Role of erythroid Kruppel-like factor in human γ- to β-globin gene switching

Erythroid Kruppel-like factor (EKLF) is an erythroid-specific transcription factor that contains zinc finger domains similar to the Kruppel protein of Drosophila melanogaster. Previous studies demonstrated that EKLF binds to the CACCC box in the human β-globin gene promoter and activates transcription. CACCC box mutations that cause severe β-thalassemias in humans inhibit EKLF binding. Results described in this paper suggest that EKLF functions predominately in adult erythroid tissue. The EKLF gene is expressed at a 3-fold higher level in adult erythroid tissue than in fetal erythroid tissue, and the EKLF protein binds to the human β-globin promoter 8-fold more efficiently than to the human γ-globin promoter. Co-transfection experiments in the human fetal-like erythroleukemia cell line K562 demonstrate that over-expression of EKLF activates a β-globin reporter construct 1000-fold; a linked γ-globin reporter is activated only 3-fold. Mutation of the β-globin CACCC box severely inhibits activation. These results demonstrate that EKLF is a developmental stage-enriched protein that preferentially activates human β-globin gene expression. The data strongly suggest that EKLF is an important factor involved in human γ- to β-globin gene switching

Atividade de peroxidase de três populações de "Switchgrass" (Panicum virgatum L.) em resposta à injúria do pulgão-verdedos-cereais (Schizaphis graminum) (Hemiptera: Aphididae).

?Switchgrass? é uma gramínea que tem recebido atenção especial como uma cultura bioenergética nos EUA. Os pulgões foram relatados como uma praga potencial em populações desta gramínea e não está claro como a manipulação genética para uma melhor produção de bioenergia afetará a capacidade da planta em tolerar a injuria deste inseto. Portanto, os objetivos deste estudo foram comparar o teor de proteína total e atividade de peroxidase de três populações de ?switchgrass? (Kanlow, KxS e Summer) a injuria pelo pulgão-verde-dos-cereais

A Single Heterochromatin Boundary Element Imposes Position-Independent Antisilencing Activity in Saccharomyces cerevisiae Minichromosomes

Chromatin boundary elements serve as cis-acting regulatory DNA signals required to protect genes from the effects of the neighboring heterochromatin. In the yeast genome, boundary elements act by establishing barriers for heterochromatin spreading and are sufficient to protect a reporter gene from transcriptional silencing when inserted between the silencer and the reporter gene. Here we dissected functional topography of silencers and boundary elements within circular minichromosomes in Saccharomyces cerevisiae. We found that both HML-E and HML-I silencers can efficiently repress the URA3 reporter on a multi-copy yeast minichromosome and we further showed that two distinct heterochromatin boundary elements STAR and TEF2-UASrpg are able to limit the heterochromatin spreading in circular minichromosomes. In surprising contrast to what had been observed in the yeast genome, we found that in minichromosomes the heterochromatin boundary elements inhibit silencing of the reporter gene even when just one boundary element is positioned at the distal end of the URA3 reporter or upstream of the silencer elements. Thus the STAR and TEF2-UASrpg boundary elements inhibit chromatin silencing through an antisilencing activity independently of their position or orientation in S. cerevisiae minichromosomes rather than by creating a position-specific barrier as seen in the genome. We propose that the circular DNA topology facilitates interactions between the boundary and silencing elements in the minichromosomes

Genetically tagged TRE5-A retrotransposons reveal high amplification rates and authentic target site preference in the Dictyostelium discoideum genome

Retrotransposons contribute significantly to the evolution of eukaryotic genomes. They replicate by producing DNA copies of their own RNA, which are integrated at new locations in the host cell genome. In the gene-dense genome of the social amoeba Dictyostelium discoideum, retrotransposon TRE5-A avoids insertional mutagenesis by targeting the transcription factor (TF) IIIC/IIIB complex and integrating ∼50 bp upstream of tRNA genes. We generated synthetic TRE5-A retrotransposons (TRE5-Absr) that were tagged with a selection marker that conferred resistance to blasticidin after a complete retrotransposition cycle. We found that the TRE5-Absr elements were efficiently mobilized in trans by proteins expressed from the endogenous TRE5-A population found in D. discoideum cells. ORF1 protein translated from TRE5-Absr elements significantly enhanced retrotransposition. We observed that the 5′ untranslated region of TRE5-A could be replaced by an unrelated promoter, whereas the 3′ untranslated region of TRE5-A was essential for retrotransposition. A predicted secondary structure in the RNA of the 3′ untranslated region of TRE5-A may be involved in the retrotransposition process. The TRE5-Absr elements were capable of identifying authentic integration targets in vivo, including formerly unnoticed, putative binding sites for TFIIIC on the extrachromosomal DNA element that carries the ribosomal RNA genes

Evaluation of Greenbug and Yellow Sugarcane Aphid Feeding Behavior on Resistant and Susceptible Switchgrass Cultivars

Switchgrass (Panicum virgatum L.) is an emerging biofuel crop that serves as host for aphids. To discern the effects of plant age and possible resistance mechanisms, the feeding behavior of greenbugs (Schizaphis graminum Rondani.) and the yellow sugarcane aphid (Sipha flava Forbes.) was monitored on three diverse switchgrasses by the electrical penetration graph (EPG) technique. Callose deposition and genes associated with callose metabolism were also analyzed to discern their association with plant resistance. There was a strong host effect on greenbugs feeding on lowland cultivar Kanlow at the V3 stage of development, as compared to the greenbug-susceptible upland cultivar Summer and plants derived from Kanlow (♂) × Summer (♀) (K×S) crosses. These data confirmed that Kanlow at the V3 stage had antibiosis to greenbugs, which was absent in the Summer and K×S plants. In contrast, similar effects were not observed for yellow sugarcane aphids, excluding significant differences in the time to first probe on Kanlow plants at the V1 stage and reduction in time spent on pathway processes on Kanlow plants at the V3 stage. These data demonstrated that Kanlow plants may have multiple sources of resistance to the two aphids, and possibly some were phloem based. Microscopy of leaf sections stained with aniline blue for callose was suggestive of increased callose deposition in the sieve elements in Kanlow plants relative to Summer and K×S plants. RT-qPCR analysis of several genes associated with callose metabolism in infested plants was equivocal. Overall, these studies suggest the presence of multiple defense mechanisms against aphids in Kanlow plants, relative to Summer and K×S plants

The Impact of Local Genome Sequence on Defining Heterochromatin Domains

Characterizing how genomic sequence interacts with trans-acting regulatory factors to implement a program of gene expression in eukaryotic organisms is critical to understanding genome function. One means by which patterns of gene expression are achieved is through the differential packaging of DNA into distinct types of chromatin. While chromatin state exerts a major influence on gene expression, the extent to which cis-acting DNA sequences contribute to the specification of chromatin state remains incompletely understood. To address this, we have used a fission yeast sequence element (L5), known to be sufficient to nucleate heterochromatin, to establish de novo heterochromatin domains in the Schizosaccharomyces pombe genome. The resulting heterochromatin domains were queried for the presence of H3K9 di-methylation and Swi6p, both hallmarks of heterochromatin, and for levels of gene expression. We describe a major effect of genomic sequences in determining the size and extent of such de novo heterochromatin domains. Heterochromatin spreading is antagonized by the presence of genes, in a manner that can occur independent of strength of transcription. Increasing the dosage of Swi6p results in increased heterochromatin proximal to the L5 element, but does not result in an expansion of the heterochromatin domain, suggesting that in this context genomic effects are dominant over trans effects. Finally, we show that the ratio of Swi6p to H3K9 di-methylation is sequence-dependent and correlates with the extent of gene repression. Taken together, these data demonstrate that the sequence content of a genomic region plays a significant role in shaping its response to encroaching heterochromatin and suggest a role of DNA sequence in specifying chromatin state

Non-invasive quantitative imaging of hepatocellular carcinoma growth in mice by micro-CT using liver-targeted iodinated nano-emulsions

Hepatocellular carcinoma (HCC) is the only cancer for which non-invasive diagnosis is recognized by international guidelines. Contrast agent free ultrasound imaging, computed tomography (CT) and/or magnetic resonance imaging are techniques used for early detection and confirmation. Clinical evidence depicts that CT is 30% less precise as compared to MRI for detection of small tumors. In our work, we have reported some novel tools that can enhance the sensitivity and precision of CT applied to preclinical research (micro-CT). Our system, containing non-toxic nano-droplets loaded with iodine has high contrasting properties, liver and hepatocyte specificity and strong liver persistence. Micro-CT was performed on HCC model implanted in nude mice by intrahepatic injection. Contrast agent was administrated intravenously. This method allows an unprecedented high precision of detection, quantitative measurement of tumor volume and quantitative follow-up of the tumor development.PMC565532

Nucleoporin Mediated Nuclear Positioning and Silencing of HMR

The organization of chromatin domains in the nucleus is an important factor in gene regulation. In eukaryotic nuclei, transcriptionally silenced chromatin clusters at the nuclear periphery while transcriptionally poised chromatin resides in the nuclear interior. Recent studies suggest that nuclear pore proteins (NUPs) recruit loci to nuclear pores to aid in insulation of genes from silencing and during gene activation. We investigated the role of NUPs at a native yeast insulator and show that while NUPs localize to the native tDNA insulator adjacent to the silenced HMR domain, loss of pore proteins does not compromise insulation. Surprisingly we find that NUPs contribute to silencing at HMR and are able to restore silencing to a silencing-defective HMR allele when tethered to the locus. We show that the perinuclear positioning of heterochromatin is important for the NUP-mediated silencing effect and find that loss of NUPs result in decreased localization of HMR to the nuclear periphery. We also show that loss of telomeric tethering pathways does not eliminate NUP localization to HMR, suggesting that NUPs may mediate an independent pathway for HMR association with the nuclear periphery. We propose that localization of NUPs to the tDNA insulator at HMR helps maintain the intranuclear position of the silent locus, which in turn contributes to the fidelity of silencing at HMR

Cohesin is required for higher-order chromatin conformation at the imprinted IGF2-H19 locus

Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi-mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF-mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesin's function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion