Biology subjects

Humphreys, N.

Publications and source records attributed to Humphreys, N..

4 recordsLinked to original sources

bioRxiv2024

Genetic gradual reduction of OGT activity unveils the essential role of O-GlcNAc in the mouse embryo

The reversible glycosylation of nuclear and cytoplasmic proteins (O-GlcNAcylation) is catalyzed by a single enzyme, namely O-GlcNAc transferase (OGT). The mammalian Ogt gene is X-linked and it is essential for embryonic development and for the viability of proliferating cells. We perturbed OGTs function in vivo by creating a murine allelic series of four single amino acid substitutions reducing OGTs catalytic activity to a range of degrees. The severity of the embryonic lethality was proportional to the degree of impairment of OGTs catalysis, demonstrating that the O-GlcNAc modification itself is required for early development. We identified milder hypomorphic Ogt alleles that perturb O-GlcNAc homeostasis while being compatible with embryogenesis. The analysis of the transcriptomes of the mutant embryos at different stages suggested a sexually-dimorphic developmental delay caused by the decrease in O-GlcNAc. Furthermore, a mild reduction of OGTs enzymatic activity was sufficient to loosen the silencing of endogenous retroviruses in vivo.

developmental biology↗

bioRxiv2023

Overexpression of IkappaBalpha modulates NF-kappaB activation of inflammatory target gene expression

Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-{kappa}B (NF-{kappa}B) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm to mediate immune response. We investigate the relationship between p65 and inhibitor-{kappa}B (I{kappa}B) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of I{kappa}B-eGFP protein overexpression in a native genomic context. We find that cells with high levels of the negative regulator I{kappa}B remain responsive to inflammatory stimuli and maintain dynamics for both p65 and I{kappa}B. In contrast, canonical target gene expression is dramatically reduced by overexpression of I{kappa}B, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of I{kappa}B also suppresses canonical target gene expression, suggesting a mechanism in which nuclear I{kappa}B accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immune precipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both I{kappa}B and p65, and that transcription can be partially decoupled from p65 protein dynamics. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response.

cell biology↗

bioRxiv2021

A kinase-dead Csf1r mutation associated with adult-onset leukoencephalopathy has a dominant-negative impact on CSF1R signaling.

Amino acid substitutions in the kinase domain of the human CSF1R gene are associated with autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). To model the human disease, we created a disease-associated mutation (Glu631Lys; E631K) in the mouse Csf1r locus. Homozygous mutation (Csf1rE631K/E631K) phenocopied the Csf1r knockout; with prenatal mortality or severe postnatal growth retardation and hydrocephalus. Heterozygous mutation delayed the postnatal expansion of tissue macrophage populations in most organs. Bone marrow cells from Csf1rE631K/+ mice were resistant to CSF1 stimulation in vitro, and Csf1rE631K/+ mice were unresponsive to administration of a CSF1-Fc fusion protein which expands tissue macrophage populations in controls. In the brain, microglial cell numbers and dendritic arborization were reduced in the Csf1rE631K/+ mice as in ALSP patients. The microglial phenotype is the opposite of microgliosis observed in Csf1r+/- mice. However, we found no evidence of brain pathology or impacts on motor function in aged Csf1rE631K/+ mice. We conclude that disease-associated CSF1R mutations encode dominant negative repressors of CSF1R signaling. We speculate that leukoencephalopathy associated with human CSF1R mutations requires an environmental trigger and/or epistatic interaction with common neurodegenerative disease-associated alleles. Summary StatementThis study describes the effect of a human disease-associated mutation in the mouse CSF1R gene on postnatal development and growth factor responsiveness of cells of the macrophage lineage.

developmental biology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗