Myb-binding protein 1A (MYBBP1A) is essential for early embryonic development, controls cell cycle and mitosis, and acts as a tumor suppressor

MYBBP1A is a predominantly nucleolar transcriptional regulator involved in rDNA synthesis and p53 activation via acetylation. However little further information is available as to its function. Here we report that MYBBP1A is developmentally essential in the mouse prior to blastocyst formation. In cell culture, down-regulation of MYBBP1A decreases the growth rate of wild type mouse embryonic stem cells, mouse embryo fibroblasts (MEFs) and of human HeLa cells, where it also promotes apoptosis. HeLa cells either arrest at G2/M or undergo delayed and anomalous mitosis. At mitosis, MYBBP1A is localized to a parachromosomal region and gene-expression profiling shows that its down-regulation affects genes controlling chromosomal segregation and cell cycle. However, MYBBP1A down-regulation increases the growth rate of the immortalized NIH3T3 cells. Such Mybbp1a down-regulated NIH3T3 cells are more susceptible to Ras-induced transformation and cause more potent Ras-driven tumors. We conclude that MYBBP1A is an essential gene with novel roles at the pre-mitotic level and potential tumor suppressor activity.NHMRC: This work was supported by Associazione Italiana Ricerche sul Cancro (AIRC) grant 8929 and European Community FP7 201681 ‘‘Prepobedia’’ to FB, the Australian National Health and Medical Research Council to RK and TJG (project ID000115). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Gait Pattern and Motor Performance During Discrete Gait Perturbation in Children With Autism Spectrum Disorders

Quantitative evaluation of gait has been considered a useful tool with which to identify subtle signs of motor system peculiarities in autism spectrum disorder (ASD). However, there is a paucity of studies reporting gait data in ASD as well as investigating learning processes of locomotor activity. Novel advanced technologies that couple treadmills with virtual reality environments and motion capture systems allows the evaluation of gait patterns on multiple steps and the effects of induced gait perturbations, as well as the ability to manipulate visual and proprioceptive feedbacks. This study aims at describing the gait pattern and motor performance during discrete gait perturbation of drug-naïve, school-aged children with ASD compared to typically developing (TD) peers matched by gender and age. Gait analysis was carried out in an immersive virtual environment using a 3-D motion analysis system with a dual-belt, instrumented treadmill. After 6 min of walking, 20 steps were recorded as baseline. Then, each participant was exposed to 20 trials with a discrete gait perturbation applying a split-belt acceleration to the dominant side at toe-off. Single steps around perturbations were inspected. Finally, 20 steps were recorded during a post-perturbation session. At baseline, children with ASD had reduced ankle flexion moment, greater hip flexion at the initial contact, and greater pelvic anteversion. After the discrete gait perturbation, variations of peak of knee extension significantly differed between groups and correlated with the severity of autistic core symptoms. Throughout perturbation trials, more than 60% of parameters showed reliable adaptation with a decay rate comparable between groups. Overall, these findings depicted gait peculiarities in children with ASD, including both kinetic and kinematic features; a motor adaptation comparable to their TD peers, even though with an atypical pattern; and a motor adaptation rate comparable to TD children but involving different aspects of locomotion. The platform showed its usability with children with ASD and its reliability in the definition of paradigms for the study of motor learning while doing complex tasks, such as gait. The additional possibility to accurately manipulate visual and proprioceptive feedback will allow researchers to systematically investigate motor system features in people with ASD

Timed pregnancy analysis of heterozygous <i>MYBBP1A</i>^+/− crosses.

<p>N = Total number.</p><p>NP = No PCR product.</p

Tumorigenesis by RasVal12 in NIH3T3 cells infected with an empty vector (C) or with a retrovirus carrying an shRNA specific for <i>MYBBP1A</i> (Sh3).

#<p>Total number of mice in the group.</p>○<p>volume expressed in cm³.</p>*<p>SEM = Standard Error of the Mean.</p>$<p>Student's t-test.</p

<i>MYBBP1A</i> down-regulation induces apoptosis.

<p>(<b>A</b>) HeLa cells were transiently transfected with siRNA1, 2 or 3, or with control High-GC or Medium-GC oligonucleotides, or with Lipofectamine only, for 48 h. The figure shows the determination of early apoptotic cells by flow cytometry (i.e. Annexin V positive and 7AAD-negative) (left panel, circled gate) 48 h post transfection. The histogram on the right shows the quantification in the various samples at the different times after transfection. In untreated cells only 1% of the cells were in apoptosis. (<b>B</b>) Western-blot analysis of HeLa cells transiently transfected for 48 h with CTL (untreated cells), LIPO (treated only with lipofectamine), HIGH GC (transfected with High-GC control), siRNA1 (transfected with MYBBP1A-specific siRNA1). The immunoblot was performed against MYBBP1A, active Caspase 3 and Caspase 9; tubulin is shown as loading control.</p

Genes differentially expressed in <i>MYBBP1A</i>-down-regulated HeLa cells^*.

*<p>Data obtained with a human Affymetrix ST 1.0 chip. Analysis of gene expression profiling on HeLa cells transfected with siRNA3, harvested 48 h after transfection.</p>§<p>Genes whose expression level varied by more than 50% (p<0.0001).</p

Gene Onthology Analysis of the differentially expressed genes following <i>MYBBP1A</i>-down-regulation in HeLa cells.

<p>N = total number of differentially expressed genes within the GO cathegory.</p><p>%: percent of the total number of differentially expressed genes.</p><p><i>p-value</i>: calculated on the basis of the enrichment of the category (ie number of genes affected/number of genes in the category).</p

Specific down-regulation of <i>Mybbp1a</i> in wild type ES cells blocks proliferation and induces activation of caspase-3.

<p>(<b>A</b>) ES cells were infected with the <i>Mybbp1a</i>-specific shRNA3 or with an empty non-target (NT) lentivirus vector (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039723#s2" target="_blank">Materials and Methods</a>), and the proliferation rate was measured starting two days after infection (0 h) every 24 h, using a cell counter. (<b>B</b>). At the end of the experiment (72 h) the cells were lysed and the extract immunoblotted with Mybbp1a-specific antibodies (anti-p160C) using Tubulin as loading control. (<b>C, D</b>). The extracts were also immunoblotted against Oct4 (for specificity of the down-regulation) and cleaved-Caspase 3 for apoptosis using Vinculin as loading control.</p

Down-regulation of <i>Mybbp1a</i> in primary wild type MEFs and in NIH3T3 cells has opposite effects on cell proliferation.

<p>(<b>A</b>) Down regulation of <i>Mybbp1a</i> in MEFs with the specific lentiviral vector shRNA3 induces early senescence, as opposed to a non-target empty (NT) vector. (<b>B</b>) Immunoblot of the cells of panel A showing down regulation of MYBBP1A (p160), using tubulin as control. (<b>C</b>) Growth rate determination (crystal violet assay, see Methods) in Mybbp1a down-regulated NIH3T3 cells with lentiviral vectors expressing specific Sh1RNA or Sh3RNA, as opposed with control empty Non Target vector (NT). Crystal violet assays were performed over 10 days counting the cells in triplicate every 2 days. The p-value of the difference between Sh3 and NT-treated cells was ≤0.001 (t test). (<b>D</b>) Immunoblot of the cells of panel C. (<b>E</b>) Growth rate determination of <i>MYBBP1A</i> down-regulated HeLa cells by specific siRNAs (see Methods). Ctl: untreated cells. Lipo: transfection control with lipofectamine only. High GC and Medium GC: two control oligonucleotides (indicated by the siRNAs manufacturer) of high and, respectively, medium GC content. siRNA1, siRNA2 and siRNA3: specific MYBBP1A siRNAs (see Methods for sequences). (<b>F</b>) Immunoblots of the cells of panel B upon culturing for 24, 48, 72 and 96 h after transfection.</p

Connection between <i>MYBBP1A</i>, cell cycle genes and their function.

<p>The figure depicts the connection between <i>MYBBP1A</i>, the genes regulating the cell cycle that are altered by <i>MYBBP1A</i> down-regulation and their function. In particular, the expression of red genes is increased in the absence of MYBBP1A, while those in blue are decreased. Thus, MYBBP1A induces the inhibitors of Cdc2/cyclinB and down-regulates the activators. Note that although the figure focuses on the G2/M phase, some of the genes function also at the G1/S (in particular CDKN1A). MYBBP1A might act by regulating the synthesis and/or activity of one or more of its partner transcription factors (cMyb, cJun, NF-kB). In fact, the level of cJun mRNA is increased upon <i>MYBBP1A</i> down-regulation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039723#pone-0039723-t004" target="_blank">Table 4</a>).</p