scholarly journals Proerythroblast Cells of Diamond-Blackfan Anemia Patients With RPS19 and CECR1 Mutations Have Similar Transcriptomic Signature

2021 ◽  
Vol 12 ◽  
Author(s):  
Beren Karaosmanoglu ◽  
M. Alper Kursunel ◽  
Duygu Uckan Cetinkaya ◽  
Fatma Gumruk ◽  
Gunes Esendagli ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Mononuclear Cells ◽  
Erythroid Differentiation ◽  
Cell Types ◽  
Ribosomal Protein Genes ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Altered Expression ◽  
Diamond Blackfan Anemia ◽  
Transcriptomic Profile

Diamond Blackfan Anemia (DBA) is an inherited bone marrow (BM) failure syndrome, characterized by a paucity of erythroid differentiation. DBA is mainly caused by the mutations in ribosomal protein genes, hence classified as ribosomopathy. However, in approximately 30% of patients, the molecular etiology cannot be discovered. RPS19 germline mutations caused 25% of the cases. On the other hand, CECR1 mutations also cause phenotypes similar to DBA but not being a ribosomopathy. Due to the blockade of erythropoiesis in the BM, we investigated the transcriptomic profile of three different cell types of BM resident cells of DBA patients and compared them with healthy donors. From BM aspirates BM mononuclear cells (MNCs) were isolated and hematopoietic stem cells (HSC) [CD71–CD34+ CD38mo/lo], megakaryocyte–erythroid progenitor cells (MEP) [CD71–CD34+ CD38hi] and Proerythroblasts [CD71+ CD117+ CD38+] were sorted and analyzed with a transcriptomic approach. Among all these cells, proerythroblasts had the most different transcriptomic profile. The genes associated with cellular stress/immune responses were increased and some of the transcription factors that play a role in erythroid differentiation had altered expression in DBA proerythroblasts. We also showed that gene expression levels of ribosomal proteins were decreased in DBA proerythroblasts. In addition to these, colony formation assay (CFU-E) provided functional evidence of the failure of erythroid differentiation in DBA patients. According to our findings that all patients resembling both RPS19 and CECR1 mutations have common transcriptomic signatures, it may be possible that inflammatory BM niche may have a role in DBA pathogenesis.

scholarly journals Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia

2020 ◽  
Vol 12 (566) ◽  
pp. eabb5831 ◽  
Author(s):  
Alison M. Taylor ◽  
Elizabeth R. Macari ◽  
Iris T. Chan ◽  
Megan C. Blair ◽  
Sergei Doulatov ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Ribosomal Proteins ◽  
P53 Protein ◽  
Erythroid Differentiation ◽  
Normal Activity ◽  
Small Subunit ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Calmodulin Inhibitors

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration–approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

scholarly journals Targeting Autophagy as a Therapeutic Pathway in Diamond-Blackfan Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-28-SCI-28
Author(s):  
Sergei Doulatov
Keyword(s):  
Oxidative Stress ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Autophagic Flux ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Stem And Progenitor Cells ◽  
Induced Pluripotent

Abstract Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. We previously used unbiased drug screens in induced pluripotent stem cells (iPSCs) which identified SMER28 as a potential therpauetic for DBA. SMER28 acts by selectively modulating autophagy, but has distinct effects from the mTOR inhibitor rapamycin, highlighting the need for further study. Autophagy and mitophagy are critical metabolic pathways that mediate turnover of damaged organelles and mitochondria. Autophagy has been linked to regulation of hematopoietic stem cell function and terminal erythroid differentiation. However, the mechanisms by which autophagy regulates hematopoiesis are still poorly understood. We utilize primary cord blood and adult human progenitors and iPSCs to circumvent the paucity of primary patient blood stem and progenitor cells. To understand the role of autophagy, we have developed a lentiviral LC3-based reporter which allows real-time quantitation of autophagic flux. Using this reporter, we show that autophagy is dynamically regulated during erythroid differentiation and closely parallels mitochondrial mass and levels of reactive oxygen species. In our model, oxidative stress in erythroid precursors drives a stress response which involves activation of autophagy and mitophagy pathways. The interplay between oxidative stress and autophagy regulates erythropoiesis in normal and multiple disease contexts, including DBA and myelodysplastic syndromes (MDS). SMER28 and mitochondrial uncouplers promote homeostasis by facilitating mitochondrial clearance. In summary, therpauetic modulation of autophagy may be a broadly applicable therpauetic strategy in both inherited and acquired anemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals SATB1 Regulates GATA1 Protein Expression in Early Hematopoiesis and Is Deregulated in Diamond Blackfan Anemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Mark C Wilkes ◽  
Hee-Don Chae ◽  
Ethan Patrick Wentworth ◽  
Toshinobu Nishimura ◽  
Anupama Narla ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fetal Liver ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Ribosomal Gene ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Enhancer Element ◽  
Diamond Blackfan Anemia

The induction of the master erythroid transcription factor, GATA1 during early erythropoiesis is critical for efficient red blood cell production. However, GATA1 is expressed at low levels in hematopoietic stem cells (HSCs) and is moderately induced at both the common myeloid progenitor (CMP) and megakaryocyte/erythroid progenitor (MEP) stages prior to lineage commitment. Diamond Blackfan Anemia is a rare disease, usually associated with ribosomal gene mutations, leading to significant decrease in GATA1 expression and block in early committed erythroid differentiation. Mild defects in other myeloid lineages are also observed, with limited clinical relevance. The importance of GATA1 downregulation in disease pathogenesis is manifested by rare patients with DBA carrying GATA1 mutations. To understand signaling pathways that contribute to the pathogenesis DBA, we perform RNA-seq with mRNA from human CD34+ fetal liver cells and found that the chromatin organizer, Special AT-rich sequence binding protein 1 (SATB1) was prematurely downregulated. Our results further demonstrated that sustained SATB1 expression is critical to maintain required levels of GATA1 protein at both the CMP and MEP stages of differentiation, but not in committed erythroid progenitors. In mice, SATB1 is modestly expressed in HSCs and upregulated during lymphopoiesis. SATB1 is downregulated during myeloid and erythroid differentiation and antagonizes myeloid and erythroid expansion. However, in human hematopoietic stem and progenitor cells (HSPCs), SATB1 is required for efficient expansion of these lineages. SATB1 maintains 78% expression in human MEPs, but is undetectable in early committed erythroid progenitors. In RPS19-insufficient human HSPCs, SATB1 was downregulated to 22% in MEPs (p=0.02). Re-expression of SATB1 corrected a significant subset of deregulated mRNAs, including GATA1 regulators. In the absence of SATB1, one such GATA1 regulator, heat shock protein 70 (HSP70), failed to be induced in ribosome-competent human MEPs, reducing GATA1 protein expression by 35.7% (p= 0.026). Concurrently, MEP expansion was inhibited by 64.5% (p=0.023), reducing erythroid and megakaryocyte expansion by 18.2% (p=0.024) and 20.4% (p=0.183) respectively. SATB1 facilitated the formation of chromatin loops linking together an enhancer element with HSP70 promoters required for HSP70 induction in early differentiation. Although GATA1 is significantly upregulated in committed erythroid progenitors, RPS19-insufficient human CD235+ erythrocytes express GATA1 28.4% of controls (p= 0.011). SATB1 re-expression increased GATA1 expression to 31.4% (p=0.089). Similarly, SATB1 re-expression increased CD235+ expansion from 13.9% to 39.5% (p=0.02) compared to controls. Our data indicate that premature SATB1 downregulation contributes to erythroid failure in DBA by reducing MEP expansion, but aberrant GATA1 expression observed in more mature erythrocytes is predominantly SATB1-independent. However, SATB1-re-expression improved CD11b+ myeloid expansion from 81.2% to 90.4% (p=0.045) and CD41a+ megakaryocyte expansion from 76.7% to 214.7% (p=0.038) respectively. Our results demonstrate that SATB1 plays an important role in human hematopoiesis and is an important regulator of GATA1. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.

scholarly journals Human leukemia mutations corrupt but do not abrogate GATA-2 function

2018 ◽  
Vol 115 (43) ◽  
pp. E10109-E10118 ◽  
Author(s):  
Koichi R. Katsumura ◽  
Charu Mehta ◽  
Kyle J. Hewitt ◽  
Alexandra A. Soukup ◽  
Isabela Fraga de Andrade ◽  
...  
Keyword(s):  
Dna Binding ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Erythroid Differentiation ◽  
Cell Types ◽  
Myeloid Differentiation ◽  
Human Leukemia ◽  
Hematopoietic Stem ◽  
Amino Terminal ◽  
Disease Mutations

By inducing the generation and function of hematopoietic stem and progenitor cells, the master regulator of hematopoiesis GATA-2 controls the production of all blood cell types. Heterozygous GATA2 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA2 disease mutations commonly disrupt amino acid residues that mediate DNA binding or cis-elements within a vital GATA2 intronic enhancer, suggesting a haploinsufficiency mechanism of pathogenesis. Mutations also occur in GATA2 coding regions distinct from the DNA-binding carboxyl-terminal zinc finger (C-finger), including the amino-terminal zinc finger (N-finger), and N-finger function is not established. Whether distinct mutations differentially impact GATA-2 mechanisms is unknown. Here, we demonstrate that N-finger mutations decreased GATA-2 chromatin occupancy and attenuated target gene regulation. We developed a genetic complementation assay to quantify GATA-2 function in myeloid progenitor cells from Gata2 −77 enhancer-mutant mice. GATA-2 complementation increased erythroid and myeloid differentiation. While GATA-2 disease mutants were not competent to induce erythroid differentiation of Lin−Kit+ myeloid progenitors, unexpectedly, they promoted myeloid differentiation and proliferation. As the myelopoiesis-promoting activity of GATA-2 mutants exceeded that of GATA-2, GATA2 disease mutations are not strictly inhibitory. Thus, we propose that the haploinsufficiency paradigm does not fully explain GATA-2–linked pathogenesis, and an amalgamation of qualitative and quantitative defects instigated by GATA2 mutations underlies the complex phenotypes of GATA-2–dependent pathologies.

scholarly journals Molecular Analysis of Diamond Blackfan Anemia and Genotype-Phenotype Correlation: Experience from the Canadian Inherited Marrow Failure Registry

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3621-3621
Author(s):  
Omri Avraham Arbiv ◽  
Bozana Zlateska ◽  
Robert J. Klaassen ◽  
Conrad Fernandez ◽  
Rochelle Yanofsky ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Research Funding ◽  
Gene Panel ◽  
Ribosomal Protein Genes ◽  
Severe Phenotype ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Genetic Groups ◽  
Number Of Patients ◽  
The Impact

Abstract Background/Objectives: Diamond Blackfan anemia (DBA) is an inherited disorder characterized by chronic hypoproductive anemia, physical malformations, and an increased risk of malignancies. At least 12 DBA genes have been identified, which include various ribosomal protein genes and the transcription factor GATA1. The aims of our study were (1) to identify the mutation spectrum of DBA patients, utilizing a cohort of patients enrolled on the Canadian Inherited Marrow Failure Registry (CIMFR) and (2) to determine whether specific hematological abnormalities, malformations, and outcomes are associated with specific mutations. Methods: Patients were enrolled on the CIMFR, which is a multicenter cohort study of inherited bone marrow failure syndromes (IBMFS). Genetic testing was performed using one or more of the following tests: Sanger sequencing, next generation sequencing (NGS) DBA gene panel, a comprehensive NGS IBMFS gene panel developed in our laboratory, or comparative genetic hybridization (CGH). Severity of the hematological disease was dichotomized according to a patient's requirement for chronic treatment: those who were maintained on corticosteroids, blood transfusions, or received a hematopoietic stem cell transplantation were considered to have a more severe phenotype than those who did not require hematological treatment. Chi-square tests with a Fisher's exact test correction were used to compare genetic groups with at least 5 patients on observed phenotypes. Results: 71 patients with DBA have been enrolled in our registry. A causal mutation has been identified in 36 of these patients, with the following rates: RPS19 (n=11), RPL11 (n=7), RPL5 (n=6), RPS26 (n=5), RPL35a (n=2), RPS24 (n=2), and one of each RPS7, RPS29, RPS17. Remarkably, a substantial number of patients in our population-based cohort (19.4%) had mild hematological phenotype requiring no therapy. Patients with RPL11 mutations tended to have a less severe DBA phenotype, while patients with RPS19 mutations tended to have a more severe phenotype (p=0.04). In terms of non-hematological malformations, we found no differences in cardiac, stature and craniofacial malformations across the groups compared (all p>0.1). However, patients with RPL5 mutations had significantly more hand malformations (p=0.02), and patients with RPS26 mutations had more genitourinary malformations (p=0.04). To control for the impact of mutation severity on the observed phenotype, we compared the prevalence of mutations that are predicted to result in truncated or lack of protein from the respective allele (large copy-number variation, nonsense, or indel frameshift) to mutations that are predicted to be hypomorphic or affect function (splicing, indel/inframe and, missense) between mutation categories. There were no differences among genetic groups in the severity of their mutations (p=0.58). Conclusions: Mutations in a wide spectrum of ribosomal protein genes underlie DBA cases in Canada, which approximate those observed by other registries in Western countries. Patients with DBA caused by RPL11 mutations tended to have a milder hematological phenotype, while patients with RPS19 mutation tended to have a more severe phenotype. Mutations in RPS26 and RPL5 are associated with genitourinary and hand malformations, respectively. Our findings may help improve counseling of DBA patients and their family. Future studies are needed to replicate our results and determine whether these findings can help personalize care. Disclosures Lipton: Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

p53 Dependent and Dose Dependent Effects of Rps29 Mutation In the Zebrafish Embryo.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1170-1170
Author(s):  
Alison M. Taylor ◽  
Jessica M. Humphries ◽  
Richard M. White ◽  
Ryan D. Murphey ◽  
Caroline E. Burns ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Board Of Directors ◽  
Ribosomal Proteins ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Ribosomal Protein Genes ◽  
Advisory Committees ◽  
Cardinal Vein ◽  
Hematopoietic Stem ◽  
Equity Ownership

Abstract Abstract 1170 Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by red cell aplasia and craniofacial abnormalities. Ribosomal protein genes are often mutated in patients with this disease, but the mechanism of action is still being investigated. To elucidate the effect of mutations in ribosomal proteins, we are studying a zebrafish rps29 mutant with hematopoietic and endothelial defects. Hematopoietic stem cells (HSCs) in rps29-/- embryos are significantly decreased, as assayed by runx1 and cmyb expression. Although the aorta and posterior cardinal vein form in the mutant, intersomitic vessel formation is affected. To test whether decreased p53 levels can rescue these defects, we crossed fish with mutated p53 into the rps29 background. In rps29-/-;p53-/- embryos, the vascular and HSC phenotypes are rescued, demonstrating that p53 may be required for these effects of rps29 knockdown. We performed a microarray comparing rps29-/- embryos and their siblings to identify genes that are differentially expressed in the mutant. Using gene set enrichment analysis (GSEA), we determined that the list of genes up-regulated in the rps29 mutant is enriched for genes up-regulated by p53 in response to irradiation. Many of the genes identified have known roles in apoptosis and stress response. We have also identified genes whose expression correlates with the number of wildtype copies of rps29. Orthopedia homolog a (otpa), which is specifically expressed in forebrain and hindbrain tissues at 24 hours post fertilization (hpf), is decreased in heterozygous siblings and further decreased in homozygous siblings. In addition, p53 knockdown partially increases otpa levels in the mutant. These data support a model where p53 activation is one of the critical downstream mediators of rps29 knockdown in several tissues, but the mechanism of tissue specificity remains unclear. The otpa phenotype suggests that regulation of some genes is dependent on rps29 levels. The zebrafish rps29 mutant will be a useful model for understanding how a decrease in ribosomal protein levels can cause specific defects in hematopoietic and neural tissues. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Ribosomal protein gene deletions in Diamond-Blackfan anemia

Blood ◽  
2011 ◽  
Vol 118 (26) ◽  
pp. 6943-6951 ◽  
Author(s):  
Jason E. Farrar ◽  
Adrianna Vlachos ◽  
Eva Atsidaftos ◽  
Hannah Carlson-Donohoe ◽  
Thomas C. Markello ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Single Nucleotide Polymorphism Array ◽  
Ribosomal Protein Gene ◽  
Small Subunit ◽  
Ribosomal Protein Genes ◽  
Gene Deletions ◽  
Diamond Blackfan Anemia ◽  
Genome Wide

Abstract Diamond-Blackfan anemia (DBA) is a congenital BM failure syndrome characterized by hypoproliferative anemia, associated physical abnormalities, and a predisposition to cancer. Perturbations of the ribosome appear to be critically important in DBA; alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, at present, only 50% to 60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide single-nucleotide polymorphism array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9 of 51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A. No recurrent regions of copy variation at novel loci were identified. Because RPS17 is a duplicated gene with 4 copies in a diploid genome, we demonstrate haploinsufficient RPS17 expression and a small subunit ribosomal RNA processing abnormality in patients harboring RPS17 deletions. Finally, we report the novel identification of variable mosaic loss involving known DBA gene regions in 3 patients from 2 kindreds. These data suggest that ribosomal protein gene deletion is more common than previously suspected and should be considered a component of the initial genetic evaluation in cases of suspected DBA.

scholarly journals SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2189-2189
Author(s):  
Mark C Wilkes ◽  
Aya Shibuya ◽  
Vanessa M Scanlon ◽  
Hee-Don Chae ◽  
Anupama Narla ◽  
...  
Keyword(s):  
Cord Blood ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Culture Media ◽  
Hsp70 Expression ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Enhancer Element ◽  
Diamond Blackfan Anemia

Abstract Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulatory association of long noncoding RNAs and chromatin accessibility facilitates erythroid differentiation

2021 ◽  
Author(s):  
Yunxiao Ren ◽  
Junwei Zhu ◽  
Yuanyuan Han ◽  
Pin Li ◽  
Jing Wu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Chromatin Accessibility ◽  
Human Cord Blood ◽  
Erythroid Progenitor ◽  
Integrated Network ◽  
Hematopoietic Stem ◽  
Multipotent Progenitor Cells ◽  
Tf Gene ◽  
Terminal Erythroid Differentiation

Erythroid differentiation is a dynamic process regulated by multiple factors, while the interaction between long non-coding RNAs and chromatin accessibility and its influence on erythroid differentiation remains unclear. To elucidate this interaction, we employed hematopoietic stem cells, multipotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, and erythroblasts from human cord blood as an erythroid differentiation model to explore the coordinated regulatory functions of lncRNAs and chromatin accessibility by integrating RNA-Seq and ATAC-Seq data. We revealed that the integrated network of chromatin accessibility and lncRNAs exhibits stage-specific changes throughout the erythroid differentiation process, and that the changes at the EB stage of maturation are dramatic. We identified a subset of stage-specific lncRNAs and transcription factors (TFs) that associate with chromatin accessibility during erythroid differentiation, in which lncRNAs are key regulators of terminal erythroid differentiation via a lncRNA-TF-gene network. LncRNA PCED1B-AS1 was revealed to regulate terminal erythroid differentiation by coordinating GATA1 dynamically binding to the chromatin and interacting with cytoskeleton network during erythroid differentiation. DANCR, another lncRNA that is highly expressed at the MEP stage, was verified to promote erythroid differentiation by compromising megakaryocyte differentiation and coordinating with chromatin accessibility and TFs, such as RUNX1. Overall, our results identified the associated network of lncRNAs and chromatin accessibility in erythropoiesis and provide novel insights into erythroid differentiation and abundant resources for further study.

scholarly journals Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 455-455
Author(s):  
Mark C Wilkes ◽  
Jacqueline D Mercado ◽  
Mallika Saxena ◽  
Jun Chen ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fold Increase ◽  
Healthy Controls ◽  
In Vitro Activity ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Mtor Activity

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions or infections from immunosuppressive drugs and hematopoietic stem cell transplantation. Human hematopoietic stem and progenitor cells (HSPCs) from cord blood were transduced with lentiviral shRNA against a number of ribosomal genes associated with DBA, reducing the specific ribosomal protein expression by approximately 50%. During differentiation, these cells demonstrated a DBA-like phenotype with significantly reduced differentiation of erythroid progenitors (over 80%), yet only modest (15-30%) reduction of other hematopoietic lineages. NLK was immunopurifed from differentiating HSPCs and activity was assessed by the extent of in vitro phosphorylation of 3 known NLK substrates NLK, c-Myb and Raptor. As NLK activation requires phosphorylation at Thr298, we also showed that in vitro activity correlated with intracellular NLK phosphorylation by Western blot analysis. Nemo-like Kinase (NLK) was hyperactivated in the erythroid progenitors (but not other lineages), irrespective of the type of ribosomal gene insufficiency. We extended these studies using other sources of HSPCs (fetal liver, whole blood and bone marrow), along with RPS19- and RPL11-insufficient mouse models of the disease, as well as DBA patient samples. NLK was hyperactivated in erythroid progenitors from mice (5.3- and 7.2-fold increase in Raptor phosphorylation in RPS19- and RPL-11 insufficiency respectively) and from humans (7.3- and 9.0-fold in RPS19- and RPL11-insufficiency respectively) as well as HSPCs from three DBA patient (4.8-, 4.1- and 4.2-fold increase above controls). In RPS19-insufficient human HSPCs, genetic silencing of NLK increased erythroid expansion by 2.2-fold (p=0.0065), indicating that aberrant NLK activation contributes to disease pathogenesis. Furthermore, a high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increases erythroid expansion in murine (5.4-fold) and human (6.3-fold) models of DBA without effects on normal erythropoiesis (EC50: 0.7 µM). Identical results were observed in bone marrow CD34+ progenitors from three DBA patients with a 2.3 (p=0.0009), 1.9 (p=0.0007) and 2.1-fold (p=0.0001) increase in CD235+ erythroid progenitor population following NLK inhibition. In erythroid progenitors, RPS19-insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR in vitro activity by 82%. This was restored close to basal levels (93.8% of healthy control) upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. In early clinical trials, not all DBA patients have responded to leucine therapy. We hypothesize that one of the reasons might be due to NLK phosphorylation of Raptor. While leucine treatment increased mTOR activity in both RPS19-insufficient and control cells (164% of healthy controls: p=0.007 and 24% to 42% of healthy controls: p=0.0064), combining leucine with NLK inhibition increased mTOR activity in RPS19-insufficiency from 24% to 142% of control (p=0.0012). This translated to improvements in erythroid expansion of RPS19-insufficient HSPCs from 8.4% to 16.3% with leucine treatment alone, 28.4% with NLK inhibition alone, but 68.6% when leucine and NLK inhibition were combined. This 8.2-fold improvement in erythroid progenitor production indicates that identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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