In somatic cells of female placental mammals, one X chromosome is inactivated to minimize sex-related dosage differences of X-encoded genes. Random X chromosome inactivation (XCI) in the embryo is a stochastic process, in which each X has an independent probability to initiate XCI, triggered by the nuclear concentration of one or more X-encoded XCI-activators. Here, we identify the E3 ubiquitin ligase RNF12 as an important XCI-activator. Additional copies of mouse Rnf12 or human RNF12 result in initiation of XCI in male mouse ES cells and on both X chromosomes in a substantial percentage of female mouse ES cells. This activity is dependent on an intact open reading frame of Rnf12 and correlates with the transgenic expression level of RNF12. Initiation of XCI is markedly reduced in differentiating female heterozygous Rnf12(+/-) ES cells. These findings provide evidence for a dose-dependent role of RNF12 in the XCI counting and initiation process.

During meiotic prophase in male mammals, the X and Y chromosomes are incorporated in the XY body. This heterochromatic body is transcriptionally silenced and marked by increased ubiquitination of histone H2A. This led us to investigate the relationship between histone H2A ubiquitination and chromatin silencing in more detail. First, we found that ubiquitinated H2A also marks the silenced X chromosome of the Barr body in female somatic cells. Next, we studied a possible relationship between H2A ubiquitination, chromatin silencing, and unpaired chromatin in meiotic prophase. The mouse models used carry an unpaired autosomal region in male meiosis or unpaired X and Y chromosomes in female meiosis. We show that ubiquitinated histone H2A is associated with transcriptional silencing of large chromatin regions. This silencing in mammalian meiotic prophase cells concerns unpaired chromatin regions and resembles a phenomenon described for the fungus Neurospora crassa and named meiotic silencing by unpaired DNA.Chromatin remodeling is at the basis of control of cellspecific gene expression, cell determination, and differentiation. The nucleosome units of chromatin consist of two each of the histones H2A, H2B, H3, and H4. The N-terminal ends of these core histones extend from the nucleosome and can undergo posttranslational covalent modifications, such as methylation, acetylation, phosphorylation, and ADP-ribosylation of specific amino acid residues. Together, these modifications constitute the so-called histone code (45). Interaction of other nuclear proteins with chromatin is dependent on the histone code at specific chromatin regions and determines local chromatin structure, which can be open or closed. A remarkable component of the histone code is ubiquitination of C-terminal lysine residues of histones H2A and H2B. Ubiquitin, a protein of 7 kDa, can be attached to lysine residues of a specific protein substrate through the action of a multienzyme complex containing ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. Polyubiquitination can target proteins for degradation by the proteasome (34, 35). Monoubiquitination of histones, however, is a stable modification that does not decrease the half-life of the target histone (56).In the yeast Saccharomyces cerevisiae, histone H2A ubiquitination is not required for cell growth or sporulation (47), but histone H2B ubiquitination is an essential mechanism involved in sporulation (37). Most importantly, it has been shown that ubiquitination of H2B by the ubiquitin-conjugating enzyme RAD6, interacting with the ubiquitin ligase BRE1, is a prerequisite for dimethylation of histone H3 at lysine residues 4 and 79 (5,12,37,46). This mechanism is thought to be associated with potentiation of gene activation. It is not known whether this "trans-histone" mechanism is conserved between yeast and mammals. RAD6 shows marked evolutionary conservation. The two mammalian homologs of yeast RAD6, Hr6a/Ube2a and Hr6b/Ube2b, both show approximately 70% amino...

Evolution of the mammalian sex chromosomes has resulted in a heterologous X and Y pair, where the Y chromosome has lost most of its genes. Hence, there is a need for X-linked gene dosage compensation between XY males and XX females. In placental mammals, this is achieved by random inactivation of one X chromosome in all female somatic cells. Upregulation of Xist transcription on the future inactive X chromosome acts against Tsix antisense transcription, and spreading of Xist RNA in cis triggers epigenetic changes leading to X-chromosome inactivation. Previously, we have shown that the X-encoded E3 ubiquitin ligase RNF12 is upregulated in differentiating mouse embryonic stem cells and activates Xist transcription and X-chromosome inactivation. Here we identify the pluripotency factor REX1 as a key target of RNF12 in the mechanism of X-chromosome inactivation. RNF12 causes ubiquitination and proteasomal degradation of REX1, and Rnf12 knockout embryonic stem cells show an increased level of REX1. Using chromatin immunoprecipitation sequencing, REX1 binding sites were detected in Xist and Tsix regulatory regions. Overexpression of REX1 in female embryonic stem cells was found to inhibit Xist transcription and X-chromosome inactivation, whereas male Rex1(+/-) embryonic stem cells showed ectopic X-chromosome inactivation. From this, we propose that RNF12 causes REX1 breakdown through dose-dependent catalysis, thereby representing an important pathway to initiate X-chromosome inactivation. Rex1 and Xist are present only in placental mammals, which points to co-evolution of these two genes and X-chromosome inactivation.

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.

Male infertility in HR6B knockout mice is associated with impairment of spermatogenesis. The HR6B gene is a mammalian, autosomal homolog of the Saccharomyces cerevisiae gene Rad6 encoding a ubiquitin-conjugating enzyme. In addition, X-chromosomal HR6A has been identified, in human and mouse. RAD6 in yeast is required for a variety of cellular functions, including sporulation, DNA repair, and mutagenesis. Since RAD6 and its mammalian homologs can ubiquitinate histones in vitro, we have investigated the pattern of histone ubiquitination in mouse testis. By immunoblot and immunohistochemical analysis of wild-type mouse testis, a high amount of ubiquitinated H2A (uH2A) was detected in pachytene spermatocytes. This signal became undetectable in round spermatids, but then increased again during a relatively short developmental period, in elongating spermatids. No other ubiquitinated histones were observed. In the HR6B knockout mice, we failed to detect an overt defect in the overall pattern of histone ubiquitination. For somatic cell types, it has been shown that histone ubiquitination is associated with destabilization of nucleosomes, in relation to active gene transcription. Unexpectedly, the most intense uH2A signal in pachytene spermatocytes was detected in the sex body, an inactive nuclear structure that contains the heterochromatic X and Y chromosomes. The postmeiotic uH2A immunoexpression in elongating spermatids indicates that nucleosome destabilization induced by histone ubiquitination may play a facilitating role during histone-to-protamine replacement.