scholarly journals Transcriptional Regulation of the SCL Locus: Identification of an Enhancer That Targets the Primitive Erythroid Lineage In Vivo

2005 ◽  
Vol 25 (12) ◽  
pp. 5215-5225 ◽  
Author(s):  
E. Delabesse ◽  
S. Ogilvy ◽  
M. A. Chapman ◽  
S. G. Piltz ◽  
B. Gottgens ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Cell Types ◽  
Regulatory Elements ◽  
Hematopoietic Cell ◽  
Erythroid Cell ◽  
Sequence Comparisons ◽  
Helix Loop Helix ◽  
Primitive Erythropoiesis

ABSTRACT The stem cell leukemia (SCL) gene, also known as TAL-1, encodes a basic helix-loop-helix protein that is essential for the formation of all hematopoietic lineages, including primitive erythropoiesis. Appropriate transcriptional regulation is essential for the biological functions of SCL, and we have previously identified five distinct enhancers which target different subdomains of the normal SCL expression pattern. However, it is not known whether these SCL enhancers also regulate neighboring genes within the SCL locus, and the erythroid expression of SCL remains unexplained. Here, we have quantitated transcripts from SCL and neighboring genes in multiple hematopoietic cell types. Our results show striking coexpression of SCL and its immediate downstream neighbor, MAP17, suggesting that they share regulatory elements. A systematic survey of histone H3 and H4 acetylation throughout the SCL locus in different hematopoietic cell types identified several peaks of histone acetylation between SIL and MAP17, all of which corresponded to previously characterized SCL enhancers or to the MAP17 promoter. Downstream of MAP17 (and 40 kb downstream of SCL exon 1a), an additional peak of acetylation was identified in hematopoietic cells and was found to correlate with expression of SCL but not other neighboring genes. This +40 region is conserved in human-dog-mouse-rat sequence comparisons, functions as an erythroid cell-restricted enhancer in vitro, and directs β-galactosidase expression to primitive, but not definitive, erythroblasts in transgenic mice. The SCL +40 enhancer provides a powerful tool for studying the molecular and cellular biology of the primitive erythroid lineage.

Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of Hematopoietic Cell Types and Immortalization in the Myeloid Lineage

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3226-3239 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Christine K. Bieszczad ◽  
Randolph Noelle ◽  
Michael Binder ◽  
...  
Keyword(s):  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Cell Number ◽  
Hematopoietic Cell ◽  
Viable Cell Number ◽  
Wide Range ◽  
Transgenic Cells

Abstract Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addition, among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell number, but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at “viability gates” along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization. © 1998 by The American Society of Hematology.

Stat5 and Stat3 Differentially Regulate Early and Late Stages of Primary Embryonic Erythroid Cell Maturation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3877-3877
Author(s):  
Zachary C. Murphy ◽  
Paul D. Kingsley ◽  
Kathleen E. McGrath ◽  
James Palis
Keyword(s):  
Cell Size ◽  
Late Stage ◽  
Conflicts Of Interest ◽  
Janus Kinase ◽  
Erythroid Cell ◽  
Null Mouse ◽  
Delayed Maturation ◽  
Primitive Erythropoiesis

Erythropoietin is the critical regulator of adult (definitive) red cell production, acting through its cognate receptor (EpoR) and downstream Janus kinase (Jak)2 and signal transducer and activator of transcription (Stat)5 signaling. Mutant JAK2 activation causes hyperproliferation of erythroid precursors in polycythemia vera, highlighting the importance of Jak2-STAT signaling in pathologic states in addition to normal development. EpoR-null mouse embryos die at midgestation of profound anemia and we have recently determined that EpoR signaling is also specifically required for the survival and terminal maturation of primitive erythroblasts. Unlike definitive erythropoiesis, Stat3 is significantly expressed in primitive erythroblasts but its function in erythropoiesis remains poorly understood. We find that Stat3, in addition to Stat5, is phosphorylated downstream of EpoR in primitive erythroblasts. Interestingly, phospho-Stat5 intensity decreases during primitive erythroblast maturation while phospho-Stat3 levels remain constant, resulting in an increase in relative phospho-Stat3 in late-stage primitive erythroblasts. Knockdown of Jak2 and Stat5 each results in accelerated maturation, decreased cell size, and increased apoptosis of primary primitive erythroblasts. In contrast, Stat3 knockdown results in delayed maturation and increased cell size of late-stage primary primitive erythroblasts, consistent with an increased role of phospho-Stat3 once phospho-Stat5 signal intensity has diminished. Stat3 knockdown, both in vitro and in vivo, also increased apoptosis of late-stage primitive erythroblasts. While Stat3 and Stat5 bind many apoptosis-related genes in common, Stat3 specifically binds several caspase genes and their regulators. We find that activated caspase3 levels increase in late-stage primitive erythroblasts, and that knockdown of Stat3 results in decreased levels of caspases and their regulators. In addition, caspase inhibition, like Stat3 knockdown, results in delayed maturation of late-stage primitive erythroblasts. Taken together, our data support a model where Stat3 and Stat5 differentially regulate erythroid maturation and Stat3 preferentially regulates terminal stages of primitive erythropoiesis through its activation and regulation of apoptotic machinery. Disclosures No relevant conflicts of interest to declare.

Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of Hematopoietic Cell Types and Immortalization in the Myeloid Lineage

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3226-3239 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Christine K. Bieszczad ◽  
Randolph Noelle ◽  
Michael Binder ◽  
...  
Keyword(s):  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Cell Number ◽  
Hematopoietic Cell ◽  
Viable Cell Number ◽  
Wide Range ◽  
Transgenic Cells

Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addition, among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell number, but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at “viability gates” along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization. © 1998 by The American Society of Hematology.

scholarly journals Nonmyristoylated Abl proteins transform a factor-dependent hematopoietic cell line.

1992 ◽  
Vol 12 (4) ◽  
pp. 1864-1871 ◽  
Author(s):  
G Q Daley ◽  
R A Van Etten ◽  
P K Jackson ◽  
A Bernards ◽  
D Baltimore
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Hematopoietic Cell ◽  
Myelogenous Leukemia ◽  
3T3 Fibroblasts ◽  
Nih 3T3

N-terminal myristoylation can promote the association of proteins with the plasma membrane, a property that is required for oncogenic variants of Src and Abl to transform fibroblastic cell types. The P210bcr/abl protein of chronic myelogenous leukemia cells is not myristoylated and does not stably transform NIH 3T3 fibroblasts; however, it will transform lymphoid and myeloid cell types in vitro and in vivo, suggesting that myristoylation is not required for Abl variants to transform hematopoietic cells. To test this hypothesis, we introduced point mutations that disrupt myristoylation into two activated Abl proteins, v-Abl and a deletion mutant of c-Abl (delta XB), and examined their ability to transform an interleukin-3-dependent lymphoblastoid cell line, Ba/F3. Neither of the nonmyristoylated Abl proteins transformed NIH 3T3 fibroblasts, but like P210bcr/abl, both were capable of transforming the Ba/F3 cells to factor independence and tumorigenicity. Nonmyristoylated Abl variants did not associate with the plasma membrane in the transformed Ba/F3 cells. These results demonstrate that Abl proteins can transform hematopoietic cells in the absence of membrane association and suggest that distinct functions of Abl are required for transformation of fibroblast and hematopoietic cell types.

scholarly journals Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors.

1994 ◽  
Vol 14 (3) ◽  
pp. 2159-2169 ◽  
Author(s):  
P A Garrity ◽  
D Chen ◽  
E V Rothenberg ◽  
B J Wold
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cyclosporin A ◽  
Interleukin 2 ◽  
Cell Types ◽  
Regulatory Elements ◽  
In Vitro Assays ◽  
Nuclear Extracts

Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including EL4 T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using nuclear extracts. We conclude that binding activities of all classes fail to stably occupy their cognate sites in IL-2, except following activation of T cells, and that specificity of IL-2 transcription is enforced at the level of chromosomal occupancy, which appears to be an all-or-nothing phenomenon.

scholarly journals Transcriptional Regulation of vav, a Gene Expressed Throughout the Hematopoietic Compartment

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 419-430 ◽  
Author(s):  
Sarah Ogilvy ◽  
Andrew G. Elefanty ◽  
Jane Visvader ◽  
Mary L. Bath ◽  
Alan W. Harris ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cell Types ◽  
Regulatory Elements ◽  
Erythroid Cell ◽  
Transcription Start ◽  
Specific Expression ◽  
Fibroblast Line ◽  
Hematopoietic Organs ◽  
Dna Fragment

Abstract The vav gene is expressed in all hematopoietic but few other cell types. To explore its unusual compartment-wide regulation, we cloned the murine gene, sequenced its promoter region, identified DNase I hypersensitive (HS) sites in the chromatin, and tested their promoter activity with a β-galactosidase (β-gal) reporter gene in cell lines and transgenic mice. Whereas fibroblasts had no HS sites, a myeloid and an erythroid cell line contained five, located 0.2 kb (HS1), 1.9 kb (HS2), and 3.6 kb (HS3) upstream from the transcription start and 0.6 kb (HS4) and 10 kb (HS5) downstream. A vav DNA fragment including HS1 promoted β-gal expression in a myeloid but not a fibroblast line. Expression in leukocytes of transgenic mice also required HS2 and HS5. Only hematopoietic organs contained β-gal, but virtually all β-gal+ cells were B or T lymphocytes. Expression was always variegated (mosaic), and the proportion of β-gal+ cells declined with lymphoid maturation and animal age. Thus, these vav regulatory elements promoted hematopoietic-specific expression in vivo, at least in lymphocytes, but the transgene was sporadically silenced. Maintaining pan-hematopoietic expression may require additional vavelements or an alternative reporter.

scholarly journals The basic helix-loop-helix transcription factors LIN-32 and HLH-2 function together in multiple steps of a C. elegans neuronal sublineage

Development ◽  
2000 ◽  
Vol 127 (24) ◽  
pp. 5415-5426 ◽  
Author(s):  
D.S. Portman ◽  
S.W. Emmons
Keyword(s):  
Transcription Factors ◽  
Neuronal Development ◽  
Cell Types ◽  
Gene Encoding ◽  
Structural Cell ◽  
C Elegans ◽  
Helix Loop Helix ◽  
Bhlh Genes

bHLH transcription factors function in neuronal development in organisms as diverse as worms and vertebrates. In the C. elegans male tail, a neuronal sublineage clonally gives rise to the three cell types (two neurons and a structural cell) of each sensory ray. We show here that the bHLH genes lin-32 and hlh-2 are necessary for the specification of multiple cell fates within this sublineage, and for the proper elaboration of differentiated cell characteristics. Mutations in lin-32, a member of the atonal family, can cause failures at each of these steps, resulting in the formation of rays that lack fully-differentiated neurons, neurons that lack cognate rays, and ray cells defective in the number and morphology of their processes. Mutations in hlh-2, the gene encoding the C. elegans E/daughterless ortholog, enhance the ray defects caused by lin-32 mutations. In vitro, LIN-32 can heterodimerize with HLH-2 and bind to an E-box-containing probe. Mutations in these genes interfere with this activity in a manner consistent with the degree of ray defects observed in vivo. We propose that LIN-32 and HLH-2 function as a heterodimer to activate different sets of targets, at multiple steps in the ray sublineage. During ray development, lin-32 performs roles of proneural, neuronal precursor, and differentiation genes of other systems.

scholarly journals Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors

1994 ◽  
Vol 14 (3) ◽  
pp. 2159-2169
Author(s):  
P A Garrity ◽  
D Chen ◽  
E V Rothenberg ◽  
B J Wold
Keyword(s):  
T Cells ◽  
Dna Binding ◽  
Cyclosporin A ◽  
Interleukin 2 ◽  
Cell Types ◽  
Regulatory Elements ◽  
In Vitro Assays ◽  
Nuclear Extracts

Interleukin-2 (IL-2) transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. Prior studies have shown that this stringent two-tiered regulation is mediated through a transcriptional promoter/enhancer DNA segment which is composed of diverse recognition elements. Factors binding to some of these elements are present constitutively in many cell types, while others are signal dependent, T cell specific, or both. This raises several questions about the molecular mechanism by which IL-2 expression is regulated. Is the developmental commitment of T cells reflected molecularly by stable interaction between available factors and the IL-2 enhancer prior to signal-dependent induction? At which level, factor binding to DNA or factor activity once bound, are individual regulatory elements within the native enhancer regulated? By what mechanism is developmental and physiological specificity enforced, given the participation of many relatively nonspecific elements? To answer these questions, we have used in vivo footprinting to determine and compare patterns of protein-DNA interactions at the native IL-2 locus in cell environments, including EL4 T-lymphoma cells and 32D clone 5 premast cells, which express differing subsets of IL-2 DNA-binding factors. We also used the immunosuppressant cyclosporin A as a pharmacological agent to further dissect the roles played by cyclosporin A-sensitive factors in the assembly and maintenance of protein-DNA complexes. Occupancy of all site types was observed exclusively in T cells and then only upon excitation of signal transduction pathways. This was true even though partially overlapping subsets of IL-2-binding activities were shown to be present in 32D clone 5 premast cells. This observation was especially striking in 32D cells because, upon signal stimulation, they mobilized a substantial set of IL-2 DNA-binding activities, as measured by in vitro assays using nuclear extracts. We conclude that binding activities of all classes fail to stably occupy their cognate sites in IL-2, except following activation of T cells, and that specificity of IL-2 transcription is enforced at the level of chromosomal occupancy, which appears to be an all-or-nothing phenomenon.

scholarly journals Casein kinase II increases the transcriptional activities of MRF4 and MyoD independently of their direct phosphorylation.

1996 ◽  
Vol 16 (4) ◽  
pp. 1604-1613 ◽  
Author(s):  
S E Johnson ◽  
X Wang ◽  
S Hardy ◽  
E J Taparowsky ◽  
S F Konieczny
Keyword(s):  
Protein Function ◽  
Regulatory Elements ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
E Protein ◽  
Protein Partners

The myogenic regulatory factors (MRFs) are a subclass of a much larger group of basic helix-loop-helix transcription factors which includes members of the E protein such as E47, E2-2, and HEB. Although the MRFs are unique in their ability to confer a myogenic phenotype on nonmuscle cells, they require E protein partners to form a MRF-E protein heterodimer, which represents the functional myogenesis-inducing complex. The mechanisms controlling homodimer and heterodimer formation in vivo remain largely unknown, although it is likely that posttranslational modification of one or both basic helix-loop-helix partners is critical to this regulatory event. In this respect, MyoD and MRF4, both members of the MRF family, exist in vivo as phosphoproteins and contains multiple consensus phosphorylation sites, including sites for casein kinase II (CKII) phosphorylation. In this study, we demonstrate that overexpression of CKII increases the transcriptional activities of MRF4 and MyoD in vivo. Interestingly, mutation of the individual CKII sites within MRF4 and MyoF does not alter the ability of CKII to enhance MRF transcriptional activity, suggesting that the effect of CKII expression on the MRFs is indirect. Given that the MRFs require dimerization with E protein partners to activate muscle-specific transcription, the effects of CKII expression on E protein function also were examined. Our studies show that E47 serves as an in vitro substrate for CKII and that CKII-phosphorylated E-47 proteins no longer bind to DNA. These observations were confirmed by in vivo experiments showing that overexpressing of CKII produces a dramatic reduction in E47 homodimer-directed transcription. We conclude from these studies that CKII may act as a positive regulator of myogenesis by preventing E protein homodimers from binding to muscle gene regulatory elements.

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