Self-assembly of DNA parallel double-crossover motifs.

DNA double-crossover motifs, including parallel and antiparallel crossovers, serve as the structural foundation for the creation of diverse nanostructures and dynamic devices in DNA nanotechnology. Parallel crossover motifs have unique advantages over the widely used antiparallel crossover design but have not developed as substantially due to the difficulties in assembly. Here we created 29 designs of parallel double-crossover motifs varying in hybridization pathways, central domain lengths, and crossover locations to investigate their assembly mechanism. Arrays were successfully formed in four distinct designs, and large tubular structures were obtained in seven designs with predefined pathways and central domains appoximately 16 nucleotides in length. The nanotubes obtained from parallel crossover design showed improved nuclease resistance than the ones from the antiparallel counterpart design. Overall, our study provides a basis for the development of generalized assembly rules of DNA parallel crossover systems and opens new opportunities for their potential use in biological systems.

TET3-mediated DNA oxidation is essential for intestinal epithelial cell response to stressors.

DNA methylation functions as a repressive epigenetic mark that can be reversed by the Ten-eleven translocation (TET) family of DNA dioxygenases that sequentially oxidize 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised by DNA base-excision repair factors leading to unmodified cytosines. TET enzymes were recently implicated as potential risk factors for inflammatory bowel disease (IBD), but the contribution of TET-mediated DNA oxidation to intestinal homeostasis and response to environmental stressors are unknown. Here, we show prominent roles of TET3 in regulating mouse intestinal epithelial differentiation and response to luminal stressors. Compared with wild-type littermates, mice with intestinal epithelial cell-specific ablation of Tet3 (Tet3ΔIEC) demonstrated a decreased transcriptome involved in innate immune response, Paneth cell differentiation, and epithelial regeneration. Tet3IEC mice exhibited an elevated susceptibility to enteric pathogen infection that is correlated with a decreased epithelial 5hmC abundance. Infection of human enterocytes or mice with the pathogenic bacteria acutely increased 5hmC abundance. Genome-wide 5hmC profiling revealed a shift of genomic enrichment of 5hmC toward genes involved in activating Notch, Wnt, and autophagy pathways. Furthermore, chemical stressor dextran sulfate sodium (DSS) represses epithelial 5hmC abundance in a temporal fashion, and Tet3IEC mice exhibited increased susceptibility to DSS experimental colitis with reduced regenerative capacity. TET3 is a critical regulator of gut epithelial DNA methylome and transcriptome, especially in response to luminal stressors, for the maintenance of tissue homeostasis.

Low dose acetyl salicylic acid (LDA) mediates epigenetic changes in preeclampsia placental mesenchymal stem cells similar to cells from healthy pregnancy.

INTRODUCTION: Preeclampsia (PE) affects 2-8% of all pregnancies, and is the leading cause of maternal and fetal morbidity and mortality. We reported on pathophysiological changes in placenta mesenchymal stem cells (P-MSCs) in PE. P-MSCs can be isolated from different layers of the placenta at the interface between the fetus and mother. The ability of MSCs from other sources to be immune licensed as immune suppressor cells indicated that P-MSCs could mitigate fetal rejection. Acetylsalicylic acid (aspirin) is indicated for treating PE. Indeed, low-dose aspirin is recommended to prevent PE in high risk patients. METHODS: We conducted robust computational analyses to study changes in gene expression in P-MSCs from PE and healthy term pregnancies as compared with PE-MSCs treated with low dose acetyl salicylic acid (LDA). Confocal microscopy studied phospho-H2AX levels in P-MSCs. RESULTS: We identified changes in >400 genes with LDA, similar to levels of healthy pregnancy. The top canonical pathways that incorporate these genes were linked to DNA repair damage - Basic excision repair (BER), Nucleotide excision repair (NER) and DNA replication. A role for the sumoylation (SUMO) pathway, which could regulate gene expression and protein stabilization was significant although reduced as compared to BER and NER pathways. Labeling for phopho-H2AX indicated no evidence of double strand break in PE P-MSCs. DISCUSSION: The overlapping of key genes within each pathway suggested a major role for LDA in the epigenetic landscape of PE P-MSCs. Overall, this study showed a new insight into how LDA reset the P-MSCs in PE subjects around the DNA.

Aspirin-Mediated Reset of Preeclamptic Placental Stem Cell Transcriptome - Implication for Stabilized Placental Function.

Preeclampsia (PE) is a pregnancy-specific disease, occurring in ~ 2-10% of all pregnancies. PE is associated with increased maternal and perinatal morbidity and mortality, hypertension, proteinuria, disrupted artery remodeling, placental ischemia and reperfusion, and inflammation. The mechanism of PE pathogenesis remains unresolved explaining limited treatment. Aspirin is used to reduce the risk of developing PE. This study investigated aspirin's effect on PE-derived placenta mesenchymal stem cells (P-MSCs). P-MSCs from chorionic membrane (CM), chorionic villi, membranes from the maternal and amniotic regions, and umbilical cord were similar in morphology, phenotype and multipotency. Since CM-derived P-MSCs could undergo long-term passages, the experimental studies were conducted with this source of P-MSCs. Aspirin (1 mM) induced significant functional and transcriptomic changes in PE-derived P-MSCs, similar to healthy P-MSCs. These include cell cycle quiescence, improved angiogenic pathways, and immune suppressor potential. The latter indicated that aspirin could induce an indirect program to mitigate PE-associated inflammation. As a mediator of activating the DNA repair program, aspirin increased p53, and upregulated genes within the basic excision repair pathway. The robust ability for P-MSCs to maintain its function with high dose aspirin contrasted bone marrow (M) MSCs, which differentiated with eventual senescence/aging with 100 fold less aspirin. This difference cautions how data from other MSC sources are extrapolated to evaluate PE pathogenesis. Dysfunction among P-MSCs in PE involves a network of multiple pathways that can be restored to an almost healthy functional P-MSC. The findings could lead to targeted treatment for PE.

Restoration of aged hematopoietic cells by their young counterparts through instructive microvesicles release.

This study addresses the potential to reverse age-associated morbidity by establishing methods to restore the aged hematopoietic system. Parabiotic animal models indicated that young secretome could restore aged tissues, leading us to establish a heterochronic transwell system with aged mobilized peripheral blood (MPB), co-cultured with young MPB or umbilical cord blood (UCB) cells. Functional studies and omics approaches indicate that the miRNA cargo of microvesicles (MVs) restores the aged hematopoietic system. The in vitro findings were validated in immune deficient (NSG) mice carrying an aged hematopoietic system, improving aged hallmarks such as increased lymphoid:myeloid ratio, decreased inflammation and cellular senescence. Elevated MYC and E2F pathways, and decreased p53 were key to hematopoietic restoration. These processes require four restorative miRs that target the genes for transcription/differentiation, namely PAX and phosphatase PPMIF. These miRs when introduced in aged cells were sufficient to restore the aged hematopoietic system in NSG mice. The aged MPBs were the drivers of their own restoration, as evidenced by the changes from distinct baseline miR profiles in MPBs and UCB to comparable expressions after exposure to aged MPBs. Restorative natural killer cells eliminated dormant breast cancer cells in vivo, indicating the broad relevance of this cellular paradigm - preventing and reversing age-associated disorders such as clearance of early malignancies and enhanced responses to vaccine and infection.

NFĸB Targeting in Bone Marrow Mesenchymal Stem Cell-Mediated Support of Age-Linked Hematological Malignancies.

Mesenchymal stem cells (MSCs) can become dysfunctional in patients with hematological disorders. An unanswered question is whether age-linked disruption of the bone marrow (BM) microenvironment is secondary to hematological dysfunction or vice versa. We therefore studied MSC function in patients with different hematological disorders and found decreased MHC-II except from one sample with acute myeloid leukemia (AML). The patients' MSCs were able to exert veto properties except for AML MSCs. While the expression of MHC-II appeared to be irrelevant to the immune licensing of MSCs, AML MSCs lost their ability to differentiate upon contact and rather, continued to proliferate, forming foci-like structures. We performed a retrospective study that indicated a significant increase in MSCs, based on phenotype, for patients with BM fibrosis. This suggests a role for MSCs in patients transitioning to leukemia. NFĸB was important to MSC function and was shown to be a potential target to sensitize leukemic CD34+/CD38- cells to azacitidine. This correlated with their lack of allogeneic stimulation. This study identified NFĸB as a potential target for combination therapy to treat leukemia stem cells and showed that understanding MSC biology and immune response could be key in determining how the aging BM might support leukemia. More importantly, we show how MSCs might be involved in transitioning the high risk patient with hematological disorder to AML.

Hypomethylating Chemotherapeutic Agents as Therapy for Myelodysplastic Syndromes and Prevention of Acute Myeloid Leukemia.

Myelodysplastic Syndromes (MDSs) affect the elderly and can progress to Acute Myeloid Leukemia (AML). Epigenetic alterations including DNA methylation and chromatin modification may contribute to the initiation and progression of these malignancies. DNA hypomethylating agents such as decitabine and azacitidine are used as therapeutic treatments and have shown to promote expression of genes involved in tumor suppression, apoptosis, and immune response. Another anti-cancer drug, the proteasome inhibitor bortezomib, is used as a chemotherapeutic treatment for multiple myeloma (MM). Phase III clinical trials of decitabine and azacitidine used alone and in combination with other chemotherapeutics demonstrated their capacity to treat hematological malignancies and prolong the survival of MDS and AML patients. Although phase III clinical trials examining bortezomib's role in MDS and AML patients are limited, its underlying mechanisms in MM highlight its potential as a chemotherapeutic for such malignancies. Further research is needed to better understand how the epigenetic mechanisms mediated by these chemotherapeutic agents and their targeted gene networks are associated with the development and progression of MDS into AML. This review discusses the mechanisms by which decitabine, azacitidine, and bortezomib alter epigenetic programs and their results from phase III clinical trials.

SIRT6 transcriptionally regulates fatty acid transport by suppressing PPARγ.

Pathological lipid accumulation is often associated with enhanced uptake of free fatty acids via specific transporters in cardiomyocytes. Here, we identify SIRT6 as a critical transcriptional regulator of fatty acid transporters in cardiomyocytes. We find that SIRT6 deficiency enhances the expression of fatty acid transporters, leading to enhanced fatty acid uptake and lipid accumulation. Interestingly, the haploinsufficiency of SIRT6 is sufficient to induce the expression of fatty acid transporters and cause lipid accumulation in murine hearts. Mechanistically, SIRT6 depletion enhances the occupancy of the transcription factor PPARγ on the promoters of critical fatty acid transporters without modulating the acetylation of histone 3 at Lys 9 and Lys 56. Notably, the binding of SIRT6 to the DNA-binding domain of PPARγ is critical for regulating the expression of fatty acid transporters in cardiomyocytes. Our data suggest exploiting SIRT6 as a potential therapeutic target for protecting the heart from metabolic diseases.

Specific N-cadherin-dependent pathways drive human breast cancer dormancy in bone marrow.

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells show preference for the BM, resulting in poor prognosis. CSCs use connexin 43 (Cx43) to form gap junctional intercellular communication with BM niche cells, fibroblasts, and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy because of its role as a hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, colocalizes with Cx43 in BC cells within BM biopsies of patients, and is required for Cx43-mediated gap junctional intercellular communication with BM niche cells. Notably, CDH2 and anti-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.

Mesenchymal Stem Cell-Secreted Extracellular Vesicles Instruct Stepwise Dedifferentiation of Breast Cancer Cells into Dormancy at the Bone Marrow Perivascular Region.

In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-ß-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)-released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) in vivo. Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. SIGNIFICANCE: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell-derived exosomes, indicating a potential target for therapeutic intervention.

Non-Coding RNAs as Mediators of Epigenetic Changes in Malignancies.

Non-coding RNAs (ncRNAs) are untranslated RNA molecules that regulate gene expressions. NcRNAs include small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), circular RNAs (cRNAs) and piwi-interacting RNAs (piRNAs). This review focuses on two types of ncRNAs: microRNAs (miRNAs) or short interfering RNAs (siRNAs) and long non-coding RNAs (lncRNAs). We highlight the mechanisms by which miRNAs and lncRNAs impact the epigenome in the context of cancer. Both miRNAs and lncRNAs have the ability to interact with numerous epigenetic modifiers and transcription factors to influence gene expression. The aberrant expression of these ncRNAs is associated with the development and progression of tumors. The primary reason for their deregulated expression can be attributed to epigenetic alterations. Epigenetic alterations can cause the misregulation of ncRNAs. The experimental evidence indicated that most abnormally expressed ncRNAs impact cellular proliferation and apoptotic pathways, and such changes are cancer-dependent. In vitro and in vivo experiments show that, depending on the cancer type, either the upregulation or downregulation of ncRNAs can prevent the proliferation and progression of cancer. Therefore, a better understanding on how ncRNAs impact tumorigenesis could serve to develop new therapeutic treatments. Here, we review the involvement of ncRNAs in cancer epigenetics and highlight their use in clinical therapy.

Hypoxia-mediated changes in bone marrow microenvironment in breast cancer dormancy.

Breast cancer (BC) remains a clinical challenge despite improved treatments and public awareness to ensure early diagnosis. A major issue is the ability of BC cells (BCCs) to survive as dormant cancer cells in the bone marrow (BM), resulting in the cancer surviving for decades with the potential to resurge as metastatic cancer. The experimental evidence indicates similarity between dormant BCCs and other stem cells, resulting in the preponderance of data to show dormant BCCs being cancer stem cells (CSCs). The BM niche and their secretome support BCC dormancy. Lacking in the literature is a comprehensive research to describe how the hypoxic environment within the BM may influence the behavior of BCCs. This information is relevant to understand the prognosis of BC in young and aged individuals whose oxygen levels differ in BM. This review discusses the changing information on vascularity in different regions of the BM and the impact on endogenous hematopoietic stem cells (HSCs). This review highlights the necessary information to provide insights on vascularity of different BM regions on the behavior of BCCs, in particular a dormant phase. For instance, how the transcription factor HIF1-α (hypoxia-inducible factor 1 alpha), functioning as first responder under hypoxic conditions, affects the expression of specific gene networks involved in energy metabolism, cell survival, tumor invasion and angiogenesis. This enables cell fate transition and facilitates tumor heterogeneity, which in turn favors tumor progression and resistance to anticancer treatments Thus, HIF1-α could be a potential target for cancer treatment. This review describes epigenetic mechanisms involved in hypoxic responses during cancer dormancy in the bone marrow. The varied hypoxic environment in the BM is relevant to understand the complex process of the aging bone marrow for insights on breast cancer outcome between the young and aged.

Epigenetic dynamics in cancer stem cell dormancy.

Cancer remains one of the most challenging diseases despite significant advances of early diagnosis and therapeutic treatments. Cancerous tumors are composed of various cell types including cancer stem cells capable of self-renewal, proliferation, differentiation, and invasion of distal tumor sites. Most notably, these cells can enter a dormant cellular state that is resistant to conventional therapies. Thereby, cancer stem cells have the intrinsic potential for tumor initiation, tumor growth, metastasis, and tumor relapse after therapy. Both genetic and epigenetic alterations are attributed to the formation of multiple tumor types. This review is focused on how epigenetic dynamics involving DNA methylation and DNA oxidations are implicated in breast cancer and glioblastoma multiforme. The emergence and progression of these cancer types rely on cancer stem cells with the capacity to enter quiescence also known as a dormant cellular state, which dictates the distinct tumorigenic aggressiveness between breast cancer and glioblastomas.

The Histone Deacetylase SIRT6 Restrains Transcription Elongation via Promoter-Proximal Pausing.

Transcriptional regulation in eukaryotes occurs at promoter-proximal regions wherein transcriptionally engaged RNA polymerase II (Pol II) pauses before proceeding toward productive elongation. The role of chromatin in pausing remains poorly understood. Here, we demonstrate that the histone deacetylase SIRT6 binds to Pol II and prevents the release of the negative elongation factor (NELF), thus stabilizing Pol II promoter-proximal pausing. Genetic depletion of SIRT6 or its chromatin deficiency upon glucose deprivation causes intragenic enrichment of acetylated histone H3 at lysines 9 (H3K9ac) and 56 (H3K56ac), activation of cyclin-dependent kinase 9 (CDK9)-that phosphorylates NELF and the carboxyl terminal domain of Pol II-and enrichment of the positive transcription elongation factors MYC, BRD4, PAF1, and the super elongation factors AFF4 and ELL2. These events lead to increased expression of genes involved in metabolism, protein synthesis, and embryonic development. Our results identified SIRT6 as a Pol II promoter-proximal pausing-dedicated histone deacetylase.

A sirtuin's role in preventing senescence by protecting ribosomal DNA.

Ribosomes are encoded by many copies of ribosomal DNA (rDNA) packed into the nucleolus. High rates of transcription combined with highly repetitive sequences render rDNA loci particularly vulnerable to genomic instability, a proposed underlying cause of cellular senescence. The molecular mechanisms that maintain rDNA stability have remained unclear. A new paper elucidates a sirtuin-dependent mechanism that protects rDNA loci from genomic instability and prevents cellular senescence via heterochromatin silencing mediated by the chromatin remodeler SNF2H. This finding extends our understanding of chromatin dynamics within rDNA regions and offers new mechanistic insights into aging-related pathologies associated with genomic instability.

An inactivating mutation in the histone deacetylase SIRT6 causes human perinatal lethality.

It has been well established that histone and DNA modifications are critical to maintaining the equilibrium between pluripotency and differentiation during early embryogenesis. Mutations in key regulators of DNA methylation have shown that the balance between gene regulation and function is critical during neural development in early years of life. However, there have been no identified cases linking epigenetic regulators to aberrant human development and fetal demise. Here, we demonstrate that a homozygous inactivating mutation in the histone deacetylase SIRT6 results in severe congenital anomalies and perinatal lethality in four affected fetuses. In vitro, the amino acid change at Asp63 to a histidine results in virtually complete loss of H3K9 deacetylase and demyristoylase functions. Functionally, SIRT6 D63H mouse embryonic stem cells (mESCs) fail to repress pluripotent gene expression, direct targets of SIRT6, and exhibit an even more severe phenotype than Sirt6-deficient ESCs when differentiated into embryoid bodies (EBs). When terminally differentiated toward cardiomyocyte lineage, D63H mutant mESCs maintain expression of pluripotent genes and fail to form functional cardiomyocyte foci. Last, human induced pluripotent stem cells (iPSCs) derived from D63H homozygous fetuses fail to differentiate into EBs, functional cardiomyocytes, and neural progenitor cells due to a failure to repress pluripotent genes. Altogether, our study described a germline mutation in SIRT6 as a cause for fetal demise, defining SIRT6 as a key factor in human development and identifying the first mutation in a chromatin factor behind a human syndrome of perinatal lethality.

DUSP9 Modulates DNA Hypomethylation in Female Mouse Pluripotent Stem Cells.

Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs. Cell fusion experiments further reveal that the ratio of X chromosomes to autosomes dictates methylation levels, with female hybrids being hypomethylated and male hybrids being hypermethylated. We show that the X-linked MAPK phosphatase DUSP9 is upregulated in female compared to male ESCs, and its heterozygous loss in female ESCs leads to male-like methylation levels. However, male and female blastocysts are similarly hypomethylated, indicating that sex-specific methylation differences arise in culture. Collectively, our data demonstrate the epigenetic similarity of sex-matched ESCs and EGCs and identify DUSP9 as a regulator of female-specific hypomethylation.

Interplay between Metabolism and Epigenetics: A Nuclear Adaptation to Environmental Changes.

The physiological identity of every cell is maintained by highly specific transcriptional networks that establish a coherent molecular program that is in tune with nutritional conditions. The regulation of cell-specific transcriptional networks is accomplished by an epigenetic program via chromatin-modifying enzymes, whose activity is directly dependent on metabolites such as acetyl-coenzyme A, S-adenosylmethionine, and NAD+, among others. Therefore, these nuclear activities are directly influenced by the nutritional status of the cell. In addition to nutritional availability, this highly collaborative program between epigenetic dynamics and metabolism is further interconnected with other environmental cues provided by the day-night cycles imposed by circadian rhythms. Herein, we review molecular pathways and their metabolites associated with epigenetic adaptations modulated by histone- and DNA-modifying enzymes and their responsiveness to the environment in the context of health and disease.

The histone deacetylase SIRT6 controls embryonic stem cell fate via TET-mediated production of 5-hydroxymethylcytosine.

How embryonic stem cells (ESCs) commit to specific cell lineages and yield all cell types of a fully formed organism remains a major question. ESC differentiation is accompanied by large-scale histone and DNA modifications, but the relations between these epigenetic categories are not understood. Here we demonstrate the interplay between the histone deacetylase sirtuin 6 (SIRT6) and the ten-eleven translocation enzymes (TETs). SIRT6 targets acetylated histone H3 at Lys 9 and 56 (H3K9ac and H3K56ac), while TETs convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC). ESCs derived from Sirt6 knockout (S6KO) mice are skewed towards neuroectoderm development. This phenotype involves derepression of OCT4, SOX2 and NANOG, which causes an upregulation of TET-dependent production of 5hmC. Genome-wide analysis revealed neural genes marked with 5hmC in S6KO ESCs, thereby implicating TET enzymes in the neuroectoderm-skewed differentiation phenotype. We demonstrate that SIRT6 functions as a chromatin regulator safeguarding the balance between pluripotency and differentiation through Tet-mediated production of 5hmC.