Rapid isolation of yeast genomic DNA: Bust n' Grab

BACKGROUND: Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates. RESULTS: Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC. CONCLUSION: An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples

O-GlcNAc regulates the mitochondrial integrated stress response by regulating ATF4

BackgroundAccumulation of mitochondrial dysfunctional is a hallmark of age-related neurodegeneration including Alzheimer’s disease (AD). Impairment of mitochondrial quality control mechanisms leading to the accumulation of damaged mitochondria and increasing neuronal stress. Therefore, investigating the basic mechanisms of how mitochondrial homeostasis is regulated is essential. Herein, we investigate the role of O-GlcNAcylation, a single sugar post-translational modification, in controlling mitochondrial stress-induced transcription factor Activating Transcription Factor 4 (ATF4). Mitochondrial dysfunction triggers the integrated stress response (ISRmt), in which the phosphorylation of eukaryotic translation initiation factor 2α results in the translation of ATF4.MethodsWe used patient-derived induced pluripotent stem cells, a transgenic mouse model of AD, SH-SY5Y neuroblastoma and HeLa cell-lines to examine the effect of sustained O-GlcNAcase inhibition by Thiamet-G (TMG) on ISRmt using biochemical analyses.ResultsWe show that TMG elevates ATF4 protein levels upon mitochondrial stress in SH-SY5Y neuroblastoma and HeLa cell-lines. An indirect downstream target of ATF4 mitochondrial chaperone glucose-regulated protein 75 (GRP75) is significantly elevated. Interestingly, knock-down of O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc, in SH-SY5Y increases ATF4 protein and mRNA expression. Additionally, ATF4 target gene Activating Transcription Factor 5 (ATF5) is significantly elevated at both the protein and mRNA level. Brains isolated from TMG treated mice show elevated levels of ATF4 and GRP75. Importantly, ATF4 occupancy increases at the ATF5 promoter site in brains isolated from TMG treated mice suggesting that O-GlcNAc is regulating ATF4 targeted gene expression. Interestingly, ATF4 and GRP75 are not induced in TMG treated familial Alzheimer’s Disease mice model. The same results are seen in a human in vitro model of AD.ConclusionTogether, these results indicate that in healthy conditions, O-GlcNAc regulates the ISRmt through regulating ATF4, while manipulating O-GlcNAc in AD has no effect on ISRmt

Systematic documentation and analysis of human genetic variation in hemoglobinopathies using the microattribution approach

We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases

Deletion of the human β-globin LCR 5′HS4 or 5′HS1 differentially affects β-like globin gene expression in β-YAC transgenic mice

Photograph of a scene on Quanah Parker Lake, in the Wichita Wildlife Refuge

Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site▿ †

Autonomous silencing of γ-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the Aγ-globin gene was identified between −730 and −378 relative to the mRNA start site. A marked copy of the Aγ-globin gene inserted between locus control region 5′ DNase I-hypersensitive site 1 and the ɛ-globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the −566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low γ-globin levels, whereas only a weak signal was detected when γ-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the Aγ-globin −566 or Gγ-globin −567 GATA site when γ-globin expression was low (day 18) but not when γ-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, γ-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the −566/−567 GATA sites of the proximal γ-globin promoters

Mi2β Is Required for γ-Globin Gene Silencing: Temporal Assembly of a GATA-1-FOG-1-Mi2 Repressor Complex in β-YAC Transgenic Mice

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author’s publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the −566 GATA motif of the Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the −566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the −566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting −566 Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis

GATA-2 binding at the −566 GATA site of the ^Aγ-globin gene in wild-type β-YAC.

<p>A) GATA-2 protein occupancy at the −566 ^Aγ-globin GATA site by ChIP analysis. B) GATA-2 protein occupancy at the murine Gata-2 gene −2.8 Kb binding site by ChIP analysis. Binding of GATA-2 at the murine Gata-2 locus was used as a positive control for GATA-2 occupancy. ChIP was performed as described in the legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003155#pgen-1003155-g004" target="_blank">Figure 4</a>; labeling is also as illustrated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003155#pgen-1003155-g004" target="_blank">Figure 4</a>.</p

RP-HPLC determination of γ-globin chain expression as a percentage of total human β-like globin chain expression in −566 ^Aγ-globin HPFH β-YAC transgenic mice.

<p>RP-HPLC determination of γ-globin chain expression as a percentage of total human β-like globin chain expression in −566 ^Aγ-globin HPFH β-YAC transgenic mice.</p

Human β-like globin expression in adult −566 ^Aγ-globin HPFH β-YAC transgenic mice.

<p>(A and B) Expression of the fetal γ- and adult β-globin was analyzed by real-time qRT-PCR and the data was normalized to mouse α-globin or Gapdh gene expression. (A) Human fetal γ-globin mRNA expression; B) Adult β-globin mRNA expression. Lines are indicated at the bottom of the plot. Results are the average of triplicate experiments from three animals of lines 20. * indicates P<0.05. (C–F) Flow cytometry analysis was performed using a mouse monoclonal anti-γ-globin antibody to determine the percentage of F cells. C) Wild-type β-YAC; D) −117 Greek HPFH β-YAC; E) −566 ^Aγ-globin HPFH β-YAC transgenic line 20 (1), F) −566 ^Aγ-globin HPFH β-YAC transgenic line 20 (2). Flow cytometry controls are identical to those shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003155#pgen-1003155-g002" target="_blank">Figure 2A, 2B and 2G</a> since the experiments were performed together. (G–I) Cytospins were prepared using an anti-human hemoglobin F FITC-conjugated antibody. G) Wild-type β-YAC; H) −117 Greek HPFH β-YAC; I) −566 ^Aγ-globin HPFH β-YAC transgenic line 20. Top panels, visible light images of cytospin cells; bottom panels, immunofluorescent images of same fields.</p