Human Chromosome Research Articles

The p-Arms of Human Acrocentric Chromosomes Play by a Different Set of Rules.

The p-arms of the five human acrocentric chromosomes bear nucleolar organizer regions (NORs) comprising ribosomal gene (rDNA) repeats that are organized in a homogeneous tandem array and transcribed in a telomere-to-centromere direction. Precursor ribosomal RNA transcripts are processed and assembled into ribosomal subunits, the nucleolus being the physical manifestation of this process. I review current understanding of nucleolar chromosome biology and describe current exploration into a role for the NOR chromosomal context. Full DNA sequences for acrocentric p-arms are now emerging, aided by the current revolution in long-read sequencing and genome assembly. Acrocentric p-arms vary from 10.1 to 16.7 Mb, accounting for ∼2.2% of the genome. Bordering rDNA arrays, distal junctions, and proximal junctions are shared among the p-arms, with distal junctions showing evidence of functionality. The remaining p-arm sequences comprise multiple satellite DNA classes and segmental duplications that facilitate recombination between heterologous chromosomes, which is likely also involved in Robertsonian translocations. Expected final online publication date for the Annual Review of Genomics and Human Genetics, Volume 24 is August 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Why does the X chromosome lag behind autosomes in GWAS findings?

The X-chromosome is among the largest human chromosomes. It differs from autosomes by a number of important features including hemizygosity in males, an almost complete inactivation of one copy in females, and unique patterns of recombination. We used data from the Catalog of Published Genome Wide Association Studies to compare densities of the GWAS-detected SNPs on the X-chromosome and autosomes. The density of GWAS-detected SNPs on the X-chromosome is 6-fold lower compared to the density of the GWAS-detected SNPs on autosomes. Differences between the X-chromosome and autosomes cannot be explained by differences in the overall SNP density, lower X-chromosome coverage by genotyping platforms or low call rate of X-chromosomal SNPs. Similar differences in the density of GWAS-detected SNPs were found in female-only GWASs (e.g. ovarian cancer GWASs). We hypothesized that the lower density of GWAS-detected SNPs on the X-chromosome compared to autosomes is not a result of a methodological bias, e.g. differences in coverage or call rates, but has a real underlying biological reason-a lower density of functional SNPs on the X-chromosome versus autosomes. This hypothesis is supported by the observation that (i) the overall SNP density of X-chromosome is lower compared to the SNP density on autosomes and that (ii) the density of genic SNPs on the X-chromosome is lower compared to autosomes while densities of intergenic SNPs are similar.

The Telomeric Repeats of HHV-6A Do Not Determine the Chromosome into Which the Virus Is Integrated

Human herpes virus 6A (HHV-6A) is able to integrate into the telomeric and subtelomeric regions of human chromosomes representing chromosomally integrated HHV-6A (ciHHV-6A). The integration starts from the right direct repeat (DRR) region. It has been shown experimentally that perfect telomeric repeats (pTMR) in the DRR region are required for the integration, while the absence of the imperfect telomeric repeats (impTMR) only slightly reduces the frequency of HHV-6 integration cases. The aim of this study was to determine whether telomeric repeats within DRR may define the chromosome into which the HHV-6A integrates. We analysed 66 HHV-6A genomes obtained from public databases. Insertion and deletion patterns of DRR regions were examined. We also compared TMR within the herpes virus DRR and human chromosome sequences retrieved from the Telomere-to-Telomere consortium. Our results show that telomeric repeats in DRR in circulating and ciHHV-6A have an affinity for all human chromosomes studied and thus do not define a chromosome for integration.

Chemoproteomic discovery of a human RNA ligase

RNA ligases are present across all forms of life. While enzymatic RNA ligation between 5′-PO4 and 3′-OH termini is prevalent in viruses, fungi, and plants, such RNA ligases are yet to be identified in vertebrates. Here, using a nucleotide-based chemical probe targeting human AMPylated proteome, we have enriched and identified the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29) as a human enzyme promoting RNA ligation between 5′-PO4 and 3′-OH termini. C12orf29 catalyses ATP-dependent RNA ligation via a three-step mechanism, involving tandem auto- and RNA AMPylation. Knock-out of C12ORF29 gene impedes the cellular resilience to oxidative stress featuring concurrent RNA degradation, which suggests a role of C12orf29 in maintaining RNA integrity. These data provide the groundwork for establishing a human RNA repair pathway.

Synthetic analysis of chromatin tracing and live-cell imaging indicates pervasive spatial coupling between genes.

The role of the spatial organization of chromosomes in directing transcription remains an outstanding question in gene regulation. Here, we analyze two recent single-cell imaging methodologies applied across hundreds of genes to systematically analyze the contribution of chromosome conformation to transcriptional regulation. Those methodologies are (1) single-cell chromatin tracing with super-resolution imaging in fixed cells; and (2) high-throughput labeling and imaging of nascent RNA in living cells. Specifically, we determine the contribution of physical distance to the coordination of transcriptional bursts. We find that individual genes adopt a constrained conformation and reposition toward the centroid of the surrounding chromatin upon activation. Leveraging the variability in distance inherent in single-cell imaging, we show that physical distance - but not genomic distance - between genes on individual chromosomes is the major factor driving co-bursting. By combining this analysis with live-cell imaging, we arrive at a corrected transcriptional correlation of [Formula: see text] for genes separated by < 400 nm. We propose that this surprisingly large correlation represents a physical property of human chromosomes and establishes a benchmark for future experimental studies.

ChroSegNet: An Attention-Based Model for Chromosome Segmentation with Enhanced Processing

In modern medical diagnosis, the karyotype analysis for human chromosome is clinically significant for the diagnosis and treatment of genetic diseases. In such an analysis, it is critically important to segment the banded chromosomes. Chromosome segmentation, however, is technically challenging due to the variable chromosome features, the complex background noise, and the uneven image quality of the chromosome images. Owing to these technical challenges, the existing deep-learning-based algorithms would have severe overfitting problems and are ineffective in the segmentation task. In this paper, we propose a novel chromosome segmentation model with our enhanced chromosome processing, namely ChroSegNet. First, we develop enhanced chromosome processing techniques to realize the quality and quantity enhancement of the chromosome data, leading to the chromosome segmentation dataset for our subsequent network training. Second, we propose our novel chromosome segmentation model “ChroSegNet" based on U-Net. According to the characteristics of chromosome data, we have not only improved the baseline structure but also incorporate the hybrid attention module to ChroSegNet, which can extract the key feature information and location information of chromosome. Finally, we evaluated ChroSegNet on our chromosome segmentation dataset and obtained the MPA of 93.31% and the F1-score of 92.99%. Experimental results show that ChroSegNet not only outperforms the representative segmentation models in chromosome segmentation performance but also has a lightweight model structure. We believe that our proposed ChroSegNet is highly promising in future applications of genetic measurement and diagnosis.

Human Satellite 1A analysis provides evidence of pericentromeric transcription

BackgroundPericentromeric regions of human chromosomes are composed of tandem-repeated and highly organized sequences named satellite DNAs. Human classical satellite DNAs are classified into three families named HSat1, HSat2, and HSat3, which have historically posed a challenge for the assembly of the human reference genome where they are misrepresented due to their repetitive nature. Although being known for a long time as the most AT-rich fraction of the human genome, classical satellite HSat1A has been disregarded in genomic and transcriptional studies, falling behind other human satellites in terms of functional knowledge. Here, we aim to characterize and provide an understanding on the biological relevance of HSat1A.ResultsThe path followed herein trails with HSat1A isolation and cloning, followed by in silico analysis. Monomer copy number and expression data was obtained in a wide variety of human cell lines, with greatly varying profiles in tumoral/non-tumoral samples. HSat1A was mapped in human chromosomes and applied in in situ transcriptional assays. Additionally, it was possible to observe the nuclear organization of HSat1A transcripts and further characterize them by 3′ RACE-Seq. Size-varying polyadenylated HSat1A transcripts were detected, which possibly accounts for the intricate regulation of alternative polyadenylation.ConclusionAs far as we know, this work pioneers HSat1A transcription studies. With the emergence of new human genome assemblies, acrocentric pericentromeres are becoming relevant characters in disease and other biological contexts. HSat1A sequences and associated noncoding RNAs will most certainly prove significant in the future of HSat research.

Targeting Overexpressed Cyclin Dependent Kinase 1 (CDK1) in Human Cancers: Kamalachalcone A Emerged as Potential Inhibitor of CDK1 Kinase Through in Silico Docking Study

Background: CDK1 gene located in human chromosome 10, encodes CDK1 (tyrosine) kinase, overexpressed in multiple cancer cells. Upon phosphorylation of tyrosine kinases by adenosine triphosphate, CDK1 kinase is activated in cell cycle progression. Nonetheless, CDK1 kinase excessively partakes in the development and prognosis of specific types of aggressive cancers. Therefore, CDK1 kinase inhibition therapy is a primary target for the treatment of explicitly expressed CDK1 kinase cancers. At present, dinaciclib is the only FDA approved orphan CDK1 kinase inhibitor drug in the treatment of cancers. In molecular docking study, kamalachalcone A with significant higher binding energy competitively bound to the active receptor site of CDK1 kinase, as comparable to dinaciclib. In this framework, kamalachalcone A could emerge as a potential candidate for restricting the over expression of CDK1 kinase in multiple cancers. Objective: The present study aimed to compare the inhibitory activity of dinaciclib (CDK1 kinase inhibitor drug) with kamalachalcone A, a novel chalcone found in kamala dye. Methods: Dinaciclib and kamalachalcone A were docked on human CDK1 kinase 3D structure in molecular docking using AutoDock 4.2.6, MGL tools and molecular dynamic studies with iMOD server (iMODS). Results: These studies revealed that kamalachalcone A was a competitive CDK1 kinase inhibitor alike dinaciclib. The docking results suggested binding site similarities of dinaciclib and kamalachalcone A. The binding energies of docked dinaciclib and kamalachalcone A complexes with CDK1 kinase were -8.52 kcal/mol and -12.81 kcal/mol, respectively. The docking results were substantiated by molecular simulation studies with iMODS. Conclusion: The in silico docking simulation of dinaciclib and kamalachalcone A suggested that kamalachalcone A is likewise a competitive CDK1 kinase inhibitor.

Effect of berberine on serum hormonal levels and endometrial gene and protein signaling in a rat model of polycystic ovary syndrome

Purpose: To study the effect of berberine on serum hormonal levels, endometrial gene, and protein signaling in a rat model of polycystic ovary syndrome (PCOS).Methods: Eighty-seven healthy female Sprague Dawley (SD) rats were assigned to control group, PCOS model group, and berberine group, each with 29 rats. The rats were assigned to three groups: control, model, and berberine groups, each with 29 rats. A rat model of PCOS was established via the administration of letrozole (1 mg/kg) to rats in the model group continuously for 21 days through gavage. Rats in berberine group were also given letrozole at the same dose for the same duration through gavage, while rats in control group received physiological saline in place of berberine. Estrus cycle, serum hormonal levels, ovarian tissue morphology, estrogen receptor α (ERα) levels, and mRNA expression levels of protein kinase B subtype 3 (Akt-3β) were evaluated. RAS-C3 botulinum toxin substrate 1 (Rac1), human chromosome 10 deleted phosphatase (PTEN) and mouse sarcoma-like virus oncogene (KRAS) were also determined using appropriate procedures.Results: Berberine treatment resulted in a significant decrease in the mean body weight of PCOS rats, relative to model rats (p &lt; 0.05). Estrus interval in model rats was significantly prolonged. Levels of testosterone and estradiol, and mRNA expression levels of ERα, Akt3, Racl, PTEN, and KRAS were significantly increased, while progesterone level was significantly decreased, relative to control (p &lt; 0.05). Estrus interval in the berberine group was shortened and estrus cycle gradually returned to normal, while the levels of testosterone and estradiol, and mRNA levels of ERα were gradually restored, relative to untreated PCOS rats (p &lt; 0.05). Moreover, mRNA levels of Akt3, RAC1, PTEN, and KRAS were significantly decreased, while progesterone level was significantly increased (p &lt; 0.05).Conclusion: Berberine appreciably restores heat cycle and ovary morphology in PCOS rats, and regulates serum hormonal levels, and endometrium-related gene and protein signaling.

Random Integration Analysis of Recombinant Adeno-Associated Virus 6 Packaged in Sf9 Insect Cells

Recently, there have been growing concerns over the integration of recombinant adeno-associated virus (rAAV) used in gene therapy. Wild-type adeno-associated virus (AAV) site specifically integrates into AAVS1 site of human genome, while rAAV randomly integrates into host chromosomes at low frequencies. This research aims to study the random integration events of rAAV6-EGFP packaged in Sf9 insect cells. Baculo-Sf9 manufacturing platform has the advantages of high-density suspension culture of Sf9 insect cells and large-scale production of rAAV vectors. In this study, we used different doses of Baculo-Sf9 produced rAAV6-EGFP to transduce HEK293T cells and A549-implanted tumors in vitro and in vivo. Using flow cytometry and fluorescence microscopy, we studied their EGFP gene expression efficiencies and EGFP fluorescence intensities. Using inverse nested PCR and DNA sequencing, random integration sites of rAAV6-EGFP genome into human chromosomes were identified. In vitro results showed that gene expression efficiencies became stable after 20 days and random integration frequencies were 0.2-4.2%. Both in vitro and in vivo results indicated that random integration of Baculo-Sf9 rAAV6 was dose-dependent. Sequencing results showed two random integration sites, which were on human chromosomes 8 and 12. The findings suggest that we should use as low dose of rAAV vector as possible for safe gene therapy.

Genetic Variants in ARHGEF6 Cause Congenital Anomalies of the Kidneys and Urinary Tract in Humans, Mice, and Frogs.

About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant. To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models. We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.

3D genome architecture regulates the traffic of transcription factors throughout human chromosomes.

Transcription factors (TFs) are proteins that control the rate of transcription by binding to specific DNA sequences. The time needed for a TF to find its target sites is one of many components contributing to the genetic response mechanism. While TF target site search is a well studied problem, the effect of genome 3D architecture on such process is poorly understood. We use accurate and cell-specific 3D structures of human chromosomes to investigate how the dynamics of TFs is influenced by the spatial organization of binding sites. We use ChIP-Seq data to map the position of binding sites on chromosomal structures and we then simulate the dynamics of individual TF as they move from one binding site to another diffusing throughout chromosomal territories. We find that the distribution of binding sites along chromosomes cooperates with the 3D folding of the chromatin fiber to induce a dynamics in which TF tend to visit sites distributed sequentially along the genome. In this way, genome 3D architecture appears to reduce the time each TF spends in the unbound state while commuting from from one target site to the other. At the same time, genome 3D architecture further reduces the flux of TFs between binding sites that are already well separated along the genome. We compare the TF traffic patterns generated by the 3D structures of human chromosomes with those generated by several alternative structural models characterized by an increasing amount of randomness. Finally, we study the effect of lengthwise compaction and phase separation, known architectural features of the human genome, in TF target search. In short, our analysis demonstrates that genome architecture regulates the traffic of TF within chromosomal territories and reduces the time each TF spends commuting between binding sites.

Phase separation of AB compartments are modulated by lamina-chromatin interaction.

Recent experiments reveal that chromatin is able to bind to nuclear lamina proteins such as lamin A/C and lamin B1. Loci with high binding affinity to the lamina are called Lamin Associated Domains (LADs), primarily made up of inactive chromatin. This connection between heterochromatin and the nuclear envelope points to the possibility of further chromatin organization based on nucleus placement. Here we explore such a phenomenon by simulating five different human chromosomes in close proximity to a spherical confinement, which is attractive only to loci marked as LADs. Utilizing the chromatin types taken from the cell line GM12878, the chromosomes were modeled using the Minimal Chromatin Model (MiChroM) at 50 Kbp resolution. This model ensures that the main fold of the chromatin is due to compartmentalization, while the lamina interaction falls into the perturbative regime. According to the simulations, introducing lamina interaction alone can drive the chromatin conformation from the inverted nucleus to the conventional nucleus, and vice versa. Also, the presence of lamina interaction enhances compartmentalization at the intrachromosomal level. Further, depleted lamina simulations show that only chromatin-chromatin interactions are not enough to reproduce the experimental interchromosomal Hi-C maps. In contrast, the addition of nuclear lamina-chromatin interaction increases the frequency of interchromosomal euchromatin contact. This indicates that the nuclear lamina plays an important role in chromatin folding and dynamics.

In vivo disruption of nuclear HSATII RNA biomolecular condensates.

Emerging studies have begun highlighting the significance of cellular regulation in the context of biomolecular condensates. These membraneless entities, formed by liquid-liquid phase separation (LLPS), compartmentalize regulatory elements that include proteins and long non-coding RNAs (lncRNAs). Biogenesis of these condensates includes molecular scaffolds called architectural lncRNAs (arcRNAs); which have the ability to nucleate regulatory elements. Tandemly repeating satellite sequences account for 3% of the human genome. One of them, Human Satellite II (HSATII), has a loosely defined monomer sequence and is located near centromeres of several human chromosomes. HSATII lncRNAs have growing evidence of important regulatory functions in epithelial cancer cells. Pericentromeric HSATII DNA is transcriptionally repressed in normal cells, however misregulation of these heterochromatic sequences in cancer cells allows for its aberrant transcription. The repetitive HSATII lncRNAs aggregate into large foci that remain in cis at their sites of transcription, and recruit regulatory proteins. Using NEAT1 and paraspeckles as models we present evidence that HSATII assembles into LLPS biomolecular condensates in vivo, that can be dispersed by doxorubicin into the nucleus and cytoplasm. Treatment with doxorubicin and 1,6-Hexanediol, drugs known to disrupt other condensates, resulted in significant redistribution of HSATII RNA. While this study serves to demonstrate that HSATII foci are condensates, it may also have broader implications for drug mechanisms in cancer therapeutics.

Loss of RanGAP1 drives chromosome instability and rapid tumorigenesis of osteosarcoma.

Chromothripsis is a catastrophic event of chromosomal instability that involves intensive fragmentation and rearrangements within localized chromosomal regions. However, its cause remains unclear. Here, we show that reduction and inactivation of Ran GTPase-activating protein 1 (RanGAP1) commonly occur in human osteosarcoma, which is associated with a high rate of chromothripsis. In rapidly expanding mouse osteoprogenitors, RanGAP1 deficiency causes chromothripsis in chr1q, instant inactivation of Rb1 and degradation of p53, consequent failure in DNA damage repair, and ultrafast osteosarcoma tumorigenesis. During mitosis, RanGAP1 anchors to the kinetochore, where it recruits PP1-γ to counteract the activity of the spindle-assembly checkpoint (SAC) and prevents TOP2A degradation, thus safeguarding chromatid decatenation. Loss of RanGAP1 causes SAC hyperactivation and chromatid decatenation failure. These findings demonstrate that RanGAP1 maintains mitotic chromosome integrity and that RanGAP1 loss drives tumorigenesis through its direct effects on SAC and decatenation and secondary effects on DNA damage surveillance.

Transcription-induced active forces suppress chromatin motion by inducing a transient disorder-to-order transition.

Recent experiments have shown that the mobility of human interphase chromosome decreases during transcription, and increases upon inhibiting transcription, a finding that is counter-intuitive because it is thought that the active mechanical force ($F$) generated by RNA polymerase II (RNAPII) on chromatin would render it more open and mobile. Inspired by these observations, we use a copolymer model to investigate how $F$ affects the dynamical properties of a single chromatin. The movements of the loci in the gene-rich region are suppressed in an intermediate range of $F$, and are enhanced at small and large $F$ values. In the intermediate $F$, the bond length between consecutive loci increases, becoming commensurate with the location of the minimum in the attractive interaction between the active loci. This results in a transient disorder-to-order transition, leading to the decreased mobility during transcription. Transient ordering of the loci in the gene-rich region might be a mechanism for nucleating a dynamic network involving transcription factors, RNAPII, and chromatin.

Revealing and manipulating the structure of mitotic chromosomes using atomic force microscopy

Although the mitotic cell cycle has been extensively characterized, very little is known about the highly condensed structure of metaphase chromosomes. The hierarchical organization of the chromatin fibers and their extrusion into complex loop structures by the Structural Maintenance of Chromosome (SMC) proteins are widely acknowledged features of the chromosomal architecture; however, the small size and complex topology of these structures are hindering their unequivocal detection and characterization. Even though Atomic Force Microscopy (AFM) has the potential to access these features, chromosome deformability and susceptibility to alterations to their external environment have so far limited the application of this technique. In the present work we overcome these issues and employ AFM to study the structure of human metaphase chromosomes in a liquid environment that closely resembles their physiological conditions, without the use of fixation agents and enzymatic treatments. Our analysis reveals the finer details of the unperturbed chromosome structure, shedding light on the organization of the chromatin fiber network and testing its stability against changes in the environment ionic conditions. Moreover, by complementing the image analysis with AFM-based Force Spectroscopy we probe the response of mitotic chromosomes to nanoscale deformations in the presence and absence of specific SMC proteins, namely Condensin I and II, thereby providing new insights into their contribution to the chromosome structural stability. The presented results enrich our current understanding of chromosome organization and provide new valuable tools to study the structure of mitotic chromosomes in both healthy and pathological conditions.

Single molecule manipulation and imaging of complex DNA-protein interactions.

The genetic information of an organism is encoded in the base pair sequence of its DNA. Many specialized proteins are involved in organizing, preserving and processing the vast amounts of information on the DNA. In order to do this swiftly and correctly these proteins have to move quickly and accurately along and/or around the DNA constantly rearranging it. In this presentation I will show (Super-resolution) Correlative Tweezers-Fluorescence Microscopy (CTFM), a single-molecule approach capable of visualizing individual DNA-binding proteins on densely covered DNA and in presence of high protein concentrations. Moreover, proteins on DNA can be visualized on multiple DNA strand. Next, using this instrument we have investigated human non-homologous end joining (NHEJ). NHEJ is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells. Here we show that the XRCC4-XLF complexes robustly bridge two independent DNA molecules and that these bridges are able to slide along the DNA. These observations suggest that XRCC4-XLF complexes form mobile sleeve-like structures around DNA that can reconnect the broken ends very rapidly and hold them together. I will also show how we can use this instrument for the study of mitotic chromosomes. These structures are highly dynamic throughout the cell cycle, and undergo compaction during mitosis to adopt the characteristic “X-shape”. Here I introduce a workflow to interrogate the organization of human chromosomes based on optical trapping and manipulation. This allows high-resolution force measurements and fluorescence visualization of native metaphase chromosomes to be conducted under tightly controlled experimental conditions. The methods described here open the door to a wide array of investigations into the structure and dynamics of both normal and disease-associated chromosomes.

Regional redistribution of CB1 cannabinoid receptors in human foetal brains with Down's syndrome and their functional modifications in Ts65Dn+/+ mice.

The endocannabinoid system with its type 1 cannabinoid receptor (CB1 R) expressed in postmitotic neuroblasts is a critical chemotropic guidance module with its actions cascading across neurogenic commitment, neuronal polarisation and synaptogenesis in vertebrates. Here, we present the systematic analysis of regional CB1 R expression in the developing human brain from gestational week 14 until birth. In parallel, we diagrammed differences in CB1 R development in Down syndrome foetuses and identified altered CB1 R signalling. Foetal brains with normal development or with Down's syndrome were analysed using standard immunohistochemistry, digitalised light microscopy and image analysis (NanoZoomer). CB1 R function was investigated by in vitro neuropharmacology from neonatal Ts65Dn transgenic mice brains carrying an additional copy of ~90 conserved protein-coding gene orthologues of the human chromosome 21. We detected a meshwork of fine-calibre, often varicose processes between the subventricular and intermediate zones of the cortical plate in the late first trimester, when telencephalic fibre tracts develop. The density of CB1 Rs gradually decreased during the second and third trimesters in the neocortex. In contrast, CB1 R density was maintained, or even increased, in the hippocampus. We found the onset of CB1 R expression being delayed by ≥1month in age-matched foetal brains with Down's syndrome. In vitro, CB1 R excitation induced excess microtubule stabilisation and, consequently, reduced neurite outgrowth. We suggest that neuroarchitectural impairments in Down's syndrome brains involve the delayed development and errant functions of the endocannabinoid system, with a particular impact on endocannabinoids modulating axonal wiring.

Investigating human chromosome organization by whole-genome simulations.

Both theoreticians and experimentalists have been dedicating significant effort to studying chromosome organization and its relation to gene expression and regulation. Many experimental procedures were designed, such as Hi-C or ChIA-PET, to capture structural information of chromosomes. Several theoretical models were proposed to understand the principles underlying chromosome compartmentalization. MiChroM is a model that describes the compartmentalization behavior of interphase chromosomes. Compared to experimental data, the agreement of the in silico contact maps suggested that phase separation of A/B compartments is a key feature determining the structure of individual chromosomes. In this work, we use the Open-MiChroM software to simulate the 46 chromosomes of a GM12878 human cell at 50 kb resolution, seeking to understand whether the nuclear organization of the whole genome would rely on the same principles observed for the organization of a single chromosome. The ensemble of structures generated by our simulations captures chromosome organization within territories and phase separation of chromatin compartments. Inside a chromosome territory, active chromatin localizes preferentially in the periphery, while inactive chromatin lies on the inside. The intra-chromosomal contact maps and the shape metrics of each territory are consistent with experimental data. The resulting genome architecture resembles the inverted nucleus configuration, with active chromatin towards the periphery. In the interface between chromosomes, we also observe phase separation of compartments. However, the inter-chromosomal maps present higher contact probabilities for heterochromatin loci when compared to the experimental data. These results suggest that the interactions of chromatin with other nuclear features like the lamina greatly influences inter-chromosomal interactions while also affecting the overall positioning of chromosomes in the nucleus.