Nuclear Chromatin Structure Research Articles

Nuclear expansion and chromatin structure remodeling in mouse aging neurons

Abstract Aging, particularly in the brain, involves impairments in multiple cellular functions, many regulated at the nucleus. Chromatin structure plays a critical role in regulating gene expression and the maintenance of genomic stability. During differentiation, each cell type acquire their unique topology, this should be kept for a lifetime, but may deteriorate as we age. However, the effects of aging on the chromatin 3D structure of neurons remain largely unknown and much has been inferred from senescent cells. By combining chromosome conformation capture and microscopy techniques, we investigated cortical neurons of young and aged mice and discovered signs of neuronal nuclear expansion during neuronal aging, leading to increased distances between chromosomes. This expansion alters the topology of compartments, topologically associating domains (TADs) and chromatin loops. While larger TADs tend to dissociate, smaller TADs and loops exhibit strengthened interactions of chromatin in aged neurons. These topological changes impact the borders of TADs, resulting in weakening, this is parallel to Lamin-B weakening and nuclear envelope alterations. We attribute these alterations to changes in physical forces of an expanding nucleus, driving the distancing of chromosomes filling a growing nuclear area, affecting gene expression and topology, contributing to the functional declines observed during aging.

Illuminating the link between nuclear geometry and chromatin structures through 4D nucleus modeling

Illuminating the link between nuclear geometry and chromatin structures through 4D nucleus modeling

DNA Damage and Nuclear Morphological Changes in Cardiac Hypertrophy Are Mediated by SNRK Through Actin Depolymerization.

Proper nuclear organization is critical for cardiomyocyte function, because global structural remodeling of nuclear morphology and chromatin structure underpins the development and progression of cardiovascular disease. Previous reports have implicated a role for DNA damage in cardiac hypertrophy; however, the mechanism for this process is not well delineated. AMPK (AMP-activated protein kinase) family of proteins regulates metabolism and DNA damage response (DDR). Here, we examine whether a member of this family, SNRK (SNF1-related kinase), which plays a role in cardiac metabolism, is also involved in hypertrophic remodeling through changes in DDR and structural properties of the nucleus. We subjected cardiac-specific Snrk-/- mice to transaortic banding to assess the effect on cardiac function and DDR. In parallel, we modulated SNRK in vitro and assessed its effects on DDR and nuclear parameters. We also used phosphoproteomics to identify novel proteins that are phosphorylated by SNRK. Last, coimmunoprecipitation was used to verify Destrin (DSTN) as the binding partner of SNRK that modulates its effects on the nucleus and DDR. Cardiac-specific Snrk-/- mice display worse cardiac function and cardiac hypertrophy in response to transaortic banding, and an increase in DDR marker pH2AX (phospho-histone 2AX) in their hearts. In addition, in vitro Snrk knockdown results in increased DNA damage and chromatin compaction, along with alterations in nuclear flatness and 3-dimensional volume. Phosphoproteomic studies identified a novel SNRK target, DSTN, a member of F-actin depolymerizing factor proteins that directly bind to and depolymerize F-actin. SNRK binds to DSTN, and DSTN downregulation reverses excess DNA damage and changes in nuclear parameters, in addition to cellular hypertrophy, with SNRK knockdown. We also demonstrate that SNRK knockdown promotes excessive actin depolymerization, measured by the increased ratio of G-actin to F-actin. Last, jasplakinolide, a pharmacological stabilizer of F-actin, rescues the increased DNA damage and aberrant nuclear morphology in SNRK-downregulated cells. These results indicate that SNRK is a key player in cardiac hypertrophy and DNA damage through its interaction with DSTN. This interaction fine-tunes actin polymerization to reduce DDR and maintain proper cardiomyocyte nuclear shape and morphology.

Analysis of a rare progeria variant of Barrier-to-autointegration factor in Drosophila connects centromere function to tissue homeostasis

Barrier-to-autointegration factor (BAF/BANF) is a nuclear lamina protein essential for nuclear integrity, chromatin structure, and genome stability. Whereas complete loss of BAF causes lethality in multiple organisms, the A12T missense mutation of the BANF1 gene in humans causes a premature aging syndrome, called Néstor-Guillermo Progeria Syndrome (NGPS). Here, we report the first in vivo animal investigation of progeroid BAF, using CRISPR editing to introduce the NGPS mutation into the endogenous Drosophila baf gene. Progeroid BAF adults are born at expected frequencies, demonstrating that this BAF variant retains some function. However, tissue homeostasis is affected, supported by studies of the ovary, a tissue that depends upon BAF for stem cell survival and continuous oocyte production. We find that progeroid BAF causes defects in germline stem cell mitosis that delay anaphase progression and compromise chromosome segregation. We link these defects to decreased recruitment of centromeric proteins of the kinetochore, indicating dysfunction of cenBAF, a localized pool of dephosphorylated BAF produced by Protein Phosphatase PP4. We show that DNA damage increases in progenitor germ cells, which causes germ cell death due to activation of the DNA damage transducer kinase Chk2. Mitotic defects appear widespread, as aberrant chromosome segregation and increased apoptosis occur in another tissue. Together, these data highlight the importance of BAF in establishing centromeric structures critical for mitosis. Further, these studies link defects in cenBAF function to activation of a checkpoint that depletes progenitor reserves critical for tissue homeostasis, aligning with phenotypes of NGPS patients.

Hmga2 protein loss alters nuclear envelope and 3D chromatin structure

BackgroundThe high-mobility group Hmga family of proteins are non-histone chromatin-interacting proteins which have been associated with a number of nuclear functions, including heterochromatin formation, replication, recombination, DNA repair, transcription, and formation of enhanceosomes. Due to its role based on dynamic interaction with chromatin, Hmga2 has a pathogenic role in diverse tumors and has been mainly studied in a cancer context; however, whether Hmga2 has similar physiological functions in normal cells remains less explored. Hmga2 was additionally shown to be required during the exit of embryonic stem cells (ESCs) from the ground state of pluripotency, to allow their transition into epiblast-like cells (EpiLCs), and here, we use that system to gain further understanding of normal Hmga2 function.ResultsWe demonstrated that Hmga2 KO pluripotent stem cells fail to develop into EpiLCs. By using this experimental system, we studied the chromatin changes that take place upon the induction of EpiLCs and we observed that the loss of Hmga2 affects the histone mark H3K27me3, whose levels are higher in Hmga2 KO cells. Accordingly, a sustained expression of polycomb repressive complex 2 (PRC2), responsible for H3K27me3 deposition, was observed in KO cells. However, gene expression differences between differentiating wt vs Hmga2 KO cells did not show any significant enrichments of PRC2 targets. Similarly, endogenous Hmga2 association to chromatin in epiblast stem cells did not show any clear relationships with gene expression modification observed in Hmga2 KO. Hmga2 ChIP-seq confirmed that this protein preferentially binds to the chromatin regions associated with nuclear lamina. Starting from this observation, we demonstrated that nuclear lamina underwent severe alterations when Hmga2 KO or KD cells were induced to exit from the naïve state and this phenomenon is accompanied by a mislocalization of the heterochromatin mark H3K9me3 within the nucleus. As nuclear lamina (NL) is involved in the organization of 3D chromatin structure, we explored the possible effects of Hmga2 loss on this phenomenon. The analysis of Hi-C data in wt and Hmga2 KO cells allowed us to observe that inter-TAD (topologically associated domains) interactions in Hmga2 KO cells are different from those observed in wt cells. These differences clearly show a peculiar compartmentalization of inter-TAD interactions in chromatin regions associated or not to nuclear lamina.ConclusionsOverall, our results indicate that Hmga2 interacts with heterochromatic lamin-associated domains, and highlight a role for Hmga2 in the crosstalk between chromatin and nuclear lamina, affecting the establishment of inter-TAD interactions.

Ankle2 deficiency-associated microcephaly and spermatogenesis defects in zebrafish are alleviated by heterozygous deletion of vrk1

Autosomal recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by a below average brain volume at birth and is associated with neurodevelopmental disorders such as growth retardation and intellectual disability. Mutations in ANKLE2 have been identified as one of the causes of MCPH (MCPH16). ANKLE2 is a target molecule of the Zika virus NS4a protein that interferes with ANKLE2 function, resulting in severe microcephaly. ANKLE2 is essential for organizing the nuclear envelope and chromatin structures during the mitotic-end process via barrier to autointegration factor (BAF) dephosphorylation. However, the precise mechanism by which the loss of ANKLE2 function causes the pathogenesis of microcephaly remains unclear. In this study, we generated Ankle2-deficient zebrafish (ankle2−/−) with a significant reduction in brain size compared with that of their control siblings. The ankle2−/− brain showed a significant decrease in the number of radial glial progenitor cells, suggesting that Ankle2 deficiency in zebrafish causes neurogenesis defects. Furthermore, ankle2−/− male zebrafish showed infertility owing to defects in spermatogenesis. Notably, microcephaly was overcome by vrk1 morpholino knockdown or vrk1 heterozygous deletion. In addition, spermatogenesis in ankle2−/− zebrafish males was partially restored by the vrk1 heterozygous deletion, although infertility was not resolved. These results indicate that ANKLE2 and VRK1 coordinate with each other for BAF phosphorylation to maintain normal mitosis during neurogenesis and spermatogenesis.

Lipodystrophy-associated progeroid syndromes.

With the exception of HIV-associated lipodystrophy, lipodystrophy syndromes are rare conditions characterized by a lack of adipose tissue, which is not generally recovered. As a consequence, an ectopic deposition of lipids frequently occurs, which usually leads to insulin resistance, atherogenic dyslipidemia, and hepatic steatosis. These disorders include certain accelerated aging syndromes or progeroid syndromes. Even though each of them has unique clinical features, most show common clinical characteristics that affect growth, skin and appendages, adipose tissue, muscle, and bone and, in some of them, life expectancy is reduced. Although the molecular bases of these Mendelian disorders are very diverse and not well known, genomic instability is frequent as a consequence of impairment of nuclear organization, chromatin structure, and DNA repair, as well as epigenetic dysregulation and mitochondrial dysfunction. In this review, the main clinical features of the lipodystrophy-associated progeroid syndromes will be described along with their causes and pathogenic mechanisms, and an attempt will be made to identify which of López-Otín's hallmarks of aging are present.

Abstract 14247: Myocardial Histopathology In Atrial Cardiomyopathy

Background: Atrial cardiomyopathy (ACM) is characterized by multiple histopathological features but the entity lacks systematic validation. Objectives: We sought to correlate atrial histopathology with clinical characteristics of patients with ACM and AF and outcomes following cardiac surgery. Methods: The pathological findings of the atrial myocardial biopsies from 178 patients who underwent any cardiac surgery between 2016-2018. Atrial cell nuclear structure and morphology, myocyte hypertrophy and interstitial fibrosis were graded and analyzed quantitatively. Study population was divided in three groups based on preoperative ECG: 1) patients in sinus rhythm with no signs of ACM (P wave duration <120ms)(SRn); 2) patients in sinus rhythm and ACM (P>=120 or biphasic waves in inferior leads)(SRa); and 3) atrial fibrillation (AF). Results: SRa patients had decreased number of nuclei/area and smaller total cardiomyocyte area compared to SRn patients (p=0.04 forboth). Patients with AF had bigger, less solid and circular nuclei compared to SRn patients (p<0.01 for all). AF patients had increased area of interstitial fibrosis (p<0.01) compared to SRn patients. Conclusions: Histopathological characteristics of ACM include changes in nuclear chromatin structure.

Methods of In Situ Quantitative Root Biology.

When dealing with plant roots, a multiscale description of the functional root structure is needed. Since the beginning of 21st century, new devices such as laser confocal microscopes have been accessible for coarse root structure measurements, including three-dimensional (3D) reconstruction. Most researchers are familiar with using simple 2D geometry visualization that does not allow quantitative determination of key morphological features from an organ-like perspective. We provide here a detailed description of the quantitative methods available for 3D analysis of root features at single-cell resolution, including root asymmetry, lateral root analysis, cell size and nuclear organization, cell-cycle kinetics, and chromatin structure analysis. Quantitative maps of the root apical meristem (RAM) are shown for different species, including Arabidopsis thaliana (L.), Heynh, Nicotiana tabacum L., Medicago sativa L., and Setaria italica (L.) P. Beauv. The 3D analysis of the RAM in these species showed divergence in chromatin organization and cell volume distribution that might be used to study root zonation for each root tissue. Detailed protocols and possible pitfalls in the usage of the marker lines are discussed. Therefore, researchers who need to improve their quantitative root biology portfolio can use them as a reference.

Metacyclogenesis defects and gene expression hallmarks of histone deacetylase 4-deficient Trypanosoma cruzi cells

Trypanosoma cruzi—the causative agent of Chagas disease—like other kinetoplastids, relies mostly on post-transcriptional mechanisms for regulation of gene expression. However, trypanosomatids undergo drastic changes in nuclear architecture and chromatin structure along their complex life cycle which, combined with a remarkable set of reversible histone post-translational modifications, indicate that chromatin is also a target for control of gene expression and differentiation signals in these organisms. Chromatin-modifying enzymes have a direct impact on gene expression programs and DNA metabolism. In this work, we have investigated the function of T. cruzi histone deacetylase 4 (TcHDAC4). We show that, although TcHDAC4 is not essential for viability, metacyclic trypomastigote TcHDAC4 null mutants show a thin cell body and a round and less condensed nucleus located very close to the kinetoplast. Sixty-four acetylation sites were quantitatively evaluated, which revealed H2AT85ac, H4K10ac and H4K78ac as potential target sites of TcHDAC4. Gene expression analyses identified three chromosomes with overrepresented regions of differentially expressed genes in the TcHDAC4 knockout mutant compared with the wild type, showing clusters of either up or downregulated genes. The adjacent chromosomal location of some of these genes indicates that TcHDAC4 participates in gene expression regulation during T. cruzi differentiation.

Plant long non-coding RNAs in the regulation of transcription

Eukaryotic genomes are pervasively transcribed, producing large numbers of non-coding RNAs (ncRNAs), including tens of thousands of long ncRNAs (lncRNAs), defined as ncRNAs longer than 200 nucleotides. Recent studies have revealed the important roles lncRNAs play in the regulation of gene expression at various levels in all eukaryotes; moreover, emerging research in plants has identified roles for lncRNAs in key processes such as flowering time control, root organogenesis, reproduction, and adaptation to environmental changes. LncRNAs participate in regulating most steps of gene expression, including reshaping nuclear organization and chromatin structure; governing multiple steps of transcription, splicing, mRNA stability, and translation; and affecting post-translational protein modifications. In this review, I present the latest progress on the lncRNA-mediated regulatory mechanisms modulating transcription in Arabidopsis thaliana, focusing on their functions in regulation of gene expression via chromatin structure and interactions with the transcriptional machinery.

Nuclear Dynamics and Chromatin Structure: Implications for Pancreatic Cancer.

Changes in nuclear shape have been extensively associated with the dynamics and functionality of cancer cells. In most normal cells, nuclei have a regular ellipsoid shape and minimal variation in nuclear size; however, an irregular nuclear contour and abnormal nuclear size is often observed in cancer, including pancreatic cancer. Furthermore, alterations in nuclear morphology have become the ‘gold standard’ for tumor staging and grading. Beyond the utility of altered nuclear morphology as a diagnostic tool in cancer, the implications of altered nuclear structure for the biology and behavior of cancer cells are profound as changes in nuclear morphology could impact cellular responses to physical strain, adaptation during migration, chromatin organization, and gene expression. Here, we aim to highlight and discuss the factors that regulate nuclear dynamics and their implications for pancreatic cancer biology.

Abstract 2096: Genome wide remodeling of DNA methylation and CpG hypermethylation is tightly linked to acquired resistance in testicular cancer

Abstract Acquired resistance to chemotherapy is perhaps the major barrier to cure of advanced cancers. Testicular germ cell tumors (TGCTs) are the most common cancers of young males. TGCTs are one of the few solid tumors that are cured at a high rate with standard cisplatin-based chemotherapy. However, TGCTs become resistance to cisplatin resulting in poor outcomes. While the mechanisms accounting for cisplatin hypersensitivity in TGCT remain elusive, it has been suggested that DNA hypermethylation may play a role. However, there is little direct experimental evidence to support this claim. In the present study, we have leveraged unique isogenic cell models to provide strong evidence for net DNA hypermethylation as a key mechanism leading to acquired cisplatin resistance in TGCT cells. Genome-wide DNA methylation analysis revealed that hypomethylated CpG loci were enriched for SUZ12 and EZH2 binding and H3K27me3 sites. In contrast hypermethylated CpG loci were significantly enriched for CTCF and RAD21 domains as well as repressive DNA segments indicating the global remodeling of nuclear chromatin structure in resistant cells. Integrative computational analysis of RNA-seq and EPIC DNA methylation array data revealed a strong inverse correlation between gene expression and CpG gene promoter methylation. This analysis also revealed a strong bidirectional shift between gene promoter and gene body DNA methylation that correlated closely with upregulated expression of PRC2 target genes and down regulation of tumor suppression genes in resistant cells. Hence the data further suggests a functional interplay between DNA methylation and the PRC2 pathway that modulates cisplatin sensitivity of TGCTs. Further studies including H3K27me3 ChIP-seq are ongoing to further elucidate this relationship. In conclusion, our finding supports the hypothesis that genome wide remodeling of DNA methylation is associated with cisplatin hypersensitivity and resistance of TGCTs and further supports the use of hypomethylation therapy for relapsed TGCT patients. Citation Format: Zeeshan Fazal, Ratnakar Singh, Megan Tomlin, Doha Shokry, Aleyah Hattab, Sarah Spinella, Michael Spinella. Genome wide remodeling of DNA methylation and CpG hypermethylation is tightly linked to acquired resistance in testicular cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2096.

Genome-Wide Chromatin Analysis of FFPE Tissues Using a Dual-Arm Robot with Clinical Potential.

Simple SummaryFormalin-fixed paraffin-embedded (FFPE) specimens, which are pathological specimens of human tissues, are of high clinical value because they are associated with clinical information such as drug sensitivity and side effects and exist in huge numbers worldwide. However, the quality of DNA and RNA extracted from FFPE specimens is generally poor, and it is still difficult to perform ChIP-seq. Here, we describe an experimental procedure for FFPE ChIP-seq called RCRA ChIP-seq that allows identification of the genome-wide distributions of key histone modifications and binding sites of the insulator transcription factor CTCF. We have also succeeded in obtaining accurate and stable results even for the analysis of a large number of FFPE samples by using an industrial robot. Thus, routine ChIP-seq analysis of FFPE specimens could lead to new epigenomic mechanisms in various diseases.Although chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) using formalin-fixed paraffin-embedded tissue (FFPE) has been reported, it remained elusive whether they retained accurate transcription factor binding. Here, we developed a method to identify the binding sites of the insulator transcription factor CTCF and the genome-wide distribution of histone modifications involved in transcriptional activation. Importantly, we provide evidence that the ChIP-seq datasets obtained from FFPE samples are similar to or even better than the data for corresponding fresh-frozen samples, indicating that FFPE samples are compatible with ChIP-seq analysis. H3K27ac ChIP-seq analyses of 69 FFPE samples using a dual-arm robot revealed that driver mutations in EGFR were distinguishable from pan-negative cases and were relatively homogeneous as a group in lung adenocarcinomas. Thus, our results demonstrate that FFPE samples are an important source for epigenomic research, enabling the study of histone modifications, nuclear chromatin structure, and clinical data.

Nanoscale insight into chromatin remodeling and DNA repair complex in HeLa cells after ionizing radiation

The dynamic structure of nuclear chromatin and its regulation in the formation of repair complex is essential in DNA damage response and repair. Using single molecule localization microscopy STORM this work discovered that the nuclear chromatin organization was relaxed from 200 to 400 nm thick irregular frame and remodeled to dispersed sub-100 nm structure in HeLa cells after X-ray irradiation. The DSB repair factors (γ-H2AX, MDC1, 53BP1) showed distribution as microscale-colocalized and nanoscale interlaced substructure in the DSB repair complex. The dual-color nanoscopic imaging of γ-H2AX and chromatin at the DSB sites suggest that DNA damage response and repair cascade are chromatin structure-dependent and also partly dependent on the distance to the DSB sites. The sub-100 nm structure of fibers and nanoclusters of the relaxed nuclear chromatin and the DSB repair factors highly resembled the cross-section view of chromatin organization. These data demonstrated the polymorphic and dynamic behavior of the chromatin organization in vivo, and provided nanoscale insight into the interplay between chromatin remodeling and DNA damage response and DNA repair.

Spatial patterns of CTCF sites define the anatomy of TADs and their boundaries

BackgroundTopologically associating domains (TADs) are genomic regions of self-interaction. Additionally, it is known that TAD boundaries are enriched in CTCF binding sites. In turn, CTCF sites are known to be asymmetric, whereby the convergent configuration of a pair of CTCF sites leads to the formation of a chromatin loop in vivo. However, to date, it has been unclear how to reconcile TAD structure with CTCF-based chromatin loops.ResultsWe approach this problem by analysing CTCF binding site strengths and classifying clusters of CTCF sites along the genome on the basis of their relative orientation. Analysis of CTCF site orientation classes as a function of their spatial distribution along the human genome reveals that convergent CTCF site clusters are depleted while divergent CTCF clusters are enriched in the 5- to 100-kb range. We then analyse the distribution of CTCF binding sites as a function of TAD boundary conservation across seven primary human blood cell types. This reveals divergent CTCF site enrichment at TAD boundaries. Furthermore, convergent arrays of CTCF sites separate the left and right sections of TADs that harbour internal CTCF sites, resulting in unequal TAD ‘halves’.ConclusionsThe orientation-based CTCF binding site cluster classification that we present reconciles TAD boundaries and CTCF site clusters in a mechanistically elegant fashion. This model suggests that the emergent structure of nuclear chromatin in the form of TADs relies on the obligate alternation of divergent and convergent CTCF site clusters that occur at different length scales along the genome.Graphical abstract

Characteristics of Hematopoiesis in Follicular Lymphoma Patients

Aim. To assess hematopoiesis in follicular lymphoma (FL) patients at different disease stages with different morphologic structures of tumor and bone marrow microenvironment. Materials & Methods. The trial included 152 FL patients treated from 2006 to 2016. In all of them the diagnosis was based on immunohistochemical analysis of extramedullar tumor as well as the analysis of bone marrow aspirates and core biopsy samples. In cases of bone marrow lesions (n = 33) a detailed morphoimmunophenotypic evaluation of tumor cells was carried out by means of flow cytometry, and lymphocyte subset panel evaluation was performed. Results. Anemia, thrombocytopenia, and monocytosis in blood of FL patients are not associated with bone marrow lesions. In the absence of signs of these lesions anemia was detected in 23 (19 %) patients, thrombocytopenia was identified in 8 (7 %) patients, and 11 (9.1 %) patients showed monocytosis. Among patients with bone marrow lesions 9 (27.2 %) anemia, 11 (33.8 %) thrombocytopenia, and 7 (21 %) monocytosis cases were reported. Depth of cytopenia was determined by the degree of bone marrow tumor infiltration. Based on lymphocyte subset panel evaluation the following types of tumor cells in bone marrow aspirates were characterized: elements with blastic structure of nuclear chromatin, atypical lymphoid cells, and those similar to normal lymphocytes. Immunophenotypic heterogeneity of tumor cells in bone marrow was demonstrated. The trial showed that hemoglobin level, the count of blood thrombocytes and monocytes as well as the count of bone marrow T-cells are not associated with types of tumor cells. Conclusion. Arrest of hematopoiesis and increasing number of monocytes in blood correlate with the degree of bone marrow tumor infiltration and are not affected by morphoimmunological characteristics of FL tumor cells.

Processes regulating early lymphocyte differentiation

Lymphocytes play pivotal roles in innate and adaptive immunity. The differentiation process by which hematopoietic stem cells (HSCs) acquire specific functions has been extensively investigated and is considered the paradigm of cell differentiation. It has been widely accepted that highly enriched HSCs are heterogeneous with respect to their lymphopoietic potential, and aged or stressed HSCs are skewed to the myeloid lineage. Several transcription factors and cytokine signaling pathways have been reported as essential to lymphocyte differentiation. However, the molecular mechanism that modulates the earliest stage remains unclear. Furthermore, the origin and characteristics of early T-lymphoid progenitors that migrate from the bone marrow to the thymus are still unknown in this field. Epigenetic mechanisms likely influence early lineage specification through the regulation of mitochondrial function and modification of nuclear chromatin structure. This review summarizes previous and recent findings on the processes involved in early lymphocyte differentiation. Thus, it provides a foundation for the understanding of the physiology of HSC aging and the pathology of intractable acute lymphocytic leukemia.

Calcitriol Prevents RAD51 Loss and cGAS-STING-IFN Response Triggered by Progerin.

Hutchinson Gilford progeria syndrome (HGPS) is a devastating accelerated aging disease caused by LMNA gene mutation. The truncated lamin A protein produced "progerin" has a dominant toxic effect in cells, causing disruption of nuclear architecture and chromatin structure, genomic instability, gene expression changes, oxidative stress, and premature senescence. It was previously shown that progerin-induced genomic instability involves replication stress (RS), characterized by replication fork stalling and nuclease-mediated degradation of stalled forks. RS is accompanied by activation of cGAS/STING cytosolic DNA sensing pathway and STAT1-regulated interferon (IFN)-like response. It is also found that calcitriol, the active hormonal form of vitamin D, rescues RS and represses the cGAS/STING/IFN cascade. Here, the mechanisms underlying RS in progerin-expressing cells and the rescue by calcitriol are explored. It is found that progerin elicits a marked downregulation of RAD51, concomitant with increased levels of phosphorylated-RPA, a marker of RS. Interestingly, calcitriol prevents RS and activation of the cGAS/STING/IFN response in part through maintenance of RAD51 levels in progerin-expressing cells. Thus, loss of RAD51 is one of the consequences of progerin expression that can contribute to RS and activation of the IFN response. Stabilization of RAD51 helps explain the beneficial effects of calcitriol in these processes.

The distributions of protein coding genes within chromatin domains in relation to human disease

BackgroundOur understanding of the nuclear chromatin structure has increased hugely during the last years mainly as a consequence of the advances in chromatin conformation capture methods like Hi-C. The unprecedented resolution of genome-wide interaction maps shows functional consequences that extend the initial thought of an efficient DNA packaging mechanism: gene regulation, DNA repair, chromosomal translocations and evolutionary rearrangements seem to be only the peak of the iceberg. One key concept emerging from this research is the topologically associating domains (TADs) whose functional role in gene regulation and their association with disease is not fully untangled.ResultsWe report that the lower the number of protein coding genes inside TADs, the higher the tendency of those genes to be associated with disease (p-value = 4 × 10^{-54}). Moreover, housekeeping genes are less associated with disease than other genes. Accordingly, they are depleted in TADs containing less than three protein coding genes (p-value = 3.9 × 10^{-34}). We observed that TADs with higher ratios of enhancers versus genes contained higher numbers of disease-associated genes. We interpret these results as an indication that sharing enhancers among genes reduces their involvement in disease. Larger TADs would have more chances to accommodate many genes and select for enhancer sharing along evolution.ConclusionsGenes associated with human disease do not distribute randomly over the TADs. Our observations suggest general rules that confer functional stability to TADs, adding more evidence to the role of TADs as regulatory units.