Polyamine Dysregulation and Nucleolar Disruption in Alzheimer's Disease.

A hypothesis of Alzheimer's disease etiology is proposed describing how cellular stress induces excessive polyamine synthesis and recycling which can disrupt nucleoli. Polyamines are essential in nucleolar functions, such as RNA folding and ribonucleoprotein assembly. Changes in the nucleolar pool of anionic RNA and cationic polyamines acting as counterions can cause significant nucleolar dynamics. Polyamine synthesis reduces S-adenosylmethionine which, at low levels, triggers tau phosphorylation. Also, polyamine recycling reduces acetyl-CoA needed for acetylcholine, which is low in Alzheimer's disease. Extraordinary nucleolar expansion and/or contraction can disrupt epigenetic control in peri-nucleolar chromatin, such as chromosome 14 with the presenilin-1 gene; chromosome 21 with the amyloid precursor protein gene; chromosome 17 with the tau gene; chromosome 19 with the APOE4 gene; and the inactive X chromosome (Xi; aka "nucleolar satellite") with normally silent spermine synthase (polyamine synthesis) and spermidine/spermine-N1-acetyltransferase (polyamine recycling) alleles. Chromosomes 17, 19 and the Xi have high concentrations of Alu elements which can be transcribed by RNA polymerase III if positioned nucleosomes are displaced from the Alu elements. A sudden flood of Alu RNA transcripts can competitively bind nucleolin which is usually bound to Alu sequences in structural RNAs that stabilize the nucleolar heterochromatic shell. This Alu competition leads to loss of nucleolar integrity with leaking of nucleolar polyamines that cause aggregation of phosphorylated tau. The hypothesis was developed with key word searches (e.g., PubMed) using relevant terms (e.g., Alzheimer's, lupus, nucleolin) based on a systems biology approach and exploring autoimmune disease tautology, gaining synergistic insights from other diseases.

Genes that escape from X-chromosome inactivation and sexual dimorphism of systemic lupus erythematosus.

X chromosome inactivation can balance the effects of the two X chromosomes in females, and emerging evidence indicates that numerous genes on the inactivated X chromosome have the potential to evade inactivation. The mechanisms of escape include modification of DNA, RNA, histone, epitope, and various regulatory proteins, as well as the spatial structure of chromatin. The study of X chromosome inactivation escape has paved the way for investigating sex dimorphism in human diseases, particularly autoimmune diseases. It has been demonstrated that the presence of TLR7, CD40L, IRAK-1, CXCR3, and CXorf21 significantly contributes to the prevalence of SLE (systemic lupus erythematosus) in females. This article mainly reviews the molecular mechanisms underlying these genes that escape from X-chromosome inactivation and sexual dimorphism of systemic lupus erythematosus. Therefore, elucidating the molecular mechanisms underlying sexual dimorphism in SLE is not only crucial for diagnosing and treating the disease, but also holds theoretical significance in comprehensively understanding the development and regulatory mechanisms of the human immune system.

Chromosomal and environmental contributions to sex differences in the vulnerability to neurological and neuropsychiatric disorders: Implications for therapeutic interventions.

Neurological and neuropsychiatric disorders affect men and women differently. Multiple sclerosis, Alzheimer's disease, anxiety disorders, depression, meningiomas and late-onset schizophrenia affect women more frequently than men. By contrast, Parkinson's disease, autism spectrum condition, attention-deficit hyperactivity disorder, Tourette's syndrome, amyotrophic lateral sclerosis and early-onset schizophrenia are more prevalent in men. Women have been historically under-recruited or excluded from clinical trials, and most basic research uses male rodent cells or animals as disease models, rarely studying both sexes and factoring sex as a potential source of variation, resulting in a poor understanding of the underlying biological reasons for sex and gender differences in the development of such diseases. Putative pathophysiological contributors include hormones and epigenetics regulators but additional biological and non-biological influences may be at play. We review here the evidence for the underpinning role of the sex chromosome complement, X chromosome inactivation, and environmental and epigenetic regulators in sex differences in the vulnerability to brain disease. We conclude that there is a pressing need for a better understanding of the genetic, epigenetic and environmental mechanisms sustaining sex differences in such diseases, which is critical for developing a precision medicine approach based on sex-tailored prevention and treatment.

Visualization of Hidden Epitopes at the Inactive X Chromosome.

Immunodetection of nuclear antigens is often complicated by epitope masking, so that proteins known to function in the nucleus are sometimes not easily detected at their sites of action. Moreover, protein populations that are detected before unmasking can be very different to those seen after removal of nucleic acids. This is particularly true for components of the nuclear matrix, including those known to function at the inactive X chromosome. Here we describe an unmasking protocol that reveals previously undetected proteins at the inactive X chromosome in mouse fibroblasts.

Practical Analysis of Hi-C Data: Generating A/B Compartment Profiles.

Recent advances in next-generation sequencing (NGS) and chromosome conformation capture (3C) analysis have led to the development of Hi-C, a genome-wide version of the 3C method. Hi-C has identified new levels of chromosome organization such as A/B compartments, topologically associating domains (TADs) as well as large megadomains on the inactive X chromosome, while allowing the identification of chromatin loops at the genome scale. Despite its powerfulness, Hi-C data analysis is much more involved compared to conventional NGS applications such as RNA-seq or ChIP-seq and requires many more steps. This presents a significant hurdle for those who wish to implement Hi-C technology into their laboratory. On the other hand, genomics data repository sites sometimes contain processed Hi-C data sets, allowing researchers to perform further analysis without the need for high-spec workstations and servers. In this chapter, we provide a detailed description on how to calculate A/B compartment profiles from processed Hi-C data on the autosomes and the active/inactive X chromosomes.

Live cell imaging of X chromosome reactivation during somatic cell reprogramming.

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.

Loss of SETDB1 decompacts the inactive X chromosome in part through reactivation of an enhancer in the IL1RAPL1 gene.

BACKGROUND: The product of dosage compensation in female mammals is the inactive X chromosome (Xi). Xi facultative heterochromatin is organized into two different types, one of which is defined by histone H3 trimethylated at lysine 9 (H3K9me3). The rationale for this study was to assess SET domain bifurcated 1 (SETDB1) as a candidate for maintaining this repressive modification at the human Xi. RESULTS: Here, we show that loss of SETDB1 does not result in large-scale H3K9me3 changes at the Xi, but unexpectedly we observed striking decompaction of the Xi territory. Close examination revealed a 0.5 Mb region of the Xi that transitioned from H3K9me3 heterochromatin to euchromatin within the 3' end of the IL1RAPL1 gene that is part of a common chromosome fragile site that is frequently deleted or rearranged in patients afflicted with intellectual disability and other neurological ailments. Centrally located within this interval is a powerful enhancer adjacent to an ERVL-MaLR element. In the absence of SETDB1, the enhancer is reactivated on the Xi coupled with bidirectional transcription from the ERVL-MaLR element. Xa deletion of the enhancer/ERVL-MaLR resulted in loss of full-length IL1RAPL1 transcript in cis, coupled with trans decompaction of the Xi chromosome territory, whereas Xi deletion increased detection of full-length IL1RAPL1 transcript in trans, but did not impact Xi compaction. CONCLUSIONS: These data support a critical role for SETDB1 in maintaining the ERVL-MaLR element and adjacent enhancer in the 3' end of the IL1RAPL1 gene in a silent state to facilitate Xi compaction.

When the Lyon(ized chromosome) roars: ongoing expression from an inactive X chromosome.

A tribute to Mary Lyon was held in October 2016. Many remarked about Lyon's foresight regarding many intricacies of the X-chromosome inactivation process. One such example is that a year after her original 1961 hypothesis she proposed that genes with Y homologues should escape from X inactivation to achieve dosage compensation between males and females. Fifty-five years later we have learned many details about these escapees that we attempt to summarize in this review, with a particular focus on recent findings. We now know that escapees are not rare, particularly on the human X, and that most lack functionally equivalent Y homologues, leading to their increasingly recognized role in sexually dimorphic traits. Newer sequencing technologies have expanded profiling of primary tissues that will better enable connections to sex-biased disorders as well as provide additional insights into the X-inactivation process. Chromosome organization, nuclear location and chromatin environments distinguish escapees from other X-inactivated genes. Nevertheless, several big questions remain, including what dictates their distinct epigenetic environment, the underlying basis of species differences in escapee regulation, how different classes of escapees are distinguished, and the roles that local sequences and chromosome ultrastructure play in escapee regulation.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.

A Review of Autoimmune Disease Hypotheses with Introduction of the "Nucleolus" Hypothesis.

Numerous hypotheses have been proposed in order to explain the complexity of autoimmune diseases. These hypotheses provide frameworks towards understanding the relations between triggers, autoantigen development, symptoms, and demographics. However, testing and refining these hypotheses are difficult tasks since autoimmune diseases have a potentially overwhelming number of variables due to the influence on autoimmune diseases from environmental factors, genetics, and epigenetics. Typically, the hypotheses are narrow in scope, for example, explaining the diseases in terms of genetics without defining detailed roles for environmental factors or epigenetics. Here, we present a brief review of the major hypotheses of autoimmune diseases including a new one related to the consequences of abnormal nucleolar interactions with chromatin, the "nucleolus" hypothesis which was originally termed the "inactive X chromosome and nucleolus nexus" hypothesis. Indeed, the dynamic nucleolus can expand as part of a cellular stress response and potentially engulf portions of chromatin, leading to disruption of the chromatin. The inactive X chromosome (a.k.a. the Barr body) is particularly vulnerable due to its close proximity to the nucleolus. In addition, the polyamines, present at high levels in the nucleolus, are also suspected of contributing to the development of autoantigens.

Human Embryonic Stem Cells Do Not Change Their X Inactivation Status during Differentiation.

Applications of embryonic stem cells (ESCs) require faithful chromatin changes during differentiation, but the fate of the X chromosome state in differentiating ESCs is unclear. Female human ESC lines either carry two active X chromosomes (XaXa), an Xa and inactive X chromosome with or without XIST RNA coating (XiXIST+Xa;XiXa), or an Xa and an eroded Xi (XeXa) where the Xi no longer expresses XIST RNA and has partially reactivated. Here, we established XiXa, XeXa, and XaXa ESC lines and followed their X chromosome state during differentiation. Surprisingly, we found that the X state pre-existing in primed ESCs is maintained in differentiated cells. Consequently, differentiated XeXa and XaXa cells lacked XIST, did not induce X inactivation, and displayed higher X-linked gene expression than XiXa cells. These results demonstrate that X chromosome dosage compensation is not required for ESC differentiation. Our data imply that XiXIST+Xa ESCs are most suited for downstream applications and show that all other X states are abnormal byproducts of our ESC derivation and propagation method.

A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation.

Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms.

Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture.

During interphase, the inactive X chromosome (Xi) is largely transcriptionally silent and adopts an unusual 3D configuration known as the "Barr body." Despite the importance of X chromosome inactivation, little is known about this 3D conformation. We recently showed that in humans the Xi chromosome exhibits three structural features, two of which are not shared by other chromosomes. First, like the chromosomes of many species, Xi forms compartments. Second, Xi is partitioned into two huge intervals, called "superdomains," such that pairs of loci in the same superdomain tend to colocalize. The boundary between the superdomains lies near DXZ4, a macrosatellite repeat whose Xi allele extensively binds the protein CCCTC-binding factor. Third, Xi exhibits extremely large loops, up to 77 megabases long, called "superloops." DXZ4 lies at the anchor of several superloops. Here, we combine 3D mapping, microscopy, and genome editing to study the structure of Xi, focusing on the role of DXZ4 We show that superloops and superdomains are conserved across eutherian mammals. By analyzing ligation events involving three or more loci, we demonstrate that DXZ4 and other superloop anchors tend to colocate simultaneously. Finally, we show that deleting DXZ4 on Xi leads to the disappearance of superdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks. Thus, DXZ4 is essential for proper Xi packaging.

Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci.

BACKGROUND: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super-resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). RESULTS: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an 'autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. CONCLUSIONS: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi.

A new light on DNA replication from the inactive X chromosome.

While large portions of the mammalian genome are known to replicate sequentially in a distinct, tissue-specific order, recent studies suggest that the inactive X chromosome is duplicated rapidly via random, synchronous DNA synthesis at numerous adjacent regions. The rapid duplication of the inactive X chromosome was observed in high-resolution studies visualizing DNA replication patterns in the nucleus, and by allele-specific DNA sequencing studies measuring the extent of DNA synthesis. These studies conclude that inactive X chromosomes complete replication earlier than previously thought and suggest that the strict order of DNA replication detected in the majority of genomic regions is not preserved in non-transcribed, "silent" chromatin. These observations alter current concepts about the regulation of DNA replication in non-transcribed portions of the genome in general and in the inactive X-chromosome in particular.

Species conserved DNA damage response at the inactive human X chromosome.

Chromatin modifications are long known as an essential part of the orchestrated response resulting in the repair of radiation-induced DNA double-strand breaks (DSBs). Only recently, however, the influence of the chromatin architecture itself on the DNA damage response has been recognised. Thus for heterochromatic DSBs the sensing and early recruitment of repair factors to the lesion occurs within the heterochromatic compartments, but the damage sites are subsequently relocated from the inside to the outside of the heterochromatin. While previous studies were accomplished at the constitutive heterochromatin of centromeric regions in mouse and flies, here we examine the DSB repair at the facultative heterochromatin of the inactive X chromosome (Xi) in humans. Using heavy ion irradiation we show that at later times after irradiation the DSB damage streaks bend around the Xi verifying that the relocation process is conserved between species and not specialised to repetitive sequences only. In addition, to measure chromatin relaxation at rare positions within the genome, we established live cell microscopy at the GSI microbeam thus allowing the aimed irradiation of small nuclear structures like the Xi. Chromatin decondensation at DSBs within the Xi is clearly visible within minutes as a continuous decrease of the DNA staining over time, comparable to the DNA relaxation revealed at DSBs in mouse chromocenters. Furthermore, despite being conserved between species, slight differences in the underlying regulation of these processes in heterochromatic DSBs are apparent.