Antibodies used in immunohistochemistry.

<p>Antibodies used in immunohistochemistry.</p

Neonatal lethality in <i>UBR2^−/−</i> newborn pups associated with defects in lung expansion and neural development.

<p>(<b>A</b>) The majority of <i>UBR2^−/−</i> mice in the C57 genetic background die neonatally. Shown is gross morphology of neonatal pups enriched in the C57 genetic background at P1. Surviving <i>UBR2^−/−</i> neonates weighed slightly less than their +/+ and <i>UBR2^+/−</i> littermates of the same gender. No gross morphological differences were observed between <i>UBR2^−/−</i> and control mice during embryogenesis and after birth. Arrowheads mark stomachs with or without milk, which indicate the feeding from mother. (<b>B</b>) The lungs of <i>UBR2^−/−</i> newborn pups are not properly expanded. Shown are H&E-stained cross sections from +/+ and <i>UBR2^−/−</i> lungs at P0. Arrowheads indicate alveoli. (<b>C</b>) <i>In situ</i> hybridization of <i>UBR2</i> mRNA on cross sections of +/+ and <i>UBR2^−/−</i> brains at P1. (<b>D</b>) Dilated ventricles in <i>UBR2^−/−</i> brain (arrowhead). Shown are Nissl-stained cross sections of +/+ and <i>UBR2^−/−</i> brains at P1. (<b>E</b>) Defective gliogenesis and neuronal differentiation in hippocampus of <i>UBR2^−/−</i> brains at P1. Cross sections of a mildly affected <i>UBR2^−/−</i> brain, together with its littermate control, were subjected to Nissl or DAPI staining, or immunofluorescent staining of GFAP or NeuN.</p

Total ubiquitylation activities are significantly reduced in <i>UBR2^−/−</i> spermatocytes at pachytene.

<p>Surface-spread meiotic chromosomes were coimmunostained with FK2 antibody (red) which recognizes both monoubiquitin and polyubiquitin conjugates and an antibody to SCP3 (green), a component of the synaptonemal complex. (<b>A</b>, <b>B</b>) Leptotene. (<b>C</b>, <b>D</b>) Zygotene. (<b>E</b>, <b>F</b>) Early pachytene. (<b>G</b>, <b>H</b>) Mid-pachytene. (<b>I</b>, <b>J</b>) Late pachytene. Compared with controls, the FK2 staining in <i>UBR2^−/−</i> chromosomes is relatively weak in the XY body at mid-pachytene and throughout the entire chromosomal regions at mid-late pachytene. Scale bar: 10 µm.</p

UBR2 is associated with chromatin during cell cycle of somatic cells.

<p>(<b>A</b>) UBR2 is enriched in the nucleus of MEFs. MEFs were stained for UBR2 without (top) and with a peptide that has been used to raise the antibody. (<b>B</b>) UBR2 is associated with chromatin in MEFs. Control and <i>UBR2^−/−</i> cells were separated into cytosolic, nuclear soluble, and chromatin-bound fractions in the presence or absence of iodoacetamide, followed by immunoblotting for proteins indicated. (<b>C</b>) Chromatin association of UBR2 is cell cycle-dependent. HeLa cells were synchronized at the G1-S border using the double thymidine block, released from G1-S arrest, and subjected to time-course fractionation and immunoblotting. Cell cycle stages were verified using flow cytometry and based on behaviors of cell cycle regulators, including down-regulation of chromatin-associated cyclin A and CDC6.</p

Pachytene arrest of <i>UBR2^−/−</i> spermatocytes at stage IV.

<p>(<b>A</b>–<b>D</b>) Testis sections from 8-week (A–C) and 3-week (D) old <i>UBR2^−/−</i> tubules. (<b>A</b>) Tubules that did not yet reach epithelial stage IV. A large number of spermatocytes (thick arrow) are present, indicating that the spermatogonial compartment keeps forming spermatocytes. Arrowhead, diplotene spermatocyte; thin arrow, round spermatid. The predecessors of these cells survived the stage IV arrest. (<b>B</b>) Tubules in epithelial stage IV as evidenced by the presence of large, G2 phase intermediate (In) spermatogonia (blue arrow) about to or dividing into B spermatogonia (yellow arrowhead). There is massive apoptosis of spermatocytes (asterisks). (<b>C</b>) Tubules after stage IV. A variable number of spermatocytes survive the passage through stage IV. The left tubule shows only one spermatocyte (arrowhead) and a few round spermatids (thin arrow) that stem from spermatocytes that survived stage IV one epithelial cycle earlier. The tubule on the right shows more spermatocytes (arrowhead) and round spermatids (black arrow) and even a few elongated spermatids (yellow arrow). (<b>D</b>) Stage IV arrest at the age of 3 weeks. The lower tubule shows massive apoptosis (asterisk). The upper tubule is after stage IV and shows two surviving spermatocytes (arrowhead), indicating that the arrest was already present before three weeks. (<b>E</b>) Surface-spread chromosomes of 781 control and 691 <i>UBR2^−/−</i> spermatocytes isolated from mice at P17 were stained with SCP3 and staged based on the morphology of SCP3-positive chromosomes. (<b>F</b>) Surface-spread chromosomes of 344 +/+ and 161 <i>UBR2^−/−</i> pachytene spermatocytes were substaged.</p

UBR2 mediates monoubiquitylation and polyubiquitylation of H2A and H2B but not H3 and H4.

<p>(<b>A</b>) <i>In vitro</i> ubiquitylation assay (20 µL) with 1 µg of histone H2A, H2B, H1, or H3. The reaction contains 100 ng E3-F (or E3-V) prepared from rat testes, 30 ng UbcH2, and Ub activating reagents, including 1 µg flag-Ub and 100 ng E1. E3-F and E3-V represent protein mixtures that have been captured by Phe-peptide and Val-peptide, respectively. (<b>B</b>) <i>In vitro</i> binding assay in which UBR2 (as a mixture with UBR1) immobilized on Phe-peptide-beads was mixed with histone H2A, H2B, or H3 in the presence of HR6B, E1, and Ub activating reagents, followed by immunoblotting of histones retained by X-peptide (X = Phe or Val). (<b>C</b>) The screening of E2s which can support E3-F-mediated ubiquitylation of H2A. <i>In vitro</i> ubiquitylation assays were performed as (A) with different E2s indicated above. In this screening, UbcH2 and UbcH5b showed reproducibly the E2 activity in H2A ubiquitylation. (<b>D</b>–<b>F</b>) Allosteric modulation, an additional E2, and synthetic ligands for UBR2. (<b>D</b>) The interaction between UBR2 and HR6B is cooperatively accelerated by H2A and Arg-Ala. UBR2 (0.2 µg) from 10 mg rat testes extracts were immobilized with Phe-peptide conjugated with beads. Precipitated E3-peptide beads were mixed with 60 ng HR6B, 1 µg H2A, and/or 2 mM Arg-Ala, followed by immunoblotting analysis. (<b>E</b>) The HR6B-H2A interaction is cooperatively facilitated by UBR2 and Arg-Ala. GST-pulldown assays were done with 200 ng GST-HR6B, 200 ng UBR2, 1 µg H2A, and/or 2 mM Arg-Ala. (<b>F</b>) UBR2-dependent H2A ubiquitylation is synergistically activated by type-1 and type-2 N-end rule ligands.</p

Chromosome instability and hypersensitivity to DNA damage of UBR2-deficient somatic cells.

<p>(<b>A</b>) UBR2-knockdown induces hyperproliferation in HeLa cells. (<b>B</b>) Metaphase chromosomes of <i>UBR2^−/−</i> MEFs show increased chromosomal aberrations, including breaks and fragmentations, compared with control cells. Arrowhead, break; Arrow, fragmentation (<b>C</b>) Quantitation of chromosomal abnormalities (breaks and fragments) observed in metaphase chromosomes from +/+ and <i>UBR2^−/−</i> MEFs. (<b>D</b>) <i>UBR2^−/−</i> MEFs are hypersensitive to hydroxyurea, or methyl methanesulfonate (Sigma).</p

Genome-wide polyubiquitylation activities on meiotic chromosomes are reduced in <i>UBR2^−/−</i> spermatocytes.

<p>Surface-spread meiotic chromosomes were coimmunostained with FK1 antibody (red) which specifically recognizes polyubiquitin conjugates and an antibody to SCP3 (green), a component of the synaptonemal complex. (<b>A</b>) Zygotene. (<b>B</b>) Early pachytene. (<b>C</b>) Mid-pachytene. Arrowhead indicates the sex chromosome. Note that polyubiquitin signals are virtually nondetectible in <i>UBR2^−/−</i> chromosomes. Scale bar: 10 µm.</p

The localization of polyubiquitin conjugates on meiotic chromosomes in comparison with UBR2.

<p>Surface-spread meiotic chromosomes were coimmunostained with FK1 antibody (red) which specifically recognizes polyubiquitin conjugates (poly-Ub) and an antibody to UBR2 (green). (<b>A</b>) Zygotene. (<b>B</b>) Early pachytene. (<b>C</b>) Mid-pachytene. (<b>D</b>) Mid-late pachytene. Different from the FK2 staining, polyubiquitin conjugates are enriched in sex chromosomes until mid-pachytene. At mid-pachytene, both polyubiquitin and UBR2 signals are drastically induced in the majority of chromosomal regions except that the chromatin domain of sex chromosomes is relatively devoid of the UBR2 staining. Scale bar: 10 µm.</p