Association of FGF4L1 Retrogene Insertion with Prolapsed Gland of the Nictitans (Cherry Eye) in Dogs.

Cherry eye is the common name for prolapse of the nictitans gland, a tear-producing gland situated under the third eyelid of dogs. Cherry eye is characterized by a red fleshy protuberance in the corner of the eye, resembling a cherry. This protrusion is a displacement of the normal gland of the third eyelid, thought to be caused by a defect in the connective tissue that secures the gland in place. Options for treatment may include anti-inflammatory medications in mild cases, but surgical replacement of the gland is usually indicated. Cherry eye is most often seen in dogs under the age of two years, with certain breeds having a higher incidence, suggesting a potential genetic association. Integration of panel genetic testing into routine clinical practice allows for the generation of large numbers of genotyped individuals paired with clinical records and enables the investigation of common disorders using a genome-wide association study (GWAS) approach at scale. In this investigation, several thousand cases and controls for cherry eye in both purebred dogs and mixed breeds are used for a large-scale GWAS, revealing a single peak of genome-wide significance on canine chromosome 18, directly at the location of the previously identified FGF4 insertion known to cause chondrodysplasia in several breeds.

Mechanism for DNA transposons to generate introns on genomic scales.

The discovery of introns four decades ago was one of the most unexpected findings in molecular biology. Introns are sequences interrupting genes that must be removed as part of messenger RNA production. Genome sequencing projects have shown that most eukaryotic genes contain at least one intron, and frequently many. Comparison of these genomes reveals a history of long evolutionary periods during which few introns were gained, punctuated by episodes of rapid, extensive gain. However, although several detailed mechanisms for such episodic intron generation have been proposed, none has been empirically supported on a genomic scale. Here we show how short, non-autonomous DNA transposons independently generated hundreds to thousands of introns in the prasinophyte Micromonas pusilla and the pelagophyte Aureococcus anophagefferens. Each transposon carries one splice site. The other splice site is co-opted from the gene sequence that is duplicated upon transposon insertion, allowing perfect splicing out of the RNA. The distributions of sequences that can be co-opted are biased with respect to codons, and phasing of transposon-generated introns is similarly biased. These transposons insert between pre-existing nucleosomes, so that multiple nearby insertions generate nucleosome-sized intervening segments. Thus, transposon insertion and sequence co-option may explain the intron phase biases and prevalence of nucleosome-sized exons observed in eukaryotes. Overall, the two independent examples of proliferating elements illustrate a general DNA transposon mechanism that can plausibly account for episodes of rapid, extensive intron gain during eukaryotic evolution.

Dnmt1-independent CG methylation contributes to nucleosome positioning in diverse eukaryotes.

Dnmt1 epigenetically propagates symmetrical CG methylation in many eukaryotes. Their genomes are typically depleted of CG dinucleotides because of imperfect repair of deaminated methylcytosines. Here, we extensively survey diverse species lacking Dnmt1 and show that, surprisingly, symmetrical CG methylation is nonetheless frequently present and catalyzed by a different DNA methyltransferase family, Dnmt5. Numerous Dnmt5-containing organisms that diverged more than a billion years ago exhibit clustered methylation, specifically in nucleosome linkers. Clustered methylation occurs at unprecedented densities and directly disfavors nucleosomes, contributing to nucleosome positioning between clusters. Dense methylation is enabled by a regime of genomic sequence evolution that enriches CG dinucleotides and drives the highest CG frequencies known. Species with linker methylation have small, transcriptionally active nuclei that approach the physical limits of chromatin compaction. These features constitute a previously unappreciated genome architecture, in which dense methylation influences nucleosome positions, likely facilitating nuclear processes under extreme spatial constraints.

Role of the Mce1 transporter in the lipid homeostasis of Mycobacterium tuberculosis.

Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the causative agent of human tuberculosis, has developed several strategies involving proteins and other compounds known collectively as virulence factors to subvert human host defences and invade the human host. The Mce proteins are among these virulence-related proteins and are encoded by the mce1, mce2, mce3 and mce4 operons in the genome of M. tuberculosis. It has been proposed that these operons encode ABC-like lipid transporters; however, the nature of their substrates has only been revealed in the case of the Mce4 proteins. Here we found that the knockout of the mce1 operon alters the lipid profile of M. tuberculosis H37Rv and the uptake of palmitic acid. Thin layer chromatography and liquid chromatography-mass spectrometry analysis showed that the mce1 mutant accumulates more mycolic acids than the wild type and complemented strains. Interestingly, this accumulation of mycolic acid is exacerbated when bacteria are cultured in the presence of palmitic acid or arachidonic acid. These results suggest that the mce1 operon may serve as a mycolic acid re-importer.

Regulation of biological accuracy, precision, and memory by plant chromatin organization.

Accumulating evidence points toward diverse functions for plant chromatin. Remarkable progress has been made over the last few years in elucidating the mechanisms for a number of these functions. Activity of the histone demethylase IBM1 accurately targets DNA methylation to silent repeats and transposable elements, not to genes. A genetic screen uncovered the surprising role of H2A.Z-containing nucleosomes in sensing precise differences in ambient temperature and consequent gene regulation. Precise maintenance of chromosome number is assured by a histone modification that suppresses inappropriate DNA replication and by centromeric histone H3 regulation of chromosome segregation. Histones and noncoding RNAs regulate FLOWERING LOCUS C, the expression of which quantitatively measures the duration of cold exposure, functioning as memory of winter. These findings are a testament to the power of using plants to research chromatin organization, and demonstrate examples of how chromatin functions to achieve biological accuracy, precision, and memory.

Expression of the ompATb operon accelerates ammonia secretion and adaptation of Mycobacterium tuberculosis to acidic environments.

Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis in macrophages. However, mechanisms by which M. tuberculosis adapts to acidic environments are poorly understood. In this study, we analysed the physiological functions of OmpATb, a surface-accessible protein of M. tuberculosis. OmpATb did not complement the permeability defects of a Mycobacterium smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of M. tuberculosis indicating that OmpATb is not a general porin. Chemical analysis of low-pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of M. tuberculosis. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which M. tuberculosis neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that M. tuberculosis has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of M. tuberculosis to acidic environments in vitro but not in mice.

Reciprocal intronic and exonic histone modification regions in humans.

While much attention has been focused on chromatin at promoters and exons, human genes are mostly composed of intronic sequences. Analyzing published surveys of nucleosomes and 41 chromatin marks in humans, we identified histone modifications specifically associated with 5' intronic sequences, distinguishable from promoter marks and bulk nucleosomes. These intronic marks were spatially reciprocal to trimethylated histone H3 Lys36 (H3K36me3), typically transitioning near internal exons. Several marks transitioned near bona fide exons, but not near nucleosomes at exon-like sequences. Therefore, we examined whether splicing affects histone marking. Even with considerable changes in regulated alternative splicing, histone marks were stable. Notably, these findings are consistent with exon definition influencing histone marks. In summary, we show that the location of many intragenic marks in humans can be distilled into a simple organizing principle: association with 5' intronic or 3' exonic regions.

Taking phage integration to the next level as a genetic tool for mycobacteria.

Genes must be stably integrated into bacterial chromosomes for complementation of gene deletion mutants in animal infection experiments or to express antigens in vaccine strains. However, with currently available vectors it is cumbersome to create multiple, stable, unmarked chromosomal integrations in mycobacteria. Here, we have constructed a novel integration vector for mycobacteria that enables expression of genes from a cassette protected from transcriptional interference by bi-directional transcriptional terminators proven to be highly efficient in in vitro transcription termination assays. Removal of the integrase gene by a site-specific recombinase, easily identifiable by loss of a backbone reporter gene, stabilizes the integration cassette and makes this vector ideally suitable for infection experiments. This integration vector can be easily adapted to different mycobacteriophage attachment sites (attB) due to its modular design. Integration of a gfp expression cassette at the L5, Giles and Ms6 attB sites in the chromosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis yielded identical gfp expression levels, indicating that none of these sites are compromised for gene expression. The copy number of pAL5000-based extrachromosomal plasmids is 23 in M. smegmatis as determined by quantitative real-time PCR and accounts for the previously observed drastic reduction of gene expression upon integration of plasmids into the chromosome of mycobacteria. Gfp expression and fluorescence of M. smegmatis and M. tuberculosis strains with multiple integrations of gfp increased concomitantly with the copy number demonstrating that these vectors can be used to generate stronger phenotypes and/or to analyze several genes simultaneously in vivo.

Hit-and-run stimulation: a novel concept to reactivate latent HIV-1 infection without cytokine gene induction.

Current antiretroviral therapy (ART) efficiently controls HIV-1 replication but fails to eradicate the virus. Even after years of successful ART, HIV-1 can conceal itself in a latent state in long-lived CD4(+) memory T cells. From this latent reservoir, HIV-1 rebounds during treatment interruptions. Attempts to therapeutically eradicate this viral reservoir have yielded disappointing results. A major problem with previously utilized activating agents is that at the concentrations required for efficient HIV-1 reactivation, these stimuli trigger high-level cytokine gene expression (hypercytokinemia). Therapeutically relevant HIV-1-reactivating agents will have to trigger HIV-1 reactivation without the induction of cytokine expression. We present here a proof-of-principle study showing that this is a possibility. In a high-throughput screening effort, we identified an HIV-1-reactivating protein factor (HRF) secreted by the nonpathogenic bacterium Massilia timonae. In primary T cells and T-cell lines, HRF triggered a high but nonsustained peak of nuclear factor kappa B (NF-kappaB) activity. While this short NF-kappaB peak potently reactivated latent HIV-1 infection, it failed to induce gene expression of several proinflammatory NF-kappaB-dependent cellular genes, such as those for tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), and gamma interferon (IFN-gamma). Dissociation of cellular and viral gene induction was achievable, as minimum amounts of Tat protein, synthesized following application of a short NF-kappaB pulse, triggered HIV-1 transactivation and subsequent self-perpetuated HIV-1 expression. In the absence of such a positive feedback mechanism, cellular gene expression was not sustained, suggesting that strategies modulating the NF-kappaB activity profile could be used to selectively trigger HIV-1 reactivation.

Mycobacterial outer membranes: in search of proteins.

The cell wall is a major virulence factor of Mycobacterium tuberculosis and contributes to its intrinsic drug resistance. Recently, cryo-electron microscopy showed that mycobacterial cell wall lipids form an unusual outer membrane. Identification of the components of the uptake and secretion machinery across this membrane will be crucial for understanding the physiology and pathogenicity of M. tuberculosis and for the development of better anti-tuberculosis drugs. Although the genome of M. tuberculosis appears to encode over 100 putative outer membrane proteins, only a few have been identified and characterized. Here, we summarize the current knowledge on the structure of the mycobacterial outer membrane and its known proteins. Through comparison to transport processes in Gram-negative bacteria, we highlight several hypothetical outer membrane proteins of M. tuberculosis that await discovery.

Accelerated fractionation head and neck intensity-modulated radiation therapy and concurrent chemotherapy in the community setting: safety and efficacy considerations.

BACKGROUND: This article reviews our community cancer center's experience treating head and neck cancer primarily with accelerated fractionation intensity-modulated radiation therapy (IMRT), with or without concurrent chemotherapy, focusing on acute toxicity and efficacy. METHODS: Fifty-two patients treated with IMRT at the Penrose Cancer Center between 2002 and 2007 constitute the cohort. The majority (75%) received an accelerated, altered fractionation regimen, typically concomitant boost to 7200 cGy. Concurrent chemotherapy was delivered to 32 (62%). The median follow-up was 24 months. RESULTS: The 2-year actuarial rates of local control, regional control, and distant metastasis-free survival were 100%, 91%, and 94%, respectively. Relapse-free survival and overall survival at 2 years were 89% and 91%, respectively. Overall, 32 of 52 patients (62%) experienced at least 1 type of grade 3 or 4 acute toxicity. CONCLUSION: Accelerated fractionation IMRT, with or without chemotherapy, can be given safely and effectively in a community cancer center setting.

Functions of the periplasmic loop of the porin MspA from Mycobacterium smegmatis.

MspA is the major porin of Mycobacterium smegmatis and mediates diffusion of small and hydrophilic solutes across the outer membrane. The octameric structure of MspA, its sharply defined constriction zone, and a large periplasmic loop L6 represent novel structural features. L6 consists of 13 amino acids and is directly adjacent to the constriction zone. Deletion of 3, 5, 7, 9, and 11 amino acids of the L6 loop resulted in functional pores that restored glucose uptake and growth of a porin mutant of M. smegmatis. Lipid bilayer experiments revealed that all mutant channels were noisier than wild type (wt) MspA, indicating that L6 is required for pore stability in vitro. Voltage gating of the Escherichia coli porin OmpF was attributed to loops that collapse into the channel in response to a strong electrical field. Here, we show that deletion mutants Delta7, Delta9, and Delta11 had critical voltages similar to wt MspA. This demonstrated that the L6 loop is not the primary voltage-dependent gating mechanism of MspA. Surprisingly, large deletions in L6 resulted in 3-6-fold less extractable pores, whereas small deletions did not alter expression levels of MspA. Pores with large deletions in L6 were more permissive for glucose than smaller deletion mutants, whereas their single channel conductance was similar to that of wt MspA. These results indicate that translocation of ions through the MspA pore is governed by different mechanisms than that of neutral solutes. This is the first study identifying a molecular determinant of solute translocation in a mycobacterial porin.

Chromatin regulation Tip(60)s the balance in embryonic stem cell self-renewal.

Histone modifications affect chromatin dynamics on several levels by serving as binding sites for regulatory proteins. In many cell types, including embryonic stem cells (ESCs), a subset of genes is marked with histone modifications thought to be both activating and repressing: H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3), respectively. As a result, genes bearing this "bivalent" mark are transcribed at low levels, but are primed for activation, should the cell receive the appropriate cues during differentiation. Recently, we found that the Tip60-p400 acetyltransferase and histone exchange complex is necessary to maintain normal self-renewal in mouse ESCs. While Tip60-p400 has histone acetyltransferase activity, which is generally associated with transcriptional activation, it acts predominantly as a repressor of genes expressed during differentiation. Surprisingly, in ESCs Tip60-p400 localizes to the promoters of genes marked by H3K4me3, which include both highly expressed genes and "bivalent" genes expressed at low levels. Tip60-p400 acetylates histones at these targets, including the promoters for developmental regulators it helps to silence in ESCs. This suggests that the effect of chromatin modifications on transcription is not always simply positive or negative. Rather, we propose that the impact of specific modifications at each promoter is determined by the chromatin context in which they are found.

An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity.

Proper regulation of chromatin structure is necessary for the maintenance of cell type-specific gene expression patterns. The embryonic stem cell (ESC) expression pattern governs self-renewal and pluripotency. Here, we present an RNAi screen in mouse ESCs of 1008 loci encoding chromatin proteins. We identified 68 proteins that exhibit diverse phenotypes upon knockdown (KD), including seven subunits of the Tip60-p400 complex. Phenotypic analyses revealed that Tip60-p400 is necessary to maintain characteristic features of ESCs. We show that p400 localization to the promoters of both silent and active genes is dependent upon histone H3 lysine 4 trimethylation (H3K4me3). Furthermore, the Tip60-p400 KD gene expression profile is enriched for developmental regulators and significantly overlaps with that of the transcription factor Nanog. Depletion of Nanog reduces p400 binding to target promoters without affecting H3K4me3 levels. Together, these data indicate that Tip60-p400 integrates signals from Nanog and H3K4me3 to regulate gene expression in ESCs.

Glucocorticoid signaling defines a novel commitment state during adipogenesis in vitro.

Differentiation of 3T3-L1 preadipocytes can be induced by a 2-d treatment with a factor "cocktail" (DIM) containing the synthetic glucocorticoid dexamethasone (dex), insulin, the phosphodiesterase inhibitor methylisobutylxanthine (IBMX) and fetal bovine serum (FBS). We temporally uncoupled the activities of the four DIM components and found that treatment with dex for 48 h followed by IBMX treatment for 48 h was sufficient for adipogenesis, whereas treatment with IBMX followed by dex failed to induce significant differentiation. Similar results were obtained with C3H10T1/2 and primary mesenchymal stem cells. The 3T3-L1 adipocytes differentiated by sequential treatment with dex and IBMX displayed insulin sensitivity equivalent to DIM adipocytes, but had lower sensitivity to ISO-stimulated lipolysis and reduced triglyceride content. The nondifferentiating IBMX-then-dex treatment produced transient expression of adipogenic transcriptional regulatory factors C/EBPbeta and C/EBPdelta, and little induction of terminal differentiation factors C/EBPalpha and PPARgamma. Moreover, the adipogenesis inhibitor preadipocyte factor-1 (Pref-1) was repressed by DIM or by dex-then-IBMX, but not by IBMX-then-dex treatment. We conclude that glucocorticoids drive preadipocytes to a novel intermediate cellular state, the dex-primed preadipocyte, during adipogenesis in cell culture, and that Pref-1 repression may be a cell fate determinant in preadipocytes.

RSC regulates nucleosome positioning at Pol II genes and density at Pol III genes.

Nucleosomes can restrict the access of transcription factors to chromatin. RSC is a SWI/SNF-family chromatin-remodeling complex from yeast that repositions and ejects nucleosomes in vitro. Here, we examined these activities and their importance in vivo. We utilized array-based methods to examine nucleosome occupancy and positioning at more than 200 locations in the genome following the controlled destruction of the catalytic subunit of RSC, Sth1. Loss of RSC function caused pronounced and general reductions in new transcription from Pol I, II, and III genes. At Pol III genes, Sth1 loss conferred a general reduction in RNA Pol III occupancy and a gain in nucleosome density. Notably at the one Pol III gene examined, histone restoration was partly replication-dependent. In contrast, at Pol II promoters we observed primarily single nucleosome changes, including movement. Importantly, alterations near the transcription start site were more common at RSC-occupied promoters than at non-occupied promoters. Thus, RSC action affects both nucleosome density and positioning in vivo, but applies these remodeling modes differently at Pol II and Pol III genes.

Copper deficiency causes reversible myelodysplasia.

Copper deficiency is a recognized but often overlooked cause of anemia and neutropenia. We began checking serum copper levels on patients referred for evaluation for unexplained anemia and neutropenia or myelodysplasia. Eight patients were identified as copper deficient (serum copper less than 70 microg/dL). The anemia was normochromic and normocytic in seven patients. Neutropenia was present in seven patients. Seven patients had been referred for evaluation of myelodysplasia. Three were seen for consideration for allogenic stem cell transplant. Five patients had concomitant peripheral neurological symptoms. Seven patients were treated with oral copper gluconate. All treated patients demonstrated a hematological response; seven had a complete remission. The improvement in anemia and neutropenia was rapid with normalization of blood counts within three to four weeks. In one patient, normalization of the underlying marrow dysplasia was demonstrated by bone marrow histology eight months after copper replacement. The cause of copper deficiency was felt to be gastrointestinal malabsorption in five of our patients. We conclude that copper deficiency should be considered in all patients with unexplained anemia and neutropenia or myelodysplasia.

Isolated antiplasmin deficiency presenting as a spontaneous bleeding disorder in a 63-year-old man.

Spontaneous bleeding in adults is a major problem, and in a significant number of these patients no cause is found. A 63-year-old Caucasian man presented to our hematology clinic with a large hematoma of his left thigh. Initial investigations did not show any conclusive abnormalities of primary or secondary hemostasis. Subsequent tests demonstrated a type 1 deficiency of antiplasmin. Treatment with low doses of epsilon-aminocaproic acid resulted in resolution of the hematoma and control of bleeding. We sought to determine the cause of the patient's isolated antiplasmin deficiency but no explanation was found. Three heterozygous polymorphisms were identified in his antiplasmin gene, ruling out major gene deletions. Each of these three polymorphisms has been previously reported in healthy blood donors. Finally, since response to antifibrinolytics can be dramatic, deficiencies of antiplasmin must be considered in patients presenting at any age with a spontaneous bleeding disorder.

Dephosphorylation and genome-wide association of Maf1 with Pol III-transcribed genes during repression.

Nutrient deprivation and various stress conditions repress RNA polymerase III (Pol III) transcription in S. cerevisiae. The signaling pathways that relay stress and nutrient conditions converge on the conserved protein Maf1, but how Maf1 integrates environmental conditions and couples them to transcriptional repression is largely unknown. Here, we demonstrate that Maf1 is phosphorylated in favorable conditions, whereas diverse unfavorable conditions lead to rapid Maf1 dephosphorylation, nuclear localization, physical association of dephosphorylated Maf1 with Pol III, and Maf1 targeting to Pol III-transcribed genes genome wide. Furthermore, Maf1 mutants defective in full dephosphorylation display maf1Delta phenotypes and are compromised for both nuclear localization and Pol III association. Repression conditions also promote TFIIIB-TFIIIC interactions in crosslinked chromatin. Taken together, Maf1 appears to integrate environmental conditions and signaling pathways through its phosphorylation state, with stress leading to dephosphorylation, association with Pol III at target loci, alterations in basal factor interactions, and transcriptional repression.