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FASEB J ; 35(9): e21802, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383984


Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3Sci/+ ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3Sci/+ mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3Sci/+ retina show an increased sensitivity of ipRGC light responses.

Ritmo Circadiano/fisiologia , Proteínas de Homeodomínio/metabolismo , Retina/metabolismo , Células Amácrinas/metabolismo , Animais , Luz , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Luminosa/métodos , Células Ganglionares da Retina/metabolismo , Visão Ocular/fisiologia
Proc Natl Acad Sci U S A ; 113(10): 2756-61, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26903623


The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2(Edo/Edo) mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2(Edo) complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2(Edo/Edo); Csnk1e(Tau/Tau) mice and the SCN. These periods are unprecedented in mice. Thus, Per2(Edo) reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.

Relógios Circadianos/genética , Ritmo Circadiano/genética , Mutação de Sentido Incorreto , Proteínas Circadianas Period/genética , Sequência de Aminoácidos , Animais , Western Blotting , Células COS , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Chlorocebus aethiops , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Atividade Motora/genética , Atividade Motora/fisiologia , Proteínas Circadianas Period/química , Proteínas Circadianas Period/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiopatologia
Cell ; 162(3): 607-21, 2015 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-26232227


We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

Ritmo Circadiano , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Neuropeptídeos/genética , Núcleo Supraquiasmático/metabolismo , Sequência de Aminoácidos , Animais , Regulação para Baixo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Alinhamento de Sequência , Transcrição Genética
J Neurosci ; 33(17): 7145-53, 2013 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-23616524


The circadian clock of the suprachiasmatic nucleus (SCN) drives daily rhythms of behavior. Cryptochromes (CRYs) are powerful transcriptional repressors within the molecular negative feedback loops at the heart of the SCN clockwork, where they periodically suppress their own expression and that of clock-controlled genes. To determine the differential contributions of CRY1 and CRY2 within circadian timing in vivo, we exploited the N-ethyl-N-nitrosourea-induced afterhours mutant Fbxl3(Afh) to stabilize endogenous CRY. Importantly, this was conducted in CRY2- and CRY1-deficient mice to test each CRY in isolation. In both CRY-deficient backgrounds, circadian rhythms of wheel-running and SCN bioluminescence showed increased period length with increased Fbxl3(Afh) dosage. Although both CRY proteins slowed the clock, CRY1 was significantly more potent than CRY2, and in SCN slices, CRY1 but not CRY2 prolonged the interval of transcriptional suppression. Selective CRY-stabilization demonstrated that both CRYs are endogenous transcriptional repressors of clock-controlled genes, but again CRY1 was preeminent. Finally, although Cry1(-/-);Cry2(-/-) mice were behaviorally arrhythmic, their SCN expressed short period (~18 h) rhythms with variable stability. Fbxl3(Afh/Afh) had no effect on these CRY-independent rhythms, confirming its circadian action is mediated exclusively via CRYs. Thus, stabilization of both CRY1 and CRY2 are necessary and sufficient to explain circadian period lengthening by Fbxl3(Afh/Afh). Both CRY proteins dose-dependently lengthen the intrinsic, high-frequency SCN rhythm, and CRY2 also attenuates the more potent period-lengthening effects of CRY1. Incorporation of CRY-mediated transcriptional feedback thus confers stability to intrinsic SCN oscillations, establishing periods between 18 and 29 h, as determined by selective contributions of CRY1 and CRY2.

Relógios Circadianos/genética , Criptocromos/fisiologia , Proteínas F-Box/fisiologia , Mutação/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Animais Recém-Nascidos , Ritmo Circadiano/genética , Criptocromos/genética , Proteínas F-Box/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos