Chromatin Packaging Research Articles

Modeling High-Order Chromatin Structure in Single Cells

Chromosome conformation capture techniques have discovered territorial organization of chromatin at an ensemble-averaged level. However, how chromatin folds at the single-cell level still remains elusive. Here, we introduce a self-returning random walk to describe the high-order structure of chromatin in single cells. Based on a simple folding algorithm, our model unifies the high contact frequency discovered by genomic techniques, and the high structural heterogeneity revealed by imaging techniques, which two chromatin properties we theoretically prove to be irreconcilable within a fractal polymer framework. Our model provides a holistic view of chromatin folding, in which the topologically associating domains are liquid-tree-like structures, linked and isolated by stretched out, transcriptionally active chromatin backbone to form a secondary structure of chromatin that further folds into a “3D forest” under confinement. Our theoretical predictions compare favorably with experimental observations of chromatin scaling and packing heterogeneity, which leads to the hypothesis that well-organized 3D structures such as domains and compartments are intrinsic chromatin characteristics at the single-cell level rather than merely statistical patterns of a large population. Based on a global folding parameter, the model reveals a unique structure-function relation that couples many chromatin structural properties under genomic response to environmental stress. Our results suggest the existence of universal folding principles for chromatin to fulfill its regulatory functions.

Quantifying Three-Dimensional Chromatin Packing through Electron Tomography

Quantifying Three-Dimensional Chromatin Packing through Electron Tomography

Influence of Micropillar Induced Deformation on Chromatin Architecture in Regulating Stem Cell Differentiation

Surface topography induced nuclear deformation in stem cells has previously shown to influence differentiation outcomes. Mechanical forces resulting from topographical interventions get transduced into the nucleus through the cytoskeleton, causing changes in chromatin architecture and gene expression. However, their effect on genome architecture and gene expression in regulating stem cell differentiation is not well understood. Here, we study the influence of nuclear deformation induced by micropillar surfaces composed of methacrylated poly (1,8-octamethylene citrate) on chromatin architecture and gene expression during human mesenchymal stem cell differentiation (hMSC) towards osteogenic and adipogenic lineages. Differentiation assays reveal that these micropillars facilitate osteogenic differentiation, and attenuate adipogenic differentiation. It has been shown that changes in chromatin nanoarchitecture regulate global gene expression patterns and that an increase in chromatin packing scaling corresponds to an increase in the dynamic range and variations in gene expression. We hypothesized that micropillars enhance osteogenic differentiation by limiting the transcriptional accessibility of the initially plastic stem cells to explore their genomic landscape, primarily, by modulating their chromatin packing scaling. Using Partial Wave Spectroscopic (PWS) microscopy, we show that hMSCs cultured on micropillars have decreased chromatin packing scaling compared to those cultured on flat surfaces. Moreover, corresponding RNA-Seq studies show significant downregulation of genes associated with cell proliferation, and, of those involved in inhibition of osteogenic differentiation in MSCs seeded on micropillars compared to the flat surface. Therefore, by combining nanoscale-imaging experiments involving PWS and Electron Microscopy with enhanced chromatin contrast (ChromEM), and sequencing performed on stem cells induced with osteogenic and adipogenic medium, this study aims to understand the relationship between nuclear deformation induced by micropillars, chromatin architecture, and expression of differentiation-specific transcriptional programs.

Progresses in the plant 3D chromatin architecture.

Chromatin architecture involves the patterns of chromatin coiling and packing as well as the mutual relative allocations of different chromatins. Besides the canonical microscopic observations, the chromatin architectural capture techniques, including the Hi-C and ChIA-PET, have been widely applied in characterization of chromatin architecture in various plant and animal model species, in which chromatin architectural features, such as the chromosome territory, compartment A/B, topological associated domains (TADs) and chromatin loops, were defined. As for the studies in plant species, replying on the two techniques above (with differences in experimental techniques and data structures), scientists have compared the variation of specific chromatin architecture features across species and/or in different cell types of the same plant species, besides detailed analyses in each individual model. Here, we mainly review the recent progresses in studies of plant chromatin architectures, in which their composition, establishing mechanism and effective factors were described and discussed. We also propose the main technical bottlenecks, describe the breaking-through progresses, and anticipate future research directions, which may offer more theoretical references for related researches in the field.

Disordered chromatin packing regulates phenotypic plasticity.

Three-dimensional supranucleosomal chromatin packing plays a profound role in modulating gene expression by regulating transcription reactions through mechanisms such as gene accessibility, binding affinities, and molecular diffusion. Here, we use a computational model that integrates disordered chromatin packing (CP) with local macromolecular crowding (MC) to study how physical factors, including chromatin density, the scaling of chromatin packing, and the size of chromatin packing domains, influence gene expression. We computationally and experimentally identify a major role of these physical factors, specifically chromatin packing scaling, in regulating phenotypic plasticity, determining responsiveness to external stressors by influencing both intercellular transcriptional malleability and heterogeneity. Applying CPMC model predictions to transcriptional data from cancer patients, we identify an inverse relationship between patient survival and phenotypic plasticity of tumor cells.

Nucleolar dynamics and interactions with nucleoplasm in living cells.

Liquid-liquid phase separation (LLPS) has been recognized as one of the key cellular organizing principles and was shown to be responsible for formation of membrane-less organelles such as nucleoli. Although nucleoli were found to behave like liquid droplets, many ramifications of LLPS including nucleolar dynamics and interactions with the surrounding liquid remain to be revealed. Here, we study the motion of human nucleoli in vivo, while monitoring the shape of the nucleolus-nucleoplasm interface. We reveal two types of nucleolar pair dynamics: an unexpected correlated motion prior to coalescence and an independent motion otherwise. This surprising kinetics leads to a nucleolar volume distribution, [Formula: see text], unaccounted for by any current theory. Moreover, we find that nucleolus-nucleoplasm interface is maintained by ATP-dependent processes and susceptible to changes in chromatin transcription and packing. Our results extend and enrich the LLPS framework by showing the impact of the surrounding nucleoplasm on nucleoli in living cells.

Irregular Chromatin: Packing Density, Fiber Width, and Occurrence of Heterogeneous Clusters

How chromatin is folded on the length scale of a gene is an open question. Recent experiments have suggested that, in vivo, chromatin is folded in an irregular manner and not as an ordered fiber with a width of 30 nm that is expected from theories of higher order packaging. Using computational methods, we examine how the interplay between DNA-bending nonhistone proteins, histone tails, intrachromatin electrostatic, and other interactions decide the nature of the packaging of chromatin. We show that although the DNA-bending nonhistone proteins make the chromatin irregular, they may not alter the packing density and size of the fiber. We find that the length of the interacting region and intrachromatin electrostatic interactions influence the packing density, clustering of nucleosomes, and the width of the chromatin fiber. Our results suggest that the heterogeneity in the interaction pattern will play an important role in deciding the nature of the packaging of chromatin.

Relationship between Sperm Parameters with Sperm Function Tests in Infertile Men with at Least One Failed Cycle after Intracytoplasmic Sperm Injection Cycle.

BackgroundImbalance between production of reactive oxygen species (ROS) and total antioxidant capacity in testis, epididymis, and seminal fluid can eventually lead to infertility. Abnormal sperm chromatin packaging, and DNA fragmentation is considered as the main underlying causes of infertility. Therefore, we aimed to assess relationship between sperm parameters with DNA damage, protamine deficiency, persistent histones, and lipid peroxidation in infertile men with at least one failed cycle after intracytoplasmic sperm injection (ICSI). Materials and MethodsIn this experimental study, semen samples were collected from infertile men with at least one failed intracytoplasmic sperm injection (ICSI) cycle (n=20). Sperm parameters, DNA damage, protamine de- ficiency, persistent histones, and lipid peroxidation were assessed using computer-assisted sperm analysis (CASA) system, sperm chromatin structure assay (SCSA) and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, chromomycin A3, aniline blue, and BODIPY C11 staining, respectively.ResultsA negative significant correlation was observed between sperm concentration with percentage of sperm persistent histones (r=-0.56, P=0.02), while positive significant correlations were found between percentage of sperm persistent histones with percentage of abnormal morphology (r=0.54, P=0.02), CMA3-positive spermatozoa (r=o.6, P=0.008) and intensity of lipid peroxidation (r=0.6, P=0.01). In addition, significant correlation was observed be- tween sperm DNA damage with intensity and percentage of lipid peroxidation (r=0.62, P=0.009). Correlation between CMA3-positive spermatozoa and intensity of lipid peroxidation (r=0.5, P=0.03) were also significant.ConclusionObserved significant correlations between sperm functional tests in infertile men with at least one failed ICSI cycle, indicated that the reduction of oxidative stress by antioxidant supplementation may be considered as one therapy approach that can improve sperm function and increase the chance of successful clinical outcomes in next assisted reproductive cycle.

Evaluation of sperm DNA fragmentation and chromatin structure in infertile men with immotile short‐tail sperm defect

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients' samples (22.6±6.9) compared with controls (16.3±4.2) (p<.05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45±4.60 vs. 7.03±2.86, p<.05) and SCSA (24.80±13.1 vs. 15.2±7.2, p<.05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis.

DNA Damage Changes Distribution Pattern and Levels of HP1 Protein Isoforms in the Nucleolus and Increases Phosphorylation of HP1β-Ser88.

The family of heterochromatin protein 1 (HP1) isoforms is essential for chromatin packaging, regulation of gene expression, and repair of damaged DNA. Here we document that γ-radiation reduced the number of HP1α-positive foci, but not HP1β and HP1γ foci, located in the vicinity of the fibrillarin-positive region of the nucleolus. The additional analysis confirmed that γ-radiation has the ability to significantly decrease the level of HP1α in rDNA promoter and rDNA encoding 28S rRNA. By mass spectrometry, we showed that treatment by γ-rays enhanced the HP1β serine 88 phosphorylation (S88ph), but other analyzed modifications of HP1β, including S161ph/Y163ph, S171ph, and S174ph, were not changed in cells exposed to γ-rays or treated by the HDAC inhibitor (HDACi). Interestingly, a combination of HDACi and γ-radiation increased the level of HP1α and HP1γ. The level of HP1β remained identical before and after the HDACi/γ-rays treatment, but HDACi strengthened HP1β interaction with the KRAB-associated protein 1 (KAP1) protein. Conversely, HP1γ did not interact with KAP1, although approximately 40% of HP1γ foci co-localized with accumulated KAP1. Especially HP1γ foci at the periphery of nucleoli were mostly absent of KAP1. Together, DNA damage changed the morphology, levels, and interaction properties of HP1 isoforms. Also, γ-irradiation-induced hyperphosphorylation of the HP1β protein; thus, HP1β-S88ph could be considered as an important marker of DNA damage.

Nucleolar dynamics and interactions with nucleoplasm in living cells

Liquid-liquid phase separation (LLPS) has been recognized as one of the key cellular organizing principles and was shown to be responsible for formation of membrane-less organelles such as nucleoli. Although nucleoli were found to behave like liquid droplets, many ramifications of LLPS including nucleolar dynamics and interactions with the surrounding liquid remain to be revealed. Here, we study the motion of human nucleoli in vivo, while monitoring the shape of the nucleolus-nucleoplasm interface. We reveal two types of nucleolar pair dynamics: an unexpected correlated motion prior to coalescence and an independent motion otherwise. This surprising kinetics leads to a nucleolar volume distribution, , unaccounted for by any current theory. Moreover, we find that nucleolus-nucleoplasm interface is maintained by ATP-dependent processes and susceptible to changes in chromatin transcription and packing. Our results extend and enrich the LLPS framework by showing the impact of the surrounding nucleoplasm on nucleoli in living cells.

SEPT14 Mutations and Teratozoospermia: Genetic Effects on Sperm Head Morphology and DNA Integrity.

The main objective of this study was to evaluate the potential genetic effects of SEPT14 on male infertility through sequencing the SEPT14 coding region. To address this research gap, 254 men with sperm abnormalities and 116 normozoospermic men were recruited, and the whole-coding regions of SEPT14 were sequenced. Two heterozygous mutations, p.Ala123Thr (3/254 vs. 0/116) and p.Ile333Thr (3/254 vs. 0/116), were identified in these cases. A high percentage of defective sperm heads was found in sperm with mutated SEPT14. Both mutations are highly evolutionarily conserved among vertebrates. The results of a fine morphological and chromatin structural analysis indicated severely malformed sperm heads with abnormal chromatin packaging through transmission electron microscopy and Toluidine blue staining. Compared with controls, high DNA fragmentation was demonstrated in sperm from cases carrying SEPT14 mutations using the comet assay. In addition, these two mutations in SEPT14 affected its polymerization ability in vitro. These data revels that the two SEPT14 missense mutations impaired sperm head morphology and induced DNA damage. Our study suggests that genetic variant of SEPT14 is one of the effects for human sperm formation and male fertility.

Canthaxanthin protects human sperm parameters during cryopreservation.

Different antioxidants have been introduced to reduce oxidative stress during the cryopreservation. The main goal of this study was to evaluate the effects of canthaxanthin on human sperm parameters during the freeze-thaw process. This study was performed on 25 normozoospermic semen samples dividing into five groups including 0, 0.1, 1, 10, and 25µM of canthaxanthin. The prepared spermatozoa were cryopreserved by rapid freezing technique. Sperm motility, viability (eosin-nigrosin), morphology (Papanicolaou), acrosome reaction (double staining), DNA denaturation (acridine orange), chromatin packaging (aniline blue and toluidine blue), and DNA fragmentation (sperm chromatin dispersion test) were evaluated before freezing and after thawing. All sperm parameters after thawing significantly were decreased compared to before freezing. Twenty-five micromolar canthaxanthin could significantly improve the progressive and total motility, viability, normal morphology, chromatin packaging, acrosome integrity and DNA denaturation and fragmentation. Ten micromolar canthaxanthin significantly improved total motility, viability, normal morphology, chromatin packaging, acrosome integrity and DNA denaturation and fragmentation. Whereas, in 1µM group, there were significant differences only in improvement of acrosome integrity, chromatin packaging (toluidine blue) and DNA denaturation and fragmentation. But, in 0.1µM group, there were no significant differences in any of measured parameters. It seems that canthaxanthin ameliorates detrimental effects of cryopreservation on human sperm parameters.

Is there any relationship between human sperm parameters and protamine deficiency in different groups of infertile men?

ObjectiveAbnormality in Histone-Protamine replacements has been indicated to cause sperm DNA damage and infertility. The aim of the present study was to investigate the relationships between sperm parameters in oligospermia, asthenospermia, and teratospermia with protamine deficiency in infertile men. Material and methodIn this case–control study, we had three experimental groups including oligospermia (n=100), asthenospermia (n=100), and teratospermia (n=100) as well as normospermia (n=100) as controls. Sperm analyses were performed according to the recommendations of the World Health Organization (WHO, 2010) and sperm chromatin quality was assessed using Chromomycin A3 (CMA3) staining for each sample. ResultsThe comparison of the data between groups indicated that the percentage of spermatozoa with protamine deficiency was significantly different in patients with oligospermia, asthenospermia, and teratospermia when compared with control ones. However, there was no significant correlation between sperm nuclear protamine deficiency and their parameters of the men with teratospermia using CMA3 test. Regarding the oligospermia and asthenospermia semen samples, the findings showed the negative correlations between the sperm nuclear protamine deficiency and progressive motility as well as immobility (p<0.001). ConclusionThe higher proportion of spermatozoa with abnormal chromatin packaging was observed in asthenospermic samples than those from other experimental groups as well as controls. It seems that normal morphology cannot have a valuable predictive value for good chromatin quality of spermatozoa, as much as normal motility characteristics, since samples with high mobility rates often have lower protamine deficiencies. The findings may provide a supportable promoting the future wider clinical application of chromatin/DNA integrity testing along with the semen analysis in male infertility.

Mechanosensing at the Nuclear Envelope by Nuclear Pore Complex Stretch Activation and Its Effect in Physiology and Pathology.

Cell fate is correlated to mechanotransduction, in which forces transmitted by the cytoskeleton filaments alter the nuclear shape, affecting transcription factor import/export, cells transcription activity and chromatin distribution. There is in fact evidence that stem cells cultured in 3D environments mimicking the native niche are able to maintain their stemness or modulate their cellular function. However, the molecular and biophysical mechanisms underlying cellular mechanosensing are still largely unclear. The propagation of mechanical stimuli via a direct pathway from cell membrane integrins to SUN proteins residing in the nuclear envelop has been demonstrated, but we suggest that the cells’ fate is mainly affected by the force distribution at the nuclear envelope level, where the SUN protein transmits the stimuli via its mechanical connection to several cell structures such as chromatin, lamina and the nuclear pore complex (NPC). In this review, we analyze the NPC structure and organization, which have not as yet been fully investigated, and its plausible involvement in cell fate. NPC is a multiprotein complex that spans the nuclear envelope, and is involved in several key cellular processes such as bidirectional nucleocytoplasmic exchange, cell cycle regulation, kinetochore organization, and regulation of gene expression. As several connections between the NPC and the nuclear envelope, chromatin and other transmembrane proteins have been identified, it is reasonable to suppose that nuclear deformations can alter the NPC structure. We provide evidence that the transmission of mechanical forces may significantly affects the basket conformation via the Nup153-SUN1 connection, both altering the passage of molecules through it and influencing the state of chromatin packing. Finally, we review the known correlations between a pathological NPC structure and diseases such as cancer, autoimmune disease, aging and laminopathies.

Abstract 429: Increased chromatin heterogeneity in circulating tumor cells (CTCs) is associated with high levels of HER2 expression in metastatic breast cancer (MBC)

Abstract Introduction: Molecular and genomic characterization of CTCs may help to understand MBC prognosis and predict treatment benefit. We previously reported that overexpression of HER2 in CTCs is associated with detection of clusters, more aggressive clinical features and prognosis in MBC (2018 AACR #5195). Herein, we report a new finding that a widespread variation on chromatin distribution in CTCs is associated with HER2 expression in MBC which may indicate more aggressive tumor. Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV BCa patients before therapy. CTC enumeration were performed in FDA approved CELLTRACKS ANALYZERII® System (Menarini) by targeting the Epithelial Cell Adhesion Molecule antigen for capturing CTCs. After confirming CTCs were positive by CellSearch system, live CTCs enrichment was performed by using Parsortix system (ANGLE) utilizes microfluidic based 10μm separation cassettes. The captured CTCs were stained by Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes), and anti-HER2-FITC. CTCs chromatin packing density was scanned by Partial Wave Spectroscopic (PWS) microscopy which is a label-free spectroscopic microscopy method that resolves structures in cells and quantify the cell nucleus heterogeneity of chromatin packing density scaling between 20-350 nm, or from the kb-pair to 10 Mb-pair range. For each nuclei, the nanoscale heterogeneity of chromatin packing was analyzed as nuclear statistical parameter standard deviation Σ (sigma, RMS). Kruskal-Wallis test was used for statistics. Results: We identified 400 CTCs by CellSearch system, including 115 HER2+ CTCs and 285 HER2- CTCs. The live CTCs were sorted by Parsortix system and were proceeded with multiple staining and scanning by PWS microscopy. CTCs were classified based on morphology and correct phenotype as CK+, DAPI+ and CD45-. There were 97 acquired nuclei that were scanned and evaluated successfully, including 17 HER2+ CTCs, 12 HER2- CTCs and 80 non-CTC cells. According to the images, stronger nanoscopic variations at each pixel in internal structure represents higher chromatin distribution heterogeneity quantified by RMS. The average RMS was 0.05125 in CTC cells which is significantly higher than the non-CTC population (RMS=0.0381, p&amp;lt;0.01). Moreover, the HER2+ CTCs demonstrated the highest nuclear sigma (RMS=0.05366) among all the subgroups when compared to HER2- CTCs (RMS=0.04546, p&amp;lt;0.01). Conclusion: This is the first report of chromatin heterogeneity in CTCs, and the association between HER2 expression and high variations of chromatin distribution in MBC patients. This novel property of CTCs describing nanoscale heterogeneity of chromatin packing requires further evaluation and validation but, it may offer a novel dimension in the understanding of the metastatic process. Citation Format: Qiang Zhang, Lorenzo Gerratana, Ami N. Shah, Andrew Adam Davis, Lisa Flaum, Xiang Zhou, Youbin Zhang, Vadim Backman, Amir Behdad, Firas Wehbe, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased chromatin heterogeneity in circulating tumor cells (CTCs) is associated with high levels of HER2 expression in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 429.

Characterization of Structural Variations in the Context of 3D Chromatin Structure.

Chromosomes located in the nucleus form discrete units of genetic material composed of DNA and protein complexes. The genetic information is encoded in linear DNA sequences, but its interpretation requires an understanding of three-dimensional (3D) structure of the chromosome, in which distant DNA sequences can be juxtaposed by highly condensed chromatin packing in the space of nucleus to precisely control gene expression. Recent technological innovations in exploring higher-order chromatin structure have uncovered organizational principles of the 3D genome and its various biological implications. Very recently, it has been reported that large-scale genomic variations may disrupt higher-order chromatin organization and as a consequence, greatly contribute to disease-specific gene regulation for a range of human diseases. Here, we review recent developments in studying the effect of structural variation in gene regulation, and the detection and the interpretation of structural variations in the context of 3D chromatin structure.

Distribution of DNA damage in the human sperm nucleus: implications of the architecture of the sperm head.

The sperm nucleus is prone to sustain DNA damage before and after ejaculation. Distribution of the damage is not homogeneous, and the factors determining differential sensitivity among nuclear regions have not yet been characterized. Human sperm chromatin contains three structural domains, two of which are considered the most susceptible to DNA damage: the histone bound domain, harboring developmental related genes, and the domain associated with nuclear matrix proteins. Using a quantitative polymerase chain reaction (qPCR) approach, we analyzed the number of lesions in genes homeobox A3 (HOXA3), homeobox B5 (HOXB5), sex-determining region Y (SRY)-box 2 (SOX2), β-GLOBIN, rDNA 18S, and rDNA 28S in human sperm after ultraviolet irradiation (400 μW cm−2, 10 min), H2O2 treatment (250 mmol l−1, 20 min), and cryopreservation, which showed differential susceptibility to genetic damage. Differential vulnerability is dependent on the genotoxic agent and independent of the sperm nuclear proteins to which the chromatin is bound and of accessibility to the transcription machinery. Immunodetection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that the highest level of oxidation was observed after H2O2 treatment. The distribution of oxidative lesions also differed depending on the genotoxic agent. 8-OHdG did not colocalize either with histone 3 (H3) or with type IIα + β topoisomerase (TOPO IIα + β) after H2O2 treatment but matched perfectly with peroxiredoxin 6 (PRDX6), which is involved in H2O2 metabolism. Our study reveals that the characteristics of the sperm head domains are responsible for access of the genotoxicants and cause differential degree of damage to nuclear areas, whereas chromatin packaging has a very limited relevance. The histone-enriched genes analyzed cannot be used as biomarkers of oxidative DNA damage.

A New crucial role for complement receptor CD46 in chromatin packaging and direct gene regulation

Abstract Chromatin is the condensed form of DNA ‘packaged’ by positively charged histones in the nucleus. Post-translational modifications of histones regulate nucleosome dynamics, chromatin accessibility and gene expression. Signals mediated by autocrine activation of the human-specific complement regulator/receptor CD46 during T cell receptor stimulation are vital to Th1 induction in CD4+ T cells. CD46 is expressed in different isoforms that arise through alternative-splicing of a single gene - with two distinct cytoplasmic tails: CYT-1 or CYT-2. CYT-1 nuclear translocation is critical for the expression of genes that are involved in the metabolic adaptations underlying Th1 activity. Because CD46-CYT-1 lacks direct DNA-binding domains, we hypothesized that CYT-1 interacts with transcription factor (TF) activator and/or repressor complexes to control gene expression. ChIP-seq analyses of human CD4+ T cells using anti-CYT-1 antibody revealed 327 genes as direct CD46-CYT-1 targets and five conserved TF-binding motifs allowing us to define CYT-1-interacting TFs in T cells. Interestingly, pathway analysis of CYT-1-regulated genes identified broad nucleosome regulation as top ‘CYT-1 activity’. Indeed, we confirmed that CYT-1 directly regulates the expression and post-translational modification of core histones including H3 and H4 at lysine (4, 8, 9, 12, 20 and 27) in a time-dependent manner. Such CD46-driven nucleosome regulation was aberrant in T cells from CD46-deficient patients, explaining now mechanistically their inability to mount Th1 responses. These data define a novel role for CD46 in chromatin packaging and support our notion that intracellular/autocrine complement regulates basic cell-physiological processes.

Identification of Two Novel Mutations in KDM3A Regulatory Gene in Iranian Infertile Males

Background:KDM3A is a key epigenetic regulator expressed in the testis and is required for packaging and condensation of sperm chromatin. To this point, the association of the KDM3A gene with infertility has not been studied in human. The aim of this study was to screen any new mutation in KDM3A gene to explore more details of human male infertility.Methods:In this work, 150 infertile men (oligozoospermia and azoospermia) and 150 normal healthy fathers were studied. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing were used to screen any mutation in exons 12, 22, and 24 of KDM3A.Results:The infertile men showed various SSCP patterns for the exons 12 and 24, but not for exon 22. A transversion point mutation in exon 12 and a single nucleotide deletion in exon 24 were detected using sequencing analysis. The transversion mutation was located in the preceding exon of lysine-specific demethylase1 and Jumonji (Jmj)-C domain and the later one (deletion) in the cupin-like motif of KDM3A protein. Neither Y chromosome microdeletions nor partial azoospermia factor deletion was found in these patients.Conclusion:The mutations found in infertile men with otherwise unexplained severe spermatogenic failure could be considered as the origin of their abnormalities.