bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Candidate Regulators of Hematopoietic Stem Cell Aging Using a Combined QTL Mapping and Microarray Analysis Approach.
Authors: Oakley Erin J. , Van Zant Gary 
Published: April 2020

Abstract
A combination of quantitative trait locus (QTL) mapping and microarray expression profiling was used to identify five novel candidate genes for regulating hematopoietic stem cell aging in two strains of inbred mice, C57BL/6 (B6) and DBA/2 (D2). Despite their ability to self-renew and maintain essentially normal levels of hematopoietic function throughout the lifetime of an organism, it has become increasingly evident that hematopoietic stem cells (HSCs) are subject to qualitative and quantitative changes over time, and that these changes may affect both the rate of aging and longevity of organisms. In mice, the effect of aging on stem cells is highly strain-specific, thus suggesting that genetic regulation plays a role in HSC aging. Our lab has identified a QTL on murine chromosome 2 that is associated with the variation in frequency of HSCs between aged B6 and D2 mice. In B6 mice, the frequency of HSCs increases with age, whereas in D2 mice the population of HSCs decreases as the animals age. Using a congenic mouse model in which the region surrounding the QTL in D2 mice was introgressed onto a B6 background, genome wide gene expression analyses were performed using sorted lineage negative hematopoietic cells, which are enriched for primitive stem and progenitor hematopoietic cells. RNA isolated from lineage negative cells from chromosome 2 congenic and B6 background mice at young and old ages was reverse transcribed, labeled, and hybridized to Affymetrix Murine Genome 430 arrays. Using an overall p-value cut-off of ≤ 0.01, a total of 4,368 transcripts were differentially expressed between background and congenic mice, out of 39,000 transcripts present on the mouse genome chip. Following a ‘QTL to Gene’ strategy, we identified 123 differentially expressed transcripts located in the congenic interval on chromosome 2. We then assessed for hematopoietic tissue specific expression patterns, the presence of single nucleotide polymorphisms (SNPs) in the transcripts, and cis-regulation of transcript expression. Five genes were identified that met these criteria, and thus are considered candidate regulators of hematopoietic stem cell aging. We used real time PCR to validate differential expression of a subset of the identified genes. Interestingly, a majority of the genes are associated with cell cycle control and/or protein targeting and transport processes. Additionally, all five cis-regulated genes were found to be down-regulated in aged congenic mice compared to aged B6 mice, thus suggesting coordinated regulation by a common promoter region(s) located within the chromosome 2 QTL. These findings suggest that stem cell aging is associated with specifc intrinsic cellular events and provide insight into the genetic mechanisms underlying hematopoietic stem cell aging.
An Early Block to Erythro-Megakaryocytic Development Conferred by Loss of Transcription Factor GATA-1.
Authors: Stachura David L. , Chou Stella T. , Weiss Mitchell J. 
Published: April 2020

Abstract
Transcription factor GATA-1 is essential at multiple stages of hematopoiesis. Murine gene targeting and analysis of naturally occurring human mutations demonstrate that GATA-1 is required for the maturation of committed erythroid precursors and megakaryocytes. Prior studies also suggest additional, poorly defined, roles for GATA-1 at earlier stages of erythro-megakaryocytic development. To investigate these functions further, we studied hematopoietic differentiation of Gata1− murine embryonic stem cells on OP9 stroma with the cytokine thrombopoietin (TPO) present. Initially, the Gata1− cultures generated a wave of mutant megakaryocytes, but these were rapidly overgrown by a unique population of TPO-dependent blasts that continued to proliferate for more than 6 months in culture. These immature Gata1− cells arose reproducibly in culture without growth lag or crisis, indicating that they derive directly from loss of GATA-1 and not from random genetic events acquired during cell culture. The cells express transcription factors GATA-2, FOG-1 and PU.1 and exhibit the surface phenotype Lin−, Sca1−, IL7R−, CD41+, cKit+, CD9+, and GPIblow. Importantly, upon restoration of GATA-1 function, these cells undergo both erythroid and megakaryocytic differentiation, as assessed by morphology, ultrastructural analysis and the induction of lineage-specific markers. Clonal analysis shows that individual cells maintain the capacity for erythro-megakaryocytic differentiation. Hence, we term this unique population G1ME for Gata1− -Megakaryocyte-Erythroid. To determine if G1ME cells are present in vivo, we analyzed E13.5 fetal livers of Gata1−/Gata1wild-type chimeric embryos. Flow cytometry analysis demonstrates an expanded population of cells expressing the G1ME surface phenotype. Individual cells within this population also exhibit TPO-dependency, extensive proliferative capacity and GATA-1-dependent biphenotypic erythro-megakaryocytic maturation in vitro. Our findings indicate that the loss of GATA-1 impairs the maturation of a specific megakaryocyte-erythroid progenitor. This defines a new role for GATA-1 at a relatively early stage of hematopoiesis and provides potential insight into recent discoveries that human GATA1 mutations promote acute megakaryoblastic leukemia (AMKL), a clonal malignancy with features of both erythroid and megakaryocyte maturation.
RUNX1 Directly Regulates Band 3 Transcription.
Authors: Xie Jingping , Hiebert Scott W. , Koury Mark J. , Brandt Stephen J. 
Published: April 2020

Abstract
RUNX1 (AML1 or CBFA2) regulates the expression of a number of genes important to hematopoiesis. Gene knockout studies demonstrated that a heterodimeric complex of RUNX1 and its DNA binding partner, core binding factor-beta (CBFbeta), is essential for definitive hematopoiesis. Here, we report that RUNX1 directly represses expression of the Band 3 gene prior to terminal erythroid differentiation. Band 3 is one of four major components of the erythrocyte membrane skeleton and is important for maintenance of cytoskeletal architecture and electroneutral Cl-/HCO3− exchange across the red cell membrane. Band 3 expression, like that of beta-globin, increases dramatically with terminal erythroid differentiation. In a previous study, we identified an upstream region in the mouse Band 3 gene designated as B3URE (for Band 3 upstream regulatory region) bound by multiple transcription factors, including TAL1 (also known as SCL), RUNX1, Ldb1, and GATA1, that acts as an orientation- and position-independent and tissue-specific repressive element. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 was associated with the B3URE in intact MEL cells and electrophoretic mobility shift analysis confirmed specific RUNX1 interaction with RUNX1 binding sites in the B3URE. Together with CBFbeta, RUNX1 inhibited reporter activity from a construct linking the B3URE with 1 kb of Band 3 promoter in transiently transfected MEL but not COS cells. DNA affinity precipitation analysis with wild-type and mutant oligos established that RUNX1 and CBFbeta in MEL cell nuclear extracts could interact with the B3URE in vitro and suggested that RUNX1 recruits TAL1 and Ldb1 to DNA. Northern blot and quantitative real-time PCR analysis demonstrated that enforced expression of RUNX1 dramatically inhibited dimethylsulfoxide (DMSO)-induced Band 3 gene expression. Quantitative ChIP analysis showed that histone acetylation in the B3URE increased more than 4-fold, while histone methylation decreased ~50% after 3 days of DMSO-induced differentiation. Over the same time frame, the promoter region underwent significantly less acetylation but more extensive demethylation. Finally, changes in B3URE acetylation and methylation were attenuated and inhibited, respectively, in RUNX1-transfected MEL cells relative to vector controls. In sum, these results demonstrate that the Band 3 gene is a direct target of RUNX1 in erythroid cells and indicate that the B3URE contributes to the tightly regulated expression of this gene in differentiating erythroid progenitors. One mechanism by which RUNX1 regulates Band 3 transcription may be by influencing histone acetylation/methylation in this upstream regulatory region.
Outcomes after HLA-Matched Sibling Transplants or Chemotherapy in Children with Acute Lymphoblastic Leukemia in Second Remission: A Collaborative Study of the Children’s Oncology Group (COG) and the Center for International Blood and Marrow Transplant Research (CIBMTR).
Authors: Eapen Mary , Raetz Elizabeth , Zhang Mei-Jie , Muehlenbein Catherine , Devidas Meenakshi , Abshire Thomas , Billett Amy , Homans Alan , Camitta Bruce , Carroll William , Davies Stella 
Published: April 2020

Abstract
The best treatment for children with B-precursor acute lymphoblastic leukemia (ALL) in second remission after a marrow relapse is controversial. To address this question, we compared outcomes in 192 patients enrolled on COG chemotherapy trials 9110 (n=109), 9310 (n=59) and 9411 (n=29) and 189 HLA-matched sibling transplant recipients reported to the CIBMTR. Patients received treatment between 1991 and 1997. Patients with isolated extra-medullary relapse were excluded. Median follow-up was 103 (range 34–159) and 101 (16–160) months after chemotherapy and transplantation, respectively. Groups were similar with respect to sex, leukemia sub-type, leukocyte count at diagnosis and duration of first remission. Chemotherapy recipients were younger at diagnosis (5 vs. 8 years) and less likely to have combined marrow and extra-medullary site relapse (12% vs. 30%). To adjust for time-to transplant bias, we used left-truncated Cox regression models to examine treatment outcomes. The relative efficacy of chemotherapy and transplantation depended on the duration of first clinical remission and transplant conditioning regimen as shown in the Table below. Comparing children with early relapse (&lt;36 months from diagnosis) the 5-year probabilities of leukemia-free survival (LFS) were 23%, 44% and 8% after chemotherapy alone, transplantation with a total body radiation (TBI)-containing regimen and a non-TBI regimen, respectively. In contrast to these findings, relapse and treatment failure were similar after transplantation with a TBI-containing regimen or chemotherapy after a late relapse (≥36 months from diagnosis). The 5-year probabilities of LFS were 61% and 60% after chemotherapy alone and a TBI-containing regimen, respectively and significantly lower at 30% after a non-TBI regimen. These data support HLA-matched related donor transplantation using a TBI-containing regimen in second remission for patients with ALL and early relapse.
Early relapse (&lt;36 months) Late relapse ≥36 months) ( Outcome N Relative Risk (95% CI) P-value N Relative Risk (95% CI) P-value Leukemia recurrence Chemotherapy 115 1.00 81 1.00 TBI-containing regimen 92 0.50 (0.27–0.68) 0.0004 61 1.02 (0.62–1.68) 0.91 Non-TBI containing regimen 19 1.36 (0.81–2.31) 0.24 14 2.51 (1.23–5.16) 0.01 Treatment failure Chemotherapy 115 1.00 81 1.00 TBI-containing regimen 92 0.53 (0.37–0.75) 0.0003 61 0.93 (0.63–1.37) 0.70 Non-TBI containing regimen 19 1.49 (0.93–2.37) 0.09 14 2.87 (1.60–5.13) 0.0004
A Global Role for EKLF in Definitive and Primitive Erythropoiesis.
Authors: Perkins Andrew C. , Coghill Elise , Maguire Tina , Hartmann Belinda , McDowall Alasdair , Weiss Mitchell , Grimmond Sean , Keys Janelle , Hodge Denise 
Published: April 2020

Abstract
Erythroid Kruppel-like factor (EKLF or Klf1) is an erythroid specific C2H2 zinc-finger transcription factor which is essential for definitive erythropoiesis and β-globin gene expression. The absence of EKLF results in fatal anaemia but correction of globin chain imbalance does result in rescue, suggesting the existence of additional EKLF target genes. The aim of this study was to search for such genes by expression profiling. We performed profiling on fetal livers from wild-type versus EKLF null litter mates, and also EKLF null erythroid cell lines containing an inducible EKLF-ERTM fusion construct. Hybridisations were performed on microarray slides printed with a 23K oligo library from Compugen. Target gene validation was performed by real-time RT-PCR, chromatin immuno-precipitation (ChIP) and promoter-reporter assays. A large number of genes were down regulated in the absence of EKLF but few were up regulated, suggesting EKLF acts primarily as a transcriptional activator in vivo. One hundred genes were EKLF dependent in both systems. These include heme synthesis enzymes, red cell surface proteins including Rh and the transferrin receptor, and erythroid transcription factors. Two interesting highly EKLF-dependent genes are α-haemoglobin stabilising protein (AHSP), a key chaperone for free a-globin chains, and dematin (band 4.9) which links the cytoskeleton to the red cell membrane. A search for EKLF binding sites within the dematin and AHSP genes demonstrated a number of phylogenetically conserved CACC sites, and ChIP demonstrated in vivo EKLF occupancy at some but not all of these. Promoter-reporter assays showed EKLF directly activates dematin gene transcription through two promoters containing these sites. Lastly, investigation of EKLF target genes in the yolk sac lead to the discovery of unexpected defects in the embryonic red cell membrane and cytoskeleton. In conclusion, EKLF regulates global erythroid gene expression which is critical for development of primitive as well as definitive red cells.
Differential GATA-1 Sensitivities of Target Loci.
Authors: Johnson Kirby D. , Pal Saumen , Grass Jeffrey A. , Bresnick Emery H. 
Published: April 2020

Abstract
The transcription factor GATA-1 is a key regulator of red cell differentiation, activating numerous erythroid-specific genes while downregulating genes that permit proliferation of erythroid precursors. We have demonstrated that GATA-1 only occupies a limited number of the high affinity WGATAR motifs present within target gene loci, FOG-1 is required for GATA-1 to occupy a subset of chromatin sites, and GATA-1 occupancy often coincides with GATA-2 displacement or a “GATA switch”. Once bound, GATA-1 elicits changes in the histone modification patterns and the three dimensional structure of target gene loci via recruitment of cofactors such as FOG-1 and CBP. As detailed mechanistic studies have not been conducted with most GATA-1 target genes, it is unclear whether these genes are equally sensitive to GATA-1 or if they respond differently to the rising GATA-1 levels during erythropoiesis. Using GATA-1 fusions to the estrogen receptor ligand binding domain (ER-GATA-1) in the GATA-1-null erythroid precursor cell line, G1E, we analyzed the responses of endogenous GATA-1 target genes to varied levels of GATA-1 activity. We found that transcriptional activation of Tac-2 required higher concentrations of ER-GATA-1 than is required for other GATA-1 target genes. Previously, we showed that Tac-2, which encodes the neurokinin-B precursor protein preprotachykinin B, is regulated by GATA-1 in erythroid cell lines and is induced upon ex vivo differentiation of human CD34+ peripheral blood cells. Importantly, whereas regulation of many GATA-1 target genes is only partially disrupted by removal of the N-terminal 193 amino acids from ER-GATA-1 (ER-GATA-1 ΔN), Tac-2 expression was very sensitive to this truncation. Whereas NK-B signals through G-protein-coupled receptors to modulate neuronal function, its functions beyond the nervous system are poorly understood. Although erythroid cells do not express NK-B receptors, the receptors, but not NK-B, are expressed in certain endothelial cell subtypes, and elevated levels of NK-B are implicated in the pregnancy-associated disorder pre-eclampsia. Tac-2 represents the first GATA-1 target gene that critically requires the N-terminus. Studies are underway to elucidate mechanisms underlying the exquisite sensitivity of Tac-2 to deletion of the GATA-1 N-terminus, the relationship between Tac-2 deregulation and GATA-1 N-terminal deletions in megakaryoblastic leukemia, and the function of erythroid cell-derived NK-B.
Strikingly Homologous Immunoglobulin Gene Rearrangements and Poor Outcome in VH3-21-Utilizing Chronic Lymphocytic Leukemia Independent of Geographical Origin and Mutational Status.
Authors: Murray Fiona , Thorselius Mia , Krober Alexander , Thunberg Ulf , Tobin Gerard , Buhler Andreas , Kienle Dirk , Albesiano Emilia , Dao-Ung Lan-Phuong , Wiley James , Vilpo Juhani , Laurell Anna , Roos Goran , Karlsson Karin , Chiorazzi Nicholas , Marasca Roberto , Dohner Hartmut , Stilgenbauer Stephan , Rosenquist Richard 
Published: April 2020

Abstract
We recently reported that Swedish VH3-21-utilizing chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite VH mutation status. To investigate whether VH3-21+ CLLs have similar characteristics in different parts of the world, we analyzed the VH and VL gene rearrangements in 90 patients from Sweden, Germany, Italy, USA, Finland and Australia and correlated these data with survival. Sixty-three percent of cases exhibited mutated VH genes and 37% had unmutated VH genes. Fifty patients (56%) displayed a short and homologous heavy-chain CDR3, many of these with the amino acid motif, DANGMDV. Also, a highly biased Vλ2-14 usage was evident in 73% of patients with a restricted light-chain CDR3, QVWDS(S/G)SDHPWV. Combined restricted heavy- and light-chain CDR3s were found in patients from all included countries. Although VH3-21+ CLLs have a remarkably predominant λ-expression, analyses of kappa deleting element showed a conserved rearrangement order of the light-chain loci. The overall survival was poor in the VH3-21+ cohort (median survival 88 months) with no significant difference in relation to mutation status or homologous/non-homologous CDR3. In summary, highly restricted B-cell receptors and worse outcome characterize VH3-21+ CLLs independent of geographical origin and mutation status. VH3-21 usage should now be included in prognostic stratification of CLL when assessing mutation status.
Increased Risk for Treatment-Related Mortality of Bone Marrow Transplantation in GSTM1-Positive Recipients.
Authors: Terakura Seitaro , Murata Makoto , Nishida Tetsuya , Emi Nobuhiko , Akatsuka Yoshiki , Riddell Stanley R. , Morishima Yasuo , Kodera Yoshihisa , Naoe Tomoki 
Published: April 2020

Abstract
Treatment-related mortality (TRM) is a major obstacle to successful allogeneic hematopoietic stem cell transplantation (HSCT). A variety of drugs are used in allogeneic HSCT, such as chemotherapeutic agents, immunosuppressants, antibiotics, antifungal and antiviral drugs, thus, a genetic polymorphism in metabolic enzymes would affect the metabolism of drugs and subsequent TRM after HSCT. We recently reported a metabolic enzyme UDP glycosyltransferase 2 family, polypeptide B17 (UGT2B17) has a null phenotype, and the use of a UGT2B17-positive donor is an independent risk factor for higher TRM and lower survival after HSCT (ASH annual meeting 2004, abstract# 1837). Here, we focused attention on glutathione S-transferase (GST) M1 and GSTT1 enzymes. These enzymes mainly metabolize chemotherapeutic agents, chemical carcinogens and by-products of oxidative stress and are absent from more than 50% of some populations. To assess the significance of homozygous GSTM1 and GSTT1 gene deletion in allogeneic HSCT, we analyzed DNA from 373 patients with a hematological disease and their HLA-A, B, C, DRB1-identical unrelated bone marrow donors using PCR method. All patients received an intensive myeloablative pretransplant conditioning regimen, an unmanipulated marrow graft, and cyclosporine A or tacrolimus as GVHD prophylaxis. Homozygous GSTM1 and GSTT1 gene deletion were observed in 56% and 45% of patients with a hematological disease, respectively, and 57% and 46% of healthy donors, respectively. There was no difference in the frequencies between disease group and donor group. We next applied Cox proportional hazard model to multivariate analysis for TRM which was defined as any death that occurred while the patient was in remission, acute GVHD, chronic GVHD, relapse, disease-free survival (DFS) and overall survival (OS). The analysis showed no significant association between GSTT1 polymorphism and any outcome. However, GSTM1-positive recipient was significantly associated with higher TRM and lower survival: TRM; Odds ratio (OR), 1.49; 95% confidence interval (CI), 1.03–2.16; P=0.036: DFS; OR, 1.50; 95% CI, 1.08–1.97; P=0.013: OS; OR, 1.41; 95% CI, 1.04–1.92; P=0.030. TRM was analyzed in relation to GSTM1 genotype in the patient by the Kaplan-Meier method (Fig 1). TRM in the GSTM1-positive patients (42.9%, n=166) was significantly higher than that in the GSTM1-negative patients (29.3%, n=201) (P=0.04). These data suggest the presence of the GSTM1 null genotype in recipient provide better protection against TRM after allogeneic HSCT. Some reactive intermediates or toxic metabolites generated by GSTM1 may initiate or promote the development of TRM.
Figure Figure
Multidrug Resistance-1 Gene Polymorphism Associated with the Outcomes after Allogeneic HLA-Identical Stem Cell Transplantation.
Authors: Kim Dong Hwan , Jeon Seok Bong , Baek Jin Ho , Lee Nan Young , Kim Jong Gwang , Sohn Sang Kyun , Suh Jang Soo , Do Young Rok , Kwon Ki Kyung 
Published: April 2020

Abstract
Background: The pharmacokinetic impact of multidrug resistance-1 (MDR1) gene single nucleotide polymorphisms (SNPs) has been already investigated in solid organ transplantation field, however, data is still lacking in an allogeneic stem cell transplantation (SCT) setting.
Methods: A total of 82 patients receiving an allogeneic HLA-identical sibling (n=70) or unrelated SCT (n=12) with graft-versus-host disease (GVHD) prophylaxis of cyclosporine-A (CSA) plus methotrexate (MTX) were included in the current study. Two SNPs of MDR1 gene (C3435T and G2677T/A) were analyzed using PCR/RFLP assay.
Results: As regards G2677T/A SNP, GG genotype showed a higher incidence of NRM compared to non-GG genotype (67% vs. 32%, p=0.0073), yet not C3435T (p=0.2026) or MDR1 haplotype (p=0.2238). Accordingly, overall survival (OS) was significantly correlated with G2677T/A genotype (p=0.0048), yet not with C3435T (p=0.5041) or MDR1 haplotype (p=0.4086). However, no difference in the relapse incidence was noted according to G2677T/A, C3435T genotype or MDR1 haplotype. In a multivariate analysis, those patients without GG genotype at G2677T/A were found to have favorable prognosis in terms of OS (p=0.003) or NRM (p=0.031) along with occurrence of chronic GVHD (p&lt;0.001 for OS, p=0.001 for NRM), standard disease risk (p=0.045 for OS) or acute grade 0,1 GVHD (p=0.019 for NRM). However, no correlation was found between the blood concentrations of CSA and MDR1 genotype and CSA neurotoxicity and MDR1 genotype.
Conclusion: The G2677T/A genotype seemed to be associated with the transplantation outcomes, especially NRM. Further study is warranted to clarify its mechanism of MDR1 SNPs other than pharmacokinetic aspects.
Figure. Overall survival (A) and cumulative incidence of non-relapse mortality (B) according to G2677T/A MDR1 genotype Figure. Overall survival (A) and cumulative incidence of non-relapse mortality (B) according to G2677T/A MDR1 genotype