32 research outputs found
The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration
ObjectivesThe circadian clocks are internal timing mechanisms that drive ā¼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration.MethodsClock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containingPer2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock geneBmal1.ResultsHere we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1Ī² but not tumour necrosis factor Ī±. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout ofBmal1in their disc cells demonstrated age-related degeneration of IVDs.ConclusionsWe have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.</jats:sec
A dynamic, spatially periodic, microāpattern of HES5 underlies neurogenesis in the mouse spinal cord
From Wiley via Jisc Publications RouterHistory: received 2020-08-03, rev-recd 2021-03-30, accepted 2021-04-06, pub-print 2021-05, pub-electronic 2021-05-25Article version: VoRPublication status: PublishedFunder: Wellcome; Id: http://dx.doi.org/10.13039/100004440; Grant(s): 106185/Z/14/Z, 103986/Z/14/Z, 215189/Z/19/Z, 220001/Z/19/Z, 204057/Z/16/Z, 220001/Z/19/ZAbstract: Ultradian oscillations of HES Transcription Factors (TFs) at the singleācell level enable cell state transitions. However, the tissueālevel organisation of HES5 dynamics in neurogenesis is unknown. Here, we analyse the expression of HES5 ex vivo in the developing mouse ventral spinal cord and identify microclusters of 4ā6 cells with positively correlated HES5 level and ultradian dynamics. These microclusters are spatially periodic along the dorsoventral axis and temporally dynamic, alternating between high and low expression with a supraāultradian persistence time. We show that Notch signalling is required for temporal dynamics but not the spatial periodicity of HES5. Few Neurogenin 2 cells are observed per cluster, irrespective of high or low state, suggesting that the microcluster organisation of HES5 enables the stable selection of differentiating cells. Computational modelling predicts that different cell coupling strengths underlie the HES5 spatial patterns and rate of differentiation, which is consistent with comparison between the motoneuron and interneuron progenitor domains. Our work shows a previously unrecognised spatiotemporal organisation of neurogenesis, emergent at the tissue level from the synthesis of singleācell dynamics
Cardiac mitochondrial function depends on BUD23 mediated ribosome programming.
Efficient mitochondrial function is required in tissues with high energy demand such as the heart, and mitochondrial dysfunction is associated with cardiovascular disease. Expression of mitochondrial proteins is tightly regulated in response to internal and external stimuli. Here we identify a novel mechanism regulating mitochondrial content and function, through BUD23-dependent ribosome generation. BUD23 was required for ribosome maturation, normal 18S/28S stoichiometry and modulated the translation of mitochondrial transcripts in human A549 cells. Deletion of Bud23 in murine cardiomyocytes reduced mitochondrial content and function, leading to severe cardiomyopathy and death. We discovered that BUD23 selectively promotes ribosomal interaction with low GC-content 5'UTRs. Taken together we identify a critical role for BUD23 in bioenergetics gene expression, by promoting efficient translation of mRNA transcripts with low 5'UTR GC content. BUD23 emerges as essential to mouse development, and to postnatal cardiac function
Extending the allelic spectrum at noncoding risk loci of orofacial clefting
Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology
Adipocyte NR1D1 dictates adipose tissue expansion during obesity
From eLife via Jisc Publications RouterHistory: received 2020-09-22, collection 2021, accepted 2021-07-30, pub-electronic 2021-08-05Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; FundRef: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/I018654/1Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265; Grant(s): MR/N021479/1Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265; Grant(s): MR/P00279X/1Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265; Grant(s): MR/P011853/1Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265; Grant(s): MR/P023576/1Funder: Wellcome Trust; FundRef: http://dx.doi.org/10.13039/100004440; Grant(s): 107849/Z/15/ZFunder: Wellcome Trust; FundRef: http://dx.doi.org/10.13039/100004440; Grant(s): 107851/Z/15/ZThe circadian clock component NR1D1 (REVERBĪ±) is considered a dominant regulator of lipid metabolism, with global Nr1d1 deletion driving dysregulation of white adipose tissue (WAT) lipogenesis and obesity. However, a similar phenotype is not observed under adipocyte-selective deletion (Nr1d1Flox2-6:AdipoqCre), and transcriptional profiling demonstrates that, under basal conditions, direct targets of NR1D1 regulation are limited, and include the circadian clock and collagen dynamics. Under high-fat diet (HFD) feeding, Nr1d1Flox2-6:AdipoqCre mice do manifest profound obesity, yet without the accompanying WAT inflammation and fibrosis exhibited by controls. Integration of the WAT NR1D1 cistrome with differential gene expression reveals broad control of metabolic processes by NR1D1 which is unmasked in the obese state. Adipocyte NR1D1 does not drive an anticipatory daily rhythm in WAT lipogenesis, but rather modulates WAT activity in response to alterations in metabolic state. Importantly, NR1D1 action in adipocytes is critical to the development of obesity-related WAT pathology and insulin resistance
Role of Estrogen Response Element in the Human Prolactin Gene:Transcriptional Response and Timing
The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering, we have manipulated a 160-kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located ā1189 bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing Luc under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to 17Ī²-estradiol (E2) treatment compared with cells expressing Luc from the wild-type (WT) ERE hPRL-Luc promoter (ERE-WT). The ā1189 ERE controls not only the response to E2 treatment but also the acute transcriptional response to TNFĪ±, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response after TNFĪ± treatment, the acute phase of which was blocked after treatment with the estrogen receptor antagonist 4-hydroxy-tamoxifen. Unexpectedly, we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, real-time bioluminescence imaging showed that transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the ā1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFĪ± and, crucially, that cycles of hPRL promoter activity are independent of estrogen receptor binding
Response to correspondence on Reproducibility of CRISPR-Cas9 Methods for Generation of Conditional Mouse Alleles: A Multi-Center Evaluation
A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord
CM was supported by a Sir Henry Wellcome Fellowship (103986/Z/14/Z) and University of Manchester Presidential Fellowship. VB was supported by a Wellcome Trust Senior Research Fellowship to NP (106185/Z/14/Z). JH (220001/Z/19/Z), EJ (204057/Z/16/Z) and DH (Wellcome Trust Grant No. 215189/Z/19/Z) were supported by Wellcome Trust PhD studentships. JK was supported by Wellcome Trust Senior Research Fellowship to NP and a University of St Andrews Lectureship.Ultradian oscillations of HES Transcription Factors (TFs) at the single-cell level enable cell state transitions. However, the tissue-level organisation of HES5 dynamics in neurogenesis is unknown. Here, we analyse the expression of HES5 ex vivo in the developing mouse ventral spinal cord and identify microclusters of 4?6 cells with positively correlated HES5 level and ultradian dynamics. These microclusters are spatially periodic along the dorsoventral axis and temporally dynamic, alternating between high and low expression with a supra-ultradian persistence time. We show that Notch signalling is required for temporal dynamics but not the spatial periodicity of HES5. Few Neurogenin 2 cells are observed per cluster, irrespective of high or low state, suggesting that the microcluster organisation of HES5 enables the stable selection of differentiating cells. Computational modelling predicts that different cell coupling strengths underlie the HES5 spatial patterns and rate of differentiation, which is consistent with comparison between the motoneuron and interneuron progenitor domains. Our work shows a previously unrecognised spatiotemporal organisation of neurogenesis, emergent at the tissue level from the synthesis of single-cell dynamics.Publisher PDFPeer reviewe