Roles of the Rlim-Rex1 axis during X chromosome inactivation in mice.

In female mice, the gene dosage from X chromosomes is adjusted by a process called X chromosome inactivation (XCI) that occurs in two steps. An imprinted form of XCI (iXCI) that silences the paternally inherited X chromosome (Xp) is initiated at the 2- to 4-cell stages. As extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI require the lncRNA Xist, which is expressed from the X to be inactivated. The X-linked E3 ubiquitin ligase Rlim (Rnf12) in conjunction with its target protein Rex1 (Zfp42), a critical repressor of Xist, have emerged as major regulators of iXCI. However, their roles in rXCI remain controversial. Investigating early mouse development, we show that the Rlim-Rex1 axis is active in pre-implantation embryos. Upon implantation Rex1 levels are downregulated independently of Rlim specifically in epiblast cells. These results provide a conceptual framework of how the functional dynamics between Rlim and Rex1 ensures regulation of iXCI but not rXCI in female mice.

How does the Xist activator Rlim/Rnf12 regulate Xist expression?

The long non-coding RNA (lncRNA) Xist is crucially involved in a process called X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female mammals to achieve X dosage compensation between the sexes. Because Xist RNA silences the X chromosome from which it is transcribed, the activation of Xist transcription marks the initiation of the XCI process and thus, mechanisms and players that activate this gene are of central importance to the XCI process. During female mouse embryogenesis, XCI occurs in two steps. At the 2-4 cell stages imprinted XCI (iXCI) silences exclusively the paternally inherited X chromosome (Xp). While extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo random XCI (rXCI) around implantation. Both iXCI and rXCI are dependent on Xist. Rlim, also known as Rnf12, is an X-linked E3 ubiquitin ligase that is involved in the transcriptional activation of Xist. However, while data on the crucial involvement of Rlim during iXCI appear clear, its role in rXCI has been controversial. This review discusses data leading to this disagreement and recent evidence for a regulatory switch of Xist transcription in epiblasts of implanting embryos, partially reconciling the roles of Rlim during Xist activation.

RLIM Is a Candidate Dosage-Sensitive Gene for Individuals with Varying Duplications of Xq13, Intellectual Disability, and Distinct Facial Features.

Interpretation of the significance of maternally inherited X chromosome variants in males with neurocognitive phenotypes continues to present a challenge to clinical geneticists and diagnostic laboratories. Here we report 14 males from 9 families with duplications at the Xq13.2-q13.3 locus with a common facial phenotype, intellectual disability (ID), distinctive behavioral features, and a seizure disorder in two cases. All tested carrier mothers had normal intelligence. The duplication arose de novo in three mothers where grandparental testing was possible. In one family the duplication segregated with ID across three generations. RLIM is the only gene common to our duplications. However, flanking genes duplicated in some but not all the affected individuals included the brain-expressed genes NEXMIF, SLC16A2, and the long non-coding RNA gene FTX. The contribution of the RLIM-flanking genes to the phenotypes of individuals with different size duplications has not been fully resolved. Missense variants in RLIM have recently been identified to cause X-linked ID in males, with heterozygous females typically having normal intelligence and highly skewed X chromosome inactivation. We detected consistent and significant increase of RLIM mRNA and protein levels in cells derived from seven affected males from five families with the duplication. Subsequent analysis of MDM2, one of the targets of the RLIM E3 ligase activity, showed consistent downregulation in cells from the affected males. All the carrier mothers displayed normal RLIM mRNA levels and had highly skewed X chromosome inactivation. We propose that duplications at Xq13.2-13.3 including RLIM cause a recognizable but mild neurocognitive phenotype in hemizygous males.

Kaposi's Sarcoma-Associated Herpesvirus LANA Modulates the Stability of the E3 Ubiquitin Ligase RLIM.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. In a previous study, we have identified LANA-interacting proteins using a protein array screen. Here, we explore the effect of LANA on the stability and activity of RLIM (RING finger LIM-domain-interacting protein, encoded by the RNF12 gene), a novel LANA-interacting protein identified in that protein screen. RLIM is an E3 ubiquitin ligase that leads to the ubiquitination and degradation of several transcription regulators, such as LMO2, LMO4, LHX2, LHX3, LDB1, and the telomeric protein TRF1. Expression of LANA leads to downregulation of RLIM protein levels. This LANA-mediated RLIM degradation is blocked in the presence of the proteasome inhibitor, MG132. Therefore, the interaction between LANA and RLIM could be detected in coimmunoprecipitation assay only in the presence of MG132 to prevent RLIM degradation. A RING finger mutant RLIM is resistant to LANA-mediated degradation, suggesting that LANA promotes RLIM autoubiquitination. Interestingly, we found that LANA enhanced the degradation of some RLIM substrates, such as LDB1 and LMO2, and prevented RLIM-mediated degradation of others, such as LHX3 and TRF1. We also show that transcription regulation by RLIM substrates is modulated by LANA. RLIM substrates are assembled into multiprotein transcription regulator complexes that regulate the expression of many cellular genes. Therefore, our study identified another way KSHV can modulate cellular gene expression.IMPORTANCE E3 ubiquitin ligases mark their substrates for degradation and therefore control the cellular abundance of their substrates. RLIM is an E3 ubiquitin ligase that leads to the ubiquitination and degradation of several transcription regulators, such as LMO2, LMO4, LHX2, LHX3, LDB1, and the telomeric protein TRF1. Here, we show that the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA protein enhances the ubiquitin ligase activity of RLIM, leading to enhanced RLIM autoubiquitination and degradation. Interestingly, LANA enhanced the degradation of some RLIM substrates, such as LDB1 and LMO2, and prevented RLIM-mediated degradation of others, such as LHX3 and TRF1. In agreement with protein stability of RLIM substrates, we found that LANA modulates transcription by LHX3-LDB1 complex and suggest additional ways LANA can modulate cellular gene expression. Our study adds another way a viral protein can regulate cellular protein stability, by enhancing the autoubiquitination and degradation of an E3 ubiquitin ligase.

Knockdown of RLIM inhibits XIST expression and improves developmental competence of cloned male pig embryos.

Ectopic expression of Xist on the putative active X chromosome is a primary cause of the low developmental efficiency of cloned mouse and pig embryos. Suppression of abnormal Xist expression via gene knockout or RNA interference (RNAi) can significantly enhance the developmental competence of cloned mouse and pig embryos. RLIM is a Xist expression activator, whereas REX1 is an Xist transcription inhibitor, as RLIM triggers Xist expression by mediating the proteasomal degradation of REX1 to induce imprinted and random X chromosome inactivation in mice. This study aimed to test whether the knockdown of RLIM and overexpression of REX1 can repress aberrant Xist expression and improve the developmental ability of cloned male pig embryos. Results showed that injection of anti-RLIM small interfering RNA significantly decreased Xist messenger RNA abundance, increased REX1 protein level, and enhanced the preimplantation development of cloned male porcine embryos. These positive effects were not observed in cloned male pig embryos injected with REX1 expression plasmid, which might be due to the low expression efficiency of injected REX1 plasmid and/or the short half-life of expressed REX1 protein. The findings from this study indicated that RLIM participated in the ectopic activation of Xist expression in cloned pig embryos by targeting REX1 degradation. Furthermore, this study provided a new method to improve cloned pig embryo development by the inhibition of Xist expression via RNAi of RLIM.

Activators and repressors: A balancing act for X-inactivation.

In early female embryos X-chromosome inactivation occurs concomitant with up regulation of the non-coding RNA, Xist, on the future inactive X-chromosome. Up regulation of Xist and coating of the future inactive X is sufficient to induce silencing. Therefore unlocking the mechanisms of X-chromosome inactivation requires thorough understanding of the transcriptional regulators, both activators and repressors, which control Xist. Mouse pluripotent embryonic stem cells, which have two active X chromosomes, provide a tractable ex vivo model system for studying X-chromosome inactivation, since this process is triggered by differentiation signals in these cultured cells. Yet there are significant discrepancies found between ex vivo analyses in mouse embryonic stem cells and in vivo studies of early embryos. In this review we elaborate on potential models of how Xist is up regulated on a single X chromosome in female cells and how ex vivo and in vivo analyses enlighten our understanding of the activators and repressors that control this non-coding RNA gene.

Histone deacetylase 2 controls p53 and is a critical factor in tumorigenesis.

Histone deacetylase 2 (HDAC2) regulates biological processes by deacetylation of histones and non-histone proteins. HDAC2 is overexpressed in numerous cancer types, suggesting general cancer-relevant functions of HDAC2. In human tumors the TP53 gene encoding p53 is frequently mutated and wild-type p53 is often disarmed. Molecular pathways inactivating wild-type p53 often remain to be defined and understood. Remarkably, current data link HDAC2 to the regulation of the tumor suppressor p53 by deacetylation and to the maintenance of genomic stability. Here, we summarize recent findings on HDAC2 overexpression in solid and hematopoietic cancers with a focus on mechanisms connecting HDAC2 and p53 in vitro and in vivo. In addition, we present an evidence-based model that integrates molecular pathways and feedback loops by which p53 and further transcription factors govern the expression and the ubiquitin-dependent proteasomal degradation of HDAC2 and of p53 itself. Understanding the interactions between p53 and HDAC2 might aid in the development of new therapeutic approaches against cancer.

Knockout of Rlim Results in a Sex Ratio Shift toward Males but Superovulation Cannot Compensate for the Reduced Litter Size.

Technologies that can preselect offspring gender hold great promise for improving farm animal productivity and preventing human sex-related hereditary diseases. The maternal Rlim allele is required for imprinted X-chromosome inactivation, which is essential for the normal development of female mouse embryos. In this study, we inactivated the maternal Rlim allele in embryos by crossing a male transgenic mouse line carrying an X-linked CMV-Cre transgene with a female line carrying a loxP-flanked Rlim gene. Knockout of the maternal Rlim gene in embryos resulted in a male-biased sex ratio skew in the offspring. However, it also reduced litter size, and this effect was not compensated for by superovulation in the mother mice. In addition, we showed that siRNA-mediated knockdown of Rlim in mouse embryos leads to the birth of male-only progenies. This study provides a new promising method for male-biased sex selection, which may help to improve the productivity in livestock and prevent sex-associated hereditary diseases in humans.

Sequential stabilization of RNF220 by RLIM and ZC4H2 during cerebellum development and Shh-group medulloblastoma progression.

Sonic hedgehog (Shh) signaling is essential for the proliferation of cerebellar granule neuron progenitors (CGNPs), and its misregulation is linked to various disorders, including cerebellar cancer medulloblastoma (MB). During vertebrate neural development, RNF220, a ubiquitin E3 ligase, is involved in spinal cord patterning by modulating the subcellular location of glioma-associated oncogene homologs (Glis) through ubiquitination. RNF220 is also required for full activation of Shh signaling during cerebellum development in an epigenetic manner through targeting embryonic ectoderm development. ZC4H2 was reported to be involved in spinal cord patterning by acting as an RNF220 stabilizer. Here, we provided evidence to show that ZC4H2 is also required for full activation of Shh signaling in CGNP and MB progression by stabilizing RNF220. In addition, we found that the ubiquitin E3 ligase RING finger LIM domain-binding protein (RLIM) is responsible for ZC4H2 stabilization via direct ubiquitination, through which RNF220 is also thus stabilized. RLIM is a direct target of Shh signaling and is also required for full activation of Shh signaling in CGNP and MB cell proliferation. We further provided clinical evidence to show that the RLIM‒ZC4H2‒RNF220 cascade is involved in Shh-group MB progression. Disease-causative human RLIM and ZC4H2 mutations affect their interaction and regulation. Therefore, our study sheds light on the regulation of Shh signaling during cerebellar development and MB progression and provides insights into neural disorders caused by RLIM or ZC4H2 mutations.

Deficient spermiogenesis in mice lacking Rlim.

The X-linked gene Rlim plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. However, while females carrying a systemic Rlim knockout (KO) die around implantation, male Rlim KO mice appear healthy and are fertile. Here, we report an important role for Rlim in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the Rlim gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional Rlim cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of Rlim in male reproduction specifically in round spermatids during spermiogenesis.

Rlim/Rnf12, Rex1, and X Chromosome Inactivation.

RLIM/Rnf12 is an E3 ubiquitin ligase that has originally been identified as a transcriptional cofactor associated with LIM domain transcription factors. Indeed, this protein modulates transcriptional activities and multiprotein complexes recruited by several classes of transcription factors thereby enhancing or repressing transcription. Around 10 years ago, RLIM/Rnf12 has been identified as a major regulator for the process of X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female mice and ESCs. However, the precise roles of RLIM during XCI have been controversial. Here, we discuss the cellular and developmental functions of RLIM as an E3 ubiquitin ligase and its roles during XCI in conjunction with its target protein Rex1.

RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation.

X-linked intellectual disability (XLID) is a heterogeneous syndrome affecting mainly males. Human genetics has identified >100 XLID genes, although the molecular and developmental mechanisms underpinning this disorder remain unclear. Here, we employ an embryonic stem cell model to explore developmental functions of a recently identified XLID gene, the RNF12/RLIM E3 ubiquitin ligase. We show that RNF12 catalytic activity is required for proper stem cell maintenance and neural differentiation, and this is disrupted by patient-associated XLID mutation. We further demonstrate that RNF12 XLID mutations specifically impair ubiquitylation of developmentally relevant substrates. XLID mutants disrupt distinct RNF12 functional modules by either inactivating the catalytic RING domain or interfering with a distal regulatory region required for efficient ubiquitin transfer. Our data thereby uncover a key function for RNF12 E3 ubiquitin ligase activity in stem cell and neural development and identify mechanisms by which this is disrupted in intellectual disability.

RLIM suppresses hepatocellular carcinogenesis by up-regulating p15 and p21.

Hepatocellular carcinogenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation and apoptosis. p15 and p21 are cyclin-dependent kinase inhibitors, which arrest cell proliferation and serve as critical tumor suppressors. Here we report that the E3 ubiquitin ligase RLIM expression is downregulated in hepatocellular carcinoma patients, and correlated with p15 and p21 expression in clinical progression. In addition, we showed that RLIM overexpression suppresses the cell growth and arrests cell cycle progression of hepatocellular carcinoma. Mechanistically, we found that RLIM directly binds to MIZ1, disrupting the interaction between c-MYC and MIZ1, and enhancing p15 and p21 transcription. Our results demonstrate that RLIM is an important suppressor in hepatocellular carcinogenesis.

Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs.

During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

Regulation of X-linked gene expression during early mouse development by Rlim.

Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos.

Rlim, an E3 ubiquitin ligase, influences the stability of Stathmin protein in human osteosarcoma cells.

Stathmin is an oncoprotein and is expressed at high levels in a wide variety of human malignancies, which plays important roles in maintenance of malignant phenotypes. The regulation of Stathmin gene overexpression has been wildly explored, but the exact mechanism still needs to be elucidated. It is believed that regulation of an oncogene protein abundance through post-translational modifications is essential for maintenance of malignant phenotypes. Here we identified the Rlim, a Ring H2 zinc finger protein with intrinsic ubiquitin ligase activity, as a Stathmin-interacting protein that could increase Stathmin turnover through binding with this targeted protein and then induce its degradation by proteasome in a ubiquitin-dependent manner. Inhibition of endogenous Rlim expression by siRNA could increase the level of Stathmin protein, which further led to cell proliferation and cell cycle changes in human osteosarcoma cell lines. On the other hand, forced overexpression of Rlim could decrease the level of Stathmin protein. These results demonstrate that Rlim is involved in the negative regulation of Stathmin protein level through physical interaction and ubiquitin-mediated proteolysis. Hence, Rlim is a novel regulator of Stathmin protein in a ubiquitin-dependent manner, and represents a new pathway for malignant phenotype turnover by modulating the level of Stathmin protein in human osteosarcomas.