Human Chromosome Research Articles

Detecting higher-order chromosome interactions by polymer modeling

Recent experiments have revealed that human chromosomes have an intricate three-dimensional architecture in the cell nucleus, encompassing a hierarchy of interactions across multiple genomic scales. Despite those advances, detecting multiway DNA contacts at the single-molecule level remains challenging. Here, we use polymer physics models to investigate higher-order chromosomal contacts within a key genomic region in human colorectal cancer cells. By running extensive Molecular Dynamics simulations, we test model predictions against available experimental data and show that the model ensemble of single-molecule conformations significantly captures complex multiway chromosomal interactions beyond mere pairwise contacts.

Characterization of novel human endogenous retrovirus structures on chromosomes 6 and 7

Human endogenous retroviruses (HERV) represent nearly 8% of the human genome. Of these, HERV-K subtype HML-2 is a transposable element that plays a critical role in embryonic development and in the pathogenesis of several diseases. Quantification and characterization of these multiple HML-2 insertions in the human chromosome has been challenging due to their size, sequence homology with each other, and their repetitive nature. We examined a cohort of 222 individuals for HML-2 proviruses 6q14.1 and 7p22.1a, two loci that are capable of producing full-length viral proteins and have been previously implicated in several cancers, autoimmune disorders and neurodegenerative diseases, using long-read DNA sequencing. While the reference genome for both regions suggests these two loci are structurally dissimilar, we found that for both loci about 5% of individuals have a unique tandem repeat-like sequence (three long terminal repeat sequences sandwiching two internal, potentially protein coding sequences), while most individuals have a standard proviral structure (one internal region sandwiched by two long terminal repeats). Moreover, both proviruses can make full-length, or nearly full-length, HERV-K proteins in multiple transcription orientations. The amino acid sequences from different loci in the same transcriptional orientation share sequence homology with each other. These results demonstrate a clear, previously unreported, relationship between HML-2 loci 6q14.1 and 7p22.1a and highlight the utility of long-read sequencing to study repetitive elements. Future studies need to determine if these polymorphisms determine genetic susceptibility to diseases that are associated with them.

Centromeric chromatin clearings demarcate the site of kinetochore formation.

The centromere is the chromosomal locus that recruits the kinetochore, directing faithful propagation of the genome during cell division. Using cryo-ET on human mitotic chromosomes, we reveal a distinctive architecture at the centromere: clustered 20- to 25-nm nucleosome-associated complexes within chromatin clearings that delineate them from surrounding chromatin. Centromere components CENP-C and CENP-N are each required for the integrity of the complexes, while CENP-C is also required to maintain the chromatin clearing. We find that CENP-C is required in mitosis, not just for kinetochore assembly, likely reflecting its role in organizing the inner kinetochore during chromosome segregation. We further visualize the scaffold of the fibrous corona, a structure amplified at unattached kinetochores, revealing crescent-shaped parallel arrays of fibrils extending >1μm. Thus, we reveal how the organization of centromeric chromatin creates a clearing at the site of kinetochore formation as well as the nature of kinetochore amplification mediated by corona fibrils.

Titin fragment is a sensitive biomarker in Duchenne muscular dystrophy model mice carrying full-length human dystrophin gene on human artificial chromosome

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations of the dystrophin gene, which spans 2.4 Mb on the X chromosome. Creatine kinase (CK) activity in blood and titin fragment levels in urine have been identified as biomarkers in DMD to monitor disease progression and evaluate therapeutic intervention. However, the difference in the sensitivity of these biomarkers in DMD remains unclear. Previously, we generated transchromosomic mice carrying the full-length human dystrophin gene on a human artificial chromosome (DYS-HAC1) vector. The human dystrophin derived from DYS-HAC1 improved pathological phenotypes observed in DMD-null mice, which lack the entire 2.4 Mb of the dystrophin gene. In this study, we compared the values of plasma CK activity and urine/plasma titin fragment levels in wild-type (WT), DYS-HAC1, DMD-null, and DYS-HAC1; DMD-null mice. Plasma CK activity and urine/plasma titin fragment levels in DMD-null mice were significantly higher than those in WT mice. Although plasma CK activity showed no significant difference between WT and DYS-HAC1; DMD-null mice, urine/plasma titin fragment levels in DYS-HAC1; DMD-null mice were higher than those in WT mice. Human dystrophin in DYS-HAC1; DMD-null mice drastically improved muscular dystrophy phenotypes seen in DMD-null mice; however, the proportion of myofibers with central nuclei in DYS-HAC1; DMD-null mice had a tendency to be slightly higher than that in WT mice. These results suggest that urine/plasma titin fragment levels could be a more sensitive biomarker than plasma CK activity.

Post-transcriptional cross- and auto-regulation buffer expression of the human RNA helicases DDX3X and DDX3Y.

The Y-linked gene DDX3Y and its X-linked homolog DDX3X survived the evolution of the human sex chromosomes from ordinary autosomes. DDX3X encodes a multifunctional RNA helicase, with mutations causing developmental disorders and cancers. We find that, among X-linked genes with surviving Y homologs, DDX3X is extraordinarily dosage sensitive. Studying cells of individuals with sex chromosome aneuploidy, we observe that when the number of Y Chromosomes increases, DDX3X transcript levels fall; conversely, when the number of X Chromosomes increases, DDX3Y transcript levels fall. In 46,XY cells, CRISPRi knockdown of either DDX3X or DDX3Y causes transcript levels of the homologous gene to rise. In 46,XX cells, chemical inhibition of DDX3X protein activity elicits an increase in DDX3X transcript levels. Thus, perturbation of either DDX3X or DDX3Y expression is buffered: by negative cross-regulation of DDX3X and DDX3Y in 46,XY cells and by negative auto-regulation of DDX3X in 46,XX cells. DDX3X-DDX3Y cross-regulation is mediated through mRNA destabilization-as shown by metabolic labeling of newly transcribed RNA-and buffers total levels of DDX3X and DDX3Y protein in human cells. We infer that post-transcriptional auto-regulation of the ancestral (autosomal) DDX3X gene transmuted into auto- and cross-regulation of DDX3X and DDX3Y as these sex-linked genes evolved from ordinary alleles of their autosomal precursor.

Cortex-specific Tmem169 Deficiency Induces Defects in Cortical Neuron Development and Autism-like Behaviors in Mice.

The development of the nervous system is a complex process, with many challenging scientific questions yet to be resolved. Disruptions in brain development are strongly associated with neurodevelopmental disorders, such as intellectual disability and autism. While the genetic basis of autism is well established, the precise pathological mechanisms remain unclear. Variations on chromosome 2q have been linked to autism, yet the specific genes responsible for the disorder have not been identified. This study investigates the role of the transmembrane protein 169 (Tmem169) gene, located on human chromosome 2q35, which has not been previously characterized. Our findings indicate that Tmem169 is highly expressed in the nervous system, and its deletion in the male mouse dorsal forebrain results in neuronal morphological abnormalities and synaptic dysfunction. Notably, Tmem169-deficient mice, irrespective of sex, display behavioral traits resembling those observed in individuals with autism. These results suggest that Tmem169 interacts with several key neuronal proteins, many of which are implicated in neurodevelopmental diseases. Furthermore, we demonstrate that Tmem169 promotes neuronal process and synapse development through its interaction with Shank3.Significance statement The prevalence of autism worldwide is estimated to be around 1-2%, causing significant burdens for both patients and their families. Our research reveals that Tmem169 not only interacts with important protein products of genes associated with autism, but also influences the levels of many critical synaptic proteins at synapses. We also demonstrated Tmem169 modulates neuronal process and synapse development at least partially via Shank3. Specific knockout of the Tmem169 gene in the cortex of mice induce autism-like behaviors. These findings provide the first insight into the role of the Tmem169 gene in neurodevelopment and offer new avenues for potential treatments and serve as an important theoretical basis for early screening and enhancing population quality related to autism.

Characterizing executive functioning and associated behaviors in individuals with dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) syndrome.

DYRK1A, a protein kinase located on human chromosome 21, plays a role in postembryonic neuronal development and degeneration. Alterations to DYRK1A have been consistently associated with cognitive functioning and neurodevelopmental disorders (e.g., autism, intellectual disability). However, the broader cognitive and behavioral phenotype of DYRK1A syndrome requires further characterization. Specifically, executive functioning, or cognitive processes that are necessary for goal-directed behavior, has not yet been characterized in this population. Individuals with DYRK1A variants (n = 29; ages 4 to 21 years) were assessed with a standardized protocol with multiple measures of executive functioning: Delis-Kaplan Executive Function Schedule, and chronologically age-appropriate caregiver-report forms of the Behavior Rating Inventory of Executive Function (BRIEF) and Achenbach System of Empirically Based Assessment (ASEBA). We first examined the feasibility and appropriateness of established executive functioning measures among participants with DYRK1A syndrome to inform selection of executive functioning tools in future research. We then characterized executive functioning among the group, including associations with other phenotypic features. Neurocognitive assessments of executive functioning were deemed infeasible due to cognitive and verbal functioning. Caregiver-report revealed elevated executive functioning concerns related to self-monitoring, working memory, and planning/organization on the BRIEF, and attention and ADHD on the CBCL. Only two participants had existing ADHD diagnoses; however, 5 participants (out of 10 participants with data) exceeded the cutoff on the BRIEF, 13 individuals (out of 27 with data) exceeded the cutoff on the ASEBA ADHD subscale, and 18 exceeded the cutoff on the ASEBA attention subscale. There was concordance between ADHD diagnosis and the ASEBA, but not BRIEF. Executive functioning was correlated with nonverbal IQ and autism traits. Objective measures of executive functioning are needed for individuals with intellectual disability who are nonverbal and/or have motor limitations. Diagnostic overshadowing, or the tendency to attribute all problems to intellectual disability and to leave other co-existing conditions, such as executive functioning challenges or ADHD, undiagnosed, is common. Phenotypic characterization of executive functioning is therefore important for our understanding of DYRK1A syndrome and for ensuring that caregivers' concerns are addressed, and individuals receive the clinical services that best meet their needs.

The Dual Role of ADAMTS9-AS1 in Various Human Cancers: Molecular Pathogenesis and Clinical Implications.

Long non-coding RNA (lncRNA) is a type of non-coding RNA distinguished by a length exceeding 200 nucleotides. Recent studies indicated that lncRNAs participate in various biological processes, such as chromatin remodeling, transcriptional and post-transcriptional regulation, and the modulation of cell proliferation, death, and differentiation, hence influencing gene expression and cellular function. ADAMTS9-AS1, an antisense long non-coding RNA situated on human chromosome 3p14.1, has garnered significant interest due to its pivotal involvement in the advancement and spread of diverse malignant tumors. ADAMTS9-AS1 functions as a competitive endogenous RNA (ceRNA) that interacts with multiple microRNAs (miRNAs) and plays a crucial role in regulating gene expression and cellular functions by modulating essential signaling pathways, including PI3K/AKT/mTOR, Wnt/β-catenin, and Ras/MAPK pathways. Dysregulation of this factor has been linked to tumor development, migration, invasion, and resistance to apoptotic mechanisms, including as iron-induced apoptosis, underscoring its intricate function in cancer pathology. While current research has clarified certain pathways involved in cancer formation, additional clinical and in vivo investigations are necessary to enhance comprehension of its specific involvement across various cancer types. This review encapsulates the recent discoveries on the correlation of ADAMTS9-AS1 with numerous malignancies, clarifying its molecular mechanisms and its prospective role as a therapeutic target in oncology. Furthermore, it identifies ADAMTS9-AS1 as a potential early diagnostic biomarker and therapeutic target, offering novel opportunities for targeted intervention in oncology.

Human artificial chromosome carrying R-spondin1 and IL-22 expression cassettes in rejuvenated MSCs enhances therapeutic efficacy in ulcerative colitis model mice.

Ulcerative colitis (UC) is an incurable intestinal disease, with current treatments mainly focused on inflammation control and, in severe cases, surgical resection. Recent studies have highlighted the need for new therapies that promote tissue regeneration. R-spondin-1 (RSPO1) and interleukin-22 (IL-22) have shown anti-inflammatory and regenerative effects in UC models, but have short half-lives and poor targeting abilities. Another therapeutic tool, mesenchymal stem cells (MSCs), offer promising migratory and homing capabilities; however, the preparation of homogeneous therapeutic MSCs at sufficient levels in vitro is challenging. Therefore, we developed a novel line of MSCs (HAC-MSC) with significant therapeutic effects in dextran sodium sulfate (DSS)-induced colitis mice. Construction of HAC-MSC involved a two-part strategy: 1) establishment of a non-integrating human artificial chromosome (HAC) vector carrying therapeutic genes encoding IL22 and RSPO1; and 2) transfer of the HAC to previously characterized rejuvenated MSCs (rej-MSCs) prepared using Sendai virus technology for prolonged proliferation capacity in vitro. HAC-MSC stably and efficiently produced therapeutic factors in vitro and, following intraperitoneal administration to DSS-induced colitis mice, showed continuous expression of the therapeutic factors over 5 days. Additionally, HAC-MSC-treated mice showed alleviation of the disease activity index score, reduced depth of injury, and promotion of intestinal growth compared with MSC-treated mice. Furthermore, effective treatment with HAC-MSC required only a fraction (1 %-10 %) of the number of cells needed for conventional MSC therapy. These findings highlight the outstanding potential of rej-MSCs carrying therapeutic factor-loaded HACs as a cell therapy tool with prospective applications in the treatment of UC and other diseases.

InVivo CRISPR Activation Screening Reveals Chromosome 1q Genes VPS72, GBA1, and MRPL9 Drive Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) frequently undergoes regional chromosomal amplification, resulting in elevated gene expression levels. We aimed to elucidate the role of these poorly understood genetic changes by using CRISPR activation (CRISPRa) screening in mouse livers to identify which genes within these amplified loci are cancer driver genes. We used data from The Cancer Genome Atlas to identify that frequently copy number-amplified and up-regulated genes all reside on human chromosomes 1q and 8q. We generated CRISPRa screening transposons that contain oncogenic Myc to drive tumor formation. We conducted CRISPRa screens invivo in the liver to identify tumor driver genes. We extensively validated the findings in separate mice and performed RNA sequencing analysis to explore mechanisms driving tumorigenesis. We targeted genes that frequently undergo amplification in human HCC using an invivo CRISPRa screening system in mice, which induced extensive liver tumorigenesis. Human chromosome 1q genes Zbtb7b, Vps72, Gba1, and Mrpl9 emerged as drivers of liver tumorigenesis. In human HCC there is a trend in correlation between levels of MRPL9, VPS72, or GBA1 and poor survival. In validation assays, activation of Vps72, Gba1, or Mrpl9 resulted in extensive liver tumorigenesis and decreased survival in mice. RNA sequencing revealed different mechanisms driving HCC, with Mrpl9 activation altering genes functionally related to mitochondrial function, Vps72 levels altering phospholipid metabolism, and Gba1 activation enhancing endosomal-lysosomal activity, all leading to promotion of cellular proliferation. Analysis of human tumor tissues with high levels of MRPL9, VPS72, or GBA1 revealed congruent results, indicating conserved mechanisms driving HCC. This study reveals chromosome 1q genes Vps72, Gba1, and Mrpl9 as drivers of HCC. Future efforts to prevent or treat HCC can focus on these new driver genes.

Reduction of Cystatin B results in increased cathepsin B activity in disomic but not Trisomy 21 human cellular and mouse models.

Down syndrome, resulting from trisomy of human chromosome 21, is a common form of chromosomal disorder that results in intellectual disability and altered risk of several medical conditions. Individuals with Down syndrome have a greatly increased risk of Alzheimer's disease (DSAD), due to the presence of the APP gene on chromosome 21 that encodes the amyloid-β precursor protein (APP). APP can be processed to generate amyloid-β, which accumulates in plaques in the brains of people who have Alzheimer's disease and is the upstream trigger of disease. Cathepsin B has potential roles in both APP processing and amyloid-β degradation and has been suggested to contribute to amyloid-β accumulation. An endogenous inhibitor of Cathepsin B, Cystatin B (CSTB), is encoded on chromosome 21. The abundance of this protein is increased in the brains of individuals with DSAD, which may be associated with a decrease in Cathepsin B activity compared to individuals who have Alzheimer's disease in the general population. Whether targeting CSTB can modulate Cathepsin B activity in the context of trisomy of chromosome 21 is unclear. Here we test if reducing CSTB can alter Cathepsin B activity in a mouse and a cellular model of trisomy of chromosome 21. We find that reducing CSTB abundance increases Cathepsin B activity in disomic controls but not in the presence of trisomy of chromosome 21. These findings offer new insights into the role of CSTB in regulating Cathepsin B activity.

Screening Methods to Discover the FDA-Approved Cancer Drug Encorafenib as Optimally Selective for Metallothionein Gene Loss Ovarian Cancer.

All 11 metallothionein protein-coding genes are located on human chromosome 16q13. It is unique among human genetics to have an entire pathway's genes clustered in a short chromosomal region. Since solid tumors, particularly high-grade serous ovarian cancer (HGSC), exhibit high rates of monoallelic aneuploidy, this region is commonly lost. Studies have not yet been performed to determine what vulnerability may be created in cancer cells with low metallothionein expression. Here, a screen of FDA-approved cancer small molecule drugs for those best targeting low metallothionein ovarian cancer was completed. Screening methods were tested and compared using vehicle-treated negative controls and cadmium chloride, a positive control for cell loss selective for low metallothionein cells. CAOV3 cells, which are unique in their expression of only two metallothionein isoforms, were used, with or without shRNA knockdown of the predominantly expressed MT2A gene. A library of FDA-approved molecules was then screened. The optimal assay utilized Hoechst 33342 nuclear staining and mechanized fluorescent microscope counting of cell content. Encorafenib, an RAF inhibitor, was identified as the most selective for enhanced cytotoxicity in MT2A knockdown cells compared to scrambled controls. The nuclear stain Hoechst 33342, assessed by fluorescence microscopy, provides a low variance, moderate throughput platform for cancer cell loss screens. Low metallothionein ovarian cancer cells exhibit a vulnerability to the RAF inhibitor encorafenib.

A Hypothesis: Metabolic Contributions to 16p11.2 Deletion Syndrome

ABSTRACT16p11.2 deletion syndrome is a severe genetic disorder associated with the deletion of 27 genes from a Copy Number Variant region on human chromosome 16. Symptoms associated include cognitive impairment, language and motor delay, epilepsy or seizures, psychiatric disorders, autism spectrum disorder (ASD), changes in head size and body weight, and dysmorphic features, with a crucial need to define genes and mechanisms responsible for symptomatology. In this review, we analyze the clinical associations and biological pathways of 16p11.2 locus genes and identify that a majority of 16p11.2 genes relate to metabolic processes. We present a hypothesis in which changes in the dosage of 16p11.2 metabolic genes contribute to pathology through direct or indirect alterations in pathways that include amino acids or proteins, DNA, RNA, catabolism, lipid, energy (carbohydrate). This hypothesis suggests that research into the specific roles of each metabolic gene will help identify useful therapeutic targets.

LINE1 elements at distal junctions of rDNA repeats regulate nucleolar organization in human embryonic stem cells.

The nucleolus is a major subnuclear compartment where ribosomal DNA (rDNA) is transcribed and ribosomes are assembled. In addition, recent studies have shown that the nucleolus is a dynamic organizer of chromatin architecture that modulates developmental gene expression. rDNA gene units are assembled into arrays located in the p-arms of five human acrocentric chromosomes. Distal junctions (DJs) are ∼400 kb sequences adjacent to rDNA arrays that are thought to anchor them at the nucleolus, although the underlying regulatory elements remain unclear. Here we show that DJs display a dynamic chromosome conformation profile in human embryonic stem cells (hESCs). We identified a primate-specific, full-length insertion of the retrotransposon long interspersed nuclear element 1 (LINE1) in a conserved position across all human DJs. This DJ-LINE1 locus interacts with specific regions of the DJ and is upregulated in naïve hESCs. CRISPR-based deletion and interference approaches revealed that DJ-LINE1 contributes to nucleolar positioning of the DJs. Moreover, we found that the expression of DJ-LINE1 is required for maintenance of the structure and transcriptional output of the nucleolus in hESCs. Silencing of DJ-LINE1 leads to loss of self-renewal, disruption of the landscape of chromatin accessibility, and derepression of earlier developmental programs in naïve hESCs. This work uncovers specific LINE1 elements with a fundamental role in nucleolar organization in hESCs and provides new insights into how the nucleolus functions as a key genome-organizing hub.

Infantile Spasms in Pediatric Down Syndrome: Potential Mechanisms Driving Therapeutic Considerations.

Infantile spasms are common in Down Syndrome (DS), but the mechanisms by which DS predisposes to this devastating epilepsy syndrome are unclear. In general, neuronal excitability and therefore seizure predisposition results from an imbalance of excitation over inhibition in neurons and neural networks of the brain. Animal models provide clues to mechanisms and thereby provide potential therapeutic approaches. Ts65Dn mice have been the most widely used animal model of DS. In this model, there is evidence for both abnormal cerebral excitation and inhibition: infantile spasms-like clinical and electrographic activity can be elicited by the administration of gamma-aminobutyric acid (GABA)-B receptor agonist, gamma-butyrolactone (GBL), and depolarizing GABA-A responses persist beyond the age of their usual switch to hyperpolarized responses. But despite its widespread use, the Ts65Dn model may be suboptimal because of the absence of numerous genes that are triplicated in human DS and the presence of numerous genes that are not triplicated in human DS. Recently, a transchromosomic mouse artificial chromosome 21 (TcMAC21) mouse model has been developed, which carries a copy of human chromosome 21 and therefore has a genetic composition more similar to human DS. As in Ts65Dn mice, exposure of TcMAC21 mice to GBL results in epileptic spasms, and aberrant excitation has also been demonstrated. This review summarizes excitatory and inhibitory dysfunction in models of DS that may play a role in the generation of seizures and infantile spasms, providing a perspective on past studies and a prelude for future ones. Further elucidation will hopefully lead to rational therapeutic options for DS children with infantile spasms.

Locus-specific differential expression of human satellite sequences in the nuclei of cancer cells and heat-shocked cells

ABSTRACT Human satellites (HSats) are pericentric, tandemly repeating satellite DNA sequences in the human genome. While silent in normal cells, a subset of HSat2 noncoding RNA is expressed and accumulates in the nucleus of cancer cells. We developed a FISH-based approach for identification of the distribution of three subfamilies of HSat2 (A1, A2, B) sequences on individual human chromosomes. Further, using the HSat subfamily annotations in the T2T completed centromere satellite (CenSat) sequence, we isolated, defined and mapped differentially expressed sequence variants of nuclear-restricted HSat2 and HSat3 RNA from cancer cell lines and heat-shocked cells. We identified chromosome-specific and subfamily-specific expression of HSat2 and HSat3 and established a computational pipeline for differential expression analysis of tandemly repeated satellite sequences. Results suggest the differential expression of chromosome-specific HSat2 arrays in the human genome may underlie their accumulation in cancer cells and that specific HSat3 loci are upregulated upon heat shock.

Male Down syndrome Ts65Dn mice have impaired bone regeneration.

Trisomy of human chromosome 21 (Ts21) individuals present with a spectrum of low bone mineral density (BMD) that predisposes this vulnerable group to skeletal injuries. To determine the bone regenerative capacity of Down syndrome (DS) mice, male and female Dp16 and Ts65Dn DS mice underwent amputation of the digit tip (the terminal phalanx (P3)). This is a well-established mammalian model of bone regeneration that restores the amputated skeletal segment and all associated soft tissues. P3 amputation was performed in 8-week-old male and female DS mice and WT controls and followed by in vivo μCT, histology and immunofluorescence. Following P3 amputation, the bone degradation phase was attenuated in both Dp16 and Ts65Dn males. In Dp16 males, P3 regeneration was delayed but complete by 63days post amputation (DPA); however, male Ts65Dn exhibited attenuated regeneration by 63 DPA. In both Dp16 and Ts65Dn female DS mice, P3 regenerates were indistinguishable from WT by 42 DPA. In Ts65Dn males, osteoclasts and eroded bone surface were significantly reduced, and osteoblast number significantly decreased in the regenerating digit. In Ts65Dn females, no significant differences were observed in any osteoclast or osteoblast parameter. Like Ts21 individuals and DS mice with sex differences in bone mass, these data expand the characteristic sexually dimorphism to include bone resorption and regeneration in response to skeletal injury in Ts65Dn mice. These observations suggest that sex differences contribute to the poor bone healing of DS and compound the increased risk of bone injury in the Ts21 population.

The Role of Synaptic Cargo Transporters in Regulating Neuronal Excitation/Inhibition Balance in Autism Spectrum Disorders: A Focus on Syntabulin-syntaxin 1 A/B Axis

Various cargo vesicles containing presynaptic proteins are transported from the neuronal cell body to the neuronal terminal to aid in active zone formation. Researchers showed altered levels up to 25 different synaptic proteins (SNAP47, GRIA3/4, GAP43, synaptotagmin 2, LRFN2, SV2C) and syntabulin. Syntabulin is a key component gene of a kinesin motor-adaptor complex essential for the forward movement of active zone components in axons. It plays a crucial role in the assembly of presynaptic structures during neuronal development in response to activity. However, the specific membranous cargoes and motor-cargo interactions are not fully understood. Recent research identified a syntaxin-1-binding protein called syntabulin. Syntabulin links syntaxin-containing vesicles to microtubules and moves with syntaxin in neuronal brain processes. Knocking down syntabulin expression or disrupting the syntabulin-syntaxin interaction hinders the attachment of syntaxin-cargo vesicles to microtubules and reduces syntaxin-1 distribution in neuronal processes. Conventional kinesin I heavy chain binds to syntabulin and associates with syntabulin-linked syntaxin vesicles in vivo. Syntabulin could act as a linker molecule that connects syntaxin-cargo vesicles to kinesin I, facilitating the transport of syntaxin-1 to neuronal processes. Syntabulin also regulates neuronal excitatory and inhibitory imbalance by transporting syntaxin 1 B, more than syntaxin 1A, to the presynaptic membrane. The gene for Syntabulin is found on human chromosome 8q23.2, in mouse on chromosome 15. This review shed light on the role of syntabulin-syntaxin 1A/B axis and mention different synaptic cargo transporters which could play any role in autism spectrum disorders. Further research should focus to study synaptopathies in autism spectrum disorders to open new arenas for pharmaceutical interventions.

Generation of Monosomy 21q Human iPS Cells by CRISPR/Cas9-Mediated Interstitial Megabase Deletion.

Missing an entire chromosome or chromosome arm in normal diploid cells has a deleterious impact on cell viability, which may contribute to the development of specific birth defects. Nevertheless, the effects of chromosome loss in human cells have remained unexplored due to the lack of suitable model systems. Here, we developed an efficient, selection-free approach to generate partial monosomy in human induced pluripotent stem cells (iPSCs). The introduction of Cas9 proteins and a pair of gRNAs induces over megabase-sized interstitial chromosomal deletions. Using human chromosome 21 (HSA21) as a model, partial monosomy 21q (PM21q) iPSC lines with deletions ranging from 4.5 to 27.9 Mb were isolated. A 33.6 Mb deletion, encompassing all protein-coding genes on 21q, was also achieved, establishing the first 21q monosomy human iPSC line. Transcriptome and proteome analyses revealed that the abundances of mRNA and protein encoded by the majority of genes in the monosomic regions are half of the diploid expression level, indicating an absence of dosage compensation. The ability to generate customized partial monosomy cell lines on an isogenic, karyotypically normal background should facilitate the gain of novel insights into the impact of chromosome loss on cellular fitness.

Co-infection of HSV-1 amplicons containing the XPC gene and a human artificial chromosome vector into primary XPC deficient fibroblast cells

Gene therapy for xeroderma pigmentosum (XP), a rare, recessive DNA repair disease, has been considered since defects in XP genes result in severe and debilitating symptoms. Mutations in the XPC DNA repair gene result in a more that 1000-fold increased sensitivity to sunlight-induced skin cancer. The XPC gene is large (33 Kb) and the entire genomic locus is a difficult candidate for many gene therapy vectors to incorporate into their system by conventional cloning. Artificial chromosome vectors were developed to accommodate large genes and their regulatory sequences to allow full gene expression in cells. The HSV-1 human artificial chromosome (HAC) vectors we previously generated incorporated genes up to 100 Kb in a single vector. Subsequently, we modified the system to allow larger (>100 Kb) DNA gene sequences to be introduced by simultaneously infecting cells with two separate HSV-1 vector particles, one containing DNA required for HAC formation and the other with the desired gene. Following transduction, recombination of DNA formed a gene expressing HAC in vitro. The dual transduction system was successful for introduction and expression of the HPRT gene in human 3D engineered tissues and stem cells. In this study, we report the XPC gene delivery and transient gene expression via the dual transduction system in human cultured fibrosarcoma (HT1080) and primary XPC deficient patient cells.