Filippo Tamanini

Mutations in GDI1 are responsible for X-linked non-specific mental retardation.

Rab GDP-dissociation inhibitors (GDI) are evolutionarily conserved proteins that play an essential role in the recycling of Rab GTPases required for vesicular transport through the secretory pathway. We have found mutations in the GDI1 gene (which encodes αGDI) in two families affected with X-linked non-specific mental retardation. One of the mutations caused a non-conservative substitution (L92P) which reduced binding and recycling of RAB3A, the second was a null mutation. Our results show that both functional and developmental alterations in the neuron may account for the severe impairment of learning abilities as a consequence of mutations in GDI1, emphasizing its critical role in development of human intellectual and learning abilities.

Nucleocytoplasmic shuttling and mCRY‐dependent inhibition of ubiquitylation of the mPER2 clock protein

The core oscillator generating circadian rhythms in eukaryotes is composed of transcription–translation‐based autoregulatory feedback loops in which clock gene products negatively affect their own expression. A key step in this mechanism involves the periodic nuclear accumulation of clock proteins following their mRNA rhythms after ∼6 h delay. Nuclear accumulation of mPER2 is promoted by mCRY proteins. Here, using COS7 cells and mCry1/mCry2 double mutant mouse embryonic fibroblasts transiently expressing GFP‐tagged (mutant) mPER2, we show that the protein shuttles between nucleus and cytoplasm using functional nuclear localization and nuclear export sequences. Moreover, we provide evidence that mCRY proteins prevent ubiquitylation of mPER2 and subsequent degradation of the latter protein by the proteasome system. Interestingly, mPER2 in turn prevents ubiquitylation and degradation of mCRY proteins. On the basis of these data we propose a model in which shuttling mPER2 is ubiquitylated and degraded by the proteasome unless it is retained in the nucleus by mCRY proteins.

Phase Resetting of the Mammalian Circadian Clock by DNA Damage

To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.

Fragile X-Related Proteins Regulate Mammalian Circadian Behavioral Rhythms

Fragile X syndrome results from the absence of the fragile X mental retardation 1 (FMR1) gene product (FMRP). FMR1 has two paralogs in vertebrates: fragile X related gene 1 and 2 (FXR1 and FXR2). Here we show that Fmr1/Fxr2 double knockout (KO) and Fmr1 KO/Fxr2 heterozygous animals exhibit a loss of rhythmic activity in a light:dark (LD) cycle, and that Fmr1 or Fxr2 KO mice display a shorter free-running period of locomotor activity in total darkness (DD). Molecular analysis and in vitro electrophysiological studies suggest essentially normal function of cells in the suprachiasmatic nucleus (SCN) in Fmr1/Fxr2 double KO mice. However, the cyclical patterns of abundance of several core clock component messenger (m) RNAs are altered in the livers of double KO mice. Furthermore, FXR2P alone or FMRP and FXR2P together can increase PER1- or PER2-mediated BMAL1-Neuronal PAS2 (NPAS2) transcriptional activity in a dose-dependent manner. These data collectively demonstrate that FMR1 and FXR2 are required for the presence of rhythmic circadian behavior in mammals and suggest that this role may be relevant to sleep and other behavioral alterations observed in fragile X patients.

Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle

Significance In tissues such as bone marrow, intestinal mucosa, or regenerating liver, the daily rhythm of cell division is controlled by the cell’s circadian clock. Determining how this clock organizes important processes such as cell division, apoptosis, and DNA damage repair is key to understanding the links between circadian dysfunction and malignant cell proliferation. We show that in proliferating mouse fibroblasts there is more than one way in which the clock and cell cycle synchronize their oscillations and that one of them is the biological equivalent of the phase locking first discovered by Huygens in the 17th century when he coupled two clocks together. When phase-locked two coupled oscillators have a fixed relative phase and oscillate with a common frequency. Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.

Functional evolution of the photolyase/cryptochrome protein family : Importance of the C terminus of mammalian CRY1 for circadian core oscillator performance

ABSTRACT Cryptochromes (CRYs) are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of the circadian oscillator. However, the function of their C terminus remains to be resolved. Here, we show that the C-terminal extension of mCRY1 harbors a nuclear localization signal and a putative coiled-coil domain that drive nuclear localization via two independent mechanisms and shift the equilibrium of shuttling mammalian CRY1 (mCRY1)/mammalian PER2 (mPER2) complexes towards the nucleus. Importantly, deletion of the complete C terminus prevents mCRY1 from repressing CLOCK/BMAL1-mediated transcription, whereas a plant photolyase gains this key clock function upon fusion to the last 100 amino acids of the mCRY1 core and its C terminus. Thus, the acquirement of different (species-specific) C termini during evolution not only functionally separated cryptochromes from photolyase but also caused diversity within the cryptochrome family.

OLIGOMERIZATION PROPERTIES OF FRAGILE-X MENTAL-RETARDATION PROTEIN (FMRP) AND THE FRAGILE-X-RELATED PROTEINS FXR1P AND FXR2P

The absence of fragile-X mental-retardation protein (FMRP) results in fragile-X syndrome. Two other fragile-X-related (FXR) proteins have been described, FXR1P and FXR2P, which are both very similar in amino acid sequence to FMRP. Interaction between the three proteins as well as with themselves has been demonstrated. The FXR proteins are believed to play a role in RNA metabolism. To characterize a possible functional role of the interacting proteins the complex formation of the FXR proteins was studied in mammalian cells. Double immunofluorescence analysis in COS cells over-expressing either FMRP ISO12/FXR1P or FMRP ISO12/FXR2P confirmed heterotypic interactions. However, Western-blotting studies on cellular homogenates containing physiological amounts of the three proteins gave different indications. Gel-filtration experiments under physiological as well as EDTA conditions showed that the FXR proteins were in complexes of >600 kDa, as parts of messenger ribonuclear protein (mRNP) particles associated with polyribosomes. Salt treatment shifted FMRP, FXR1P and FXR2P into distinct intermediate complexes, with molecular masses between 200 and 300 kDa. Immunoprecipitations of FMRP as well as FXR1P from the dissociated complexes revealed that the vast majority of the FXR proteins do not form heteromeric complexes. Further analysis by [(35)S]methionine labelling in vivo followed by immunoprecipitation indicated that no proteins other than the FXR proteins were present in these complexes. These results suggest that the FXR proteins form homo-multimers preferentially under physiological conditions in mammalian cells, and might participate in mRNP particles with separate functions.

Histone methyltransferase MLL3 contributes to genome-scale circadian transcription

Daily cyclical expression of thousands of genes in tissues such as the liver is orchestrated by the molecular circadian clock, the disruption of which is implicated in metabolic disorders and cancer. Although we understand much about the circadian transcription factors that can switch gene expression on and off, it is still unclear how global changes in rhythmic transcription are controlled at the genomic level. Here, we demonstrate circadian modification of an activating histone mark at a significant proportion of gene loci that undergo daily transcription, implicating widespread epigenetic modification as a key node regulated by the clockwork. Furthermore, we identify the histone-remodelling enzyme mixed lineage leukemia (MLL)3 as a clock-controlled factor that is able to directly and indirectly modulate over a hundred epigenetically targeted circadian “output” genes in the liver. Importantly, catalytic inactivation of the histone methyltransferase activity of MLL3 also severely compromises the oscillation of “core” clock gene promoters, including Bmal1, mCry1, mPer2, and Rev-erbα, suggesting that rhythmic histone methylation is vital for robust transcriptional oscillator function. This highlights a pathway by which the clockwork exerts genome-wide control over transcription, which is critical for sustaining temporal programming of tissue physiology.

Light Entrainment of the Mammalian Circadian Clock by a PRKCA-Dependent Posttranslational Mechanism

Light is the most potent stimulus for synchronizing endogenous circadian rhythms with external time. Photic clock resetting in mammals involves cAMP-responsive element binding protein (CREB)-mediated transcriptional activation of Period clock genes in the suprachiasmatic nuclei (SCN). Here we provide evidence for an additional photic input pathway to the mammalian circadian clock based on Protein Kinase C alpha (PRKCA). We found that Prkca-deficient mice show an impairment of light-mediated clock resetting. In the SCN of wild-type mice, light exposure evokes a transient interaction between PRKCA and PERIOD 2 (PER2) proteins that affects PER2 stability and nucleocytoplasmic distribution. These posttranslational events, together with CREB-mediated transcriptional regulation, are key factors in the molecular mechanism of photic clock resetting.

Nucleocytoplasmic shuttling of clock proteins

The mammalian circadian clock in the neurons of suprachiasmatic nuclei (SCN) in the brain and in cells of peripheral tissues is driven by a self-sustained molecular oscillator, which generates rhythmic gene expression with a periodicity of about 24?h (Reppert and Weaver, 2002). This molecular oscillator is composed of interacting positive and negative transcription/translation feedback loops in which the heterodimeric transcription activator CLOCK?BMAL1 promotes the transcription of E-box containing Cryptochrome (Cry1 and Cry2) and Period (Per1 and Per2) genes, as well as clock-controlled output genes. After being synthesized in the cytoplasm, CRY and PER proteins feedback in the nucleus to inhibit the transactivation mediated by positive regulators. The mPER2 protein acts at the interphase between positive and negative feedback loops by indirectly promoting the circadian transcription of the Bmal1 gene (through RevErbalpha) (Preitner et al., 2002; Shearman et al., 2000) and by interacting with mCRY proteins (Kume et al., 1999; Yagita et al., 2002) (for a detailed review, see Reppert and Weaver, 2002). In addition to cyclic transcription of clock genes, immunohistochemical studies on SCN neurons have revealed that mCRY1, mCRY2, mPER1, and mPER2 proteins undergo near synchronous circadian patterns of nuclear abundance (Field et al., 2000). The delay of approximately 6h between the peak in clock mRNA production and maximal levels of protein expression in the nucleus is believed to originate from posttranslational modification steps involving phosphorylation, ubiquitination, and proteosomal degradation. Thus, the timing of entry, as well as the residence time of core clock proteins into the nucleus, is a critical step in maintaining the correct pace of the circadian clock. Several clock proteins have been shown to contain nuclear export signal, sequences, on top of nuclear import signals, that facilitate their cellular trafficking (Chopin-Delannoy et al., 2003; Miyazaki et al., 2001; Yagita et al., 2002). This type of dynamic intracellular movement not only regulates protein localization, but also often affects functions by determining interactive partners and protein turnover. Because most of the clock genes have been identified by genetic screening in Drosophila and by gene knockdown in mammals, the development of innovative cellular techniques is essential in learning the structure-function and regulation of the corresponding proteins. This article discusses approaches, limitations, and applicable protocols to study the regulation of cellular localization of mammalian clock proteins, with a particular focus on mammalian CRY1 and PER2 proteins.