Persistent Transactivation by Meis1 Replaces Hox Function in Myeloid Leukemogenesis Models: Evidence for Co-Occupancy of Meis1-Pbx and Hox-Pbx Complexes on Promoters of Leukemia-Associated Genes

ABSTRACT Homeobox transcription factors Meis1 and Hoxa9 promote hematopoietic progenitor self-renewal and cooperate to cause acute myeloid leukemia (AML). While Hoxa9 alone blocks the differentiation of nonleukemogenic myeloid cell-committed progenitors, coexpression with Meis1 is required for the production of AML-initiating progenitors, which also transcribe a group of hematopoietic stem cell genes, including Cd34 and Flt3 (defined as Meis1-related leukemic signature genes). Here, we use dominant trans-activating (Vp16 fusion) or trans-repressing (engrailed fusion) forms of Meis1 to define its biochemical functions that contribute to leukemogenesis. Surprisingly, Vp16-Meis1 (but not engrailed-Meis1) functioned as an autonomous oncoprotein that mimicked combined activities of Meis1 plus Hoxa9, immortalizing early progenitors, inducing low-level expression of Meis1-related signature genes, and causing leukemia without coexpression of exogenous or endogenous Hox genes. Vp16-Meis1-mediated transformation required the Meis1 function of binding to Pbx and DNA but not its C-terminal domain (CTD). The absence of endogenous Hox gene expression in Vp16-Meis1-immortalized progenitors allowed us to investigate how Hox alters gene expression and cell biology in early hematopoietic progenitors. Strikingly, expression of Hoxa9 or Hoxa7 stimulated both leukemic aggressiveness and transcription of Meis1-related signature genes in Vp16-Meis1 progenitors. Interestingly, while the Hoxa9 N-terminal domain (NTD) is essential for cooperative transformation with wild-type Meis1, it was dispensable in Vp16-Meis1 progenitors. The fact that a dominant transactivation domain fused to Meis1 replaces the essential functions of both the Meis1 CTD and Hoxa9 NTD suggests that Meis-Pbx and Hox-Pbx (or Hox-Pbx-Meis) complexes co-occupy cellular promoters that drive leukemogenesis and that Meis1 CTD and Hox NTD cooperate in gene activation. Chromatin immunoprecipitation confirmed co-occupancy of Hoxa9 and Meis1 on the Flt3 promoter.

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Leukemic CD34+CD38- Cells Display Conserved Differential Expression of Many ATP Binding-Cassette Transporter Genes.

Blood ◽

10.1182/blood.v106.11.1380.1380 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1380-1380

Author(s):

Marc H.G.P. Raaijmakers ◽

Elke P.L.M. de Grouw ◽

Louis T.F. van de Locht ◽

Bert A. van der Reijden ◽

Theo J.M. de Witte ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Stem Cell ◽

Abc Transporters ◽

Cell Biology ◽

Cell Fraction ◽

Atp Binding ◽

Stem Cell Biology ◽

Hematopoietic Stem ◽

Atp Binding Cassette

Abstract In most cases of acute myeloid leukemia (AML) CD34+CD38− cells are considered to be stem cells, responsible for the maintenance and relapse of AML. ATP binding cassette transporters function in the extrusion of xenobiotics and chemotherapeutical compounds, and may be involved in therapy resistance. Elucidation of mechanisms conferring drug resistance to CD34+CD38− cells is essential to provide novel targets for stem cell eradication in AML. We studied gene expression of all 45 transmembrane ABC transporters (the complete ABCA, B, C, D and G family) in human hematopoietic CD34+CD38− cells and more committed CD34+CD38+ progenitor cells, from healthy donors and patients with non-hematological diseases (N=11) and AML patients (N=11). Gene expression was assessed using a novel real-time RT-PCR approach with micro fluidic cards. In normal CD34+CD38− cells 36 ABC transporters were expressed, 22 of these displayed significant higher expression in the CD34+CD38− cell fraction compared to the CD34+CD38+ cell fraction. In addition to the known stem cell transporters (ABCB1, ABCC1 and ABCG2) these differential expressed genes included many members not previously associated with stem cell biology. In AML the ABC transporter expression profile was largely conserved, including expression of all 13 known drug transporters. These data suggest an important role for many ABC transporters in hematopoietic stem cell biology. In addition, the preferential expression of a high number of drug transport related transporters predicts that broad spectrum inhibition of ABC transporters is likely to be required for CD34+38− stem cell eradication in AML. This approach will, apart from affecting the leukemic stem cells, equally affect the normal stem cells.

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The Expression Signature of M3 AML Suggests Direct and Indirect Effects of PML-RARA on Target Genes.

Blood ◽

10.1182/blood.v110.11.719.719 ◽

2007 ◽

Vol 110 (11) ◽

pp. 719-719 ◽

Cited By ~ 1

Author(s):

Jacqueline E. Payton ◽

Nicole R. Grieselhuber ◽

Li-Wei Chang ◽

Mark A. Murakami ◽

Wenlin Yuan ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Cell Lines ◽

Binding Sites ◽

Expression Profiles ◽

Gene Expression Signature ◽

Myeloid Cell ◽

Expression Signature ◽

Signature Genes ◽

Cell Fractions

Abstract In order to better understand the pathogenesis of acute promyelocytic leukemia (APL, FAB M3), we sought to determine its gene expression signature by comparing the expression profiles of 14 APL samples to that of other AML subtypes (M0, M1, M2, M4, n=62) and to fractionated normal whole bone marrow cells (CD34 cells, promyelocytes, PMNs, n=5 each). We used ANOVA and SAM (Significance Analysis of Microarrays) to select genes that were highly expressed in APL cells and that displayed low to no expression in other AML subtypes. The APL signature was then further refined by filtering genes whose expression in APL was not significantly different from that of normal promyelocytes, yielding 1121 annotated genes that reliably distinguish APL from the other FAB subtypes using unsupervised hierarchical clustering, both in training and validation datasets. Fold change differences in expression between M3 and other AML FAB classes were striking, for example: GABRE 35.4, HGF 21.3, ANXA8 21.3, PTPRG 16.9, PTGDS 12.1, PPARG 11.1, STAB1 9.8. A large proportion of the APL versus other FAB dysregulome was recapitulated when we compared APL expression to that of the normal pattern of myeloid development. We identified 733 annotated genes with significantly different expression in APL versus normal myeloid cell fractions. These dysregulated genes were assigned to 4 classes: persistently expressed CD34 cell-specific genes, repressed promyelocyte-specific genes, prematurely expressed neutrophil-specific genes and genes with high expression in APL and low/no expression in normal myeloid cell fractions. Expression differences in several of the most dysregulated genes were validated by qRT-PCR. We then examined the expression of the APL signature genes in myeloid cell lines and tumors from a murine APL model. The bona fide M3 signature was not apparent in resting NB4 cells (which contain t(15;17), and which express PML-RARA), nor in PR-9 cells following Zn induction of PML-RARA expression, suggesting that neither cell line accurately models the gene expression signature of primary APL cells. Most of the nodal genes of the mCG-PML-RARA murine APL dysregulome (Yuan, et al, 2007) are similarly dysregulated in human M3 cells; however, the human and mouse dysregulomes do not completely coincide. Finally, we have begun investigating which APL signature genes are direct transcriptional targets of PML-RARA. The promoters of the APL signature genes were analyzed for the presence of known PML-RARA binding sites using multiple computational methods. The analyses demonstrated that several transcription factors (EBF3, TWIST1, SIX3, PPARG) have putative retinoic acid response elements (RAREs) in their upstream regulatory regions. Additionally, we examined the promoters of some of the most upregulated genes (HGF, PTGDS, STAB1) for known consensus sites of these transcription factors, and found that all have putative binding sites for at least one. These results suggest that PML-RARA may initiate a transcriptional cascade that relies not only on its own activity, but also on the actions of downstream transcription factors. In summary, our studies indicate that primary APL cells have a gene expression signature that is consistent and highly reproducible, but different from commonly used human APL cell lines and a mouse model of APL. The molecular mechanisms that govern this unique signature are currently under investigation.

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Histone Demethylase LSD1 Is Required to Repress Hematopoietic Stem Cell Signatures in Mature Blood Cells to Permit Terminal Differentiation

Blood ◽

10.1182/blood.v118.21.550.550 ◽

2011 ◽

Vol 118 (21) ◽

pp. 550-550

Author(s):

Marc A Kerenyi ◽

Jessica Hsu ◽

Zhen Shao ◽

Stuart H Orkin

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Chromatin Immunoprecipitation ◽

Expression Profiling ◽

Myeloid Cells ◽

Hematopoietic Differentiation ◽

Wild Type ◽

Hematopoietic Stem ◽

Knock Out ◽

Chromatin Immunoprecipitation Sequencing

Abstract Abstract 550 Lysine specific demethylase 1 (LSD1) is a demethylase that acts on mono- and dimethylated H3K4 (H3K4me1/2). Consistent with H3K4me2 (an active marker of transcription) as a substrate, LSD1 is part of a core complex with the co-repressor, CoREST and HDAC1/2. Previously our lab demonstrated that regulation of hematopoietic differentiation depends in part on the interaction of the growth factor independent transcription factors (= Gfi1 and Gfi1b) with the LSD1/CoREST/HDAC complex. We generated a conditional knock out mouse for LSD1 (LSD1fl/fl) to study its roles in hematopoiesis. Inducible deletion of LSD1fl/fl mice in all hematopoietic lineages with Mx-Cre resulted in severe neutropenia. Flow cytometric analysis showed that LSD1fl/fl Mx-Cre mice lacked Gr-1high Mac-1high double positive mature neutrophilic granulocytes in the bone marrow and the peripheral blood; however, the frequency of Gr-1dim Mac-1high (mainly consisting of promyelocytes and myeloblasts but not mature neutrophils) increased in frequency. To reveal the mechanism responsible for the observed neutropenia, we performed global mRNA expression profiling and chromatin immunoprecipitation sequencing (ChIPSeq) for H3K4 methylation states in Gr-1dim Mac-1high cells from LSD1fl/fl Mx-Cre and LSD1fl/fl mice. Five hundred ninety-eight genes (412 up / 186 down; p≤0.01, 2-fold cutoff) were differentially expressed in the absence of LSD1. Although we did not detect changes in expression of established myeloid transcription factors, including Pu.1, C/EBPα, C/EBPε or Gfi1, gene set enrichment analysis (GSEA) of Gr-1dim Mac-1high cells from LSD1fl/fl Mx-Cre using gene signatures for mature myeloid cells clearly showed that LSD1 deficient Gr-1dim Mac-1high cells failed to display a gene signature of differentiated myeloid cells (NES: 1.88; p≤0.003). Among the most highly upregulated genes, we observed genes highly expressed in hematopoietic stem and progenitor cells (HSPCs; i.e.: CD34 36.2-fold; HoxA9 26.3-fold; Sca-1 10.8-fold; Meis 1 2.6-fold). Therefore we performed GSEA using signatures from HSPCs (encompassing over 200 genes); the stem/progenitor gene set was highly significantly enriched (NES: −1.9; p<10−4) in LSD1 deficient Gr-1dim Mac-1high cells. Chromatin immunoprecipitation sequencing did not reveal any global changes in the amount of H3K4me2/3 histone methylation, however many genes critical for HSPCs, including Meis1 and the entire HoxA gene locus, where more strongly H3K4me2/3 marked than in control cells, which is in concord with the gene expression data. To determine if LSD1 represses stem/progenitor genes in additional lineages, we analyzed the effects of LSD1 loss in erythroid cell development through breeding with EpoR-Cre. Wild type, as well as control embryos, were recovered at Mendalian ratios up to E12.5, but no live LSD1fl/fl EpoR-Cre embryos were observed after E15.5. At E13.5, LSD1-deficient embryos were smaller and paler as compared to control embryos. Flow cytometry revealed a severe differentiation defect at the transition from pro-erythroblasts to basophilic erythroblasts, resulting in a paucity of more mature erythroid cells. To unravel molecular mechanisms responsible for this deficit, we performed gene expression profiling of wild type and knock out CD71+ c-kit+ Ter119lo pro-erythroblasts. Again, we did not detect changes in the expression levels of established erythroid transcription factors, including Gata-1, Klf1, SCL/Tal1, NF-E1, Ldb1, Lmo2 or Myb. By GSEA analysis we observed that LSD1 deficient CD71+ c-kit+ Ter119lo pro-erythroblasts displayed higher expression of the hematopoietic stem and progenitor cell gene signatures (NES: −2.4; p<10−4), a finding strikingly similar to the data in myeloid cells. Therefore, LSD1 is required in multiple hematopoietic lineages to repress stem/progenitor gene expression programs in maturing cells. We propose that repression of these early programs is essential for subsequent hematopoietic differentiation. Disclosures: No relevant conflicts of interest to declare.

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Chromatin State Shaping the Gene Expression Profiling in the Inducible Differentiation of Promyelocytic Leukemia Cells

Blood ◽

10.1182/blood.v124.21.5188.5188 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5188-5188

Author(s):

Chunhua Song ◽

Lisa Chung ◽

Xiaokang Pan ◽

Li Zhanjun ◽

Yali Ding ◽

...

Keyword(s):

Gene Expression ◽

Epigenetic Regulation ◽

Conflicts Of Interest ◽

Promyelocytic Leukemia ◽

Epigenetic Mechanism ◽

Chromatin State ◽

Hematopoietic Stem ◽

Signature Genes ◽

Lineage Differentiation ◽

On Chip

Abstract Background Epigenetic changes in DNA and chromatin are regarded as emerging major players for hematopoietic stem cells development and lineage differentiation. Epigenetic deregulation of gene expression leads to leukemia and reversibility of epigenetic modifications makes DNA and chromatin changes attractive targets for therapeutic intervention. The promyelocytic leukemia HL-60 cell can differentiate into microphage, granulocyte and monocyte with stimulation of phorbol myristic acid (PMA), dimethylsulfoxide (DMSO), and Vitamin D3, respectively, by affecting the gene expression on cell cycle and cell differentiation. However, how epigenetic regulation on gene expression in the inducible differentiation is still undetermined. Methods and Results We did the microarray analysis to identify the genome-wide gene expression profile of HL-60 cells in various time points(0h, 6h, 1d,2d,4d and 6d) under the treatment of PMA, DMSO and D3 and found that around 3000 genes are significantly altered commonly in the cells upon the 3 treatments. The percentage of down-regulated genes in the all commonly altered genes is significantly higher than that of up-regulated genes, and the significantly altered genes showed significantly physical clustering on chromosome loci, indicating the epigenetic regulation involved in the regulation of the gene expression. We did observed the expression changes of epigenetic enzymes in the process and further did the ChIP-on-ChIP analysis by using the customer array tiling the selected changed genes and 5 genomic loci hybridizing with ChIP’d DNA of histone markers (H3K4me3, H3K9me3 and H3K27me3). We found that the obvious decrease of H3K4me3 binding and increase of H3K9me3 and H3K27me3 are observed in the promoter of the commonly down-regulated genes. These data indicated histone H3K4, H3K9 and H3K27 marker play important roles in shaping the chromatin state and regulating gene expression. Conclusion We identified the signature genes controlling the inducible differentiation of promyelocytic leukemiacells and found the epigenetic mechanism regulating the gene expression in the process. Disclosures No relevant conflicts of interest to declare.

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Ezh2 and Runx1 Mutations Targeted to Early Lymphoid Progenitors Collaborate to Promote Early Thymic Progenitor Leukemia

Blood ◽

10.1182/blood.v126.23.846.846 ◽

2015 ◽

Vol 126 (23) ◽

pp. 846-846

Author(s):

Christopher Booth ◽

Nikolas Barkas ◽

Wen Hao Neo ◽

Elizabeth Soilleux ◽

Hanane Boukarabila ◽

...

Keyword(s):

Gene Expression ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

High Sensitivity ◽

Response To Therapy ◽

Ras Signaling ◽

Specific Cell ◽

Cell Of Origin ◽

Hematopoietic Stem ◽

Lymphoid Progenitors

Abstract Understanding the specific cell populations responsible for propagation of leukemia is an important step for development of effective targeted therapies. Recently, the lymphoid-primed multipotent progenitor (LMPP) has been proposed to be a key propagating population in acute myeloid leukemia (AML; PMID 21251617). We have also shown that LMPPs share many functional and gene expression properties with early thymic progenitors (ETPs; PMID 22344248). This finding is of particular interest as ETP leukemias have recently been described: a distinct and poor prognostic disease entity with a transcriptional profile reminiscent of murine ETPs, showing co-expression of hematopoietic stem cell (HSC) and myeloid markers (PMID 19147408). Together, this raises the question whether ETPs can act as a leukemia-initiating/propagating cell population; however, relevant disease models to test this hypothesis are currently lacking. Analysis of the genetic landscape of ETP leukemias has revealed frequent coexistence of inactivating mutations of EZH2 and RUNX1 (PMID 22237106). We therefore generated mice with deletions of Ezh2 and Runx1 specifically targeted to early lymphoid progenitors using Rag1Cre (Ezh2fl/flRunx1fl/flRag1Cre+; DKO mice). As anticipated, HSCs lacked significant recombination in DKO mice whereas close to 100% of purified ETPs (Lin- CD4- CD8- CD44+ CD25- Kit+ Flt3+) showed deletion of Ezh2 and Runx1. Strikingly, despite a 16-fold reduction in thymus cellularity caused by a block in thymocyte maturation at the DN2-DN3 transition, absolute numbers of ETPs within the thymus of DKO mice were markedly expanded (12-fold; p<0.0001). In contrast, Ezh2 or Runx1 deletion alone had no impact on numbers of ETPs. RNA-sequencing of the expanded ETPs in DKO mice revealed upregulation of HSC- and myeloid-associated transcriptional programs, reminiscent of ETP leukaemia e.g. Pbx1 (log2FC=3.0; p<0.0001) and Csf3r (log2FC=1.9; p=0.0038). Single-cell gene expression analysis confirmed co-expression of HSC and myeloid programs with lymphoid genes within individual DKO ETPs. Further, some key regulators of T-cell maturation which are aberrantly expressed in ETP leukemia were also disrupted in DKO ETPs e.g. Tcf7 (log2FC=-9.5; p<0.0001). Gene expression associated with aberrant Ras signalling was also present. However, despite a continued expansion of the ETP population with age, we did not observe leukemia in DKO mice with over 1 year of follow-up. Since ETP leukemias frequently feature activating mutations in genes regulating RAS signaling, we hypothesised that the expanded "pre-leukemic" ETPs in DKO mice would be primed for leukemic transformation by signalling pathway mutation. We therefore crossed DKO mice with a Flt3ITD/+ knock-in mouse line, as internal tandem duplications (ITD) of FLT3 are frequent in ETP leukemias. Ezh2fl/flRunx1fl/flRag1Cre+Flt3ITD/+ (DKOITD) mice showed dramatically reduced survival (median 9.3 weeks) resulting from an aggressive, fully penetrant acute leukemia showing a predominantly myeloid phenotype (e.g. Mac1) but with co-expression of some lymphoid antigens (e.g. intracellular CD3). Crucially, this leukaemia could be propagated in wild-type recipients upon transplantation of the expanded ETPs. DKOITD ETPs were transcriptionally very similar to DKO ETPs, retaining expression of lymphoid alongside HSC- and myeloid-associated genes. Finally, in a lympho-myeloid cell line model (EML cells) we demonstrated that Ezh2 inactivation-induced loss of H3K27me3 is associated with a corresponding increase in H3K27Ac, a transcriptional activating signal that recruits bromodomain proteins. As such, we reasoned that our ETP leukemia model might be sensitive to bromodomain inhibitors such as JQ1. Indeed, we observed high sensitivity of expanded DKOITD ETPs to JQ1, raising the possibility of a new therapeutic approach for ETP leukemias. This novel mouse model of ETP-propagated leukemia, driven by clinically relevant mutations, provides intriguing evidence that leukemias with a predominant myeloid phenotype, but co-expressing lymphoid genes, may initiate within a bona fide early lymphoid progenitor population. Since the functional characteristics of the cell of origin of a leukaemia may direct its progression and response to therapy, these findings could have important implications for future stratification and treatment of both AML and ETP leukemias. Disclosures No relevant conflicts of interest to declare.

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A Transcriptomic Continuum of Differentiation Arrest in Acute Leukemia

Blood ◽

10.1182/blood-2019-125902 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2511-2511

Author(s):

Jonathan Bond ◽

Aleksandra Krzywon ◽

Ludovic Lhermitte ◽

Christophe Roumier ◽

Anne Roggy ◽

...

Keyword(s):

Gene Expression ◽

Gene Selection ◽

Early Stage ◽

Myeloid Cell ◽

Clustering Methods ◽

Humanized Mouse Model ◽

Acute Leukemias ◽

Hematopoietic Stem ◽

Lymphoid Progenitors

Introduction: Traditional classification of acute lymphoblastic and myeloid leukemias (ALLs and AMLs) remains heavily based on phenotypic resemblance to normal hematopoietic precursors of the respective lineages. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, which often have poor responses to treatment. This system also takes little account of modern concepts of hematopoietic identity that are mainly based on transcriptional signature identification and functional assays. Recent advances in genome-wide analytical methods developed to reconstruct landscapes of normal differentiation now provide an opportunity to re-evaluate traditional binary approaches to myeloid and lymphoid lineage assignment in leukemia. Methods: We used novel computational tools, including the recently described Iterative Clustering and Guide Gene Selection (ICGS) method to perform transcriptional analyses of a series of 125 T-ALLs and AMLs, which comprised a high proportion of phenotypically immature cases (53.1% and 40.8% respectively). The leukemias were additionally characterized by targeted next generation sequencing (NGS). ICGS was also used to analyze independent adult and pediatric T-ALL cohorts. Results: There was significant overlap in gene expression between leukemias of different diagnostic categories. In contrast to traditional clustering methods, ICGS analysis permitted unbiased classification of acute leukemias along a continuum of hematopoietic differentiation, according to the expression of a limited number of lineage-discriminating guide genes that defined hematopoietic cell expression modules. While AMLs and T-ALLs at either end of the differentiation spectrum showed specific enrichment for transcriptional signatures of the corresponding lineage precursors, leukemias that were arrested at the myeloid/ T-lymphoid interface either showed no clear evidence of mature T-lymphoid or mature myeloid identity, or had incomplete Hematopoietic Stem and Progenitor Cell (HSPC) and mature myeloid cell profiles. NGS analysis revealed that the spectrum of differentiation arrest defined by ICGS is only partially paralleled by underlying mutational genotype. Notably, interface leukemias originally diagnosed as T-ALL were significantly more likely to have PTEN mutations than the rest of the T-ALL cohort (60% v 6.7%, p=0.0151), while RUNX1-mutated AMLs were restricted to interface clusters. We found that interface leukemias shared gene expression programs with a series of multi- or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a-CD7- subset of early thymic precursors (ETPs). Within interface leukemias, enrichment for lymphoid progenitor population signatures including multi-lymphoid progenitors (MLPs), lymphoid-mono-dendritic progenitors (LMDPs), T-oriented CD127- and B-oriented CD127+ early lymphoid progenitors (ELPs) from an umbilical cord blood humanized mouse model and early B-cell progenitors, was more likely in cases that were originally diagnosed as AML, rather than T-ALL. In addition, transcriptional resemblance to both B/myeloid and T/myeloid mixed phenotype acute leukemias (MPALs) was primarily driven by AMLs within these interface clusters, suggesting that these cases demonstrate significant lymphoid transcriptional orientation. Conclusion: Our results suggest that traditional binary approaches to leukemia categorization are reductive, and that leukemias arrested at the T-lymphoid/ myeloid interface exhibit significant transcriptional heterogeneity. These data also provide evidence that a subset of leukemias originally diagnosed as AML may be more likely to arise from lymphoid-oriented progenitors and/or be arrested at an early stage of lymphoid orientation than is currently recognized. We believe that better identification of interface acute leukemias will allow improved evaluation of appropriate therapeutic options for these cases. Disclosures Boissel: NOVARTIS: Consultancy. Laurenti:GSK: Research Funding.

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A Two-Step, PU.1-Dependent Mechanism for Developmentally Regulated Chromatin Remodeling and Transcription of the c-fms Gene

Molecular and Cellular Biology ◽

10.1128/mcb.01915-06 ◽

2006 ◽

Vol 27 (3) ◽

pp. 878-887 ◽

Cited By ~ 71

Author(s):

Hanna Krysinska ◽

Maarten Hoogenkamp ◽

Richard Ingram ◽

Nicola Wilson ◽

Hiromi Tagoh ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Regulatory Element ◽

Gene Activation ◽

Myeloid Cell ◽

Developmentally Regulated ◽

Enhancer Activity ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Ets Family

ABSTRACT Hematopoietic stem cells and multipotent progenitors exhibit low-level transcription and partial chromatin reorganization of myeloid cell-specific genes including the c-fms (csf1R) locus. Expression of the c-fms gene is dependent on the Ets family transcription factor PU.1 and is upregulated during myeloid differentiation, enabling committed macrophage precursors to respond to colony-stimulating factor 1. To analyze molecular mechanisms underlying the transcriptional priming and developmental upregulation of the c-fms gene, we have utilized myeloid progenitors lacking the transcription factor PU.1. PU.1 can bind to sites in both the c-fms promoter and the c-fms intronic regulatory element (FIRE enhancer). Unlike wild-type progenitors, the PU.1−/− cells are unable to express c-fms or initiate macrophage differentiation. When PU.1 was reexpressed in mutant progenitors, the chromatin structure of the c-fms promoter was rapidly reorganized. In contrast, assembly of transcription factors at FIRE, acquisition of active histone marks, and high levels of c-fms transcription occurred with significantly slower kinetics. We demonstrate that the reason for this differential activation was that PU.1 was required to promote induction and binding of a secondary transcription factor, Egr-2, which is important for FIRE enhancer activity. These data suggest that the c-fms promoter is maintained in a primed state by PU.1 in progenitor cells and that at FIRE PU.1 functions with another transcription factor to direct full activation of the c-fms locus in differentiated myeloid cells. The two-step mechanism of developmental gene activation that we describe here may be utilized to regulate gene activity in a variety of developmental pathways.

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Efficient differentiation and function of human macrophages in humanized CSF-1 mice

Blood ◽

10.1182/blood-2010-12-326926 ◽

2011 ◽

Vol 118 (11) ◽

pp. 3119-3128 ◽

Cited By ~ 99

Author(s):

Chozhavendan Rathinam ◽

William T. Poueymirou ◽

Jose Rojas ◽

Andrew J. Murphy ◽

David M. Valenzuela ◽

...

Keyword(s):

Cell Biology ◽

Fetal Liver ◽

Myeloid Cell ◽

Human Monocytes ◽

Humanized Mouse ◽

Newborn Mice ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Immunocompromised Mice ◽

And Function

Abstract Humanized mouse models are useful tools to understand pathophysiology and to develop therapies for human diseases. While significant progress has been made in generating immunocompromised mice with a human hematopoietic system, there are still several shortcomings, one of which is poor human myelopoiesis. Here, we report that human CSF-1 knockin mice show augmented frequencies and functions of human myeloid cells. Insertion of human CSF1 into the corresponding mouse locus of Balb/c Rag2−/− γc−/− mice through VELOCIGENE technology resulted in faithful expression of human CSF-1 in these mice both qualitatively and quantitatively. Intra-hepatic transfer of human fetal liver derived hematopoietic stem and progenitor cells (CD34+) in humanized CSF-1 (CSF1h/h) newborn mice resulted in more efficient differentiation and enhanced frequencies of human monocytes/macrophages in the bone marrow, spleens, peripheral blood, lungs, liver and peritoneal cavity. Human monocytes/macrophages obtained from the humanized CSF-1 mice show augmented functional properties including migration, phagocytosis, activation and responses to LPS. Thus, humanized mice engineered to express human cytokines will significantly help to overcome the current technical challenges in the field. In addition, humanized CSF-1 mice will be a valuable experimental model to study human myeloid cell biology.

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A histidine cluster determines YY1-compartmentalized coactivators and chromatin elements in phase-separated super-enhancers

10.1101/2021.09.15.460559 ◽

2021 ◽

Author(s):

Wenmeng Wang ◽

Shiyao Qiao ◽

Guangyue Li ◽

Cuicui Yang ◽

Chen Zhong ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Phase Separation ◽

Rna Polymerase Ii ◽

Target Gene ◽

Gene Activation ◽

Transactivation Domain ◽

Yin Yang 1 ◽

Oncogenic Transcription Factor ◽

Liquid Liquid Phase Separation

As an oncogenic transcription factor, Yin Yang 1 (YY1) regulates enhancer and promoter connection. However, gaps still exist in understanding how YY1 coordinates coactivators and chromatin elements to assemble super-enhancers. Here, we demonstrate that YY1 activates FOXM1 gene expression through forming liquid-liquid phase separation to compartmentalize both coactivators and enhancer elements. In the transactivation domain of YY1, a histidine cluster is essential for its activities of forming phase separation, which can be extended to additional proteins. Coactivators EP300, BRD4, MED1 and active RNA polymerase II are components of YY1-rich nuclear puncta. Consistently, histone markers for gene activation, but not repression, colocalize with YY1. Importantly, multiple enhancer elements and the FOXM1 promoter are bridged by YY1 to form super-enhancers. These studies propose that YY1 is a general transcriptional activator, and promotes phase separation with incorporation of major coactivators and stabilization by distal enhancers to activate target gene expression.

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A partially disordered region connects gene repression and activation functions of EZH2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914866117 ◽

2020 ◽

Vol 117 (29) ◽

pp. 16992-17002 ◽

Cited By ~ 4

Author(s):

Lianying Jiao ◽

Murtada Shubbar ◽

Xin Yang ◽

Qi Zhang ◽

Siming Chen ◽

...

Keyword(s):

Gene Expression ◽

Cancer Cells ◽

Gene Activation ◽

Activation Function ◽

Binary Complex ◽

Protein Oligomerization ◽

Dependent Manner ◽

Transactivation Domain ◽

Transcriptional Coactivator ◽

H3k27 Methylation

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), which minimally requires two other subunits, EED and SUZ12, for enzymatic activity. EZH2 has been traditionally known to mediate histone H3K27 trimethylation, a hallmark of silent chromatin. Emerging evidence indicates that EZH2 also activates gene expression in cancer cells in a context distinct from canonical PRC2. The molecular mechanism underlying the functional conversion of EZH2 from a gene repressor to an activator is unclear. Here, we show that EZH2 harbors a hidden, partially disordered transactivation domain (TAD) capable of interacting with components of active transcription machinery, mimicking archetypal acidic activators. The EZH2 TAD comprises the SRM (Stimulation-Responsive Motif) and SANT1 (SWI3, ADA2, N-CoR, and TFIIIB 1) regions that are normally involved in H3K27 methylation. The crystal structure of an EZH2−EED binary complex indicates that the EZH2 TAD mediates protein oligomerization in a noncanonical PRC2 context and is entirely sequestered. The EZH2 TAD can be unlocked by cancer-specific EZH2 phosphorylation events to undergo structural transitions that may enable subsequent transcriptional coactivator binding. The EZH2 TAD directly interacts with the transcriptional coactivator and histone acetyltransferase p300 and activates gene expression in a p300-dependent manner in cells. The corresponding TAD may also account for the gene activation function of EZH1, the paralog of EZH2. Distinct kinase signaling pathways that are known to abnormally convert EZH2 into a gene activator in cancer cells can now be understood in a common structural context of the EZH2 TAD.

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