RINGs, DUBs and Abnormal Brain Growth—Histone H2A Ubiquitination in Brain Development and Disease

During mammalian neurodevelopment, signaling pathways converge upon transcription factors (TFs) to establish appropriate gene expression programmes leading to the production of distinct neural and glial cell types. This process is partially regulated by the dynamic modulation of chromatin states by epigenetic systems, including the polycomb group (PcG) family of co-repressors. PcG proteins form multi-subunit assemblies that sub-divide into distinct, yet functionally related families. Polycomb repressive complexes 1 and 2 (PRC1 and 2) modify the chemical properties of chromatin by covalently modifying histone tails via H2A ubiquitination (H2AK119ub1) and H3 methylation, respectively. In contrast to the PRCs, the Polycomb repressive deubiquitinase (PR-DUB) complex removes H2AK119ub1 from chromatin through the action of the C-terminal hydrolase BAP1. Genetic screening has identified several PcG mutations that are causally associated with a range of congenital neuropathologies associated with both localised and/or systemic growth abnormalities. As PRC1 and PR-DUB hold opposing functions to control H2AK119ub1 levels across the genome, it is plausible that such neurodevelopmental disorders arise through a common mechanism. In this review, we will focus on advancements regarding the composition and opposing molecular functions of mammalian PRC1 and PR-DUB, and explore how their dysfunction contributes to the emergence of neurodevelopmental disorders

Impaired p53-Mediated DNA Damage Response Contributes to Microcephaly in Nijmegen Breakage Syndrome Patient-Derived Cerebral Organoids

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive genetic disorder caused by mutations within nibrin (NBN), a DNA damage repair protein. Hallmarks of NBS include chromosomal instability and clinical manifestations such as growth retardation, immunodeficiency, and progressive microcephaly. We employed induced pluripotent stem cell-derived cerebral organoids from two NBS patients to study the etiology of microcephaly. We show that NBS organoids carrying the homozygous 657del5 NBN mutation are significantly smaller with disrupted cyto-architecture. The organoids exhibit premature differentiation, and Neuronatin (NNAT) over-expression. Furthermore, pathways related to DNA damage response and cell cycle are differentially regulated compared to controls. After exposure to bleomycin, NBS organoids undergo delayed p53-mediated DNA damage response and aberrant trans-synaptic signaling, which ultimately leads to neuronal apoptosis. Our data provide insights into how mutations within NBN alters neurogenesis in NBS patients, thus providing a proof of concept that cerebral organoids are a valuable tool for studying DNA damage-related disorders

Dynamics and Structure-Function Relationships of the Lamin B Receptor (LBR)

<div><p>The lamin B receptor (LBR) is a multi-spanning membrane protein of the inner nuclear membrane that is often employed as a “reporter” of nuclear envelope dynamics. We show here that the diffusional mobility of full-length LBR exhibits significant regional variation along the nuclear envelope, consistent with the existence of discrete LBR microdomains and the occurrence of multiple, asymmetrically-spaced anastomoses along the nuclear envelope-endoplasmic reticulum interface. Interestingly, a commonly used fusion protein that contains the amino-terminal region and the first transmembrane domain of LBR exhibits reduced mobility at the nuclear envelope, but behaves similarly to full-length LBR in the endoplasmic reticulum. On the other hand, carboxy-terminally truncated mutants that retain the first four transmembrane domains and a part or the whole of the amino-terminal region of LBR are generally hyper-mobile. These results suggest that LBR dynamics is structure and compartment specific. They also indicate that native LBR is probably “configured” by long-range interactions that involve the loops between adjacent transmembrane domains and parts of the amino-terminal region.</p></div

A provisional model explaining regional variation of LBR mobility.

<p>The “scanning probe” cartoon explains how LBR mobility could vary from one region of the NE to the next, depending on the relative abundance of immobile (<i>dark green</i>) or mobile (<i>light green</i>) LBR molecules. The basic assumptions in this model are that: a) ROI selection is entirely random and therefore, the probability of including or not including LBR microdomains is proportional to the abundance of the latter; b) LBR microdomains contain many more molecules than “free” LBR; c) the areas of the INM neighbouring to the NPCs, where the lamina meshwork is interrupted, contain exclusively free LBR. The same applies for the outer nuclear membrane and the surface of the ER. The histogram shown is based on an entirely hypothetical example, where the ROIs include from 0 to 2 microdomains each, depending on size. Arbitrarily, we have assumed that regions containing free LBR regain 80% of the initial fluorescence, whereas areas containing 0.5 to 2 microdomains regain from 20% to 70% of the fluorescence, respectively. Notice that increase of ROI size results in the broadening of the <i>Mf</i> frequency distribution (“homogenization”) and shifting of the average <i>Mf</i> towards lower values. ROIs are indicated by a circle. (<i>ER</i>): ER cisterna; (<i>ONM</i>): outer nuclear membrane; (<i>INM</i>): inner nuclear membrane; (<i>NPC</i>): nuclear pore complex. For further details see text.</p

LBR mobility differences at the level of cell population.

<p>(A) FRAP assays using circular ROIs with diameters of 1.0 μm or 1.5 μm and an arc-shaped ROI approximately 1.7 μm wide and 10 μm long. (B) <i>Mf</i> and <i>t</i>_<i>1/2</i> box plots for all samples presented in (A). Statistical analysis based on the Kolmogorov-Smirnov test, sample number and coefficients of variation (<i>CV</i>) are specified. Red lettering indicates statistically significant differences with a threshold of 0.005. <i>CVctrl</i> indicates variation due to experimental error as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169626#pone.0169626.s002" target="_blank">S2A Fig</a>. (C) Frequency distribution of <i>Mf</i>. (D) Frequency distribution of <i>t</i>_<i>1/2</i>.</p

Properties of a “soluble” protein corresponding to the amino-terminal region of LBR.

<p>(A) Localization pattern and FRAP data obtained with the Nt mutant, after transfection of Hela cells. <i>Mf</i> and <i>t</i>_<i>1/2</i> box plots comparing the dynamic parameters of FL-LBR and Nt in FRAP assays with circular ROIs ranging from 1.5 to 3.0 μm in diameter. Statistical analysis based on the K-S test, sample number and coefficients of variation (<i>CVs</i>) are specified. <i>CVctrl</i> indicates variation due to experimental error as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169626#pone.0169626.s002" target="_blank">S2A Fig</a>. Notice that variation is much lower with Nt than with FL-LBR. (B) A typical FLIP experiment with Nt (10 successive bleaching-recovery cycles). The histograms show the changes in fluorescence intensity (<i>I</i>_<i>final</i> <i>/I</i>_{<i>initial</i>}) in the nucleoplasm (<i>NUC</i>) and the cytoplasm (<i>CYT</i>). Bars in all panels, 6 μm.</p

ROI geometries and bleach volume.

<p>(A) ROIs employed in the FRAP assays. The panels show FL-LBR distribution before and after bleaching different parts of the NE, as indicated in the images. Bar, 6 μm. (B) Intensity profiles before and after bleaching a circular spot with a diameter of 1.5 μm and a curvilinear strip (half-rim) in fixed Hela cells. Notice the nearly Gaussian profiles and the moderate “halo” effect (Δx ~1.0 μm). (C) Bleach depth after focusing the laser beam at the top (triangle, <i>pink</i>), equator (curvilinear segment, <i>green</i>), or the bottom (square, <i>violet</i>) of the cell. The series show successive optical sections at the <i>xy</i> and <i>xz</i> level, or rotated images (<i>rot</i>. <i>xyz</i>), revealing an axial bleach half-depth of 1.4–2.6 μm (distally and proximally to the beam, respectively). Pseudo-color according to sample depth was applied using the <i>LAS-AF</i> program.</p

Properties of membrane-spanning LBR mutants.

<p>(A) Localization patterns of FL-LBR, LBR mutants and Sec61β (an ER membrane protein) after transfection of Hela cells. (B) Staining of Gr–expressing cells with anti-lamin B antibodies. Notice the cytoplasmic accumulations that stain positive for lamin Β in some of the cells. Bars in (A) and (B), 6 μm. (C-D) <i>Mf</i> and <i>t</i>_<i>1/2</i> box plots for all proteins using an arc-shaped ROI. The panel shows two separate sets of data obtained by probing either the NE or the peripheral ER. Sample number and coefficients of variation (<i>CVs</i>) are specified. <i>CVctrl</i> indicates variation due to experimental error as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169626#pone.0169626.s002" target="_blank">S2A Fig</a>. (E) Statistical evaluation and comparison of the data shown in (C-D) by the K-S test. The threshold of statistical significance is 0.005. Red lettering indicates significant differences; black lettering corresponds to differences close to the statistical threshold; and grey lettering denotes differences that are not significant.</p

LBR mobility differences at the level of single cells.

<p>(A) FL-LBR recovery in the central area and the edges of the same arc-shaped ROI (see image). The table underneath shows the <i>CVs</i> for <i>Mf</i> and <i>t</i>_<i>1/2</i> in each experiment. The <i>CVs</i> of the control (<i>ctrl</i>), calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169626#pone.0169626.s002" target="_blank">S2A Fig</a>, are also included for a comparison. (B) A profile FRAP experiment with a sample exhibiting significant <i>Mf</i> and <i>t</i>_<i>1/2</i> variation across the same ROI. The upper panel depicts successive snapshots of the photobleached area at various time intervals. Details of the ROI at higher zoom and contrast are shown underneath. The bleach and recovery profiles along adjacent sub-ROIs (no. 1–17) are shown separately in the right panel. (<i>I</i>): fluorescence intensity; (<i>x</i>): contour length; (<i>R</i>): fluorescence intensity across successive sub-ROIs at various time intervals in relation to the fluorescence intensity immediately after the bleach. The bleach was done at <i>t</i> = 35 s and the recovery process was completed at <i>t</i> = 308 s. (C) Variation of LBR mobility in different sectors of the same NE (see images). The results are presented in the same format as in (<i>A</i>). Bars are 6 μm and 1 μm in (<i>B</i>) “detail”.</p