Browsing by Subject "CLOCK Proteins"
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Item Architecture of the Mammalian Circadian Repressive Complex(2017-07-27) Rosensweig, Clark Jeffrey; Hibbs, Ryan E.; Green, Carla B.; Takahashi, Joseph; Konopka, GenevieveIntricate timing systems have evolved to help organisms in all walks of life organize their physiology to the solar day. Mammalian circadian clocks are driven by a transcription/translation feedback loop composed of positive regulators (CLOCK/BMAL1) and repressors (CRY1/2 and PER1/2). To understand what drives periodicity within this clock, I took structural approaches with the hope of identifying atomic-level details that inform behavioral outputs. Despite high sequence identity, null mutations of Cry1 or Cry2 have divergent effects on periodicity, accelerating and decelerating the clock speed, respectively. To understand the unique roles of CRY1 and CRY2, we used statistical coupling analysis to identify co-evolving residues within the CRY protein family. We identified an evolutionary hotspot, an ancestral secondary cofactor-binding pocket, which has been repurposed for direct interaction with CLOCK and BMAL1. Mutations weakening binding between CLOCK/BMAL1 and CRY1 lead to acceleration of the clock, revealing a novel mode of period regulation in the mammalian clock. Subtle divergence between CRY1 and CRY2 at the secondary pocket underlies differences in affinity for CLOCK/BMAL1. The lower affinity interaction with CRY2 is strengthened by co-expression of PER2, suggesting that PER expression limits the length of the repressive phase in CRY2-driven rhythms. In order to better understand PER's role, we collaborated with another lab to solve and validate a structure of CRY2 bound to a fragment of PER. In so doing, we discovered that interaction between PER and CRY is necessary for rhythmic derepression, providing insight into the role of a key interaction in the molecular clockwork.Item Evolution and Function of the Genomic Landscape in the Human Brain(2019-01-22) Fontenot, Miles Ray; Green, Carla B.; Konopka, Genevieve; Johnson, Jane E.; Monteggia, Lisa; Takahashi, JosephThe molecular mechanisms underlying human brain evolution are not fully understood; however, previous work suggested that expression of the transcription factor CLOCK in the human cortex might be relevant to evolution of the human brain and human cognition and disease. In this dissertation, we investigated this novel transcriptional role for CLOCK in human neurons by performing chromatin-immunoprecipitation sequencing for endogenous CLOCK in adult neocortex and RNA-sequencing following CLOCK knockdown in differentiated human neurons in vitro. These data suggested that CLOCK regulates expression of genes involved in neuronal migration, and a functional assay showed that CLOCK knockdown increased neuronal migratory distance. Furthermore, dysregulation of CLOCK disrupts co-expressed networks of genes implicated in neuropsychiatric disorders, and the expression of these networks are driven by hub genes with human-specific patterns of expression. Thus, these data support a role for CLOCK-regulated transcriptional cascades involved in human brain evolution and function. We further created a humanized mouse model with increased neocortical expression of CLOCK to mimic the human pattern of expression, providing a novel system for in vivo mechanistic studies of CLOCK function. Finally, we have conducted preliminary gene expression analysis of the human epileptic brain compared to control tissue from donors without neuropsychiatric disease. These data will be correlated with in-patient recordings of neuronal activity to identify potential new avenues for investigation into epilepsy. In total, we have contributed a rich dataset of genomics dataset related to CLOCK function in human neurons, generated a novel humanized mouse model, and initiated an exciting study into the gene expression and function of the epileptic brain.Item The Role of CLOCK in Regulation of Dopamine Neurotransmission in the CLOCKdelta19 Mutant Mouse Model(2012-07-17) Spencer, Sade Monique; McClung, Colleen A.Mice with a mutation in the circadian gene Clock (Clockdelta19) display a behavioral profile which parallels a euphoric manic-like state including hyperactivity, disrupted activity rhythms, increased substance abuse vulnerability, and decreases in anxiety and depression-related behavior. The molecular clock has significant cross-talk with many of the brain’s neurotransmitter systems. The purpose of this dissertation is to characterize the role of CLOCK in regulating dopamine transmission in mood and reward-related circuits. We present a mechanism by which CLOCK regulates dopaminergic activity in the mesoaccumbens circuit and contributes to anxiety-related behavior. In vivo recording of ventral tegmental area (VTA) dopamine cells throughout the 24 hour cycle revealed that firing and bursting was elevated in Clockdelta19 mutants with the most significant deviations early in the light cycle. Mimicking this increase in dopaminergic activity using optogenetic targeting resulted in decreased anxiety-related behavior similar to the Clockdelta19 phenotype. Consistent with the electrophysiological findings, tyrosine hydroxylase (TH) mRNA and protein was elevated in the VTA in a daytime-specific manner leading to increased dopamine synthesis in the nucleus accumbens. CLOCK binding was detected at E-box elements within the TH promoter with greater enrichment observed during the light phase when TH expression is low. These results suggest a negative regulation of TH by CLOCK. To examine alterations in the nigrostriatal dopamine circuit, HPLC measurements of dopamine and metabolites were performed in the dorsal striatum revealing significant increases in DOPAC and HVA. Dopamine receptor agonists and antagonists were used to pharmacologically probe dopamine receptor function. An enhancement of the locomotor suppressing response to dopamine antagonists in Clockdelta19 mice suggested increased dopaminergic tone. Clockdelta19 mice were insensitive to the locomotor stimulating effects of a D1 agonist, but displayed increased levels of D1DR protein. Conversely, the Clockdelta19 mutants displayed enhanced locomotor suppression to a D2 agonist and a coincident increase in D2DR protein. Forskolin stimulation of cAMP resulted in blunted molecular responses in the Clockdelta19 mutants consistent with impairments in D1 signaling and/or enhancements in D2 signaling. In summary, normal CLOCK function appears to be involved in the regulation of dopamine transmission in the striatum.Item The Role of Codon Usage in Regulating Protein Expression, Structure and Function(2014-06-10) Zhou, Mian; Tu, Benjamin; Liu, Yi; Takahashi, Joseph; Zinn, Andrew R.Codon usage bias has been observed in the genomes of almost all organisms and is thought to result from selection for efficient translation of highly expressed genes. Many genes, however, exhibit little codon usage bias. It's not clear whether the lack of codon bias for a gene is due to lack of selection for mRNA translation or it has some biological significance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. However, unlike most genes inNeurospora, frq exhibits non-optimal codon usage across its entire open reading frame (ORF). Optimization of frq codon usage results in the abolition of both overt and molecular circadian rhythms. Codon optimization not only increases FRQ expression level but surprisingly, also results in conformational changes in FRQ protein, impaired FRQ phosphorylation, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for maintaining circadian rhythmicity in Neurospora. Interestingly, there is a correlation between codon usage score and FRQ protein structure: the regions that are predicted to be disordered preferentially uses more non-optimal codons. This negative correlation is also found in the proteasome of Neurospora, as well in yeast, Drosophila, C. elegans and E. coli. By making a series of Neurospora strains with frq optimized in different regions, we find that codon optimizations in the predicted disordered regions of FRQ have more prominent effects on FRQ activity and structure. Furthermore, codon optimization of disordered regions in several other Neurospora genes results in altered protein degradation rates, suggesting structural changes by codon optimization. Together, these results suggest that codon usage adapts to protein structures and there is a "code" within genetic codons that allow optimal co-translational protein folding.