P6045 Dine mapping a sheep genomic locus involved in viral restriction of ovine lentivirus

SAM and HD domain-containing protein 1 (SAMHD1) is a host factor that restricts reverse transcription of lentiviruses such as HIV in myeloid cells and resting T cells through its dNTP triphosphohydrolase (dNTPase) activity. Lentiviruses counteract this restriction by expressing the accessory protein Vpx or Vpr, which targets SAMHD1 for proteasomal degradation. SAMHD1 is conserved among mammals, and the feline and bovine SAMHD1 proteins (fSAM and bSAM) restrict lentiviruses by reducing cellular dNTP concentrations. However, the functional regions of fSAM and bSAM that are required for their biological functions are not well-characterized. Here, to establish alternative models to investigate SAMHD1 in vivo, we studied the restriction profile of fSAM and bSAM against different primate lentiviruses. We found that both fSAM and bSAM strongly restrict primate lentiviruses and that Vpx induces the proteasomal degradation of both fSAM and bSAM. Further investigation identified one and five amino acid sites in the C-terminal domain (CTD) of fSAM and bSAM, respectively, that are required for Vpx-mediated degradation. We also found that the CTD of bSAM is directly involved in mediating bSAM's antiviral activity by regulating dNTPase activity, whereas the CTD of fSAM is not. Our results suggest that the CTDs of fSAM and bSAM have important roles in their antiviral functions. These findings advance our understanding of the mechanism of fSAM- and bSAM-mediated viral restriction and might inform strategies for improving HIV animal models.

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SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells

Nature Communications ◽

10.1038/s41467-021-24802-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Charlotte Martinat ◽

Arthur Cormier ◽

Joëlle Tobaly-Tapiero ◽

Noé Palmic ◽

Nicoletta Casartelli ◽

...

Keyword(s):

Antiviral Activity ◽

Reverse Transcription ◽

Immune Cells ◽

Viral Restriction ◽

Antiviral Function ◽

Hiv 1 ◽

Constitutively Active

AbstractSAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

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A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽

10.1093/function/zqab012 ◽

2021 ◽

Vol 2 (3) ◽

Cited By ~ 1

Author(s):

Nelly Redolfi ◽

Elisa Greotti ◽

Giulia Zanetti ◽

Tino Hochepied ◽

Cristina Fasolato ◽

...

Keyword(s):

Transgenic Mouse ◽

Fluorescent Protein ◽

Ex Vivo ◽

Brain Slices ◽

Resonance Energy ◽

Mouse Line ◽

Isolated Cells ◽

Genomic Locus ◽

Transgenic Mouse Line

AbstractMitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

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In Situ Saturating Mutagenesis Screening Identifies a Functional Genomic Locus that Regulates Ucp1 Expression

Phenomics ◽

10.1007/s43657-020-00006-7 ◽

2021 ◽

Vol 1 (1) ◽

pp. 15-21

Author(s):

Yan Qiu ◽

Xiaojian Liu ◽

Yingmin Sun ◽

Shuang Li ◽

Yuda Wei ◽

...

Keyword(s):

Functional Genomic ◽

Genomic Locus ◽

Ucp1 Expression

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Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

Journal of Fungi ◽

10.3390/jof7020149 ◽

2021 ◽

Vol 7 (2) ◽

pp. 149

Author(s):

Sarah-Maria Wege ◽

Katharina Gejer ◽

Fabienne Becker ◽

Michael Bölker ◽

Johannes Freitag ◽

...

Keyword(s):

Genome Editing ◽

Cell Biology ◽

Gene Deletion ◽

Fluorescent Protein ◽

Point Mutations ◽

Model Organism ◽

Ustilago Maydis ◽

Genomic Locus ◽

Targeted Gene Deletion ◽

Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

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Considerations when investigating lncRNA function in vivo

eLife ◽

10.7554/elife.03058 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 222

Author(s):

Andrew R Bassett ◽

Asifa Akhtar ◽

Denise P Barlow ◽

Adrian P Bird ◽

Neil Brockdorff ◽

...

Keyword(s):

Normal Cell ◽

Regulatory Elements ◽

Genomic Locus ◽

Cellular Processes ◽

Cell Processes ◽

Non Coding Rnas ◽

Per Se ◽

Dna Regulatory Elements

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.

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Abstract 161: Identification of Genetic Variants Associated with Kidney Injury That Leads to Increased Blood Pressure

Hypertension ◽

10.1161/hyp.60.suppl_1.a161 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Ashlyn C Harmon ◽

Ashley C Johnson ◽

Santosh Atanur ◽

Klio Maratou ◽

Tim Aitman ◽

...

Keyword(s):

Blood Pressure ◽

Bone Marrow ◽

Immune Response ◽

Renal Function ◽

Kidney Disease ◽

Kidney Function ◽

Genetic Variants ◽

Kidney Injury ◽

Chromosome 11 ◽

Genomic Locus

Hypertension, diabetes and obesity, along with genetic predisposition, contribute to the growing number of chronic kidney disease patients. Our novel congenic model [S.SHR(11)] was developed through genetic modification of the Dahl salt-sensitive (S) rat, a model of hypertension related renal disease. The S.SHR(11) strain exhibits accelerated kidney injury compared to the already highly susceptible S rat. On either a low or high-salt diet, the S.SHR(11) model predominately exhibited more tubulointerstitial fibrosis compared to the S rat (17.1±1.29% vs. 12.9±1.22%). Increased α-SMA and macrophage infiltration was also observed. The S and S.SHR(11) had similar blood pressure (week 12), despite an early reduction in renal function in the S.SHR(11); however at an advanced age the S.SHR(11) demonstrated significantly higher blood pressure than the S (215±6.6 mm Hg vs. 183±5.9, respectively). This suggests that increased kidney injury is driving the development of hypertension later in life. Since these two animal models are identical with exception of chromosome 11, the causative genetic variants contributing to decreased renal function must reside within this region. The Dahl S and SHR genomes have been sequenced; this data provides a catalog of all the genetic variants between the two models. The 95% confidence interval of the genomic locus contains 28 non-synonymous SNP, with 15 of these SNP occurring within only three genes: Retnlg , Trat1 and Myh15. Two of these genes, Retnlg and Trat1, are known to play a role in immune response leading to our hypothesis that genetic variants in these genes alter protein function and lead to an increased immune response. Bone marrow transplant studies have been initiated to test our hypothesis and preliminary data shows that S rats who receive S.SHR(11) bone marrow have kidney function measurements similar to the S.SHR(11). The sequencing information has also lead to the development of nine new, more refined congenic strains. Through functional analysis of these new congenic animals, identification of the causative genetic variations will be expedited. In summary, we are employing a model of accelerated kidney disease to identify genes or genetic variants responsible for reduced kidney function and hypertension.

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Expression of Sry, the mouse sex determining gene

Development ◽

10.1242/dev.121.6.1603 ◽

1995 ◽

Vol 121 (6) ◽

pp. 1603-1614 ◽

Cited By ~ 9

Author(s):

A. Hacker ◽

B. Capel ◽

P. Goodfellow ◽

R. Lovell-Badge

Keyword(s):

Germ Cells ◽

Translational Control ◽

Direct Interaction ◽

Coding Region ◽

Genital Ridge ◽

Sry Gene ◽

Genomic Locus ◽

Rnase Protection ◽

Unique Region ◽

Adult Testis

In the mouse, Sry is expressed by germ cells in the adult testis and by somatic cells in the genital ridge. Transcripts in the former exist as circular RNA molecules of 1.23 kb, which are unlikely to be efficiently translated. We have used RNase protection to map the extent of the less abundant Sry transcript in the developing gonad. We demonstrate that it is a linear mRNA derived from a single exon. This begins in the unique region 5′ of the protein coding region and extends several kilobases into the 3′ arm of the large inverted repeat which bounds the Sry genomic locus. Knowledge of this transcript, which is very different from that of the human SRY gene, allows us to predict its protein product and reveals several features which may be involved in translational control. Our data is also consistent with there being two promoters for the Sry gene, a proximal one that gives functional transcripts in the genital ridge and a distal promoter used in germ cells in the adult testis. As RNase protection is a quantitative technique, a detailed timecourse of Sry expression was carried out using accurately staged samples. Sry transcripts are first detectable just after 10.5 days post coitum, they reach a peak at 11.5 days and then decline sharply so that none are detected 24 hours later. This was compared with anti-Mullerian hormone gene expression, an early marker of Sertoli cells and the first known downstream gene of Sry. Amh expression begins 20 hours after the onset of Sry expression at a time when Sry transcripts are at their peak. While this result does not prove a direct interaction between the two genes, it defines the critical period during which Sry must act to initiate Sertoli cell differentiation.

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