Noncoding human Y RNAs are overexpressed in tumours and required for cell proliferation

Noncoding Y RNAs have recently been identified as essential factors for chromosomal DNA replication in human cell nuclei. Here, we investigate the expression of human Y RNAs in tumours and test their requirement for cell proliferation. Relative expression levels of all four human Y RNAs (hY1, hY3, hY4 and hY5 RNA) were determined by quantitative RT–PCR in extracts from human solid tumours, corresponding nonmalignant normal tissues and derived cultured cells. On average, all four hY RNAs are significantly overexpressed in solid tumours between 4- and 13-fold, compared to the corresponding normal tissues. In particular, hY1 and hY3 RNAs are overexpressed in carcinomas (and adenocarcinomas) of the bladder, cervix, colon, kidney, lung and prostate with extremely high statistical significance (ANOVA, between groups, P<10e-22). A functional requirement of all four hY RNAs for cell proliferation was investigated in a systematic survey for loss-of-function by RNA interference (RNAi). Degradation of hY1 and hY3 RNAs in human cell lines resulted in a significant cytostatic inhibition of cell proliferation. We conclude that noncoding hY RNAs have potential both as new cancer biomarkers and as molecular targets for anti-proliferative intervention

CAF-1 Is Essential for Heterochromatin Organization in Pluripotent Embryonic Cells

During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells

Temporal Processing of Vibratory Communication Signals at the Level of Ascending Interneurons in Nezara viridula (Hemiptera: Pentatomidae)

During mating, males and females of N. viridula (Heteroptera: Pentatomidae) produce sex- and species-specific calling and courtship substrate-borne vibratory signals, grouped into songs. Recognition and localization of these signals are fundamental for successful mating. The recognition is mainly based on the temporal pattern, i.e. the amplitude modulation, while the frequency spectrum of the signals usually only plays a minor role. We examined the temporal selectivity for vibratory signals in four types of ascending vibratory interneurons in N. viridula. Using intracellular recording and labelling technique, we analyzed the neurons' responses to 30 pulse duration/interval duration (PD/ID) combinations. Two response arrays were created for each neuron type, showing the intensity of the responses either as time-averaged spike counts or as peak instantaneous spike rates. The mean spike rate response arrays showed preference of the neurons for short PDs (below 600 ms) and no selectivity towards interval duration; while the peak spike rate response arrays exhibited either short PD/long ID selectivity or no selectivity at all. The long PD/short ID combinations elicited the weakest responses in all neurons tested. No response arrays showed the receiver preference for either constant period or duty cycle. The vibratory song pattern selectivity matched the PD of N. viridula male vibratory signals, thus pointing to temporal filtering for the conspecific vibratory signals already at level of the ascending interneurons. In some neurons the responses elicited by the vibratory stimuli were followed by distinct, regular oscillations of the membrane potential. The distance between the oscillation peaks matched the temporal structure of the male calling song, indicating a possible resonance based mechanism for signal recognition

Transgenerational Stress Memory Is Not a General Response in Arabidopsis

Adverse conditions can trigger DNA damage as well as DNA repair responses in plants. A variety of stress factors are known to stimulate homologous recombination, the most accurate repair pathway, by increasing the concentration of necessary enzymatic components and the frequency of events. This effect has been reported to last into subsequent generations not exposed to the stress. To establish a basis for a genetic analysis of this transgenerational stress memory, a broad range of treatments was tested for quantitative effects on homologous recombination in the progeny. Several Arabidopsis lines, transgenic for well-established recombination traps, were exposed to 10 different physical and chemical stress treatments, and scored for the number of somatic homologous recombination (SHR) events in the treated generation as well as in the two subsequent generations that were not treated. These numbers were related to the expression level of genes involved in homologous recombination and repair. SHR was enhanced after the majority of treatments, confirming previous data and adding new effective stress types, especially interference with chromatin. Compounds that directly modify DNA stimulated SHR to values exceeding previously described induction rates, concomitant with an induction of genes involved in SHR. In spite of the significant stimulation in the stressed generations, the two subsequent non-treated generations only showed a low and stochastic increase in SHR that did not correlate with the degree of stimulation in the parental plants. Transcripts coding for SHR enzymes generally returned to pre-treatment levels in the progeny. Thus, transgenerational effects on SHR frequency are not a general response to abiotic stress in Arabidopsis and may require special conditions

The maize root stem cell niche: a partnership between two sister cell populations

Using transcript profile analysis, we explored the nature of the stem cell niche in roots of maize (Zea mays). Toward assessing a role for specific genes in the establishment and maintenance of the niche, we perturbed the niche and simultaneously monitored the spatial expression patterns of genes hypothesized as essential. Our results allow us to quantify and localize gene activities to specific portions of the niche: to the quiescent center (QC) or the proximal meristem (PM), or to both. The data point to molecular, biochemical and physiological processes associated with the specification and maintenance of the niche, and include reduced expression of metabolism-, redox- and certain cell cycle-associated transcripts in the QC, enrichment of auxin-associated transcripts within the entire niche, controls for the state of differentiation of QC cells, a role for cytokinins specifically in the PM portion of the niche, processes (repair machinery) for maintaining DNA integrity and a role for gene silencing in niche stabilization. To provide additional support for the hypothesized roles of the above-mentioned and other transcripts in niche specification, we overexpressed, in Arabidopsis, homologs of representative genes (eight) identified as highly enriched or reduced in the maize root QC. We conclude that the coordinated changes in expression of auxin-, redox-, cell cycle- and metabolism-associated genes suggest the linkage of gene networks at the level of transcription, thereby providing additional insights into events likely associated with root stem cell niche establishment and maintenance

Silencing of Chromatin Assembly Factor 1 in Human Cells Leads to Cell Death and Loss of Chromatin Assembly during DNA Synthesis

In eukaryotic cells, chromatin serves as the physiological template for gene transcription, DNA replication, and repair. Chromatin assembly factor 1 (CAF-1) is the prime candidate protein to mediate assembly of newly replicated DNA into chromatin. To investigate the physiological role of CAF-1 in vivo, we used RNA interference (RNAi) to silence the 60-kDa subunit of CAF-1 (p60) in human cells. Transfection of a small interfering RNA (siRNA) directed against p60 resulted in efficient silencing of p60 expression within 24 h. This silencing led to an induction of programmed cell death in proliferating but not in quiescent human cells. Concomitantly, proliferating cells lacking p60 accumulated DNA double-strand breaks and increased levels of the phosphorylated histone H2A.X. Nuclear extracts from cells lacking p60 exhibited a 10-fold reduction of nucleosome assembly activity during DNA synthesis, which was restored upon addition of recombinant p60 protein. Nascent chromatin in cell nuclei lacking p60 showed significantly increased nuclease sensitivity, indicating chromatin assembly defects during DNA synthesis in vivo. Collectively, these data identify CAF-1 as an essential factor not only for S-phase-specific chromatin assembly but also for proliferating cell viability

Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells

Genome stability in eukaryotic cells is maintained through efficient DNA damage repair pathways, which have to access and utilize chromatin as their natural template. Here we investigate the role of chromatin assembly factor 1 (CAF-1) and its interacting protein, PCNA, in the response of quiescent human cells to DNA double-strand breaks (DSBs). The expression of CAF-1 and PCNA is dramatically induced in quiescent cells upon the generation of DSBs by the radiomimetic drug bleocin (a bleomycin compound) or by ionizing radiation. This induction depends on DNA-PK. CAF-1 and PCNA are recruited to damaged chromatin undergoing DNA repair of single- and double-strand DNA breaks by the base excision repair and nonhomologous end-joining pathways, respectively, in the absence of extensive DNA synthesis. CAF-1 prepared from repair-proficient quiescent cells after induction by bleocin mediates nucleosome assembly in vitro. Depletion of CAF-1 by RNA interference in bleocin-treated quiescent cells in vivo results in a significant loss of cell viability and an accumulation of DSBs. These results support a novel and essential role for CAF-1 in the response of quiescent human cells to DSBs, possibly by reassembling chromatin following repair of DNA strand breaks

Temporal pattern recognition based on instantaneous spike rate coding in a simple auditory system

Temporal pattern recognition based on instantaneous spike rate coding in a simple auditory system. J Neurophysiol 90: 2484–2493, 2003; 10.1152/jn.00259.2003. Auditory pattern recognition by the CNS is a fundamental process in acoustic communication. Because crickets communicate with stereotyped patterns of constant frequency syllables, they are established models to investigate the neuronal mechanisms of auditory pattern recognition. Here we provide evidence that for the neural processing of amplitude-modulated sounds, the instantaneous spike rate rather than the time-averaged neural activity is the appropriate coding principle by comparing both coding parameters in a thoracic interneuron (Omega neuron ON1) of the cricket (Gryllus bimaculatus) auditory system. When stimulated with different temporal sound patterns, the analysis of the instantaneous spike rate demonstrates that the neuron acts as a low-pass filter for syllable patterns. The instantaneous spike rate is low at high syllable rates, but prominent peaks in the instantaneous spike rate are generated as the syllable rate resembles that of the species-specific pattern. The occurrence and repetition rate of these peaks in the neuronal discharge are sufficient to explain temporal filtering in the cricket auditory pathway as they closely match the tuning of phonotactic behavior to different sound patterns. Thus temporal filtering or “pattern recognition ” occurs at an early stage in the auditory pathway

Clinical significance and prognostic value of chromatin assembly factor-1 overexpression in human solid tumours

International audienceAims: The chromatin assembly factor CAF-1, whose function is critical for maintaining chromatin stability during DNA replication and repair, has been identified as a proliferation marker in breast cancer. Here, we investigate the interest of CAF-1 as a proliferation marker in a wide spectrum of solid tumors, and assess its potential value in predicting clinical outcome. Methods and Results: Using immunocytochemistry on paraffin-embedded tissue sections, we compare CAF-1 labeling index with known proliferation markers Ki-67 and MCMs, and we analyze its association with clinicopathological data and patients' outcome. CAF-1 expression shows a strong positive correlation with Ki-67, routinely used to detect proliferating cells, while it generally displays weaker correlations with MCM markers, known to label cells with replicative potential. CAF-1 expression is significantly associated with histological grade in breast, cervical, endometrial and renal cell carcinomas, and with disease stage in endometrial and renal carcinomas. Furthermore, high expression of CAF-1 is an independent predictor of adverse clinical outcome in renal, endometrial and cervical carcinomas. Conclusions: These data demonstrate that CAF-1 is a proliferation marker in various malignant tumors with prognostic value in renal, endometrial and cervical carcinomas, which supports the relevance of CAF-1 as a clinical marker to monitor cancer progression