scholarly journals High Order Chromatin Structure Regulates Gene Expression in Hematopoietic Stem Cell Self-Renewal and Erythroid Differentiation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1033-1033
Author(s):  
Xiaotian Zhang ◽  
Mira Jeong ◽  
Ivan Bochkov ◽  
Muhammad Saad Shamim ◽  
Erez Lieberman Aiden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Chromatin Structure ◽  
Erythroid Differentiation ◽  
High Order ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
High Order Chromatin Structure ◽  
High Order Chromatin ◽  
Ctcf Site

Abstract High order chromatin structure is implicated in multiple developmental processes and disease. However, a global picture of chromosomal looping interaction alterations during stem cell self-renewal and differentiation is lacking. Hematopoietic stem cell (HSCs) and their differentiated progenitors (HSPCs) offer a system in which to examine this. Of the key differentiated lineages, the erythroid lineage undergoes a unique nuclear condensation process during a well-characterized differentiation process which can be induced in vitro from CD34+ HSPCs. Thus erythroid differentiation offers an ideal model system to study differentiation-associated changes in high order chromatin structure. We have thus generated the in situ Hi-C contact map for human cord blood CD34+ CD38- HSPC (CD34+) and erythroid progenitors undergoing differentiation in vitro at day 7 from CD34+ HSPCs (EryD7). In our 5kb resolution map, we identified over 2000 chromosomal loop interactions in both CD34+ and Day 7 erythroid respectively . The EryD7 sample exhibited higher random intra-chromosomal interactions in comparison with CD34+, presumably due to nuclear condensation. By comparing the chromosomal loop interactions in the 2 cell types. We identified self-renewal and erythroid differentiation-specific looping patterns in the two cell types. Strikingly, we found that a gene depleted region (GDR) 2MB upstream of the HOXA cluster forms a strong chromosome loop with the HOXA cluster exclusively in the HSPCs (Fig1A). Within this GDR site, we identified two conserved CTCF sites, which are thought to organized chromosome looping. Utilizing the CRISPR-mediated deletion of each of the two CTCF sites, we found that deletion of either site reduce the colony forming ability of CD34+, indicating a loss of stem cell self-renewal. (Fig 1B) Gene expression analysis showed that HOXA9 expression was compromised the CTCF site deletion. These data suggest that the GDR is forming a distant regulatory loop which controls the expression of HOXA9 in HSPCs. Because the GDR is implicated in controlling HOXA9 expression, a key gene in leukemogenesis, we then tested the importance of this looping site in different leukemia cell lines that are dependent on HOXA9. Of those cell lines, we found the deletion of the CTCF sites inhibit the growth of DNMT3A and NPM1 mutated OCI-AML3 and promote the apoptosis. In contrast, growth of the MLL translocation cell line MV 4:11 was not abrogated by their deletion (Fig 1C). As a control cell line which doesn't express HOXA9, HL60 cells were not sensitive to the deletion of the GDR CTCF sites. Together, these data indicate leukemic cells may adopt different strategies to activate HOXA9. MLL translocation leukemias activates HOXA9 by the direct binding of the MLL fusion protein, while the NPM1 mutated leukemia is more likely to utilize the stem cell looping to activate HOXA9 expression. Among EryD7 specific interactions, we found the β-globin locus specifically forms chromatin loops at Day7 that are not evident in the CD34+ HSPCs. Detailed examination showed that Dnase I hypersensitivity sites HS5 and 3'HS1 both contains CTCF site and form chromosomal loops. Two other loop-forming CTCF sites, both on the telomeric and centromeric side of β-globin locus were also identified. Interestingly, we found a CTCF binding site adjacent to OR52A5 gene which forms a chromosomal loop with HS5 and is not well studied. To test the role of the chromosomal looping in the regulation of hemoglobin gene expression in β-globin locus, we deleted the OR52A5-CTCF site and the 3'HS1 CTCF site in K562 and adult CD34+ HSPCs. We found the deletion of OR52A5-CTCF resulted in a decrease of HBE and increase of HBB expression in K562 cells, which suggest the OR52A5 CTCF also plays a role in regulating hemoglobin gene expression in the β-globin locus (Fig 1D). Furthermore, we found the deletion of 3'HS1 CTCF resulted in a 4-fold increase of HBG2 expression in adult CD34+ HSPC during erythroid differentiation (Fig1 E). Thus this indicating the 3'HS1 and OR52A5-CTCF CTCF sites in β-globin locus are forming loops that regulate the β-globin locus gene expression. In summary, we have mapped the higher order chromatin structure alterations during stem cell differentiation and identified the critical looping interaction essential for the self-renewal and differentiation specific functions. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1204-1204
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Nathanael G Bailey ◽  
Meiling Zhao ◽  
Kevin B Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Double Mutant ◽  
Mutant Mice ◽  
Cytokine Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Mutant ◽  
Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sparse conserved under-methylated CpGs are associated with high-order chromatin structure

2017 ◽  
Vol 18 (1) ◽  
Author(s):  
Xueqiu Lin ◽  
Jianzhong Su ◽  
Kaifu Chen ◽  
Benjamin Rodriguez ◽  
Wei Li
Keyword(s):  
Chromatin Structure ◽  
High Order ◽  
High Order Chromatin Structure ◽  
High Order Chromatin

Abstract PR07: MicroRNA-130a regulates hematopoietic stem cell self-renewal and erythroid differentiation

2017 ◽  
Author(s):  
Gabriela Krivdova ◽  
Eric R. Lechman ◽  
Erwin M. Schoof ◽  
Veronique Voisin ◽  
Olga I. Gan ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Erythroid Differentiation ◽  
Self Renewal ◽  
Hematopoietic Stem

MiR-29a is Essential in Leukemic Transformation and Maintaining Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4792-4792
Author(s):  
Wenhuo Hu ◽  
James Dooley ◽  
Luisa Cimmino ◽  
Adrian Liston ◽  
Christopher Y. Park
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Protein Translation ◽  
Epigenetic Mark ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Leukemic Transformation ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract MicroRNAs are small non-coding RNAs that interfere with gene expression by degrading messenger RNAs (mRNAs) or blocking protein translation. Expression profiling studies has identified miRNAs that regulate normal and malignant hematopoietic stem cell function. Our previous studies showed that ectopic expression of miR-29a in mouse bone marrow cells induced a myeloproliferative disorder that progressed to acute myeloid leukemia (AML). Over-expression of miR-29b in AML cell lines has been reported to induce apoptosis by negatively regulating Dnmt3a. We recently found that miR-29a positively regulates hematopoietic stem cell (HSC) self-renewal and proliferation using a knockout mouse model of miR-29ab. miR-29a null mice contained significantly lower HSC numbers and miR-29a null HSCs exhibited markedly decreased reconstitution ability in both competitive and non-competitive transplantation assays. To investigate the mechanism of miR-29a action, we performed transcriptomal profiling of miR-29a null HSCs and found that miR-29a null HSCs exhibit a gene expression pattern more similar to wild-type committed progenitors than wild-type HSCs. We identified Dnmt3a as one dysregulated miR-29a target as showing increased expression in miR-29a null HSCs, and haplodeficiency of Dnmt3a partly restores miR-29a deficient HSC function. In order to test the requirement for miR-29a in myeloid leukemogenesis, we transduced miR-29a deficient Lin-c-Kit+Sca-1+ (LSK) cells with the oncogenic MLL-AF9 fusion gene, and found that the development of AML from these cells was markedly delayed. We found that Meis1, Ccna2, Hoxa5 and Hoxa9 transcripts were significantly downregulated in miR-29a null LSK cells compared to WT LSK cells, but they were similarly induced in MLL-AF9 transformed c-Kit+Mac-1+ cells. To investigate whether the epigenetic dysregulation resulting from miR-29a deletion may underlie this transformation-resistant phenotype, we examined the distribution of the active epigenetic mark, H3K79me2, in c-Kit+Mac-1+ miR-29a null cells using a ChIP-Seq assay. After analyzing H3K39me2 peaks using model-based analysis of ChIP-Seq, we identified 4281 and 3649 genes associated with this active epigenetic mark using a duplicated ChIP-Seq analysis, with an overlap of 3164 genes (66.39%). Using public available ChIP-Seq data, we compared our results with the genes associated with the H3K79me2 mark in normal immature LSK cells (9282 genes), granulocyte-macrophage progenitors (GMPs, 8556 genes), and MLL-AF9 transformed GMP cells (L-GMP, 8578 genes), and found 4234, 4111, 4046, and 4766 genes were also identified have an active H3K79me2 mark in MLL-AF9 transformed miR-29a null cells. These data indicate that miR-29a loss inactivates a large group of genes activated by the MLL-AF9 oncogene. We also found that 379 genes were associated with H3K79me2 peaks in both normal LSK and MLL-AF9 transformed miR-29a null c-Kit+Mac-1+ cells, but were absent of this epigenetic marker in L-GMP, suggesting that these genes confer self-renewal and proliferation capacities to normal HSCs. In addition, suppression of these genes are important in leukemic transformation by MLL-AF9, and finally the reactivation of these genes in miR-29a null cells compromises the leukemogenesis ability of MLL-AF9. Interestingly, out of these 379 genes, we were able to identify 18 genes that were potential miR-29a targets including Akt3, Map4k4, Dnmt3a, et al. This suggests the direct and indirect effects from miR-29a in regulating its target gene networks at transcriptional and post-transcriptional levels. Our studies found miR-29a is essential in maintaining HSC function and loss of miR-29a abrogate the leukemogenesis capacity of MLL-AF9. Disclosures No relevant conflicts of interest to declare.

scholarly journals High-order chromatin structure and the epigenome in SAHFs

Nucleus ◽  
2013 ◽  
Vol 4 (1) ◽  
pp. 23-28 ◽  
Author(s):  
Tamir Chandra ◽  
Masashi Narita
Keyword(s):  
Chromatin Structure ◽  
High Order ◽  
High Order Chromatin Structure ◽  
High Order Chromatin

Effects of fixatives on high order chromatin structure: a calorimetric study

1992 ◽  
Vol 206 ◽  
pp. 175-179 ◽  
Author(s):  
L. Vergani ◽  
G. Mascetti ◽  
P. Gavazzo ◽  
C. Nicolini
Keyword(s):  
Chromatin Structure ◽  
High Order ◽  
Calorimetric Study ◽  
High Order Chromatin Structure ◽  
High Order Chromatin

scholarly journals Chromatin Remodeling Subunit Brm Regulates Hematopoietic Stem Cell Self-Renewal By Limiting Intracellular Valine Levels

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3702-3702
Author(s):  
Samisubbu R Naidu ◽  
Maegan L. Capitano ◽  
Scott Cooper ◽  
Xinxin Huang ◽  
Hal E. Broxmeyer
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Chromatin Remodeling ◽  
Normal Mouse ◽  
Self Renewal ◽  
Atpase Subunit ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions

Chromatin remodeling complexes facilitate gene expression and control cell fate decisions. The ATPase subunit of chromatin remodeling complex BRG1 is essential for stem cell function, but the role of its paralog Brm remains essentially unknown. To assess a role(s) for Brm in hematopoietic cell regulation in vivo, we studied hematopoietic stem (HSCs) and progenitor cells (HPCs) in bone marrow (BM) of Brm -/- vs. wildtype (WT) control mice. While BM from Brm -/- mice contain increased numbers of rigorously-defined phenotypic populations of long- and short-term repopulating HSCs and granulocyte macrophage progenitors (GMPs) and increased numbers and cycling status of functional HPC (assessed by CFU-GM, BFU-E, and CFU-GEMM colony assays), they were defective in self-renewal capacity upon in vivo serial transplantation using congenic mice (CD45.2+ donor cells, CD45.1+ competitor cells, and F1 (CD45.2+/CD45.1+) recipient mice). Increased numbers of HSCs from Brm-/- BM failed to show competitive advantage over wild type (WT) control BM cells in primary (1°) transplantation in lethally irradiated mice (based on month 4 donor cell chimerism and phenotypically defined HSC numbers). Moreover, 2° and 3° engraftment at 4 months post transplantation each, a measure of HSC self-renewal capacity, revealed much reduced engraftment of donor Brm -/- BM cell chimerism and HSC numbers compared to the extensive 2° and 3° engraftment of control WT BM. No significant differences in myeloid/lymphoid ratios were noted in 1° or 2° engrafted mice, suggesting no differentiation lineage bias of donor Brm -/- BM cells. This demonstrates a critical role for Brm in controlling in vivo self-renewal of mouse BM HSCs. Valine [(2S)-2 amino-3 methylbutanoic acid (C5H11N02)] is implicated in hematopoietic regulation, since depleting dietary valine permitted non-myeloablative mouse HSC transplantation (Taya et. al. Science 354:1152-1155, 2016). Metabolic analysis of lineage negative (lin-) cells demonstrated that valine, but not leucine, levels were very highly elevated in Brm -/- BM cells, thus linking intracellular valine levels with Brm expression. Exogenously added valine significantly increased basal oxygen consumption rates of both total WT BM and WT lin- cells, but not of total or lin-Brm -/- BM cells in vitro (via Seahorse machine analysis). To study effects of valine on HPCs, we assessed the addition of exogenously added valine on mouse BM and human cord blood (CB) cells cultured in the presence of cytokines with either non-dialyzed or dialyzed fetal bovine serum (FBS). Valine, but not leucine, dose-dependently enhanced HPC (CFU-GM, BFU-E, and CFU-GEMM) colony formation and secondary replating capacity of cytokine stimulated CFU-GM and CFU-GEMM derived colonies of normal mouse BM cells in vitro in presence of non-dialyzed FBS; additional enhanced valine effects were noted when dialyzed FBS (lacking valine and other amino acids) was used. Valine also enhanced mouse BM HPC survival in vitro in context of delayed addition of growth factors, and cytokine stimulated (SCF, FL, TPO) ex-vivo expansion of normal mouse BM HSCs and HPCs. Valine enhancement of the above noted functional mouse BM HPC assays in the presence of dialyzed FBS was also apparent with low density and CD34+ purified CB cells, demonstrating that valine effects are not species specific. Our results suggest that valine is an enhancing factor for HSC maintenance of self-renewal capacity and HPC proliferation, and that Brm gene expression limits intracellular valine levels, thereby controlling HSC self-renewal and HPC proliferation. This information is of potential use for future translation to modulate self-renewal of HSCs and survival and proliferation of HPCs for clinical advantage. Disclosures No relevant conflicts of interest to declare.

Loss Of p53 Rescues The Defective Function Of Foxo3-/- Hematopoietic Stem Cells But Enhances Their Predisposition To Malignancy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4199-4199 ◽  
Author(s):  
Carolina L. Bigarella ◽  
Pauline Rimmele ◽  
Rebeca Dieguez-Gonzalez ◽  
Raymond Liang ◽  
Brigitte Izac ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Dna Repair ◽  
Stem Cell ◽  
Gene Expression Profile ◽  
Double Mutant ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Anti Oxidant

Abstract Leukemic stem cells (LSCs) share many of the same properties of normal hematopoietic stem cells (HSCs) including their highly quiescent state, capacity to self-renew, low levels of reactive oxygen species (ROS) and enhanced DNA repair program. These properties make the efficient and specific eradication of these cells challenging. Foxo3 and p53 are two transcription factors essential for the modulation of HSC quiescence and self-renewal. While Foxo3 is inhibited by signaling from several oncoproteins but crucial for the maintenance of the LSCs in both chronic and acute myeloid leukemia (CML and AML respectively), mutations of p53 although rare, are associated with poor prognosis in advanced stages of these diseases. In vivo ROS-mediated activation of p53 is known to lead to loss of quiescence, alterations of cell cycle and exhaustion of the Foxo3-/- HSC pool. Seeking to understand the contribution of p53 to Foxo3-/- HSC cycling defects, we crossed p53+/- and Foxo3+/- mice. To our surprise we found the bone marrow (BM) frequency of both p53+/-Foxo3-/- and p53-/-Foxo3-/- LSK (Lin-Sca1+cKit+) and long-term-HSC (LT-HSC, LSK Flk2-CD34-) populations greatly increased as compared to their Foxo3-/- counterparts (n=5 mice per genotype; p<0.05). Using Ki67 and DAPI staining we found that loss of one or both alleles of p53 gradually rescued the cell cycle defect of Foxo3-/- HSC and increased the frequency of LSK cells in Go by 2-fold. Loss of p53 also rescued the impaired capacity of Foxo3-/- LSK cells to competitively repopulate multilineage blood over 16 weeks, as shown by the higher frequency of p53+/-Foxo3-/- and p53-/-Foxo3-/- donor-derived cells in the peripheral blood of recipient animals (∼47% recipients of double-mutant cells versus 20% in Foxo3-/- recipients, n=5 per group). Furthermore, loss of p53 significantly improved the compromised self-renewal of Foxo3 mutant HSC in serial BM transplantations. In our quest to identify mechanisms whereby p53 depletion improves Foxo3-/- HSC function, we noticed that the DNA damage accumulated in Foxo3-/- HSC at the steady-state was remarkably ameliorated by removal of one or both alleles of p53 from Foxo3-/- HSCs, as measured by flow cytometry levels of phospho-H2AX (gamma-H2AX) and DNA breaks by comet assay (n=3, p<0.05). Unexpectedly, ROS levels were also significantly reduced by 30% in p53+/-Foxo3-/- in comparison to Foxo3-/- LSK cells, while ROS levels in p53+/- LSK cells were similar to that in WT cells. Consistent with these results, the expression of several anti-oxidant enzymes including Sod1, Sod2, Catalase, Gpx1, Sesn1 and Sesn2 (n≥2), was highly upregulated while a number of genes implicated in mitochondrial generation of ROS were significantly deregulated as a result of loss of one or both alleles of p53. These combined findings suggest that a switch from anti-oxidant to pro-oxidant activity of p53 contributes to Foxo3-/- HSC defects. Despite their apparent normal stem cell function, p53+/-Foxo3-/- HSC were highly altered in their gene expression profile. Interestingly, Gene Set Enrichment Analysis (GSEA) of the microarray analysis (Illumina bead chip mouse-Ref8) of WT, p53+/-, Foxo3-/-, and p53+/-Foxo3-/- LSK cells showed that a cluster of genes associated with fatty acid metabolism was highly enriched in p53+/-Foxo3-/- HSCs (ES=0.746; p<0.01). In addition, from 3976 genes exclusively deregulated in p53+/-Foxo3-/- LSK cells, 201 (out of 1051) overlapped with genes downregulated, while 9 (out of 14) overlapped with genes exclusively upregulated in a LSC-gene signature. To evaluate whether this pre-leukemic profile was associated with increased susceptibility to malignancy, we compared the potential and timeline of BCR-ABL-transformed p53+/-Foxo3-/- HSC as compared to controls in establishing CML in mice. We found a shorter time to the onset of the disease and decreased survival of the recipients of p53+/-Foxo3-/- transformed HSCs (n=4 per group, p<0.05) as compared to WT and Foxo3-/- controls. We propose that the p53+/-Foxo3-/- double-mutant HSCs are enriched for preleukemic stem cells based on their quiescence and self-renewal capacity, low ROS, robust DNA repair, susceptibility to transformation and aberrant gene expression profile. These findings raise the possibility that the coordinated Foxo3 and p53 regulation of ROS wires together the stem cell program. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Prostaglandin E2 Stimulates CREB-Mediated Modification of Histone Variant Nucleosomes at Enhancers to Promote Hematopoietic Stem Cell Fate

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 530-530
Author(s):  
Audrey Sporrij ◽  
Eva M. Fast ◽  
Brejnev Muhire ◽  
Margot Manning ◽  
Marian Kalocsay ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Stem Cell ◽  
Prostaglandin E2 ◽  
Inflammatory Mediator ◽  
Specific Interaction ◽  
Micrococcal Nuclease ◽  
Open Chromatin ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Stimulation of hematopoietic stem and progenitor cells (HSPCs) with the inflammatory mediator Prostaglandin E2 (PGE2) enhances self-renewal and stem cell engraftment following transplantation. Currently, the long-acting derivative of PGE2, 16,16-dimethyl-PGE2 (dmPGE2) is in its fourth clinical trial to improve HSC engraftment and reduce graft versus host disease. To understand the effect of dmPGE2, we assessed genome-wide chromatin reorganization and gene expression changes in human CD34+ HSPCs after 2 hours of dmPGE2 treatment, the time period of treatment in the clinical trials. Enhancers are known to regulate gene expression changes in specific environmental contexts such as stress or inflammation, however the regulatory principles by which subsets of enhancers become activated are poorly understood. Here, we mapped active enhancers by ChIP-seq for H2K27ac and found that dmPGE2 activates a discrete set of enhancers in HSPCs. To investigate enhancer chromatin remodeling, we performed micrococcal nuclease digestion followed by high-throughput sequencing (MNase-seq) to map the occupancy and position of nucleosomes. We found that, contrary to the predominant assumption that open chromatin structures are essentially nucleosome-free, MNase-accessible nucleosomes are retained at inducible enhancers following dmPGE2 stimulation. Through ATAC-seq analysis we mapped changes in open chromatin and found that induced enhancers gain chromatin accessibility following stimulation while maintaining their nucleosome configuration. Surprisingly, this indicates that nucleosomes present at the center of dmPGE2-responsive enhancers play an important function in enhancer accessibility and activity. We then correlated enhancers with gene expression changes by performing RNA-seq and found that genes associated with dmPGE2-induced enhancers display higher gene expression changes after stimulation compared to genes associated with non-responsive enhancers. Transcripts upregulated after dmPGE2 treatment include previously identified regulators of self-renewal and migration such as NR4A2, EGR1 and CXCR4. Moreover, inflammatory chemokines including CXCL2 and CXCL8 as well as members of the activating protein 1 (AP-1) transcription factor gene family such as FOS, FOSL2 and JUNB are increasingly expressed upon stimulation. The gene expression profile included a signature implying CREB as the main transcription factor responsible for the acute dmPGE2 response. Western blot revealed dmPGE2-mediated activation of the signaling transcription factor CREB through phosphorylation in HSPCs. Using ChIP-seq, we found increased genomic binding of phospho-CREB (pCREB) after dmPGE2 treatment in the enhancers. Surprisingly, the binding of pCREB coincided directly with variant histone H2A.Z containing labile nucleosomes in enhancers. We validated the interaction between pCREB and H2A.Z on chromatin in dmPGE2-responsive U937 cells through chromatin fractionation followed by complex immunoprecipitation. This suggests that labile nucleosomes provide sufficient DNA access to allow for binding of pCREB at enhancers. Taken together, our study proposes a novel model for stimulus-mediated activation of enhancers by the inflammatory mediator dmPGE2. dmPGE2 induces the phosphorylation of CREB and subsequently leads to a specific interaction of pCREB with previously deposited H2A.Z-rich nucleosomes at inducible enhancers who regulate genes that promote HSPC fate. This new mechanism of variant histone deposition followed by the interaction with a signaling transcription factor at enhancers supports a rapid inducible response from the environment. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Gene Depleted DNA Methylation Canyon Maintains Hematopoietic Stem Cell Self-Renewal and NPM1c+ Leukemia By Regulating the Gene Expression of HOXA Cluster

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 649-649
Author(s):  
Xiaotian Zhang ◽  
Margaret Goodell ◽  
Mira Jeong ◽  
Haley Gore ◽  
Wanding Zhou
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cell ◽  
Cell Line ◽  
Long Range ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Hoxa Cluster ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract DNA methylation Canyons (DMC in short, also referred to as DNA methylation Valleys) are long unmethylated regions (UMR) over 3.5kb, in the mammalian genome. DMCs are associated with homeotic genes and can be classified into active DMCs marked by H3K4me3 and repressive DMCs marked by H3K27me3. We performed high resolution in situ HiC on human hematopoietic stem and progenitor cells (HSPC) and differentiated red blood cell (RBC) progenitors derived from HSPC. We found that DMCs over 7.3kb form significant 3D micro-compartment interactions with each other. These interactions are extremely long range and can occur between two loci separated by 60Mb. Thus, we name these DMCs over 7.3kb as Grand DNA methylation Canyon (GDMC). GDMCs are repressive DMCs and bear the highest level of H3K27me3 in the HSPC compared with the remaining UMRs under 7.3kb. Additionally, we found that the interacting GDMCs is organized by Polycomb mediated long range interaction but not cohesion loop extrusion. We also found GDMC interactions disappear in differentiated RBC progenitors derived from HSPC. This suggests a function of GDMC interactions in stem cell maintenance. We thus set out to test the function of GDMC interactions in stem cell self-renewal by deleting GDMC loci. We found one GDMC that lacks genes and transcription activity and enhancer activity marked by H3K4me3 and H3K27ac, is interacting with a repressive part (covered by H3K27me3) of the HOXA cluster only in HSPC. This GDMC is thus named "Geneless Canyon"- GLC in short. By deleting GLC, we found that HSPC self-renewal is impaired significantly. Moreover, expression of active HOXA9 and HOXA10 gene adjacent to the repressive part of HOXA cluster also decreased after deletion. When we checked the 3D genomic interactions around the HOXA region after deletion, we found the long range interactions with GLC disappear, and the enhancer interactions with active HOXA cluster gene promoters are also weakened. This suggests that GDMC interactions can act as the scaffold for the enhancer-promoter interactions to maintain active gene expression.In the detailed examination of regulatory elements in GLC, we found that CTCF binding sites are at the boundary of neighboring Lamin associated domain (LAD) and GLC (Figure 1A). The CTCFs are forming cohesion extrusion loops to include the whole LAD region. Deletion of the CTCF sites also result in the loss of HOXA9 and HOXA10 expression as well as the compromise of self-renewal. Since HOXA9-10 genes are important transcriptional factors for leukemias carrying NPM1c+ and MLL-X mutations. We thus performed CTCF deletions in cell lines with OCI-AML3 and MV4:11 as leukemia cell line models carrying NPM1c+ and MLL-X mutations. We surprisingly found the NPM1c+ cell leukemia cell line display the growth arrest with CTCF deletion, while the MLL-X cell line MV4:11 don't display such effect (Figure 1B). We perform in situ HiC on OCI-AML3 and MV4:11 and found in OCI-AML3 cells GLC forms interaction with HOXA9-HOTTIP regions, while in MV4:11 cells there is no such interaction (Figure 1C).Further examination on epigenomic profiles identified that GLC is activated as super enhancer in OCI-AML3 cells with the loss of Polycomb binding. This indicates that NPM1c+ leukemia may utilize the GLC region as enhancer to boost active gene expression in from HOXA9 to Hottip, with a different mechanism than HSPC. Gene expression analysis after the CTCF deletion further validates that after the CTCF deletion the expression of HOXA9, HOXA10 and HOXA11 is decreased. Interestingly, GLC is also hypermethylated in the OCI_AML3 cells. Thus, we have discovered an important DNA methylation Canyon that regulates the hematopoietic stem cell self-renewal via the structural organization of HOXA region that act as the scaffold for the enhancer-promoter interaction. This Canyon can also act as a super enhancer to activate the HOXA expression in the NPM1c+ leukemia. This suggests the versatile roles of Polycomb targeted Canyon in normal hematopoiesis and leukemia development. Figure. 1 Figure. 1. Disclosures No relevant conflicts of interest to declare.

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