Asynchronous microexon splicing of LSD1 and PHF21A during neurodevelopment.

LSD1 histone H3K4 demethylase and its binding partner PHF21A, a reader protein for unmethylated H3K4, both undergo neuron-specific microexon splicing. The LSD1 neuronal microexon weakens H3K4 demethylation activity and can alter the substrate specificity to H3K9 or H4K20. Meanwhile, the PHF21A neuronal microexon interferes with nucleosome binding. However, the temporal expression patterns of LSD1 and PHF21A splicing isoforms during brain development remain unknown. In this work, we report that neuronal PHF21A isoform expression precedes neuronal LSD1 isoform expression during human neuron differentiation and mouse brain development. The asynchronous splicing events resulted in stepwise deactivation of the LSD1-PHF21A complex in reversing H3K4 methylation. We further show that the enzymatically inactive LSD1-PHF21A complex interacts with neuron-specific binding partners, including MYT1-family transcription factors and post-transcriptional mRNA processing proteins such as VIRMA. The interaction with the neuron-specific components, however, did not require the PHF21A microexon, indicating that the neuronal proteomic milieu, rather than the microexon-encoded PHF21A segment, is responsible for neuron-specific complex formation. These results indicate that the PHF21A microexon is dispensable for neuron-specific protein-protein interactions, yet the enzymatically inactive LSD1-PHF21A complex might have unique gene-regulatory roles in neurons.

A neuron-specific microexon ablates the novel DNA-binding function of a histone H3K4me0 reader PHF21A.

How cell-type-specific chromatin landscapes emerge and progress during metazoan ontogenesis remains an important question. Transcription factors are expressed in a cell-type-specific manner and recruit chromatin-regulatory machinery to specific genomic loci. In contrast, chromatin-regulatory proteins are expressed broadly and are assumed to exert the same intrinsic function across cell types. However, human genetics studies have revealed an unexpected vulnerability of neurodevelopment to chromatin factor mutations with unknown mechanisms. Here, we report that 14 chromatin regulators undergo evolutionary-conserved neuron-specific splicing events involving microexons. Of the 14 chromatin regulators, two are integral components of a histone H3K4 demethylase complex; the catalytic subunit LSD1 and an H3K4me0-reader protein PHF21A adopt neuron-specific forms. We found that canonical PHF21A (PHF21A-c) binds to DNA by AT-hook motif, and the neuronal counterpart PHF21A-n lacks this DNA-binding function yet maintains H3K4me0 recognition intact. In-vitro reconstitution of the canonical and neuronal PHF21A-LSD1 complexes identified the neuronal complex as a hypomorphic H3K4 demethylating machinery with reduced nucleosome engagement. Furthermore, an autism-associated PHF21A missense mutation, 1285 G>A, at the last nucleotide of the common exon immediately upstream of the neuronal microexon led to impaired splicing of PHF21A -n. Thus, ubiquitous chromatin regulatory complexes exert unique intrinsic functions in neurons via alternative splicing of their subunits and potentially contribute to faithful human brain development.

Deficiency of CHAMP1, a gene related to intellectual disability, causes impaired neuronal development and a mild behavioural phenotype.

CHAMP1 is a gene associated with intellectual disability, which was originally identified as being involved in the maintenance of kinetochore-microtubule attachment. To explore the neuronal defects caused by CHAMP1 deficiency, we established mice that lack CHAMP1. Mice that are homozygous knockout for CHAMP1 were slightly smaller than wild-type mice and died soon after birth on pure C57BL/6J background. Although gross anatomical defects were not found in CHAMP1 -/- mouse brains, mitotic cells were increased in the cerebral cortex. Neuronal differentiation was delayed in CHAMP1 -/- neural stem cells in vitro, which was also suggested in vivo by CHAMP1 knockdown. In a behavioural test battery, adult CHAMP1 heterozygous knockout mice showed mild memory defects, altered social interaction, and depression-like behaviours. In transcriptomic analysis, genes related to neurotransmitter transport and neurodevelopmental disorder were downregulated in embryonic CHAMP1 -/- brains. These results suggest that CHAMP1 plays a role in neuronal development, and CHAMP1-deficient mice resemble some aspects of individuals with CHAMP1 mutations.

TORC1 inactivation promotes APC/C-dependent mitotic slippage in yeast and human cells.

Unsatisfied kinetochore-microtubule attachment activates the spindle assembly checkpoint to inhibit the metaphase-anaphase transition. However, some cells eventually override mitotic arrest by mitotic slippage. Here, we show that inactivation of TORC1 kinase elicits mitotic slippage in budding yeast and human cells. Yeast mitotic slippage was accompanied with aberrant aspects, such as degradation of the nucleolar protein Net1, release of phosphatase Cdc14, and anaphase-promoting complex/cyclosome (APC/C)-Cdh1-dependent degradation of securin and cyclin B in metaphase. This mitotic slippage caused chromosome instability. In human cells, mammalian TORC1 (mTORC1) inactivation also invoked mitotic slippage, indicating that TORC1 inactivation-induced mitotic slippage is conserved from yeast to mammalian cells. However, the invoked mitotic slippage in human cells was not dependent on APC/C-Cdh1. This study revealed an unexpected involvement of TORC1 in mitosis and provides information on undesirable side effects of the use of TORC1 inhibitors as immunosuppressants and anti-tumor drugs.

Growth Suppression of Cancer Spheroids With Mutated KRAS by Low-toxicity Compounds from Natural Products.

BACKGROUND/AIM: Among compounds from natural products selectively suppressing the growth of cancer spheroids, which have mutant (mt) KRAS, NP910 was selected and its derivatives explored. MATERIALS AND METHODS: The area of HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were measured in three-dimensional floating (3DF) cultures treated with 18 NP910 derivatives. The 50% cell growth inhibition (GI50) was determined by long-term 3DF (LT3DF) culture and nude mice assay. RESULTS: We selected NP882 (named STAR3) as the most effective inhibitor of growth of HKe3-mtKRAS spheroids with the least toxicity among NP910 derivatives. GI50s of STAR3 in LT3DF and nude mice assay were 6 µM and 30.75 mg/kg, respectively. However, growth suppression by STAR3 was observed in 50% of cell lines independent of KRAS mutation, suggesting that the target of STAR3 was not directly associated with KRAS mutation and KRAS-related signals. CONCLUSION: STAR3 is a low-toxicity compound that inhibits growth of certain tumour cells.

The transcriptional regulator Zfat is essential for maintenance and differentiation of the adipocytes.

Adipocytes play crucial roles in the control of whole-body energy homeostasis. Differentiation and functions of the adipocytes are regulated by various transcription factors. Zfat (zinc-finger protein with AT-hook) is a transcriptional regulator that controls messenger RNA expression of specific genes through binding to their transcription start sites. Here we report important roles of Zfat in the adipocytes. We establish inducible Zfat-knockout (Zfat iKO) mice where treatment with tamoxifen causes a marked reduction in Zfat expression in various tissues. Tamoxifen treatment of Zfat iKO mice reduces the white adipose tissues (WATs) mass, accompanied by the decreased triglyceride levels. Zfat is expressed in both the adipose-derived stem cells (ADSCs) and mature adipocytes in the WATs. In ex vivo assays of the mature adipocytes differentiated from the Zfat iKO ADSCs, loss of Zfat in the mature adipocytes reduces the triglyceride levels, suggesting cell autonomous roles of Zfat in the maintenance of the mature adipocytes. Furthermore, we identify the Atg13, Brf1, Psmc3, and Timm22 genes as Zfat-target genes in the mature adipocytes. In contrast, loss of Zfat in the ADSCs impairs adipocyte differentiation with the decreased expression of C/EBPα and adiponectin. Thus, we propose that Zfat plays crucial roles in maintenance and differentiation of the adipocytes.

ZFAT binds to centromeres to control noncoding RNA transcription through the KAT2B-H4K8ac-BRD4 axis.

Centromeres are genomic regions essential for faithful chromosome segregation. Transcription of noncoding RNA (ncRNA) at centromeres is important for their formation and functions. Here, we report the molecular mechanism by which the transcriptional regulator ZFAT controls the centromeric ncRNA transcription in human and mouse cells. Chromatin immunoprecipitation with high-throughput sequencing analysis shows that ZFAT binds to centromere regions at every chromosome. We find a specific 8-bp DNA sequence for the ZFAT-binding motif that is highly conserved and widely distributed at whole centromere regions of every chromosome. Overexpression of ZFAT increases the centromeric ncRNA levels at specific chromosomes, whereas its silencing reduces them, indicating crucial roles of ZFAT in centromeric transcription. Overexpression of ZFAT increases the centromeric levels of both the histone acetyltransferase KAT2B and the acetylation at the lysine 8 in histone H4 (H4K8ac). siRNA-mediated knockdown of KAT2B inhibits the overexpressed ZFAT-induced increase in centromeric H4K8ac levels, suggesting that ZFAT recruits KAT2B to centromeres to induce H4K8ac. Furthermore, overexpressed ZFAT recruits the bromodomain-containing protein BRD4 to centromeres through KAT2B-mediated H4K8ac, leading to RNA polymerase II-dependent ncRNA transcription. Thus, ZFAT binds to centromeres to control ncRNA transcription through the KAT2B-H4K8ac-BRD4 axis.

CDK phosphorylation regulates Mcm3 degradation in budding yeast.

Accurate regulation of activity and level of the MCM complex is critical for precise DNA replication and genome transmission. Cyclin-dependent kinase (CDK) negatively regulates nuclear localization of the MCM complex via phosphorylation of the Mcm3 subunit. More recently, we found that Mcm3 is degraded via the Skp1-Cullin-F-box (SCF)-proteasome axis in budding yeast. However, how Mcm3 degradation is regulated is largely unknown. Here, we show that CDK represses Mcm3 degradation. Phosphorylated Mcm3 was excluded from the nucleus, where SCF is predominantly located, although CDK-mediated phosphorylation itself generated a phosphodegron of Mcm3, stimulating the degradation of Mcm3 resident in the nucleus. Thus, CDK negatively regulated nuclear MCM levels by exclusion from the nucleus and degradation in the nucleus via Mcm3 phosphorylation. We will discuss the physiological importance of Mcm3 degradation.

Cdh1 degradation is mediated by APC/C-Cdh1 and SCF-Cdc4 in budding yeast.

Cdh1, a substrate-recognition subunit of anaphase-promoting complex/cyclosome (APC/C), is a tumor suppressor, and it is downregulated in various tumor cells in humans. APC/C-Cdh1 is activated from late M phase to G1 phase by antagonizing Cdk1-mediated inhibitory phosphorylation. However, how Cdh1 protein levels are properly regulated is ill-defined. Here we show that Cdh1 is degraded via APC/C-Cdh1 and Skp1-Cullin1-F-box (SCF)-Cdc4 in the budding yeast Saccharomyces cerevisiae. Cdh1 degradation was promoted by forced localization of Cdh1 into the nucleus, where APC/C and SCF are present. Cdk1 promoted APC/C-Cdh1-mediated Cdh1 degradation, whereas polo kinase Cdc5 elicited SCF-Cdc4-mediated degradation. Thus, Cdh1 degradation is controlled via multiple pathways.

TORC1 signaling regulates DNA replication via DNA replication protein levels.

Accurate DNA replication is at the heart of faithful genome transmission in dividing cells. DNA replication is strictly controlled by various factors. However, how environmental stresses such as nutrient starvation impact on these factors and DNA replication is largely unknown. Here we show that DNA replication is regulated by target of rapamycin complex 1 (TORC1) protein kinase, which is a central regulator of cell growth and proliferation in response to nutrients. TORC1 inactivation reduced the levels of various proteins critical for DNA replication initiation, such as Mcm3, Orc3, Cdt1, and Sld2, and retarded DNA replication. TORC1 inactivation promoted proteasome-mediated Mcm3 degradation. Skp1-Cullin-F-box (SCF)-Grr1 and PEST motif mediated Mcm3 degradation. TORC1-downstream factors PP2A-Cdc55 protein phosphatase and protein kinase A regulated Mcm3 degradation. This study showed that TORC1 signaling modulates DNA replication to coordinate cell growth and genome replication in response to nutrient availability.

When You Become a Superman: Subliminal Exposure to Death-Related Stimuli Enhances Men's Physical Force.

Research based on terror management theory (TMT) has consistently found that reminders to individuals about their mortality engender responses aimed at shoring up faith in their cultural belief system. Previous studies have focused on the critical role that the accessibility of death-related thought plays in these effects. Moreover, it has been shown that these effects occur even when death-related stimuli are presented without awareness, suggesting the unconscious effects of mortality salience. Because one pervasive cultural ideal for men is to be strong, we hypothesized that priming death-related stimuli would lead to increasing physical force for men, but not for women. Building on self-escape mechanisms from TMT, we propose that the mechanism that turns priming of death-related stimuli into physical exertion relies on the co-activation of the self with death-related concepts. To test this hypothesis, we subjected 123 participants to a priming task that enabled us to combine the subliminal priming of death-related words with briefly presented self-related words. Accordingly, three different conditions were created: a (control) condition in which only self-related stimuli were presented, a (priming) condition in which death-related words were subliminally primed but not directly paired with self-related stimuli, and a (priming-plus-self) condition in which death-related words were subliminally primed and immediately linked to self-related stimuli. We recorded handgrip force before and after the manipulations. Results showed that male participants in the priming-plus-self condition had a higher peak force output than the priming and control conditions, while this effect was absent among female participants. These results support the hypothesis that unconscious mortality salience, which is accompanied with self-related stimuli, increases physical force for men but not for women. The gender difference may reflect the cultural belief system, in which individuals are taught that men should be strong. Thus, the unconscious mortality salience produced by exposure to the death-related stimuli motivates need to conform to this internalized cultural standard.

Elucidation of novel budding yeast separase mutants.

The mitotic separase cleaves Scc1 in cohesin to allow sister chromatids to separate from each other upon anaphase onset. Separase is also required for DNA damage repair. Here, we isolated and characterized 10 temperature-sensitive (ts) mutants of separase ESP1 in the budding yeast Saccharomyces cerevisiae. All mutants were defective in sister chromatid separation at the restricted temperature. Some esp1-ts mutants were hypersensitive to the microtubule poison benomyl and/or the DNA-damaging agent bleomycin. Overexpression of securin alleviated the growth defect in some esp1-ts mutants, whereas it rather exacerbated it in others. The Drosophila Pumilio homolog MPT5 was isolated as a high-dosage suppressor of esp1-ts cells. We discuss various features of separase based on these findings.

Cdh1 is an antagonist of the spindle assembly checkpoint.

The spindle assembly checkpoint (SAC) monitors unsatisfied connections of microtubules to kinetochores and prevents anaphase onset by inhibition of the ubiquitin ligase E3 anaphase-promoting complex or cyclosome (APC/C) in association with the activator Cdc20. Another APC/C activator, Cdh1, exists permanently throughout the cell cycle but it becomes active from telophase to G1. Here, we show that Cdh1 is partially active and mediates securin degradation even in SAC-active metaphase cells. Additionally, Cdh1 mediates Cdc20 degradation in metaphase, promoting formation of the APC/C-Cdh1. These results indicate that Cdh1 opposes the SAC and promotes anaphase transition.

A near-infrared spectroscopy study of differential brain responses to one or two-handed handing actions: An implication for cultural difference in perceived politeness / Un estudio de espectroscopía casi a infraroja de respuestas cerebrales diferenciadas en acciones de una o dos manos: una implicación para la diferencia cultural en la cortesía percibida

In interpersonal communication, body posture and nonverbal behavior serve as important channels for transmitting social signals and these often vary among cultures. Specific body postures and actions have not only functional, but also affective elements. For example, in Japan, handing an object to another with both hands is considered polite behavior whereas using only one hand is not. In this study, we have examined whether handing with both hands and handing with one hand would produce indications of differential brain activities in the receiver, and whether this activity would differ among people with different cultural backgrounds. Changes in oxy-Hb concentration were measured by 48-channel near infrared spectroscopy (NIRS) from 51 female participants (25 Japanese and 26 non-Japanese). The experimenter handed a bottle to participants using both or one hand. Results showed different amounts of change in oxy-Hb concentrations in the inferior frontal regions, depending on whether one hand or both hands were used. Moreover, the pattern of brain reactions in the inferior frontal regions differed between our Japanese and non-Japanese participants. A discriminant analysis of differences in oxy-Hb values suggested that the degree of oxy-Hb reaction in the right side of inferior frontal regions could predict to which group the participants belonged. These results suggest that different cultural and habitual backgrounds may lead to different NIRS activity while interpreting another's actions, and oxy-Hb IFG concentration may reflect differential interpretations of another's actions.

En la comunicación interpersonal, la postura corporal y el comportamiento no verbal sirven como canales importantes para la transmisión de señales sociales y estas por lo general suelen variar entre culturas. Las posturas corporales específicas y las acciones tienen no sólo elementos funcionales, sino también afectivos. Por ejemplo, en Japón, la entrega de un objeto a otro con las dos manos se considera conducta cortés, mientras que con una sola mano no lo es. En este estudio, hemos examinado si la entrega con las dos manos y la entrega con una mano producen señales de actividad cerebral diferencial en el receptor, y si esta actividad sería diferente entre las personas con diferentes orígenes culturales. Los cambios en la concentración de oxi-Hb fue medida por espectroscopia de infrarrojo cercano de 48 canales (NIRS) de 51 mujeres participantes (25 japonesas y 26 no japonesas). El experimentador le entregó una botella a los participantes utilizando una o ambas manos. Los resultados mostraron diferentes cantidades de cambio en las concentraciones de oxi-Hb en las regiones frontales inferiores, dependiendo de si se utilizaba una o ambas manos. Por otra parte, el patrón de las reacciones cerebrales en las regiones frontales inferiores difiere entre los participantes japoneses y no japoneses. Un análisis discriminante de las diferencias en los valores de oxi-Hb sugirió que el grado de reacción de oxi-Hb en el lado derecho de las regiones frontales inferiores podría predecir a que grupo pertenecían las participantes. Estos resultados sugieren que los distintos orígenes culturales y de hábitos puede dar lugar a actividades diferentes en NIRS, mientras que al interpretar las acciones de los otros, y la concentración de oxi-Hb en IFG puede reflejar interpretaciones diferenciales de las acciones del otro.

Illusory motion and mislocalization of temporally offset target in apparent motion display.

When a visual target briefly appears in a display containing visual motion information, the perceived position of the target is mislocalized forward along its direction of motion. This phenomenon is assumed to be caused by the interaction between the transient onset signal of the target and motion information. However, while transient onset and offset signals are important for the establishment of our perceptual awareness, it has not been examined whether transient offset signals could be also effective for target mislocalization. Here, we demonstrate that shifts in perceived position occurred for a visual target containing a temporally transient offset signal in an apparent motion (AM) display. First, with horizontal AM, we found that illusory motion was perceived when a static target transiently and repeatedly blinked at a fixed position. The perceived direction of the illusory motion was in counter-phase with that of the AM stimuli. Further, we confirmed that illusory motion was frequently perceived when (1) the eccentricity of the target was larger, (2) offset duration was longer, and (3) smoother AM was perceived. Illusory motion was not perceived unless AM stimuli were presented after the offset signal, while illusory motion still occurred when the AM stimuli disappeared before the offset signal. In addition, we found that mislocalization of the target's perceived position actually occurred in a direction opposite to AM. These findings suggest that a transient offset signal could trigger perceptual mislocalization of static visual stimuli by interacting with motion information in a postdictive manner.

Comparing face processing strategies between typically-developed observers and observers with autism using sub-sampled-pixels presentation in response classification technique.

In the present study we modified the standard classification image method by subsampling visual stimuli to provide us with a technique capable of examining an individual's face-processing strategy in detail with fewer trials. Experiment 1 confirmed that one testing session (1450 trials) was sufficient to produce classification images that were qualitatively similar to those obtained previously with 10,000 trials (Sekuler et al., 2004). Experiment 2 used this method to compare classification images obtained from observers with autism spectrum disorders (ASD) and typically-developing (TD) observers. As was found in Experiment 1, classification images obtained from TD observers suggested that they all discriminated faces based on information conveyed by pixels in the eyes/brow region. In contrast, classification images obtained from ASD observers suggested that they used different perceptual strategies: three out of five ASD observers used a typical strategy of making use of information in the eye/brow region, but two used an atypical strategy that relied on information in the forehead region. The advantage of using the response classification technique is that there is no restriction to specific theoretical perspectives or a priori hypotheses, which enabled us to see unexpected strategies, like ASD's forehead strategy, and thus showed this technique is particularly useful in the examination of special populations.

Sound can enhance the suppression of visual target detection in apparent motion trajectory.

Detection performance is impaired for a visual target presented in an apparent motion (AM) trajectory, and this AM interference weakens when orientation information is inconsistent between the target and AM stimuli. These indicate that the target is perceptually suppressed by internal object representations of AM stimuli established along the AM trajectory. Here, we showed that transient sounds presented together with AM stimuli could enhance the magnitude of AM interference. Furthermore, this auditory effect attenuated when frequencies of the sounds were inconsistent during AM. We also confirmed that the sounds wholly elevated the magnitude of AM interference irrespective of the inconsistency in orientation information between the target and AM stimuli when the saliency of the sounds was maintained. These results suggest that sounds can contribute to the robust establishment and spatiotemporal maintenance of the internal object representation of an AM stimulus.

Inhibition of target detection in apparent motion trajectory.

Letter discrimination performance is degraded when a letter is presented within an apparent motion (AM) trajectory of a spot. This finding suggests that the internal representation of AM stimuli can perceptually interact with other stimuli. In this study, we demonstrated that AM interference could also occur for pattern detection. We found that target (Gabor patch) detection performance was degraded within an AM trajectory. Further, this AM interference weakened when the differences in orientation between the AM stimuli and target became greater. We also revealed that AM interference occurred for the target with spatiotemporally intermediate orientations of the inducers that changed their orientation during AM. In contrast, the differences in phase among the stimuli did not affect the occurrence of AM interference. These findings suggest that AM stimuli and their internal representations affect lower visual processes involved in detecting a pattern in the AM trajectory and that the internal object representation of an AM stimulus selectively reflects and maintains the stimulus attribute.

Saccadic compression of rectangle and Kanizsa figures: now you see it, now you don't.

BACKGROUND: Observers misperceive the location of points within a scene as compressed towards the goal of a saccade. However, recent studies suggest that saccadic compression does not occur for discrete elements such as dots when they are perceived as unified objects like a rectangle. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the magnitude of horizontal vs. vertical compression for Kanizsa figure (a collection of discrete elements unified into single perceptual objects by illusory contours) and control rectangle figures. Participants were presented with Kanizsa and control figures and had to decide whether the horizontal or vertical length of stimulus was longer using the two-alternative force choice method. Our findings show that large but not small Kanizsa figures are perceived as compressed, that such compression is large in the horizontal dimension and small or nil in the vertical dimension. In contrast to recent findings, we found no saccadic compression for control rectangles. CONCLUSIONS: Our data suggest that compression of Kanizsa figure has been overestimated in previous research due to methodological artifacts, and highlight the importance of studying perceptual phenomena by multiple methods.

Spatiotemporally coherent motion direction perception occurs even for spatiotemporal reversal of motion sequence.

Thus far, it has been pointed out that motion representation is completed in motion trajectory, and that motion representation interferes with a single physical input at a specific time and space in the motion trajectory. However, it has not been fully investigated whether the internal motion representation could interact with multiple physical inputs across time and space in motion trajectory. Thus, the current research investigated whether spatiotemporally coherent motion perception could be established in a situation involving the spatiotemporal reversal of motion sequences. In a five-point motion display, we found that the motion direction perception of the last two stimuli containing the spatiotemporal reversal was consistent with three preceding stimuli. This failure to perceive motion direction reversal occurred when the shapes of motion trajectory (to upper or lower) were randomly changed. Moreover, we confirmed that one preceding stimulus (three-point motion) was sufficient to produce the failure to perceive motion direction reversal, and the effect of the prior stimuli saturated when the preceding sequence contained two or more stimuli. These findings suggest that the flexible internal motion representation mechanisms reorganize the spatiotemporal irregularities in motion trajectory and establish spatiotemporally coherent motion perceptions.