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

Solving the unsolved genetic epilepsies:Current and future perspectives

Many patients with epilepsy undergo exome or genome sequencing as part of a diagnostic workup; however, many remain genetically unsolved. There are various factors that account for negative results in exome/genome sequencing for patients with epilepsy: (1) the underlying cause is not genetic; (2) there is a complex polygenic explanation; (3) the illness is monogenic but the causative gene remains to be linked to a human disorder; (4) family segregation with reduced penetrance; (5) somatic mosaicism or the complexity of, for example, a structural rearrangement; or (6) limited knowledge or diagnostic tools that hinder the proper classification of a variant, resulting in its designation as a variant of unknown significance. The objective of this review is to outline some of the diagnostic options that lie beyond the exome/genome, and that might become clinically relevant within the foreseeable future. These options include: (1) re-analysis of older exome/genome data as knowledge increases or symptoms change; (2) looking for somatic mosaicism or long-read sequencing to detect low-complexity repeat variants or specific structural variants missed by traditional exome/genome sequencing; (3) exploration of the non-coding genome including disruption of topologically associated domains, long range non-coding RNA, or other regulatory elements; and finally (4) transcriptomics, DNA methylation signatures, and metabolomics as complementary diagnostic methods that may be used in the assessment of variants of unknown significance. Some of these tools are currently not integrated into standard diagnostic workup. However, it is reasonable to expect that they will become increasingly available and improve current diagnostic capabilities, thereby enabling precision diagnosis in patients who are currently undiagnosed.</p

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

Functional dissection of the enhancer repertoire in human embryonic stem cells

Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications functionally define active enhancers remains unclear. Here, we combine chromatin immunoprecipitation with a massively parallel reporter assay (ChIP-STARR-seq) to identify functional enhancers in human embryonic stem cells (ESCs) genome-wide in a quantitative unbiased manner. Although active enhancers associate with TFs, only a minority of regions marked by NANOG, OCT4, H3K27ac, and H3K4me1 function as enhancers, with activity markedly changing under naive versus primed culture conditions. We identify an enhancer set associated with functions extending to non-ESC-specific processes. Moreover, although transposable elements associate with putative enhancers, only some exhibit activity. Similarly, within super-enhancers, large tracts are non-functional, with activity restricted to small sub-domains. This catalog of validated enhancers provides a valuable resource for further functional dissection of the regulatory genome.Barakat et al. use a combination of chromatin immunoprecipitation and a massively parallel reporter assay to identify functional enhancers in primed and naive human embryonic stem cells. This genome-wide catalog of validated enhancers provides a valuable resource for the further dissection of the regulatory genome

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

Comprehensive multi-omics integration identifies differentially active enhancers during human brain development with clinical relevance

Abstract Background Non-coding regulatory elements (NCREs), such as enhancers, play a crucial role in gene regulation, and genetic aberrations in NCREs can lead to human disease, including brain disorders. The human brain is a complex organ that is susceptible to numerous disorders; many of these are caused by genetic changes, but a multitude remain currently unexplained. Understanding NCREs acting during brain development has the potential to shed light on previously unrecognized genetic causes of human brain disease. Despite immense community-wide efforts to understand the role of the non-coding genome and NCREs, annotating functional NCREs remains challenging. Methods Here we performed an integrative computational analysis of virtually all currently available epigenome data sets related to human fetal brain. Results Our in-depth analysis unravels 39,709 differentially active enhancers (DAEs) that show dynamic epigenomic rearrangement during early stages of human brain development, indicating likely biological function. Many of these DAEs are linked to clinically relevant genes, and functional validation of selected DAEs in cell models and zebrafish confirms their role in gene regulation. Compared to enhancers without dynamic epigenomic rearrangement, DAEs are subjected to higher sequence constraints in humans, have distinct sequence characteristics and are bound by a distinct transcription factor landscape. DAEs are enriched for GWAS loci for brain-related traits and for genetic variation found in individuals with neurodevelopmental disorders, including autism. Conclusion This compendium of high-confidence enhancers will assist in deciphering the mechanism behind developmental genetics of human brain and will be relevant to uncover missing heritability in human genetic brain disorders

Inhibition of HDAC activity directly reprograms murine embryonic stem cells to trophoblast stem cells

Embryonic stem cells (ESCs) can differentiate into all cell types of the embryonic germ layers. ESCs can also generate totipotent 2C-like cells and trophectodermal cells. However, these latter transitions occur at low frequency due to epigenetic barriers, the nature of which is not fully understood. Here, we show that treating mouse ESCs with sodium butyrate (NaB) increases the population of 2C-like cells and enables direct reprogramming of ESCs into trophoblast stem cells (TSCs) without a transition through a 2C-like state. Mechanistically, NaB inhibits histone deacetylase activities in the LSD1-HDAC1/2 corepressor complex. This increases acetylation levels in the regulatory regions of both 2C- and TSC-specific genes, promoting their expression. In addition, NaB-treated cells acquire the capacity to generate blastocyst-like structures that can develop beyond the implantation stage in vitro and form deciduae in vivo. These results identify how epigenetics restrict the totipotent and trophectoderm fate in mouse ESCs.</p

Intrafamilial variability in SLC6A1-related neurodevelopmental disorders

IntroductionPhenotypic spectrum of SLC6A1-related neurodevelopmental disorders (SLC6A1-NDD) includes intellectual disability (ID), autistic spectrum disorders (ASD), epilepsy, developmental delay, beginning from early infancy or after seizure onset, and other neurological features such as hypotonia and movement disorders. Data on familial phenotypic heterogeneity have been rarely reported, thus in our study we aimed to investigate intrafamilial phenotypic variability in families with SLC6A1 variants.MethodsWe collected clinical, laboratory and genetic data on 39 individuals, including 17 probands, belonging to 13 families harboring inherited variants of SLC6A1. Data were collected through an international network of Epilepsy and Genetic Centers.ResultsMain clinical findings in the whole cohort of 39 subjects were: (a) epilepsy, mainly presenting with generalized seizures, reported in 71% of probands and 36% of siblings or first/second-degree relatives. Within a family, the same epilepsy type (generalized or focal) was observed; (b) ID reported in 100% and in 13% of probands and siblings or first/second-degree relatives, respectively; (c) learning disabilities detected in 28% of the SLC6A1 carriers, all of them were relatives of a proband; (d) around 51% of the whole cohort presented with psychiatric symptoms or behavioral disorders, including 82% of the probands. Out of the 19 patients with psychiatric symptoms, ASD were diagnosed in 40% of them; (e) neurological findings (primarily tremor and speech difficulties) were observed 38.5% of the whole cohort, including 10 probands. Our families harbored 12 different SLC6A1 variants, one was a frameshift, two stop-gain, while the remaining were missense. No genotype–phenotype associations were identified.DiscussionOur study showed that first-or second-degree relatives presented with a less severe phenotype, featuring mainly mild intellectual and/or learning disabilities, at variance with the probands who suffered from moderate to severe ID, generalized, sometimes intractable, epileptic seizures, behavioral and psychiatric disorders. These findings may suggest that a proportion of individuals with mild SLC6A1-NDD might be missed, in particular those with an older age where genetic testing is not performed. Further studies on intrafamilial phenotypic variability are needed to confirm our results and possibly to expand the phenotypic spectrum of these disorders and benefit genetic counseling