Living with two X chromosomes: of mice and women : studies on the initiation mechanisms of X chromosome inactivation in stem cells and mouse models, and the role of RNF12 herein

__Abstract__ In this thesis work, we have investigated mechanisms involved in regulation of the initiation of X chromosome inactivation (XCI). Starting point was our earlier hypothesis that the initiation of XCI is a stochastic process, controlled in trans by autosomally-encoded XCI-inhibitors and X-encoded XCI-activators. The XCI-inhibitors prevent initiation of XCI in undifferentiated cells or early embryos. Upon differentiation of these cells during development, XCI is initiated in females only, by the activity of X-linked XCI-activators, which reach a higher concentration in female cells compared to male cells, due to the location of the encoding genes on the X chromosome. As described in Chapter 2, we found that the E3 ubiquitin ligase RNF12 acts as an important X-encoded activator of X chromosome inactivation. When extra copies of Rnf12 are transgenically expressed in male ES cells, the encoded higher level of RNF12 is able to induce XCI on the single X chromosome, whereas such over-expression in female ES cells leads to ectopic XCI on both X chromosomes in a significant portion of the cells. Rnf12 becomes up-regulated in female ES cells during the developmental time window when XCI is normally occurring, and genetic ablation of one copy of Rnf12 in female ES cells results in a significant delay in the XCI process. Chapter 4 discusses this discovery in the context of the stochastic model for XCI initiation, controlled by XCI-activators and XCI-inhibitors, and further provides evidence that all features of XCI initiation can indeed be explained by this model. A novel BAC targeting strategy described in Chapter 3 enabled efficient, fast and reliable genetic modifications of mouse ES cells, and this strategy was used throughout this thesis work. Among others, it allowed us to generate the Rnf12 homozygous knockout ES cells, described in Chapter 5. Using these cells, we provide evidence that RNF12 is essential for XCI to occur, and mediates its effect mainly through activation of the Xist gene. In Chapter 6, we show that this is an indirect mechanism, in which RNF12 targets the XCI-inhibitor REX1 for proteasomal degradation. The Rnf12 gene is located in the vicinity of Xist, which ensures rapid silencing of Rnf12 transcription in cis shortly after initiation of XCI. Chapter 7 addresses the question whether other genes located near Xist and Rnf12 are also functional in the regulation of the XCI process. We found that Jpx, Ftx and the Xpr region have a cis regulatory role, in which these regions likely create an open chromatin domain, which is needed to enable activation of Xist by the trans action of RNF12. We also provide data which suggest that direct interaction between the two X chromosomes in a female nucleus, X-pairing, is not functionally required for XCI to occur. Rather, more evidence was obtained supporting an indispensible role for the trans action of RNF12, during all stages of XCI initiation. In Chapter 8, we describe an Rnf12 knockout mouse, which we have generated from the Rnf12 mutant ES cells studied in this thesis work. As expected from the observations showing loss of XCI in Rnf12 homozygous knockout ES cells, homozygous Rnf12 knockout females are not born, suggesting that RNF12 has an important role in XCI initiation also during in vivo develop

The evolution of Great Apes has shaped the functional enhancers' landscape in human embryonic stem cells

High-throughput functional assays of enhancer activity have recently enabled the genome-scale definition of molecular, structural, and biochemical features of these genomic regulatory regions. To infer the evolutionary origin of DNA sequences operating as functional enhancers in human embryonic stem cells (hESC), we examined the patterns of evolutionary conservation and divergence in the genome-wide functional enhancers' landscape of hESC. We show that a prominent majority (up to 94%) of DNA sequences identified in hESC as functional enhancers are conserved in humans and our closest evolutionary relatives, Chimpanzee and Bonobo. More than 91% of functional enhancers that are highly conserved in both Chimpanzee and Bonobo, are conserved among other Great Apes and >75% are conserved in the Rhesus genome. In striking contrast, <5% of DNA sequences operating in hESC as functional enhancers are conserved in rodents. Conserved in primates enhancers' sequences are complemented by 1619 sequences of enhancers that are specific to humans. Enhancers that harbor human-specific sequences appear enriched among the invariant enhancer module maintaining activity in different pluripotent states and these regions are associated with pluripotency- and embryonic-lineage-related genes. However, functional enhancers make up only a minority of all conserved in primates or human-specific transcription factor binding sites. Our analyses revealed that sequences that are conserved during ~8 million years of primate evolution dominate the genomic landscape of functional enhancers in both primed and naïve hESC. Collectively, these observations revealed thousands of evolutionarily conserved sequences that function as a core regulatory network in human embryonic stem cells which has recently undergone further extension after divergence of modern humans from our closest relatives, Chimpanzee and Bonobo

Beyond the Exome: The Non-coding Genome and Enhancers in Neurodevelopmental Disorders and Malformations of Cortical Development

The development of the human cerebral cortex is a complex and dynamic process, in which neural stem cell proliferation, neuronal migration, and post-migratory neuronal organization need to occur in a well-organized fashion. Alterations at any of these crucial stages can result in malformations of cortical development (MCDs), a group of genetically heterogeneous neurodevelopmental disorders that present with developmental delay, intellectual disability and epilepsy. Recent progress in genetic technologies, such as next generation sequencing, most often focusing on all proteincoding exons (e.g., whole exome sequencing), allowed the discovery of more than a 100 genes associated with various types of MCDs. Although this has considerably increased the diagnostic yield, most MCD cases remain unexplained. As Whole Exome Sequencing investigates only a minor part of the human genome (1–2%), it is likely that patients, in which no disease-causing mutation has been identified, could harbor mutations in genomic regions beyond the exome. Even though functional annotation of non-coding regions is still lagging behind that of protein-coding genes, tremendous progress has been made in the field of gene regulation. One group of non-coding regulatory regions are enhancers, which can be distantly located upstream or downstream of genes and which can mediate temporal and tissue-specific transcriptional control via long-distance interactions with promoter regions. Although some examples exist in literature that link alterations of enhancers to genetic disorders, a widespread appreciation of the putative roles of these sequences in MCDs is still lacking. Here, we summarize the current state of knowledge on cis-regulatory regions and discuss novel technologies such as massively-parallel reporter assay systems, CRISPRCas9-based screens and computational approaches that help to further elucidate the emerging role of the non-coding genome in disease. Moreover, we discuss existing literature on mutations or copy number alterations of regulatory regions involved in brain development. We foresee that the future implementation of the knowledge obtained through ongoing gene regulation studies will benefit patients and will provide an explanation to part of the missing heritability of MCDs and other genetic disorders

The Why of YY1: Mechanisms of Transcriptional Regulation by Yin Yang 1

First described in 1991, Yin Yang 1 (YY1) is a transcription factor that is ubiquitously expressed throughout mammalian cells. It regulates both transcriptional activation and repression, in a seemingly context-dependent manner. YY1 has a well-established role in the development of the central nervous system, where it is involved in neurogenesis and maintenance of homeostasis in the developing brain. In neurodevelopmental and neurodegenerative disease, the crucial role of YY1 in cellular processes in the central nervous system is further underscored. In this mini-review, we discuss the various mechanisms leading to the transcriptional activating and repressing roles of YY1, including its role as a traditional transcription factor, its interactions with cofactors and chromatin modifiers, the role of YY1 in the non-coding genome and 3D chromatin organization and the possible implications of the phase-separation mechanism on YY1 function. We provide examples on how these processes can be involved in normal development and how alterations can lead to various diseases

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice

In mice, imprinted X chromosome inactivation (iXCI) of the paternal X in the pre-implantation embryo and extraembryonic tissues is followed by X reactivation in the inner cell mass (ICM) of the blastocyst to facilitate initiation of random XCI (rXCI) in all embryonic tissues. RNF12 is an E3 ubiquitin ligase that plays a key role in XCI. RNF12 targets pluripotency protein REX1 for degradation to initiate rXCI in embryonic stem cells (ESCs) and loss of the maternal copy of Rnf12 leads to embryonic lethality due to iXCI failure. Here, we show that loss of Rex1 rescues the rXCI phenotype observed in Rnf12-/- ESCs, and that REX1 is the prime target of RNF12 in ESCs. Genetic ablation of Rex1 in Rnf12-/- mice rescues the Rnf12-/- iXCI phenotype, and results in viable and fertile Rnf12-/-:Rex1-/- female mice displaying normal iXCI and rXCI. Our results show that REX1 is the critical target of RNF12 in XCI

Isobutyryl-CoA dehydrogenase deficiency associated with autism in a girl without an alternative genetic diagnosis by trio whole exome sequencing: A case report

Background: Isobutyryl-CoA dehydrogenase (IBD) is a mitochondrial enzyme catalysing the third step in the degradation of the essential branched-chain amino acid valine and is encoded by ACAD8. ACAD8 mutations lead to isobutyryl-CoA dehydrogenase deficiency (IBDD), which is identified by increased C4-acylcarnitine levels. Affected individuals are either asymptomatic or display a variety of symptoms during infancy, including speech delay, cognitive impairment, failure to thrive, hypotonia, and emesis. Methods: Here, we review all previously published IBDD patients and describe a girl diagnosed with IBDD who was presenting with autism as the main disease feature. Results: To assess whether a phenotype-genotype correlation exists that could explain the development or absence of clinical symptoms in IBDD, we compared CADD scores, in silico mutation predictions, LoF tolerance scores and C4-acylcarnitine levels between symptomatic and asymptomatic individuals. Statistical analysis of these parameters did not establish significant differences amongst both groups. Conclusion: As in our proband, tri

Stable X chromosome reactivation in female human induced pluripotent stem cells

In placental mammals, balanced expression of X-linked genes is accomplished by X chromosome inactivation (XCI) in female cells. In humans, random XCI is initiated early during embryonic development. To investigate whether reprogramming of female human fibroblasts into induced pluripotent stem cells (iPSCs) leads to reactivation of the inactive X chromosome (Xi), we have generated iPSC lines from fibroblasts heterozygous for large X-chromosomal deletions. These fibroblasts show completely skewed XCI of the mutated X chromosome, enabling monitoring of X chromosome reactivation (XCR) and XCI using allele-specific single-cell expression analysis. This approach revealed that XCR is robust under standard culture conditions, but does not prevent reinitiation of XCI, resulting in a mixed population of cells with either two active X chromosomes (Xas) or one Xa and one Xi. This mixed population of XaXa and XaXi cells is stabilized in naive human stem cell medium, allowing expansion of clones with two Xas

Building Bridges Between the Clinic and the Laboratory: A Meeting Review – Brain Malformations: A Roadmap for Future Research

In the middle of March 2019, a group of scientists and clinicians (as well as those who wear both hats) gathered in the green campus of the Weizmann Institute of Science to share recent scientific findings, to establish collaborations, and to discuss future directions for better diagnosis, etiology modeling and treatment of brain malformations. One hundred fifty scientists from twenty-two countries took part in this meeting. Thirty-eight talks were presented and as many as twenty-five posters were displayed. This review is aimed at presenting some of the highlights that the audience was exposed to during the three-day meeting

Dynamics of gene silencing during X inactivation using allele-specific RNA-seq

Background: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. Results: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. Conclusions: The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of e