Cis-regulation of microRNA expression by scaffold/matrix-attachment regions

microRNAs (miRNAs) spatio-temporally modulate gene expression; however, very little is known about the regulation of their expression. Here, we hypothesized that the well-known cis-regulatory elements of gene expression, scaffold/matrix-attachment regions (MARs) could modulate miRNA expression. Accordingly, we found MARs to be enriched in the upstream regions of miRNA genes. To determine their role in cell type-specific expression of miRNAs, we examined four individual miRNAs (let-7b, miR-17, miR-93 and miR-221) and the miR-17–92 cluster, known to be overexpressed in neuroblastoma. Our results show that MARs indeed define the cell-specific expression of these miRNAs by tethering the chromatin to nuclear matrix. This is brought about by cell type-specific binding of HMG I/Y protein to MARs that then promotes the local acetylation of histones, serving as boundary elements for gene activation. The binding, chromatin tethering and gene activation by HMG I/Y was not observed in fibroblast control cells but were restricted to neuroblastoma cells. This study implies that the association of MAR binding proteins to MARs could dictate the tissue/context specific regulation of miRNA genes by serving as a boundary element signaling the transcriptional activation

Disruption of Mouse Cenpj, a Regulator of Centriole Biogenesis, Phenocopies Seckel Syndrome

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpjtm/tm) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpjtm/tm embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpjtm/tm embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome

Generation of a mouse model of <i>CENPJ</i>-Seckel syndrome.

<p>A. The <i>CENPJ</i> gene spans 40 kb and comprises 17 exons. The 3′ and 5′ untranslated regions are depicted in grey. Mutations in <i>CENPJ</i> have been associated with either primary microcephaly (MCPH) or Seckel syndrome (SECKEL). The mutation in intron 11 that has been associated with Seckel syndrome results in the generation of three transcripts: one lacking exon 12, one lacking 11 and 12 and one lacking exons 11,12 and 13. Disruption of mouse <i>Cenpj</i> by insertion of a cassette (depicted by the blue square) between exons 4 and 5 results in low levels of splicing over the cassette and cryptic splicing between exons 3 and 6 or 4 and 6; the latter two transcripts are predicted to result in truncated proteins. The allele was designated <i>Cenpj^{tm1a(EUCOMM)Wtsi}</i> and abbreviated to <i>Cenpj^tm</i>. Percentages show mean expression of <i>Cenpj</i> across exon boundaries as determined by quantitative RT-PCR relative to <i>Gapdh</i> for <i>Cenpj^tm/tm</i> relative to <i>Cenpj^+/+</i> for RNA extracted from n = 3 murine embryonic fibroblast (MEF) lines. B. Immunoblot to show reduction in Cenpj levels in protein extracted from <i>Cenpj^tm/tm</i> (<i>tm/tm</i>), <i>Cenpj^+/tm</i> (+/tm), and <i>Cenpj^+/+</i>(+/+) MEFs. KAP1 was used as a loading control. C. Table shows frequency of <i>Cenpj^tm/tm</i> mice born from heterozygote intercrosses. <i>Cenpj^tm/tm</i> showed partial embryonic lethality as shown by their reduced frequency at E18.5 and P14 (25% expected, *<i>P</i> = 0.02, **<i>P</i> = 0.0001, χ² test). D. Representative images of E18.5 skeletal preparations of <i>Cenpj^+/+</i> and <i>Cenpj^tm/tm</i> embryos. Staining with alcian blue (cartilage) and alizarin red (bone). <i>Cenpj^tm/tm</i> embryo with a sloping forehead and polysyndactylism of digit one of the left hindpaw (inset). E. Bodyweights of male <i>Cenpj^tm/tm</i> (n = 8), <i>Cenpj^+/tm</i> (n = 7), <i>Cenpj^+/+</i>(n = 40) and baseline wild-type controls (n = 912) from 3–16 weeks of age. Data show that <i>Cenpj^tm/tm</i> are significantly smaller than <i>Cenpj^+/+</i> mice at all ages (<i>P</i> = 2.2×10⁻¹⁶, Mann-Whitney-Wilcoxon test). F. Skeletal preparations of E18.5 <i>Cenpj^tm/tm</i> embryos showed irregular ossification of the cranium and G. sternum. H. X-Rays show that adult <i>Cenpj^tm/tm</i> mice may present with a flatter, sloping forehead (A), mild elevation of the parietal bone (B), a short humerus with a prominent deltoid tuberosity (C), prominent medial epicondyle (D), an irregular ribcage (E), short lumbar and sacral vertebrae (F), an abnormal pelvis (G), extra sacrocaudal transitional vertebrae (H), short, abnormal/fused caudal vertebrae 2/3 – caudal vertebrae 7/8 (I) and reduced intervertebral joint space (J).</p

Centrosome and mitotic spindle abnormalities are elevated in <i>Cenpj</i>-deficient cells.

<p>A. Images show examples of Cenpj staining in centrosomes of <i>Cenpj</i>^+/+ and <i>Cenpj^tm/tm</i> mouse embryonic fibroblasts (MEFs). Cells were stained with antibodies against Cenpj (green in merge) and the centrosomal protein γ-tubulin (red in merge). Framed areas are shown at higher magnification. B. Graph shows mitotic spindle phenotypes in MEFs derived from <i>Cenpj</i>^+/+, <i>Cenpj</i>^+/tm and two independent <i>Cenpj^tm/tm</i> embryos (littermates, +/+ MEFs passage 4, +/tm and <i>tm/tm</i> MEFs passage 3): <i>tm/tm</i> (1) and <i>tm/tm</i> (2). Number of mitotic cells scored are shown for each genotype. Examples for monopolar and multipolar spindle are shown. Note cell on bottom panels forming a bipolar spindle by clustering supernumerary centrosomes. Cells were stained with antibodies against α-tubulin (green in merge) and the centrosomal protein, Cdk5RAP2 (red in merge). C. Graph shows centriole numbers in mitotic MEFs of indicated genotypes (littermates, +/+ MEFs passage 4, +/tm and <i>tm/tm</i> MEFs passage 3). Cells were arrested in mitosis with monastrol that caused monopolar spindle formation and facilitated visualization of centrioles. Note that mitotic cells should normally contain a total of 4 centrioles, but even in wild-type cells we occasionally detect 3 centrioles probably due to insufficient spatial resolution, so 3 or 4 centrioles were considered a single class. Data were collected from two independent experiments; bars show mean ±SD, number of mitotic cells scored are shown for each genotype. Images below depict examples for cells with different centriole numbers (top cell with 4 centrioles is normal, all other cells have too few or too many centrioles). Cells were stained with antibodies against the microtubule-binding protein Tpx2 (green in merge) and the centriolar protein, centrin-3 (red in merge). Framed areas are shown at higher magnification. Scale bars = 5 µm.</p

Neuropathological abnormalities.

<p>A. <i>Cenpj^tm/tm</i> mouse brain weights were two standard deviations below that of control mice (n = 144 baseline control mice). *<i>P</i> = 0.0002, t-test, <i>Cenpj^tm/tm</i>, n = 6 and <i>Cenpj</i>^+/+ n = 10. The lower whisker extends to the lowest datum still within 1.5 Inter-quartile range (IQR) of the lower quartile. The upper whisker extends to the highest datum still within 1.5 IQR of the upper quartile B. The dentate gyrus was significantly shorter in <i>Cenpj^tm/tm</i> mice (n = 3) when compared to <i>Cenpj</i>^+/+ control mice (n = 30), *<i>P</i> = 0.01, t-test. Scale bar 1 mm C. Social recognition test. When tested for habituation-dishabituation, both <i>Cenpj^tm/tm</i> (n = 7) and <i>Cenpj</i>^+/+ (n = 7) mice recognized a novel stimulus mouse as shown by a decline in investigation time over the first four trials that was recovered on trial five, when they were exposed to a novel mouse (trial four <i>vs.</i> trial five, * <i>P</i> = 0.0033 and ** <i>P</i> = 0.0014, two-way ANOVA followed by post-hoc t-test). D. A discrimination test was performed 24 h later the habituation-dishabituation test. When given a choice between the familiar (same stimulus animal used for trials one to four) and a new unfamiliar mouse 24 h later, <i>Cenpj^tm/tm</i> mice could not discriminate as shown by the similar investigation time for both stimulus animals (<i>Cenpj</i>^+/+<i>P</i> = 0.0326, C<i>enpj^tm/tm P</i> = 0.957, t-test). E. Representative images of immunohistochemical stainings of E14.5 embryo sections. Cenpj was highly expressed in areas of active neurogenesis within the telencephalon. Scale bar 400 µm. There was a generalized increase in cleaved (activated) caspase-3-positive (scale bar 100 µm) and Ser139-phosphorylated H2AX (γH2AX; scale bar 200 µm) cells throughout embryos, images of striatum are shown.. The number of cells positive (as a percentage of total in two different 75 µm² areas) for cleaved (activated) caspase-3 (C3A+) and pan-nuclear Ser139-phosphorylated H2AX (γH2AX) was increased in areas of active neurogenesis within the striatum and cortex. *<i>P</i><0.05; Mann-Whitney with continuity correction, <i>Cenpj^tm/tm</i> n = 3 and <i>Cenpj</i>^+/+ n = 3. Data shows mean and SEM. F. Neuron densities were counted in three different areas (75 µm²) of active neurogenesis for each of the striatum (STR), cortex (CTX) and pro-hippocampus (HIP) and three areas of 150 µm² in the mid-striatum (M-STR) of E14.5 embryos, *<i>P</i> = 0.0008, t-test, <i>Cenpj^tm/tm</i> n = 3 and <i>Cenpj</i>^+/+ n = 3. Data shows mean and SEM.</p

Delayed onset to puberty and ocular, endocrine, haematological, and plasma abnormalities.

<p>A. Periodic acid-Schiff (PAS) staining and cleaved (activated) caspase-3 immunostaining of adrenal sections from 16 week-old virgin female <i>Cenpj^tm/tm</i> mice (n = 3) confirmed corticomedullary pigmentation and ongoing apoptosis in the X-zone, respectively (representative images, scale bars 100 µm). B. Breeding records of <i>Cenpj^tm/tm</i> females set up with <i>Cenpj^tm/tm</i> males at 6–7 weeks of age showed that <i>Cenpj^tm/tm</i> females produce their first litter around four weeks later than <i>Cenpj</i>^+/+ females. *<i>P</i> = 0.012, t-test. C. Top panel shows normal cornea from a <i>Cenpj</i>^+/+ mouse. <i>Cenpj^tm/tm</i> mice had disruption of the Descemet's membrane and corneal endothelium (arrow). Middle panel shows normal anterior segment from a <i>Cenpj</i>^+/+ mouse. The angle was displaced anteriorly in eyes from <i>Cenpj^tm/tm</i> mice and ciliary process morphology was abnormal. (a, angle; i, iris; cb, ciliary body; l, lens). Bottom panel shows normal retina from a <i>Cenpj</i>^+/+ mouse eye. The retina photoreceptor cells of <i>Cenpj^tm/tm</i> mice were reduced in number and showed columnar disorganized (arrow). (ONL, outer nuclear layer). D. Immunohistochemical staining for Cenpj in <i>Cenpj</i>^+/+ embryo eye (E14.5; RNL retinal neuroblast layer). E. Intra-peritoneal glucose tolerance test to show that female <i>Cenpj^tm/tm</i> mice have a 15 minute delay in response to glucose challenge (n = 4 <i>Cenpj^tm/tm</i> vs. n = 32 <i>Cenpj</i>^+/+, *<i>P</i> = 2×10⁻⁵, t-test). Graph also shows n = 9 <i>Cenpj^tm/+</i> and n = 670 baseline wildtype controls. F. Plasma albumin levels were decreased in <i>Cenpj^tm/tm</i> males (n = 8 <i>Cenpj^tm/tm</i> vs. n = 35 <i>Cenpj</i>^+/+, *<i>P</i> = 4.9×10⁻⁵, t-test). Graph also shows n = 7 <i>Cenpj^tm/+</i> and n = 768 baseline wildtype controls. G. Flow cytometric analysis of peripheral blood leukocytes in <i>Cenpj^tm/tm</i> mice revealed an increase in the number of CD8+CD3+ and H. total CD3+ cells. Data shows total counts per 30 000 propidium-iodide (PI) negative, CD45-positive cells from male mice. For n = 9 <i>Cenpj^tm/tm</i> vs. n = 30 <i>Cenpj</i>^+/+: CD3+CD8+ *<i>P</i> = 0.0002 and CD3 *<i>P</i> = 2.9×10⁻⁵, Mann-Whitney-Wilcoxon test. Graphs also show n = 7 <i>Cenpj</i>^+/tm and n = 356 baseline wildtype controls. For all ‘Box and Whisker’ plots, the lower whisker extends to the lowest datum still within 1.5 Inter-quartile range (IQR) of the lower quartile. The upper whisker extends to the highest datum still within 1.5 IQR of the upper quartile.</p

Genomic instability is associated with abnormal ploidy of <i>Cenpj^tm/tm</i> cells rather than an impaired DNA damage response.

<p>A. Cell cycle analysis of <i>Cenpj^tm/tm</i> mouse embryonic fibroblasts (MEFs) by flow cytometry showed an increase in the percentage of cells in G2 (4C) and cells containing >4C DNA content when compared to <i>Cenpj</i>^+/+ cells. Percentages represent means of n = 3 independent MEF lines per genotype (each pair of +/+ and <i>tm/tm</i> cells were passage-matched (passage<5) and derived from littermates), *P<0.05, t-test. PI, propidium iodide. B. Example multiplex fluorescent <i>in situ</i> hybridization (M-FISH; top) and DAPI banded (bottom) karyotype of a <i>Cenpj^tm/tm</i> MEF metaphase (passage 4). The karyotype is near tetraploid, with centric fusions (white arrows) and chromosomes that have apparently lost their centromeres (black arrows). C. Example M-FISH of a <i>Cenpj^tm/tm</i> MEF metaphase (passage 4) showing near tetraploid karyotype with a translocation (t(2;7)). D. Adult <i>Cenpj^tm/tm</i> (n = 4) mice showed increased genomic instability when compared to <i>Cenpj</i>^+/+ mice (n = 6) as determined by the increased prevalence of micronucleated normochromatic erythrocytes using a flow cytometric assay of micronucleus formation. *<i>P</i> = 0.000004, t-test. The lower whisker extends to the lowest datum still within 1.5 Inter-quartile range (IQR) of the lower quartile. The upper whisker extends to the highest datum still within 1.5 IQR of the upper quartile. E. Immunoblots show normal activation of DNA damage response markers in <i>Cenpj</i>-deficient MEFs (passage 2) before and after treatment with the DNA damaging agent camptothecin (1 µM for 1 h). KAP1 was used as a loading control.</p