Search

The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8–ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.

The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HBgene clusters is linked to the widely expressed NPRL3gene. Here we show that the nprl3-linked hblocus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HBlocus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3,which corresponds to the NPRL3intron 7 MCS-R1enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HBgenes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.

The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.

Cooperative actions of extrinsic signals and cell‐intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type β (TGFβ) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell‐type specific and context‐dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFβ system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA‐expression dynamics, gene‐gene interactions, and single‐cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA‐based perturbation of each individual component and documented the effect on steady‐state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene‐gene interactions display a marked cell‐stage specific behavior. Furthermore, single‐cell RNA‐profiling at individual stages demonstrated the presence of detailed co‐expression modules and subpopulations showing stable co‐expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions. Schematic illustration of a systems level approach to dissect the transcriptional and intradependent network of the TGFβ/BMP signaling pathway in embryonic stem cells that gradually differentiate to neural progenitors.

Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes. We recently developed targeted chromatin capture (T2C), an inexpensive method for studying the 3D organization of genomes, interactomes and structural changes associated with gene regulation, the cell cycle, and cell survival and development. Here, we present the protocol for T2C based on capture, describing all experimental steps and bio-informatic tools in full detail. T2C offers high resolution, a large dynamic interaction frequency range and a high signal-to-noise ratio. Its resolution is determined by the resulting fragment size of the chosen restriction enzyme, which can lead to sub-kilobase-pair resolution. T2C's high coverage allows the identification of the interactome of each individual DNA fragment, which makes binning of reads (often used in other methods) basically unnecessary. Notably, T2C requires low sequencing efforts. T2C also allows multiplexing of samples for the direct comparison of multiple samples. It can be used to study topologically associating domains (TADs), determining their position, shape, boundaries, and intra- and inter-domain interactions, as well as the composition of aggregated loops, interactions between nucleosomes, individual transcription factor binding sites, and promoters and enhancers. T2C can be performed by any investigator with basic skills in molecular biology techniques in ∼7–8 d. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments.

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell‐fate decisions. We generated Zeb2knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA‐sequencing (RNA‐seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast‐like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial‐to‐mesenchymal transition (EMT), and DNA‐(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1‐binding sites correlate with loss‐of‐methylation in neural‐stimulating conditions, however, after the cells initially acquired the correct DNA‐methyl marks. Interestingly, cells from such Zeb2KO EBs maintain the ability to re‐adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA‐methylation with irreversible commitment to differentiation. StemCells2017;35:611–625 Wild‐type ESCs submitted to differentiation as embryoid bodies upregulate Zeb2 levels and downregulate pluripotency genes Nanog and Oct4, and also Cdh1 and Tet1 levels, leading to EMT and DNA‐methylation, respectively. Zeb2 knockout ESCs are compromised in these regulations, display impaired differentiation, cannot maintain their initially acquired methyl‐marks on their genome and stall in an epiblast‐like state.

Cardiac stem/progenitor cells hold great potential for regenerative therapies; however, the mechanisms regulating their expansion and differentiation remain insufficiently defined. Here we show that Ldb1 is a central regulator of genome organization in cardiac progenitor cells, which is crucial for cardiac lineage differentiation and heart development. We demonstrate that Ldb1 binds to the key regulator of cardiac progenitors, Isl1, and protects it from degradation. Furthermore, the Isl1/Ldb1 complex promotes long-range enhancer-promoter interactions at the loci of the core cardiac transcription factors Mef2cand Hand2. Chromosome conformation capture followed by sequencing identified specific Ldb1-mediated interactions of the Isl1/Ldb1 responsive Mef2canterior heart field enhancer with genes that play key roles in cardiac progenitor cell function and cardiovascular development. Importantly, the expression of these genes was downregulated upon Ldb1 depletion and Isl1/Ldb1haplodeficiency. In conclusion, the Isl1/Ldb1 complex orchestrates a network for heart-specific transcriptional regulation and coordination in three-dimensional space during cardiogenesis.

An intimate relationship exists between nuclear architecture and gene activity. Unraveling the fine-scale three-dimensional structure of the genome and its impact on gene regulation is a major goal of current epigenetic research, one with direct implications for understanding the molecular mechanisms underlying human phenotypic variation and disease susceptibility. In this context, the novel revolutionary genome editing technologies and emerging new ways to manipulate genome folding offer new promises for the treatment of human disorders.

An intimate relationship exists between nuclear architecture and gene activity. Unraveling the fine-scale three-dimensional structure of the genome and its impact on gene regulation is a major goal of current epigenetic research, one with direct implications for understanding the molecular mechanisms underlying human phenotypic variation and disease susceptibility. In this context, the novel revolutionary genome editing technologies and emerging new ways to manipulate genome folding offer new promises for the treatment of human disorders.

The rearrangement of nucleosomes along the DNA fiber profoundly affects gene expression, but little is known about how signalling reshapes the chromatin landscape, in three-dimensional space and over time, to allow establishment of new transcriptional programs. Using micrococcal nuclease treatment and high-throughput sequencing, we map genome-wide changes in nucleosome positioning in primary human endothelial cells stimulated with tumour necrosis factor alpha (TNFα) - a proinflammatory cytokine that signals through nuclear factor kappa-B (NF-κB). Within 10 min, nucleosomes reposition at regions both proximal and distal to NF-κB binding sites, before the transcription factor quantitatively binds thereon. Similarly, in long TNFα-responsive genes, repositioning precedes transcription by pioneering elongating polymerases and appears to nucleate from intragenic enhancer clusters resembling super-enhancers. By 30 min, widespread repositioning throughout megabase pair-long chromosomal segments, with consequential effects on three-dimensional structure (detected using chromosome conformation capture), is seen. Whilst nucleosome repositioning is viewed as a local phenomenon, our results point to effects occurring over multiple scales. Here, we present data in support of a TNFα-induced priming mechanism, mostly independent of NF-κB binding and/or elongating RNA polymerases, leading to a plastic network of interactions that affects DNA accessibility over large domains.

Here, we show that transcription factors bound to regulatory sequences can be identified by purifying these unique sequences directly from mammalian cells in vivo. Using targeted chromatin purification (TChP), a double-pull-down strategy with a tetracycline-sensitive “hook” bound to a specific promoter, we identify transcription factors bound to the repressed γ-globin gene-associated regulatory regions. After validation of the binding, we show that, in human primary erythroid cells, knockdown of a number of these transcription factors induces γ-globin gene expression. Reactivation of γ-globin gene expression ameliorates the symptoms of β-thalassemia and sickle cell disease, and these factors provide potential targets for the development of therapeutics for treating these patients.

The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters.

The first site exhibiting hematopoietic activity in mammalian development is the yolk-sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in blast colony-forming cells to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk-sac hematopoiesis and vascular development in Ldb1−/− mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome-wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct-target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signaling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells.

The first site exhibiting hematopoietic activity in mammalian development is the yolk-sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in blast colony-forming cells to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk-sac hematopoiesis and vascular development in Ldb1−/−mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome-wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct-target genes and pathways misregulated in the absence of Ldb1.The regulation of essential developmental factors by Ldb1defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signaling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells.

B-cell lymphoma 11A (BCL11A) downregulation in human primary adult erythroid progenitors results in elevated expression of fetal γ-globin. Recent reports showed that BCL11A expression is activated by KLF1, leading to γ-globin repression. To study regulation of erythropoiesis and globin expression by KLF1 and BCL11A in an in vivo model, we used mice carrying a human β-globin locus transgene with combinations of Klf1knockout, Bcl11afloxed, and EpoRCreknockin alleles. We found a higher percentage of reticulocytes in adult Klf1wt/komice and a mild compensated anemia in Bcl11acko/ckomice. These phenotypes were more pronounced in compound Klf1wt/ko::Bcl11acko/ckomice. Analysis of Klf1wt/ko, Bcl11acko/cko, and Klf1wt/ko::Bcl11acko/ckomutant embryos demonstrated increased expression of mouse embryonic globins during fetal development. Expression of human γ-globin remained high in Bcl11acko/ckoembryos during fetal development, and this was further augmented in Klf1wt/ko::Bcl11acko/ckoembryos. After birth, expression of human γ-globin and mouse embryonic globins decreased in Bcl11acko/ckoand Klf1wt/ko::Bcl11acko/ckomice, but the levels remained much higher than those observed in control animals. Collectively, our data support an important role for the KLF1-BCL11A axis in erythroid maturation and developmental regulation of globin expression.

B-cell lymphoma 11A (BCL11A) downregulation in human primary adult erythroid progenitors results in elevated expression of fetal γ-globin. Recent reports showed that BCL11A expression is activated by KLF1, leading to γ-globin repression. To study regulation of erythropoiesis and globin expression by KLF1 and BCL11A in an in vivo model, we used mice carrying a human β-globin locus transgene with combinations of Klf1 knockout, Bcl11a floxed, and EpoRCre knockin alleles. We found a higher percentage of reticulocytes in adult Klf1wt/ko mice and a mild compensated anemia in Bcl11acko/cko mice. These phenotypes were more pronounced in compound Klf1wt/ko::Bcl11acko/cko mice. Analysis of Klf1wt/ko, Bcl11acko/cko, and Klf1wt/ko::Bcl11acko/cko mutant embryos demonstrated increased expression of mouse embryonic globins during fetal development. Expression of human γ-globin remained high in Bcl11acko/cko embryos during fetal development, and this was further augmented in Klf1wt/ko::Bcl11acko/cko embryos. After birth, expression of human γ-globin and mouse embryonic globins decreased in Bcl11acko/cko and Klf1wt/ko::Bcl11acko/cko mice, but the levels remained much higher than those observed in control animals. Collectively, our data support an important role for the KLF1-BCL11A axis in erythroid maturation and developmental regulation of globin expression.

Chromosome conformation capture (3C) technology is a powerful and increasingly popular tool for analyzing the spatial organization of genomes. Several 3C variants have been developed (e.g., 4C, 5C, ChIA-PET, Hi-C), allowing large-scale mapping of long-range genomic interactions. Here we describe multiplexed 3C sequencing (3C-seq), a 4C variant coupled to next-generation sequencing, allowing genome-scale detection of long-range interactions with candidate regions. Compared with several other available techniques, 3C-seq offers a superior resolution (typically single restriction fragment resolution; approximately 1–8 kb on average) and can be applied in a semi-high-throughput fashion. It allows the assessment of long-range interactions of up to 192 genes or regions of interest in parallel by multiplexing library sequencing. This renders multiplexed 3C-seq an inexpensive, quick (total hands-on time of 2 weeks) and efficient method that is ideal for the in-depth analysis of complex genetic loci. The preparation of multiplexed 3C-seq libraries can be performed by any investigator with basic skills in molecular biology techniques. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments. The protocol describes all materials, critical steps and bioinformatics tools required for successful application of 3C-seq technology.

Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity.

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators of microtubule behavior has been studied mainly in cultured cells. We show here that absence of murine CLASP2 in vivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2knockout mice. We propose that the CLASP2-mediated stabilization of microtubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpltranscription.

Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system.

Activation of the canonical Wnt signaling pathway synergizes with leukemia inhibitory factor (LIF) to maintain pluripotency of mouse embryonic stem cells (mESCs). However, in the absence of LIF, Wnt signaling is unable to maintain ESCs in the undifferentiated state. To investigate the role of canonical Wnt signaling in pluripotency and lineage specification, we expressed Wnt3a in mESCs and characterized them in growth and differentiation. We found that activated canonical Wnt signaling induced the formation of a reversible metastable primitive endoderm state in mESC. Upon subsequent differentiation, Wnt3a‐stimulated mESCs gave rise to large quantities of visceral endoderm. Furthermore, we determined that the ability of canonical Wnt signaling to induce a metastable primitive endoderm state was mediated by Tbx3. Our data demonstrates a specific role for canonical Wnt signaling in promoting pluripotency while at the same time priming cells for subsequent differentiation into the primitive endoderm lineage. STEMCELLS2013;31:752–764

The promyelocytic leukemia gene (PML) of acute promyelocytic leukemia is an established tumor suppressor gene with critical functions in growth suppression, induction of apoptosis, and cellular senescence. Interestingly, although less studied, PML seems to play a key role also in immune response to viral infection. Herein, we report that Pml-/- mice spontaneously develop an atypical invasive and lethal granulomatous lesion known as botryomycosis (BTM). In Pml-/- mice, BTM is the result of impaired function of macrophages, whereby they fail to become activated and are thus unable to clear pathogenic microorganisms. Accordingly, Pml-/- mice are resistant to lipopolysaccharide (LPS)–induced septic shock as a result of an ineffective production of cytokines and chemokines, suggesting a role for PML in the innate immune Toll-like receptor (TLR)/NF-?B prosurvival pathway. These results not only shed light on a new fundamental function of PML in innate immunity, but they also point to a proto-oncogenic role for PML in certain cellular and pathological contexts.

An estimated 6% to 7% of the earth's population carries a mutation affecting red blood cell function. The β-thalassemias and sickle cell disease are the most common monogenic disorders caused by these mutations. Increased levels of γ-globin ameliorate the severity of these diseases because fetal hemoglobin (HbF; α2γ2) can effectively replace adult hemoglobin (HbA; α2β2) and counteract polymerization of sickle hemoglobin (HbS; α2βS2). Therefore, understanding the molecular mechanism of globin switching is of biologic and clinical importance. Here, we show that the recently identified chromatin factor Friend of Prmt1 (FOP) is a critical modulator of γ-globin gene expression. Knockdown of FOP in adult erythroid progenitors strongly induces HbF. Importantly, γ-globin expression can be elevated in cells from β-thalassemic patients by reducing FOP levels. These observations identify FOP as a novel therapeutic target in β-hemoglobinopathies.

An estimated 6% to 7% of the earth's population carries a mutation affecting red blood cell function. The β-thalassemias and sickle cell disease are the most common monogenic disorders caused by these mutations. Increased levels of γ-globin ameliorate the severity of these diseases because fetal hemoglobin (HbF; α2γ2) can effectively replace adult hemoglobin (HbA; α2β2) and counteract polymerization of sickle hemoglobin (HbS; α2βS2). Therefore, understanding the molecular mechanism of globin switching is of biologic and clinical importance. Here, we show that the recently identified chromatin factor Friend of Prmt1 (FOP) is a critical modulator of γ-globin gene expression. Knockdown of FOP in adult erythroid progenitors strongly induces HbF. Importantly, γ-globin expression can be elevated in cells from β-thalassemic patients by reducing FOP levels. These observations identify FOP as a novel therapeutic target in β-hemoglobinopathies.

Plasma phospholipid transfer protein (PLTP) interacts with HDL particles and facilitates the transfer of phospholipids from triglyceride (TG)-rich lipoproteins to HDL. Overexpressing human PLTP in mice increases the susceptibility to atherosclerosis. In human plasma, high-active and low-active forms of PLTP exist. To elucidate the contribution of phospholipid transfer activity to changes in lipoprotein metabolism and atherogenesis, we developed mice expressing mutant PLTP, still able to associate with HDL but lacking phospholipid transfer activity. In mice heterozygous for the LDL receptor, effects of the mutant and normal human PLTP transgene (mutPLTP tg and PLTP tg, respectively) were compared. In PLTP tg mice, plasma PLTP activity was increased 2.9-fold, resulting in markedly reduced HDL lipid levels. In contrast, in mutPLTP tg mice, lipid levels were not different from controls. Furthermore, hepatic VLDL-TG secretion was stimulated in PLTP tg mice, but not in mutPLTP tg mice. When mice were fed a cholesterol-enriched diet, atherosclerotic lesion size in PLTP tg mice was increased more than 2-fold compared with control mice, whereas in mutPLTP tg mice, there was no change. Our findings demonstrate that PLTP transfer activity is essential for the development of atherosclerosis in PLTP transgenic mice, identifying PLTP activity as a possible target to prevent atherogenesis, independent of plasma PLTP concentration.

Plasma phospholipid transfer protein (PLTP) interacts with HDL particles and facilitates the transfer of phospholipids from triglyceride (TG)-rich lipoproteins to HDL. Overexpressing human PLTP in mice increases the susceptibility to atherosclerosis. In human plasma, high-active and low-active forms of PLTP exist. To elucidate the contribution of phospholipid transfer activity to changes in lipoprotein metabolism and atherogenesis, we developed mice expressing mutant PLTP, still able to associate with HDL but lacking phospholipid transfer activity. In mice heterozygous for the LDL receptor, effects of the mutant and normal human PLTP transgene (mutPLTP tg and PLTP tg, respectively) were compared. In PLTP tg mice, plasma PLTP activity was increased 2.9-fold, resulting in markedly reduced HDL lipid levels. In contrast, in mutPLTP tg mice, lipid levels were not different from controls. Furthermore, hepatic VLDL-TG secretion was stimulated in PLTP tg mice, but not in mutPLTP tg mice. When mice were fed a cholesterol-enriched diet, atherosclerotic lesion size in PLTP tg mice was increased more than 2-fold compared with control mice, whereas in mutPLTP tg mice, there was no change. Our findings demonstrate that PLTP transfer activity is essential for the development of atherosclerosis in PLTP transgenic mice, identifying PLTP activity as a possible target to prevent atherogenesis, independent of plasma PLTP concentration.

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.

Dendritic cells are key initiators and regulators of the immune response. Dendritic cell commitment and function require orchestrated regulation of transcription. Gata1 is a transcription factor expressed in several hematopoietic lineages. However, Gata1 function has not been explored in the monocytic or dendritic cell compartment. Here, we show that Gata1 is expressed in myeloid and plasmacytoid dendritic cells and that Gata1 ablation affects the survival of dendritic cells. Furthermore, lipopolysaccharide (LPS) stimulation of dendritic cells prompts Gata1 up-regulation, which is accompanied by increased levels of BclX and Ifng. Our findings show that Gata1 is a transcriptional regulator of dendritic cell differentiation and suggest that Gata1 is involved in the dendritic cell and macrophage lineage separation.

Dendritic cells are key initiators and regulators of the immune response. Dendritic cell commitment and function require orchestrated regulation of transcription. Gata1 is a transcription factor expressed in several hematopoietic lineages. However, Gata1 function has not been explored in the monocytic or dendritic cell compartment. Here, we show that Gata1 is expressed in myeloid and plasmacytoid dendritic cells and that Gata1 ablation affects the survival of dendritic cells. Furthermore, lipopolysaccharide (LPS) stimulation of dendritic cells prompts Gata1 up-regulation, which is accompanied by increased levels of BclX and Ifng. Our findings show that Gata1 is a transcriptional regulator of dendritic cell differentiation and suggest that Gata1 is involved in the dendritic cell and macrophage lineage separation.

Three Gata transcription factors (Gata1, -2, and -3) are essential for hematopoiesis. These factors are thought to play distinct roles because they do not functionally replace each other. For instance, Gata2 messenger RNA (mRNA) expression is highly elevated in Gata1-null erythroid cells, yet this does not rescue the defect. Here, we test whether Gata2and -3transgenes rescue the erythroid defect of Gata1-null mice, if expressed in the appropriate spatiotemporal pattern. Gata1, -2, and -3 transgenes driven by β-globinregulatory elements, directing expression to late stages of differentiation, fail to rescue erythropoiesis in Gata1-null mutants. In contrast, when controlled by Gata1regulatory elements, directing expression to the early stages of differentiation, Gata1, -2, and -3 do rescue the Gata1-null phenotype. The dramatic increase of endogenous Gata2 mRNA in Gata1-null progenitors is not reflected in Gata2 protein levels, invoking translational regulation. Our data show that the dynamic spatiotemporal regulation of Gata factor levels is more important than their identity and provide a paradigm for developmental control mechanisms that are hard-wired in cis-regulatory elements.

Three Gata transcription factors (Gata1, -2, and -3) are essential for hematopoiesis. These factors are thought to play distinct roles because they do not functionally replace each other. For instance, Gata2 messenger RNA (mRNA) expression is highly elevated in Gata1-null erythroid cells, yet this does not rescue the defect. Here, we test whether Gata2 and -3 transgenes rescue the erythroid defect of Gata1-null mice, if expressed in the appropriate spatiotemporal pattern. Gata1, -2, and -3 transgenes driven by β-globin regulatory elements, directing expression to late stages of differentiation, fail to rescue erythropoiesis in Gata1-null mutants. In contrast, when controlled by Gata1 regulatory elements, directing expression to the early stages of differentiation, Gata1, -2, and -3 do rescue the Gata1-null phenotype. The dramatic increase of endogenous Gata2 mRNA in Gata1-null progenitors is not reflected in Gata2 protein levels, invoking translational regulation. Our data show that the dynamic spatiotemporal regulation of Gata factor levels is more important than their identity and provide a paradigm for developmental control mechanisms that are hard-wired in cis-regulatory elements.

EBs and CLIPs are evolutionarily conserved proteins, which associate with the tips of growing microtubules, and regulate microtubule dynamics and their interactions with intracellular structures. In this study we investigated the functional relationship of CLIP-170 and CLIP-115 with the three EB family members, EB1, EB2(RP1), and EB3 in mammalian cells. We showed that both CLIPs bind to EB proteins directly. The C-terminal tyrosine residue of EB proteins is important for this interaction. When EB1 and EB3 or all three EBs were significantly depleted using RNA interference, CLIPs accumulated at the MT tips at a reduced level, because CLIP dissociation from the tips was accelerated. Normal CLIP localization was restored by expression of EB1 but not of EB2. An EB1 mutant lacking the C-terminal tail could also fully rescue CLIP dissociation kinetics, but could only partially restore CLIP accumulation at the tips, suggesting that the interaction of CLIPs with the EB tails contributes to CLIP localization. When EB1 was distributed evenly along the microtubules because of overexpression, it slowed down CLIP dissociation but did not abolish its preferential plus-end localization, indicating that CLIPs possess an intrinsic affinity for growing microtubule ends, which is enhanced by an interaction with the EBs.

Abstract The remarkable high affinity (Kd  ~ 10−15 M) of avidin/streptavidin for biotin has been extensively exploited in purification methodologies. Recently a small peptide sequence (Avi-tag) has been defined that can be specifically and efficiently biotinylated by the bacterial BirA biotin ligase. Fusion of this small peptide sequence to a protein of interest and co-expression with the BirA gene in mammalian cells allowed purification of the biotinylated protein together with its associated proteins and other molecules. Ideally, one would like to apply these technologies to purify tagged proteins directly from mouse tissues. To make this approach feasible for a large variety of proteins we developed a mouse strain that expresses the BirA gene ubiquitously by inserting it in the ROSA26 locus. We demonstrate that the BirA protein is indeed expressed in all tissues tested. In order to demonstrate functionality we show that it biotinylates the transgene-encoded Avi-tagged Gata1 and Oct6 transcription factors in erythroid cells of the foetal liver and Schwann cells of the peripheral nerve respectively. Therefore, this mouse can be crossed to any transgenic mouse to obtain efficient biotinylation of an Avi-tagged protein for the purpose of protein (complex) purification.

To study epigenetic regulation of the human β-globin locus during hematopoiesis, we investigated patterns of histone modification and chromatin accessibility along this locus in hematopoietic progenitor cells (HPCs) derived from both humans and transgenic mice. We demonstrate that the developmentally related activation of human β-like globin genes in humans and transgenic mice HPCs is preceded by a wave of gene-specific histone H3 hyperacetylation and K4 dimethylation. In erythroid cells, expression of β-like globin genes is associated with histone hyperacetylation along these genes and, surprisingly, with local deacetylation at active promoters. We also show that endogenous mouse β major and human β-like genes are subject to different epigenetic control mechanisms in HPCs. This difference is likely due to intrinsic properties of the human β-globin locus since, in transgenic mice, this locus is epigenetically regulated in the same manner as in human HPCs. Our results suggest that a defined pattern of histone H3 acetylation/dimethylation is important for specific activation of human globin promoters during development in human and transgenic HPCs. We propose that this transient acetylation/dimethylation is involved in gene-specific potentiation in HPCs (ie, before extensive chromatin remodeling and transcription take place in erythroid cells).

To study epigenetic regulation of the human β-globin locus during hematopoiesis, we investigated patterns of histone modification and chromatin accessibility along this locus in hematopoietic progenitor cells (HPCs) derived from both humans and transgenic mice. We demonstrate that the developmentally related activation of human β-like globin genes in humans and transgenic mice HPCs is preceded by a wave of gene-specific histone H3 hyperacetylation and K4 dimethylation. In erythroid cells, expression of β-like globin genes is associated with histone hyperacetylation along these genes and, surprisingly, with local deacetylation at active promoters. We also show that endogenous mouse β major and human β-like genes are subject to different epigenetic control mechanisms in HPCs. This difference is likely due to intrinsic properties of the human β-globin locus since, in transgenic mice, this locus is epigenetically regulated in the same manner as in human HPCs. Our results suggest that a defined pattern of histone H3 acetylation/dimethylation is important for specific activation of human globin promoters during development in human and transgenic HPCs. We propose that this transient acetylation/dimethylation is involved in gene-specific potentiation in HPCs (ie, before extensive chromatin remodeling and transcription take place in erythroid cells).

Deletions at the 3′ end of the human β-globin locus are associated with the hereditary persistence of fetal hemoglobin (HPFH) in adults, potentially through the juxtaposition of enhancer elements in the vicinity of the fetal γ-globin genes. We have tested how sequences at the HPFH-2, HPFH-3, and HPFH-6 breakpoints, which act as enhancers in vitro, affect the silencing of a locus control region Aγ (LCRAγ) transgene in the adult stage of mice. We found persistent Aγ expression in the adult blood of most of the multicopy HPFH-2, HPFH-3, or HPFH-6 lines, in contrast to the control LCRAγ lines which were silenced. Cre-mediated generation of single copy lines showed persistent γ gene expression maintained in some of the HPFH-2 and HPFH-6 lines, but not in any of the HPFH-3 or LCRAγ lines. In the HPFH-2 and HPFH-6 lines, persistent γ gene expression correlated with euchromatic transgene integrations. Thus, our observations provide support for a model whereby HPFH conditions arise from the juxtaposition of enhancers as well as permissive chromatin subdomains in the vicinity of the γ-globin genes.

Deletions at the 3′ end of the human β-globin locus are associated with the hereditary persistence of fetal hemoglobin (HPFH) in adults, potentially through the juxtaposition of enhancer elements in the vicinity of the fetal γ-globin genes. We have tested how sequences at the HPFH-2, HPFH-3, and HPFH-6 breakpoints, which act as enhancers in vitro, affect the silencing of a locus control region Aγ (LCRAγ) transgene in the adult stage of mice. We found persistent Aγ expression in the adult blood of most of the multicopy HPFH-2, HPFH-3, or HPFH-6 lines, in contrast to the control LCRAγ lines which were silenced. Cre-mediated generation of single copy lines showed persistent γ gene expression maintained in some of the HPFH-2 and HPFH-6 lines, but not in any of the HPFH-3 or LCRAγ lines. In the HPFH-2 and HPFH-6 lines, persistent γ gene expression correlated with euchromatic transgene integrations. Thus, our observations provide support for a model whereby HPFH conditions arise from the juxtaposition of enhancers as well as permissive chromatin subdomains in the vicinity of the γ-globin genes.

As the zinc-finger transcription factor specificity protein 3 (Sp3) has been implicated in the regulation of many hematopoietic-specific genes, we analyzed the role of Sp3 in hematopoiesis. At embryonic day 18.5 (E18.5), Sp3-/- mice exhibit a partial arrest of T-cell development in the thymus and B-cell numbers are reduced in liver and spleen. However, pre–B-cell proliferation and differentiation into immunoglobulin M–positive (IgM+) B cells in vitro are not affected. At E14.5 and E16.5, Sp3-/- mice exhibit a significant delay in the appearance of definitive erythrocytes in the blood, paralleled by a defect in the progression of differentiation of definitive erythroid cells in vitro. Perinatal death of the null mutants precludes the analysis of adult hematopoiesis in Sp3-/- mice. We therefore investigated the ability of E12.5 Sp3-/- liver cells to contribute to the hematopoietic compartment in an in vivo transplantation assay. Sp3-/- cells were able to repopulate the B- and T-lymphoid compartment, albeit with reduced efficiency. In contrast, Sp3-/- cells showed no significant engraftment in the erythroid and myeloid lineages. Thus, the absence of Sp3 results in cell-autonomous hematopoietic defects, affecting in particular the erythroid and myeloid cell lineages.

As the zinc-finger transcription factor specificity protein 3 (Sp3) has been implicated in the regulation of many hematopoietic-specific genes, we analyzed the role of Sp3 in hematopoiesis. At embryonic day 18.5 (E18.5), Sp3-/-mice exhibit a partial arrest of T-cell development in the thymus and B-cell numbers are reduced in liver and spleen. However, pre–B-cell proliferation and differentiation into immunoglobulin M–positive (IgM+) B cells in vitro are not affected. At E14.5 and E16.5, Sp3-/-mice exhibit a significant delay in the appearance of definitive erythrocytes in the blood, paralleled by a defect in the progression of differentiation of definitive erythroid cells in vitro. Perinatal death of the nullmutants precludes the analysis of adult hematopoiesis in Sp3-/-mice. We therefore investigated the ability of E12.5 Sp3-/-liver cells to contribute to the hematopoietic compartment in an in vivo transplantation assay. Sp3-/-cells were able to repopulate the B- and T-lymphoid compartment, albeit with reduced efficiency. In contrast, Sp3-/-cells showed no significant engraftment in the erythroid and myeloid lineages. Thus, the absence of Sp3 results in cell-autonomous hematopoietic defects, affecting in particular the erythroid and myeloid cell lineages.

Developmental and tissue-specific expression of higher eukaryotic genes involves activation of transcription at the appropriate time and place and keeping it silent otherwise. Unlike housekeeping genes, tissue-specific genes generally do not cluster on the chromosomes. They can be found in gene-dense regions of chromosomes as well as in regions of repressive chromatin. Depending on the location, shielding against positive or negative regulatory effects from neighboring chromatin may be required and hence insulator and boundary models were proposed. They postulate that chromosomes are partitioned into physically distinct expression domains, each containing a gene or gene cluster with its cis-regulatory elements. Specialized elements at the borders of such domains are proposed to prevent cross-talk between domains, and thus to be crucial in establishing independent expression domains. However, genes and associated cis-acting sequences often do not occupy physically distinct domains on the chromosomes. Rather, genes can overlap and cis-acting sequences can be found tens or hundreds of kilobases away from the target gene, sometimes with unrelated genes in between. Therefore the ability of a gene to communicate with positive cis-regulatory elements rather than the presence of specialized boundary elements appears to be key to establishing an independent expression profile. Our recent finding that active β-globin genes physically interact in the nuclear space with multiple cis-regulatory elements, with inactive genes looping out, has provided a potential mechanistic framework for this model. We refer to such a spatial unit of regulatory DNA elements as an active chromatin hub (ACH). We propose that productive ACH formation underlies correct gene expression, requiring the presence of protein factors with the appropriate affinities for each other bound to their cognate DNA sequences. Proximity and specificity determines which cis-acting sequences and promoter(s) form an ACH, and thus which gene will be expressed. Other regulatory sequences can interfere with transcription by blocking the appropriate physical interaction between an enhancer and promoter in the ACH. Possible mechanisms by which distal DNA elements encounter each other in the 3D nuclear space will be discussed.

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing α- and β-globin genes from the pufferfishFugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show thatFugucontains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only α-globin genes (α-locus), whereas the other also contains a β-globin gene (αβ-locus). This is the first poikilothermic species analyzed in which the physical linkage of the α- and β-globin genes has been uncoupled, supporting a model in which the separation of the α- and β-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of theFuguαβ-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian β-globin clusters, may not be present in theFuguαβ-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types.

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing α- and β-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show thatFugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only α-globin genes (α-locus), whereas the other also contains a β-globin gene (αβ-locus). This is the first poikilothermic species analyzed in which the physical linkage of the α- and β-globin genes has been uncoupled, supporting a model in which the separation of the α- and β-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu αβ-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian β-globin clusters, may not be present in the Fugu αβ-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types.

Plasma phospholipid transfer protein (PLTP) is thought to be involved in the remodeling of high density lipoproteins (HDL), which are atheroprotective. It is also involved in the metabolism of very low density lipoproteins (VLDL). Hence, PLTP is thought to be an important factor in lipoprotein metabolism and the development of atherosclerosis. We have overexpressed PLTP in mice heterozygous for the low density lipoprotein (LDL) receptor, a model for atherosclerosis. We show that increased PLTP activity results in a dose-dependent decrease in HDL, and a moderate stimulation of VLDL secretion (

In the vascular system, nitric oxide is generated by endothelial NO synthase (eNOS). NO has pleiotropic effects, most of which are believed to be atheroprotective. Therefore, it has been argued that patients suffering from cardiovascular disease could benefit from an increase in eNOS activity. However, increased NO production can cause oxidative damage, cell toxicity, and apoptosis and hence could be atherogenic rather than beneficial. To study the in vivo effects of increased eNOS activity, we created transgenic mice overexpressing human eNOS. Aortic blood pressure was approximately 20 mm Hg lower in the transgenic mice compared with control mice because of lower systemic vascular resistance. The effects of eNOS overexpression on diet-induced atherosclerosis were studied in apolipoprotein E-deficient mice. Elevation of eNOS activity decreased blood pressure ( approximately 20 mm Hg) and plasma levels of cholesterol ( approximately 17%), resulting in a reduction in atherosclerotic lesions by 40%. We conclude that an increase in eNOS activity is beneficial and provides protection against atherosclerosis.

Double-stranded RNA (dsRNA) has been shown to interfere with the function of specific genes in various invertebrate species. The application of dsRNA interference (RNAi) in vertebrates (zebrafish and mouse) is still limited to embryos and it is not clear whether the method is generally applicable. Using a transgenic mouse model we investigated whether a stably inherited dsRNA introduced as a transgene can interfere with the expression of a specific target gene in erythroid tissue during development. In our globin gene system we do not observe any specific RNA interference. We, therefore, also introduced another gene that may be involved in a mechanism of post transcriptional gene silencing (PTGS), namely RNA-dependent RNA polymerase (RdRP) that was proposed to be involved in producing RNAs that trigger PTGS in plants. However, even though the tomato RdRP is catalytically active in erythroid tissue, no RNAi was observed.