes. In many organisms, dosage compensation has thus evolved to equalize sex-linked gene expression in males and females. In mammals this is achieved by X chromosome inactivation and in flies and worms by up- or down-regulation of X-linked expression, respectively. While otherwise widespread in systems with heteromorphic sex chromosomes, the case of dosage compensation in birds (males ZZ, females ZW) remains an unsolved enigma.</p> <p>Results</p> <p>Here, we use a microarray approach to show that male chicken embryos generally express higher levels of Z-linked genes than female birds, both in soma and in gonads. The distribution of male-to-female fold-change values for Z chromosome genes is wide and has a mean of 1.4–1.6, which is consistent with absence of dosage compensation and sex-specific feedback regulation of gene expression at individual loci. Intriguingly, without global dosage compensation, the female chicken has significantly lower expression levels of Z-linked compared to autosomal genes, which is not the case in male birds.</p> <p>Conclusion</p> <p>The pronounced sex difference in gene expression is likely to contribute to sexual dimorphism among birds, and potentially has implication to avian sex determination. Importantly, this report, together with a recent study of sex-biased expression in somatic tissue of chicken, demonstrates the first example of an organism with a lack of global dosage compensation, providing an unexpected case of a viable system with large-scale imbalance in gene expression between sexes.</p

Progress and prospects toward our understanding of the evolution of dosage compensation

In many eukaryotic organisms, gender is determined by a pair of heteromorphic sex chromosomes. Degeneration of the non-recombining Y chromosome is a general facet of sex chromosome evolution. Selective pressure to restore expression levels of X-linked genes relative to autosomes accompanies Y-chromosome degeneration, thus driving the evolution of dosage compensation mechanisms. This review focuses on evolutionary aspects of dosage compensation, in light of recent advances in comparative and functional genomics that have substantially increased our understanding of the molecular mechanisms of dosage compensation and how it evolved. We review processes involved in sex chromosome evolution, and discuss the dynamic interaction between Y degeneration and the acquisition of dosage compensation. We compare mechanisms of dosage compensation and the origin of dosage compensation genes between different taxa and comment on sex chromosomes that apparently lack compensation mechanisms. Finally, we discuss how dosage compensation systems can also influence the evolution of well-established sex chromosomes

Xist regulation and function eXplored

X chromosome inactivation (XCI) is a process in mammals that ensures equal transcript levels between males and females by genetic inactivation of one of the two X chromosomes in females. Central to XCI is the long non-coding RNA Xist, which is highly and specifically expressed from the inactive X chromosome. Xist covers the X chromosome in cis and triggers genetic silencing, but its working mechanism remains elusive. Here, we review current knowledge about Xist regulation, structure, function and conservation and speculate on possible mechanisms by which its action is restricted in cis. We also discuss dosage compensation mechanisms other than XCI and how knowledge from invertebrate species may help to provide a better understanding of the mechanisms of mammalian XCI