A Comprehensive Genomic Approach Using Gain of Function and Loss of Function Cell Models and ChIP-on-Chip Technology Identifies Novel Promyelocytic Zinc Finger Protein Target Genes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1407-1407
Author(s):  
Kim L. Rice ◽  
Ari Melnick ◽  
Kenny Ye ◽  
Windy Berkofsky-Fessler ◽  
Jonathan D. Licht
Keyword(s):  
Target Genes ◽  
Rna Binding ◽  
Zinc Finger Protein ◽  
Leukemia Cells ◽  
Loss Of Function ◽  
Function Model ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Endogenous Expression ◽  
On Chip

Abstract The t(11;17)(q23;q21) form of APL involves the production of reciprocal fusion proteins, PLZF-RARα and RARα-PLZF, which mediate malignant transformation by binding to and dysregulating RARα and PLZF target genes. PLZF is expressed in hematopoietic stem cells and is downregulated as cells differentiate. The identification of PLZF target genes including cyclin A2 and MYC is consistent with the hypothesis that PLZF maintains stem cell quiescence by repressing cell cycle driving genes and provides insight into transcriptional pathways disrupted in leukemogenesis. In order to identify additional target genes of PLZF, we constructed a loss of function model in which we suppressed endogenous expression of PLZF using siRNA in KG1a leukemia cells. Our gain of function model consisted of the ectopic expression of PLZF in U937 leukemia cells which do not naturally express PLZF. Expression profiling using GeneChip™ Human Genome U133 Plus 2.0 arrays, which analyze the expression of more than 47,000 transcripts, was performed using both systems. Of the 346 genes identified in the loss of function model, 25% were also regulated by PLZF in the gain of function U937 cell line. Changes in expression of these genes could be direct (through PLZF) or indirect (through secondary effects). In order to determine which genes modulated by changes in PLZF expression are direct transcriptional targets, we immunoprecipitated chromatin using PLZF antibodies in KG1a cells, amplified the products by ligation-mediated PCR and co-hybridized these products with input chromatin to NimbleGen 1.5kB promoter arrays, which represent 24,275 human promoters. Genes bound by PLZF were identified by determining whether consecutively tiled probes were enriched in PLZF-precipitated chromatin as compared to chromatin precipitated with a non-specific antibody. Using a statistical algorithm designed to exclude those probes whose signals of PLZF enrichment might be spuriously identified, we identified 52 genes of the 24,275 on the array as potential PLZF target genes. Strikingly, correlation of these genes with expression analyses revealed that 44% of genes were also significantly regulated by PLZF in the gain of function model and 11% of genes were regulated in the loss of function model. Promoter analyses of a subset of these genes that were identified by ChIP-on-Chip and differentially expressed at least >1.3 fold in PLZF arrays (p<0.05), revealed the presence of a consensus PLZF binding site GTC(C/A)AG in 75% of genes. Analysis of gene ontology for those genes identified by ChIP-on Chip, revealed an enrichment of genes involved in RNA binding and processing as well as genes encoding small G proteins. One gene in particular, RECQL, was directly bound by PLZF in the ChIP-on-Chip assay and transcriptionally regulated by PLZF in both KG1a loss of function and U937 gain of function models. The RECQL protein is a member of the RecQ family of DNA helicases, a class of genes whose mutation is associated with genomic instability tumorigenesis and premature ageing. These data indicate a robust system for the identification of PLZF targets and suggest that PLZF may play a role in genome integrity.

scholarly journals SETDB1 mediated histone H3 lysine 9 methylation suppresses MLL-fusion target expression and leukemic transformation

Haematologica ◽  
2019 ◽  
Vol 105 (9) ◽  
pp. 2273-2285 ◽  
Author(s):  
James Ropa ◽  
Nirmalya Saha ◽  
Hsiangyu Hu ◽  
Luke F. Peterson ◽  
Moshe Talpaz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Critical Role ◽  
High Expression ◽  
Leukemia Cells ◽  
Biological Impact ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Epigenetic regulators play a critical role in normal and malignant hematopoiesis. Deregulation, including epigenetic deregulation, of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia. We recently showed that the Histone 3 Lysine 9 methyltransferase SETDB1 negatively regulates the expression of the pro-leukemic genes Hoxa9 and its cofactor Meis1 through deposition of promoter H3K9 trimethylation in MLL-AF9 leukemia cells. Here, we investigated the biological impact of altered SETDB1 expression and changes in H3K9 methylation on acute myeloid leukemia. We demonstrate that SETDB1 expression is correlated to disease status and overall survival in acute myeloid leukemia patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of leukemia cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While myeloid leukemia cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of cKit+ hematopoietic stem and progenitor cells. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of leukemia cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with acute myeloid leukemia, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in leukemia that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing acute myeloid leukemia.

scholarly journals Correction to: DNMT3A reads and connects histone H3K36me2 to DNA methylation

2019 ◽  
Vol 11 (3) ◽  
pp. 230-230
Author(s):  
Wenqi Xu ◽  
Jiahui Li ◽  
Bowen Rong ◽  
Bin Zhao ◽  
Mei Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone Mark ◽  
The Other ◽  
Therapeutic Implication ◽  
Loss Of Function ◽  
Function Model ◽  
Gain Of Function ◽  
Intergenic Dna ◽  
Preferential Recognition ◽  
Function Models

The author would like to add the below information in this correction. A similar study from Chao Lu group was published online on 5 September 2019 in Nature, entitled “The histone mark H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape” (Weinberg et al., 2019). Although both the studies reported the preferential recognition of H3K36me2 by DNMT3A PWWP, ours in addition uncovered a stimulation function by such interaction on the activity of DNMT3A. On the disease connections, we used a NSD2 gain-of-function model which led to the discovery of potential therapeutic implication of DNA inhibitors in the related cancers, while the other study only used NSD1 and DNMT3A loss-of-function models.

A Comprehensive Genomic Approach Using Gain of Function Cell Models, Patient Specimens and ChIP-on-CHIP Technologies Identifies PLZF-RARα Target Genes with Potential Roles in t(11;17) Associated APL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3168-3168
Author(s):  
Jonathan D. Licht ◽  
Kim L. Rice ◽  
Itsaso Hormaeche ◽  
Julia Meyer ◽  
Ken I. Mills ◽  
...  
Keyword(s):  
Cell Growth ◽  
Target Genes ◽  
Expression Profiles ◽  
Defense Responses ◽  
Myeloid Differentiation ◽  
Small Subset ◽  
U937 Cells ◽  
Gain Of Function ◽  
Cell Functions ◽  
On Chip

Abstract The t(11;17)(q23;q21) translocation involves the production of reciprocal fusion proteins PLZF-RARα and RARα-PLZF, which mediate malignant transformation by binding to and dysregulating RARα/RXR and PLZF target genes, respectively. In order to investigate the molecular basis for PLZF-RARα induced leukemogenesis, we used a gain of function model in which PLZF-RARα was ectopically expressed in U937 leukemia cells. After demonstrating in our system that PLZF-RARα is capable of inducing a G1 cell cycle arrest and inhibiting cell growth and myeloid differentiation, we sought to identify genes directly bound and transcriptionally regulated by PLZF-RARα. Chromatin from U937PLZF-RARα expressing cells (+10nM RA) was immunoprecipitated using PLZF antibodies, amplified by ligation-mediated PCR and biological triplicates were hybridized to NimbleGen 2.7kB promoter arrays, which represent 24,275 human promoters. We identified 1797 genes that are directly bound by PLZF-RARα in at least 2 out of 3 arrays, and the majority of these genes (89%) are also bound in the absence of exogenously added RA. Quantitative real time PCR using primary ChIP samples was used to validate ChIP-on-CHIP results and all genes tested to date (n=11) were confirmed as direct targets of PLZF-RARα. Ontological analyses of genes identified by ChIP-on-CHIP revealed enrichment for genes involved in myeloid cell functions including immune, inflammatory and defense responses, in addition to genes involved in apoptosis and signal transduction pathways. Furthermore, genes encoding nuclear proteins were also highly enriched and these included previously identified RARα/RXR target genes (ie. CEBPε, RARβ2, PRAM1, NFE-2), which are likely targeted by the PLZF-RARα oncoprotein, as well as novel PLZF-RARα targets, many of which have roles in blood cell development and have been implicated in leukemia (ie. RUNX1, MLL2, MCL1, PIM1, FANCB). Of these 1797 genes, a significant percentage (22%) are also transcriptionally regulated by PLZF-RARα (>1.5 fold, p<0.05). To identify genes specific to the PLZF-RARα fusion generated in t(11;17) APL, we compared gene expression profiles of 26 PML-RARα and 4 PLZF-RARα expressing APL patient blasts. A comparison of differentially expressed genes in the patient specimens with those both directly bound and regulated by PLZF-RARα in U937 cells, identified a small subset of genes including RUNX1, KLF10, a transcriptional regulator and inhibitor of cell growth, as well as ID1 and ID2, whose expression level has been shown to correlate with myeloid differentiation. Although the expression of these genes was variable in PML-RARα blasts, expression was consistently lower in PLZF-RARα APL blasts (>2 fold, p<0.03). In U937 cells, PLZF-RARα repressed RUNX1, KLF10 and ID1 in the absence of exogenous RA. Intriguingly, RUNX1, KLF10 and ID2 were also identified as direct target genes of PLZF in the KG1a cell line and were transcriptionally regulated by PLZF in U937 cells, suggesting that PLZF and PLZF-RARα may co-regulate a subset of target genes. Given the roles of RUNX1, KLF10, ID1 and ID2 in myeloid differentiation and growth inhibition, these genes may represent PLZF-RARα specific targets that potentially contribute to the pathogenesis of t(11;17) APL.

MicroRNA Maestros of Hematopoiesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. SCI-32-SCI-32
Author(s):  
Kara A. Scheibner ◽  
Diane Heiser ◽  
Ian M Kaplan ◽  
Wen-Chih Cheng ◽  
MinJung Kim ◽  
...  
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Human Cd34 ◽  
Leukemia Treatment ◽  
Normal Human ◽  
Target Molecules ◽  
And Function

Abstract Abstract SCI-32 MicroRNAs (miRs) inhibit stability and/or translation of mRNAs, usually by binding to specific sites in the 32′UTRs of their target mRNAs. Due to imperfect (i.e. partially complementary) miR:mRNA base-pairing, miRs can block translation of many mRNAs and serve as powerful master switches to regulate cell functions. Therefore, we profiled miR expression in human CD34+ hematopoietic stem-progenitor cells (HSPCs) and combined human HSPC miR expression, mRNA expression, and miR-mRNA target predictions to hypothesize that certain HSPC-expressed miRs (HE-miRs) target several mRNAs critical to hematopoiesis. On this informatic basis, we formulated a model of hematopoietic differentation in which many genes specifying hematopoietic differentiation are expressed by early HSPCs, but held in check by miRs [1]. In addition, we noted that the miR-23a cluster (i.e. adjacent, co-transcribed miR-23a, miR-27a, and miR-24-2) is not expressed or is expressed at levels >2-fold lower in 50% of acute myeloid leukemias and 80% of acute lymphoid leukemias tested compared to normal human HSPCs. ‘Re-expressing’ 1 or more of these miR-23a cluster members in leukemia cells promotes their apoptosis and reduces their proliferation, thus suggesting that these miRs have a tumor suppressive role. We have identified YWHAQ (14-3-3q) and several other 14-3-3 isoforms, which are anti-apoptotic and have established roles as oncogenes, as miR-23a cluster target molecules. Artificial manipulation of these HE-miRs and their target genes may lead to novel strategies for leukemia treatment and/or for expansion of normal HSPCs. Since the CD34+ HSPCs that we studied initially include rare stem cells and various stages of progenitors, we have expanded our miR profiling to more highly purified subsets of mouse HSPCs. Several previously described (e.g. miR-155 [1], miR-451 [2], miR-146 [3]) and novel HE-miRs are expressed differentially in lineages/stages of HSPCs, and their selective expression has been confirmed in human HSPC subsets. We are using cellular gain- and loss-of-function approaches with hematopoietic functional assays to determine whether these HE-miRs control human hematopoiesis. Understanding the effects of HE-miRs in hematopoiesis may elucidate hematopoietic and general stem cell biologic mechanisms. 1. Georgantas RW, 3rd, Hildreth R, Morisot S, Alder J, Liu CG, Heimfeld S, Calin GA, Croce CM, Civin CI. CD34+ hematopoietic stem-progenitor cell microRNA expression and function. A circuit diagram of differentiation control. Proc Natl Acad Sci USA. 2007;104:2750–2755. 2. Dore LC, Amigo JD, Dos Santos CO, Zhang Z, Gai X, Tobias JW, Yu D, Klein AM, Dorman C, Wu W, Hardison RC, Paw BH, Weiss MJ. A GATA-1-regulated microRNA locus essential for erythropoiesis. Proc Natl Acad Sci USA. 2008;105:3333–3338. 3. Starczynowski DT, Kuchenbauer F, Argiropoulos B, Sung S, Morin R, Muranyi A, Hirst M, Hogge D, Marra M, Wells RA, Buckstein R, Lam W, Humphries RK, Karsan A. Identification of miR-145 and miR-146a as mediators of the 5q- syndrome phenotype. Nat Med. 2010;16:49–58. Disclosures: No relevant conflicts of interest to declare.

Role of Notch Signaling in Hematopoietic Stem Cell Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-15-SCI-15
Author(s):  
Lluis Espinosa ◽  
Anna Bigas
Keyword(s):  
Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Notch Pathway ◽  
Cell Types ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
On Chip ◽  
Stem Cell Populations

Abstract Abstract SCI-15 The Notch pathway controls the generation of different cell types in most tissues including blood, and dysregulation of this pathway is strongly associated with oncogenic processes. In many systems, Notch is also required for the maintenance of the stem cell populations. However, in the adult hematopoietic system this link between Notch and stemness has not been established. Instead, work of several groups, including ours, has clearly demonstrated that Notch has a prominent role in the generation of hematopoietic stem cells (HSC) during embryonic development. Although the first wave of blood cells appears in the mouse embryo around day 7.5 of development and is independent of Notch function, embryonic HSC are formed around day 10 of development from endothelial-like progenitors that reside in the embryonic aorta surrounded by the gonad and mesonephros, also called AGM region. By analyzing different Notch pathway mutant mouse embryos, we have demonstrated the involvement of the Jagged1-Notch1-GATA2 axis in this event. However, the formal demonstration that Notch regulates the GATA2 gene during HSC generation is still lacking. We have now found that GATA2 is a direct Notch target in vivo during embryonic HSC generation. However, whereas Notch positively activates GATA2 transcription in the HSC precursors, it simultaneously activates hes1 transcription, which acts a repressor of the same GATA2 gene. This finding directly implicates hes1 in the regulation of HSC development although further studies using loss-of-function mutant embryos are still needed. Altogether, our results indicate that both Notch and hes1 are required to finely regulate the levels, distribution, and likely the timing of GATA2 expression through an incoherent feed-forward loop. In parallel, we have identified other downstream targets of Notch in the AGM region by ChIP-on-chip and expression microarray analysis that we are currently characterizing. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Loss-of-Function and Gain-of-Function Consequences of GATA2 Disease Mutations

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2519-2519
Author(s):  
Koichi Ricardo Katsumura ◽  
Peng Liu ◽  
Charu Mehta ◽  
Kyle J Hewitt ◽  
Alexandra Soukup ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Molecular Mechanisms ◽  
Differentially Expressed ◽  
Homozygous Mutation ◽  
Loss Of Function ◽  
Granulocytic Differentiation ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Disease Mutations

The master regulator of hematopoiesis GATA2 controls generation and function of hematopoietic stem and progenitor cells, and heterozygous GATA2 mutations create a predisposition to develop immunodeficiency, myelodysplasia, and acute myeloid leukemia (Spinner et al. Blood, 2014; Dickinson et al. Blood, 2014; Churpek and Bresnick J. Clin. Invest. 2019). Although mechanisms that trigger the transition of a non-pathogenic GATA2 mutation into overt pathology are enigmatic, a paradigm has arisen in which GATA2 mutations are considered to be loss-of-function. We developed a genetic rescue assay to quantify the function of wild type GATA2 and GATA2 disease mutants when expressed at near-physiological levels in primary progenitor cells and demonstrated that GATA2 disease mutations abrogate certain biological and molecular activities, while enabling others (Katsumura et al., 2018, PNAS). We isolated lineage-negative (Lin-) or Lin-Kit+ cells from fetal liver of mice with a homozygous mutation of the Gata2 -77 enhancer, which downregulates Gata2 expression by ~80%. The mutant progenitor cells are largely defective in erythroid, megakaryocytic and granulocytic differentiation and exhibit a predominant monocytic differentiation fate (Johnson et al., 2015, Science Adv.). We compared GATA2 and GATA2 disease mutant activities in the rescue system using a colony formation assay. GATA2, R307W mutant (in N-finger) and T354M mutant (in DNA-binding C-finger) rescued myeloid colony formation and promoted granulocyte proliferation. Surprisingly, R307W and T354M induced more CFU-GM than GATA2. GATA2 and R307W, but not T354M, rescued BFU-E. These data indicated that GATA2 disease mutations were not strictly inhibitory, and in certain contexts, mutant activities exceeded that of GATA2. To extend these results, we subjected -77+/+ or -77-/- Lin- cells to a short-term ex vivo liquid culture, expressed GATA2, R307W, or T354M and used RNA-seq to elucidate progenitor cell transcriptomes. While -77+/+ Lin- cells generate erythroid and myeloid cells, -77-/- Lin- cells are competent for myeloid, but not erythroid, differentiation. Comparison of -77+/+ and -77-/- cell transcriptomes revealed 3064 differentially expressed genes (>2-fold). 1824 genes were >2-fold higher in -77+/+ cells, and 1240 genes were >2-fold higher in -77-/- cells. GATA2 expression in -77-/- cells activated 834 genes >2-fold and repressed 503 genes >2-fold. 60-65% of these genes overlapped with genes differentially expressed between -77+/+ cells and -77-/- cells. R307W expression activated 661 genes >2-fold and repressed 523 genes >2-fold. T354M expression activated 468 genes >2-fold and repressed 575 genes >2-fold. The genes regulated by mutants included GATA2-regulated genes and certain genes that were not GATA2-regulated. Multiple genes were hypersensitive to the mutants, relative to GATA2, and the mutants ectopically regulated certain genes. However, R307W and T354M did not universally regulate an identical gene cohort. For example, both R307W and T354M activated Ncam1, Nrg4, and Mpo more strongly than GATA2. R307W, but not T354M, activated Ear2 and Ces1d more strongly than GATA2. By contrast, T354M, but not R307W, activated Ctsg, Epx, and Rab38 more strongly than GATA2. Both R307W and T354M repressed macrophage genes similarly to GATA2, but they lacked the capacity to activate mast cell genes, differing from GATA2. To elucidate molecular mechanisms underlying GATA2 mutant activities, we leveraged our prior discovery that p38 or ERK kinases induce multi-site GATA2 phosphorylation (Katsumura et al. Blood. 2017). We tested whether these kinases mediate the ectopic transcriptional regulatory activity of GATA2 disease mutants. p38 inhibition attenuated aberrant regulation of Ear2 and Ces1d by R307W (p < 0.05), and mutation of S192 to S192A decreased R307W-induced CFU-GM formation by 49% (p < 0.05). In aggregate, these results indicate that GATA2 disease mutants exert context-dependent activities to regulate transcription and differentiation, activities can be signal-dependent and certain activities are distinct from GATA2. It is attractive to consider the pathogenic consequences of GATA2 disease mutant gain-of-function activities, and an important implication is GATA2 mutation-associated hematologic diseases might not solely reflect haploinsufficiency. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Ribosome Biogenesis Factor Ltv1 Is Essential for Digestive Organ Development and Definitive Hematopoiesis in Zebrafish

2021 ◽  
Vol 9 ◽  
Author(s):  
Chong Zhang ◽  
Rui Huang ◽  
Xirui Ma ◽  
Jiehui Chen ◽  
Xinlu Han ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Target Genes ◽  
P53 Protein ◽  
Ribosomal Subunit ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Independent Manner ◽  
Hematopoietic Stem ◽  
Digestive Organs ◽  
Stem And Progenitor Cells

Ribosome biogenesis is a fundamental activity in cells. Ribosomal dysfunction underlies a category of diseases called ribosomopathies in humans. The symptomatic characteristics of ribosomopathies often include abnormalities in craniofacial skeletons, digestive organs, and hematopoiesis. Consistently, disruptions of ribosome biogenesis in animals are deleterious to embryonic development with hypoplasia of digestive organs and/or impaired hematopoiesis. In this study, ltv1, a gene involved in the small ribosomal subunit assembly, was knocked out in zebrafish by clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR associated protein 9 (Cas9) technology. The recessive lethal mutation resulted in disrupted ribosome biogenesis, and ltv1Δ14/Δ14 embryos displayed hypoplastic craniofacial cartilage, digestive organs, and hematopoiesis. In addition, we showed that the impaired cell proliferation, instead of apoptosis, led to the defects in exocrine pancreas and hematopoietic stem and progenitor cells (HSPCs) in ltv1Δ14/Δ14 embryos. It was reported that loss of function of genes associated with ribosome biogenesis often caused phenotypes in a P53-dependent manner. In ltv1Δ14/Δ14 embryos, both P53 protein level and the expression of p53 target genes, Δ113p53 and p21, were upregulated. However, knockdown of p53 failed to rescue the phenotypes in ltv1Δ14/Δ14 larvae. Taken together, our data demonstrate that LTV1 ribosome biogenesis factor (Ltv1) plays an essential role in digestive organs and hematopoiesis development in zebrafish in a P53-independent manner.

scholarly journals Mapping Stem Cell Dependencies in Cancer Heterogeneity and Therapy Resistance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-3-SCI-3
Author(s):  
Tannishtha Reya
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Myeloid Leukemia ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Rna Binding Proteins ◽  
Leukemia Cells ◽  
Hematopoietic Stem ◽  
New Genes ◽  
A Genome

Our research focuses on the signals that control stem cell self-renewal and how these signals are hijacked in cancer. Using a series of genetic models, we have studied how classic developmental signaling pathways play key roles in hematopoietic stem cell growth and regeneration and are dysregulated during leukemia development. Through this work we have identified Hedgehog and Wnt signaling, and more recently the cell fate determinant Musashi, as critical players in driving progression of hematologic malignancies and as targets for therapy. To search for new regulators of myeloid leukemia, we have carried out a focused screen of surface molecules that may enable leukemia cells to receive supportive cues from the microenvironment. This screen identified key new adhesion signals that are critical to leukemia growth, drug resistance and dissemination. Using high resolution in vivo imaging we have mapped how these mediate the interactions that leukemia cells make within their microenvironment. To complement this focused screen, we have also carried out a genome wide CRISPR screen to more generally define the biological determinants of myeloid leukemia establishment and propagation. This screen identified a large number of new genes and programs critically required for leukemia, including those essential for chromatin remodeling and spliceosomal assembly. Among these, RNA binding proteins (RBPs) in general, and the chromatin binding sub-family of RBPs in particular, emerged as key new dependencies of myeloid leukemia. The talk will focus in part on these new regulators. Disclosures No relevant conflicts of interest to declare.

scholarly journals Iron Deficiency in Polycythemia Vera Increases HIF Activity and Transcription of Prothrombotic Genes

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2549-2549
Author(s):  
Jihyun Song ◽  
Soo Jin Kim ◽  
Perumal Thiagarajan ◽  
Josef T. Prchal
Keyword(s):  
Iron Deficiency ◽  
Polycythemia Vera ◽  
Target Genes ◽  
Negative Regulator ◽  
Controlled Study ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Transcript Levels ◽  
Increased Risk ◽  
Hif Pathway

Abstract Polycythemia vera (PV) is characterized by elevated red cell mass due to gain-of-function mutations of the tyrosine kinase JAK2 gene. Major causes of morbidity and mortality in PV are venous and arterial thromboses. Risk factors of thrombosis in PV are age &gt; 60 years, previous thrombosis history, and leukocytosis due to increased granulocytes (PMID: 31865003). High hematocrit has been considered as a causative factor of thrombosis due to high viscosity; however, this has been challenged. Phlebotomy to maintain hematocrit below 45% is performed by most hematologists to prevent thrombosis. However, in our prospective controlled study, we reported that phlebotomies, but not high hematocrit, are associated with increased risk of thrombosis in Chuvash erythrocytosis (CE). CE is due to a hypomorphic mutation of the Von Hippel-Lindau (VHL) gene, a negative regulator of hypoxia inducible factors (HIFs) (PMID 289208), leading to augmented HIFs. We hypothesize that repeated phlebotomies may increase the risk of thrombosis in PV. We reported that HIFs, and some HIFs-regulated thrombotic genes, are upregulated in PV in both granulocytes and platelets, and even more in PV with a thrombosis history (PMID: 32203583). PV patients with mutated JAK2 have lower ferritin, serum iron, transferrin saturation, and increased soluble transferrin receptor levels compared to JAK2-wild type erythrocytosis (PMID: 30042411), indicating that PV patients with JAK2 mutation are more likely to be iron deficiency (ID). Repeated phlebotomies further augment ID, which increases the level of HIF-1 and HIF-2 by inhibiting the principal negative regulator of HIFs, prolyl hydroxylase domain 2 (PHD2) enzyme. PHD2 requires iron as a co-factor (PMID: 18066546). This led us to hypothesize that further increase of HIFs due to phlebotomy-induced ID increases expression of HIF-regulated prothrombotic genes, leading to increased risk of thrombosis in PV. We studied 17 PV with ID, 32 PV without ID, and 19 healthy control and measured their prothrombotic genes' transcript levels in the granulocytes. Of 17 PVs with ID, 9 (56.3 %) had thrombosis while 14/32 PVs without ID (43.8 %) had thrombosis history. We then measured transcript levels of prothrombotic genes, such as F3 (tissue factor), SELP (P-selectin), THBS1 (thrombospondin-1), SERPINE1(plasminogen activator inhibitor-1) and HIF-target genes VEGFA and SLC2A1 (glucose transporter-1). The transcripts of these genes were upregulated in PV with and without ID compared to the controls. However, PV with ID had higher transcripts of these genes compared to PV without ID, except SERPINE1 (Figure 1). These prothrombotic gene transcripts were inversely correlated with ferritin levels and positively correlated with HIF target genes. Thus, ID in PV further augmented HIFs activity and induced expression of prothrombotic genes. In ongoing studies of PV patients serving as their own controls, PV subjects with ID are treated with hydroxyurea to prevent rebound erythrocytosis. The hydroxyurea treatment alone did not normalize high transcripts of prothrombotic and HIF target genes. However, transcripts of these prothrombotic and HIF-target genes decreased after iron correction while their JAK2V617F allele burden did not change. We then measured transcript levels of these prothrombotic genes in the patients with germline mutations augmenting HIF activity including loss of function VHL, gain-of-function EPAS1 (HIF2a), and loss-of-function EGLN1 (PHD2). These patients have high transcript levels of prothrombotic genes due to high HIF activity although they don`t have ID. Transcripts of F3, SELP, VEGFA, and SLC2A1 of the patients with HIF-pathway mutations were increased compared to the controls and compared to PV without ID but similar to the levels in PV with ID. On the other hand, transcript levels of THBS1 and SERPINE1 in the patients with HIF-pathway mutations were higher than the levels seen in the patients with PV with ID. These data indicate that ID in PV augments HIF activity similar, although not identical, to the patients with germline HIF-pathway mutations and may facilitate propensity to thromboses. These data suggest that therapeutic phlebotomy to control high hematocrit in PV and other erythrocytoses has the potential to increase thrombosis risk. In our ongoing studies, transcript levels of these prothrombotic genes are being correlated with their functions and protein levels. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Spatial genome re-organization between fetal and adult hematopoietic stem cells

2019 ◽  
Author(s):  
C Chen ◽  
W Yu ◽  
J Tober ◽  
P Gao ◽  
B He ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Three Dimensional ◽  
Gene Promoters ◽  
Loss Of Function ◽  
Dynamic Interactions ◽  
Hematopoietic Stem ◽  
3D Genome ◽  
Chromatin Interactions

AbstractFetal hematopoietic stem cells (HSCs) undergo a developmental switch to become adult HSCs. The functional properties of the HSCs change dramatically during this switch, including their cycling behavior, hematopoietic lineage outputs and proliferation rate. The relationship between three-dimensional (3D) genome organization, epigenomic state, and transcriptome is poorly understood during this critical developmental transition. Here we conducted a comprehensive survey of the 3D genome, epigenome and transcriptome of fetal and adult HSCs in mouse. We found that chromosomal compartments and topologically associating domains (TAD) are largely conserved between fetal and adult HSCs. However, there is a global trend of increased compartmentalization and TAD boundary strength in adult HSCs. In contrast, dynamics of intra-TAD chromatin interactions is much higher and more widespread, involving over a thousand gene promoters and distal enhancers. Such dynamic interactions target genes involved in cell cycle, metabolism, and hematopoiesis. These developmental-stage-specific enhancer-promoter interactions appear to be mediated by different sets of transcription factors in fetal and adult HSCs, such as TCF3 and MAFB in fetal HSCs, versus NR4A1 and GATA3 in adult HSCs. Loss-of-function studies of TCF3 confirms the role of TCF3 in mediating condition-specific enhancer-promoter interactions and gene regulation in fetal HSCs. In summary, our data suggest that the fetal-to-adult transition is accompanied by extensive changes in intra-TAD chromatin interactions that target genes underlying the phenotypic differences between fetal and adult HSCs.

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