Henk P. Roest

Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modification.

The ubiquitin-conjugating yeast enzyme RAD6 and its human homologs hHR6A and hHR6B are implicated in postreplication repair and damage-induced mutagenesis. The yeast protein is also required for sporulation and may modulate chromatin structure via histone ubiquitination. We report the phenotype of the first animal mutant in the ubiquitin pathway: inactivation of the hHR6B-homologous gene in mice causes male infertility. Derailment of spermatogenesis becomes overt during the postmeiotic condensation of chromatin in spermatids. These findings provide a parallel between yeast sporulation and mammalian spermatogenesis and strongly implicate hHR6-dependent ubiquitination in chromatin remodeling. Since heterozygous male mice and even knockout female mice are completely normal and fertile and thus able to transmit the defect, similar hHR6B mutations may cause male infertility in man.

Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability.

The Ubiquitin-Conjugating DNA Repair Enzyme HR6A Is a Maternal Factor Essential for Early Embryonic Development in Mice

ABSTRACT The Saccharomyces cerevisiae RAD6 protein is required for a surprising diversity of cellular processes, including sporulation and replicational damage bypass of DNA lesions. In mammals, two RAD6-related genes, HR6A and HR6B, encode highly homologous proteins. Here, we describe the phenotype of cells and mice deficient for the mHR6A gene. Just like mHR6B knockout mouse embryonic fibroblasts, mHR6A-deficient cells appear to have normal DNA damage resistance properties, but mHR6A knockout male and female mice display a small decrease in body weight. The necessity for at least one functional mHR6A (X-chromosomal) or mHR6B (autosomal) allele in all somatic cell types is supported by the fact that neither animals lacking both proteins nor females with only one intact mHR6A allele are viable. In striking contrast to mHR6B knockout males, which show a severe spermatogenic defect, mHR6A knockout males are normally fertile. However, mHR6A knockout females fail to produce offspring despite a normal ovarian histology and ovulation. The absence of mHR6A in oocytes prevents development beyond the embryonic two-cell stage but does not result in an aberrant methylation pattern of histone H3 at this early stage of mouse embryonic development. These observations support redundant but dose-dependent roles for HR6A and HR6B in somatic cell types and germ line cells in mammals.

Loss of HR6B Ubiquitin-Conjugating Activity Results in Damaged Synaptonemal Complex Structure and Increased Crossing-Over Frequency during the Male Meiotic Prophase

ABSTRACT The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.

The ubiquitin system in gametogenesis

Ubiquitin is a ubiquitous and highly conserved protein of 76 amino acid residues, that can be covalently attached to cellular acceptor proteins. The attachment of ubiquitin to target proteins is achieved through a multi-step enzymatic pathway, which involves activities of ubiquitin-activating E1 enzymes, ubiquitin-conjugating E2 enzymes, and ligating E3 enzymes. Mono- or poly-ubiquitination of proteins can lead to protein degradation or modification of protein activity. Many components of the complex ubiquitin system show remarkable evolutionary conservation, from yeast to mammalian species. The ubiquitin system is essential to all eukaryotic cells. Among others, several signal transduction cascades show involvement of the ubiquitin system, but there are currently little data supporting a specific role of the ubiquitin system in hormonal control of reproduction. Interestingly, during gametogenesis, many specialized and important aspects of the ubiquitin system become apparent. Components of the ubiquitin system appear to be involved in different steps and processes during gametogenesis, including control of meiosis, and reorganization of chromatin structure.

Ubiquitin ligase Rad18Sc localizes to the XY body and to other chromosomal regions that are unpaired and transcriptionally silenced during male meiotic prophase.

In replicative damage bypass (RDB) in yeast, the ubiquitinconjugating enzyme RAD6 interacts with the ubiquitin ligase RAD18. In the mouse, these enzymes are represented by two homologs of RAD6, HR6a and HR6b, and one homolog of RAD18, Rad18Sc. Expression of these genes and the encoded proteins is ubiquitous, but there is relatively high expression in the testis. We have studied the subcellular localization by immunostaining Rad18Sc and other RDB proteins in mouse primary spermatocytes passing through meiotic prophase in spermatogenesis. The highest Rad18Sc protein level is found at pachytene and diplotene, and the protein localizes mainly to the XY body, a subnuclear region that contains the transcriptionally inactivated X and Y chromosomes. In spermatocytes that carry translocations for chromosomes 1 and 13, Rad18Sc protein concentrates on translocation bivalents that are not fully synapsed. The partly synapsed bivalents are often localized in the vicinity of the XY body, and show a very low level of RNA polymerase II, indicating that the chromatin is in a silent configuration similar to transcriptional silencing of the XY body. Thus, Rad18Sc localizes to unsynapsed and silenced chromosome segments during the male meiotic prophase. All known functions of RAD18 in yeast are related to RDB. However, in contrast to Rad18Sc, expression of UBC13 and polη, known to be involved in subsequent steps of RDB, appears to be diminished in the XY body and regions containing the unpaired translocation bivalents. Taken together, these observations suggest that the observed subnuclear localization of Rad18Sc may involve a function outside the context of RDB. This function is probably related to a mechanism that signals the presence of unsynapsed chromosomal regions and subsequently leads to transcriptional silencing of these regions during male meiotic prophase.

Knockout mouse model and gametogenic failure

To evaluate the function of a defined gene in gametogenesis, exciting opportunities are offered by the introduction of techniques to generate knockout mice. In this short article, we briefly describe a few gene knockout mouse models, which show a phenotype that involves impairment of gametogenesis and/or fertility. The focus will be on the mHR6B gene knockout mouse, which shows male infertility. The mHR6B gene encodes an ubiquitin-conjugating enzyme, and the data point to an important role of the ubiquitin pathway in gametogenesis.

HLTF and SHPRH are not essential for PCNA polyubiquitination, survival and somatic hypermutation: Existence of an alternative E3 ligase

DNA damage tolerance is regulated at least in part at the level of proliferating cell nuclear antigen (PCNA) ubiquitination. Monoubiquitination (PCNA-Ub) at lysine residue 164 (K164) stimulates error-prone translesion synthesis (TLS), Rad5-dependent polyubiquitination (PCNA-Ub(n)) stimulates error-free template switching (TS). To generate high affinity antibodies by somatic hypermutation (SHM), B cells profit from error-prone TLS polymerases. Consistent with the role of PCNA-Ub in stimulating TLS, hypermutated B cells of PCNA(K164R) mutant mice display a defect in generating selective point mutations. Two Rad5 orthologs, HLTF and SHPRH have been identified as alternative E3 ligases generating PCNA-Ub(n) in mammals. As PCNA-Ub and PCNA-Ub(n) both make use of K164, error-free PCNA-Ub(n)-dependent TS may suppress error-prone PCNA-Ub-dependent TLS. To determine a regulatory role of Shprh and Hltf in SHM, we generated Shprh/Hltf double mutant mice. Interestingly, while the formation of PCNA-Ub and PCNA-Ub(n) is prohibited in PCNA(K164R) MEFs, the formation of PCNA-Ub(n) is not abolished in Shprh/Hltf mutant MEFs. In line with these observations Shprh/Hltf double mutant B cells were not hypersensitive to DNA damage. Furthermore, SHM was normal in Shprh/Hltf mutant B cells. These data suggest the existence of an alternative E3 ligase in the generation of PCNA-Ub(n).

MicroRNA profiles in graft preservation solution are predictive of ischemic-type biliary lesions after liver transplantation

BACKGROUND & AIMS Ischemic-type biliary lesions (ITBL) are the second most common cause of graft loss after liver transplantation. Though the exact pathophysiology of ITBL is unknown, bile duct injury during graft preservation is considered to be a major cause. Here we investigated whether the release of cholangiocyte-derived microRNAs (CDmiRs) during graft preservation is predictive of the development of ITBL after liver transplantation. METHODS Graft preservation solutions (perfusates) and paired liver biopsies collected at the end of cold ischemia were analysed by RT-qPCR for CDmiR-30e, CDmiR-222, and CDmiR-296 and hepatocyte-derived miRNAs (HDmiRs) HDmiR-122 and HDmiR-148a. MicroRNAs in perfusates were evaluated on their stability by incubation and fractionation experiments. MicroRNA profiles in perfusates from grafts that developed ITBL (n=20) and grafts without biliary strictures (n=37) were compared. RESULTS MicroRNAs in perfusates were proven to be stable and protected against degradation by interacting proteins. Ratios between HDmiRs/CDmiRs were significantly higher in perfusates obtained from grafts that developed ITBL (p<0.01) and were identified as an independent risk factor by multivariate analysis (p<0.01, HR: 6.89). The discriminative power of HDmiRs/CDmiRs in perfusates was validated by analysis of separate brain death- (DBD) and cardiac death donors (DBD; p ≤ 0.016) and was superior to expression in liver biopsies (C=0.77 in perfusates vs. C<0.50 in biopsies). CONCLUSIONS This study demonstrates that differential release of CDmiRs during graft preservation is predictive of the development of ITBL after liver transplantation. This provides new evidence for the link between graft-related bile duct injury and the risk for later development of ITBL.

Congenital DNA repair deficiency results in protection against renal ischemia reperfusion injury in mice

Cockayne syndrome and other segmental progerias with inborn defects in DNA repair mechanisms are thought to be due in part to hypersensitivity to endogenous oxidative DNA damage. The accelerated aging‐like symptoms of this disorder include dysmyelination within the central nervous system, progressive sensineuronal hearing loss and retinal degeneration. We tested the effects of congenital nucleotide excision DNA repair deficiency on acute oxidative stress sensitivity in vivo. Surprisingly, we found mouse models of Cockayne syndrome less susceptible than wild type animals to surgically induced renal ischemia reperfusion injury, a multifactorial injury mediated in part by oxidative damage. Renal failure‐related mortality was significantly reduced in Csb−/– mice, kidney function was improved and proliferation was significantly higher in the regenerative phase following ischemic injury. Protection from ischemic damage correlated with improved baseline glucose tolerance and insulin sensitivity and a reduced inflammatory response following injury. Protection was further associated with genetic ablation of a different Cockayne syndrome‐associated gene, Csa. Our data provide the first functional in vivo evidence that congenital DNA repair deficiency can induce protection from acute stress in at least one organ. This suggests that while specific types of unrepaired endogenous DNA damage may lead to detrimental effects in certain tissues, they may at the same time elicit beneficial adaptive changes in others and thus contribute to the tissue specificity of disease symptoms.