John Strouboulis - Academia.edu (original) (raw)
Papers by John Strouboulis
have built an image of nuclear organization in which the molecular machines involved in transcrip... more have built an image of nuclear organization in which the molecular machines involved in transcription, RNA processing and replication assemble morphologically distinct nuclear organelles with defined functional properties. These observations indicate a very high level of structural organization for the various metabolic activities occurring within the nucleus. We discuss the possible existence of novel regulatory functions inherent to nuclear architecture itself.
Deletions at the 3 end of the human -globin locus are associated with the hereditary persistence ... more Deletions at the 3 end of the human -globin locus are associated with the hereditary persistence of fetal hemoglo- bin (HPFH) in adults, potentially through the juxtaposition of enhancer elements in the vicinity of the fetal -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 silenc- ing
Experimental Hematology, 2015
Every two years since 1978, an international group of scientists, physicians, and other researche... more Every two years since 1978, an international group of scientists, physicians, and other researchers meet to discuss the latest developments in the underlying etiology, mechanisms of action and developmental acquisition of cellular and systemic defects exhibited and elicited by the most common inherited human disorders, the hemoglobinopathies. The 19(th) Hemoglobin Switching Conference, held in September 2014 at St. John's College in Oxford, once again exceeded all expectations by describing cutting edge research in cellular, molecular, developmental and genomic advances focused on these diseases, comprised of about sixty short talks over three days by leading investigators in the field. This meeting report describes the highlights of the conference.
Molecular and cellular biology, Jan 13, 2015
The ordered assembly of a functional pre-initiation complex (PIC), composed of general transcript... more The ordered assembly of a functional pre-initiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cells in vivo we observed a differentiation block accompanied by deregulated GATA1 target genes, including Gata1 itself, suggesting functional crosstalk between GATA1 and TAF10. Additionally, we analyzed the composition of TFIID and SAGA complexes by mass spectrometry in mouse and human cells and found that their global integrity is maintained, wi...
Molecular and cellular biology, 2009
Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of pro... more Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of proteins. It binds with high affinity to E-box elements and, through interaction with coactivators, aids in the formation of transcription complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and beta-globin. Here, we show that the erythroid cell-specific expression of a dominant-negative mutant of USF, A-USF, in transgenic mice reduces the expression of all beta-type globin genes and leads to the diminished association of RNA polymerase II with locus control region element HS2 and with the beta-globin gene promoter. We further show that the expression of A-USF reduces the expression of several key erythroid cell-specific transcription factors, including EKLF and Tal-1. We provide evidence demonstrating that USF interacts with known regulatory DNA elements in the EKLF and Tal-1 gene loci in erythroid cells. Furthermore, A-USF-expre...
BMC bioinformatics, 2006
In the current era of high throughput genomics a major challenge is the genome-wide identificatio... more In the current era of high throughput genomics a major challenge is the genome-wide identification of target genes for specific transcription factors. Chromatin immunoprecipitation (ChIP) allows the isolation of in vivo binding sites of transcription factors and provides a powerful tool for examining gene regulation. Crosslinked chromatin is immunoprecipitated with antibodies against specific transcription factors, thus enriching for sequences bound in vivo by these factors in the immunoprecipitated DNA. Cloning and sequencing the immunoprecipitated sequences allows identification of transcription factor target genes. Routinely, thousands of such sequenced clones are used in BLAST searches to map their exact location in the genome and the genes located in the vicinity. These genes represent potential targets of the transcription factor of interest. Such bioinformatics analysis is very laborious if performed manually and for this reason there is a need for developing bioinformatic to...
Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2003
Proteomic approaches require simple and efficient protein purification methodologies that are ame... more Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method ...
Molecular cell, 2002
We have undertaken a detailed characterization of mouse globin gene expression patterns in the nu... more We have undertaken a detailed characterization of mouse globin gene expression patterns in the nucleus and cytoplasm of single erythroid cells. We demonstrate an imbalance of alpha- versus beta-globin expression in a significant proportion of cells both in nuclear transcription patterns and cytoplasmic mRNA levels. Clonal cell analysis showed these expression patterns to be clonally inherited, while analysis of a multicopy transgenic locus showed an all-or-none effect in the activation of all the genes in one locus. These data provide strong evidence for a stochastic basis of globin gene activation resulting in heritable all-or-none expression patterns.
Annals of the New York Academy of Sciences, 2005
We have described the application of a simple biotinylation tagging approach for the direct purif... more We have described the application of a simple biotinylation tagging approach for the direct purification of tagged transcription factor complexes, based on the use of artificial short peptide tags that are specifically and efficiently biotinylated by the bacterial BirA biotin ligase, which is co-expressed in cells with the tagged factor. We used this approach to initially characterize complexes formed by the hematopoietic transcription factor GATA-1 in erythroid cells. GATA-1 is essential for the erythroid differentiation, its functions encompassing upregulation of erythroid genes, repression of alternative transcription programs, and suppression of cell proliferation. However, it was not clear how all of these GATA-1 functions are mediated. Our work describes, for the first time, distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex, and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TA...
Molecular & cellular proteomics : MCP, 2007
Ring1B/Rnf2 is a RING finger protein member of the Polycomb group (PcG) of proteins, which form c... more Ring1B/Rnf2 is a RING finger protein member of the Polycomb group (PcG) of proteins, which form chromatin-modifying complexes essential for embryonic development and stem cell renewal and which are commonly deregulated in cancer. Ring1B/Rnf2 is a ubiquitin E3 ligase that catalyzes the monoubiquitylation of the histone H2A, one of the histone modifications needed for the transcriptional repression activity of the PcG of proteins. Ring1B/Rnf2 was shown to be part of two complexes, the PRC1 PcG complex and the E2F6.com-1 complex, which also contains non-PcG members, thus raising the prospect for additional Ring1B/Rnf2 partners and functions extending beyond the PcG. Here we used a high throughput proteomics approach based on the single step purification, using streptavidin beads, of in vivo biotinylated Ring1B/Rnf2 and associated proteins from a nuclear extract from erythroid cells and their identification by mass spectrometry. About 50 proteins were confidently identified of which 20 ...
Transgenic Research, 2007
One main determinant in high-density lipoprotein (HDL) metabolism is phospholipid transfer protei... more One main determinant in high-density lipoprotein (HDL) metabolism is phospholipid transfer protein (PLTP), a plasma protein that is associated with HDL. In transgenic mice overexpressing human PLTP we found that elevated plasma PLTP levels dose-dependently increased the susceptibility to dietinduced atherosclerosis. This could be mainly due to the fact that most functions of PLTP are potentially atherogenic, such as decreasing plasma HDL levels. To further elucidate the role of PLTP in lipoprotein metabolism and atherosclerosis we generated a novel transgenic mouse model that allows conditional expression of human PLTP. In this mouse model a human PLTP encoding sequence is controlled by a Tet-On system. Upon induction of PLTP expression, our mouse model showed a strongly increased PLTP activity (from 3.0 ± 0.6 to 11.4 ± 2.8 AU, p < 0.001). The increase in PLTP activity resulted in an acute decrease in plasma cholesterol of 33% and a comparable decrease in phospholipids. The decrease in total plasma cholesterol and phospholipids was caused by a 35% decrease in HDL-cholesterol level and a 41% decrease in HDL-phospholipid level. These results demonstrate the feasibility of our mouse model to induce an acute elevation of PLTP activity, which is easily reversible. As a direct consequence of an increase in PLTP activity, HDL-cholesterol and HDL-phospholipid levels strongly decrease. Using this mouse model, it will be possible to study the effects of acute elevation of PLTP activity on lipoprotein metabolism and pre-existing atherosclerosis.
Transgenic Research, 2005
The remarkable high affinity (Kd $ 10 )15 M) of avidin/streptavidin for biotin has been extensive... more 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.
The EMBO Journal, 2005
GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast ce... more GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/ proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.
The EMBO Journal, 2006
The passage from proliferation to terminal differentiation is critical for normal development and... more The passage from proliferation to terminal differentiation is critical for normal development and is often perturbed in malignancies. To define the molecular mechanisms that govern this process during erythropoiesis, we have used tagging/proteomics approaches and characterized protein complexes nucleated by TAL-1/SCL, a basic helix-loophelix transcription factor that specifies the erythrocytic lineage. In addition to known TAL-1 partners, GATA-1, E2A, HEB, LMO2 and Ldb1, we identify the ETO2 repressor as a novel component recruited to TAL-1 complexes through interaction with E2A/HEB. Ectopic expression and siRNA knockdown experiments in hematopoietic progenitor cells show that ETO2 actively represses erythroid TAL-1 target genes and governs the expansion of erythroid progenitors. At the onset of erythroid differentiation, a change in the stoichiometry of ETO2 within the TAL-1 complex activates the expression of known erythroidspecific TAL-1 target genes and of Gfi-1b and p21 Cip , encoding two essential regulators of erythroid cell proliferation. These results suggest that the dynamics of ETO2 recruitment within nuclear complexes couple cell proliferation to cell differentiation and determine the onset of terminal erythroid maturation.
Protein Expression and Purification, 2013
We describe here a simple procedure for greatly reducing contamination of nuclear extracts by nat... more We describe here a simple procedure for greatly reducing contamination of nuclear extracts by naturally biotinylated cytoplasmic carboxylases, which represent a major source of non-specific background when employing BirA-mediated biotinylation tagging for the purification and characterization of nuclear protein complexes by mass spectrometry. We show that the use of 0.5% of the non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is sufficient to practically eliminate contamination of nuclear extracts by carboxylases and to greatly reduce background signals in downstream mass spectrometric analyses.
PLoS Genetics, 2014
We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) eleme... more We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) elements in the eand cglobin promoters, and act as molecular anchors for the recruitment of epigenetic corepressors of the multifaceted DRED complex, thereby leading to eand c-globin transcriptional repression during definitive erythropoiesis. Other than the eand c-globin and the GATA1 genes, TR4-regulated target genes in human erythroid cells remain unknown. Here, we identified TR4 binding sites genome-wide using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) as human primary CD34 + hematopoietic progenitors differentiated progressively to late erythroid precursors. We also performed whole transcriptome analyses by RNA-seq to identify TR4 downstream targets after lentiviral-mediated TR4 shRNA knockdown in erythroid cells. Analyses from combined ChIP-seq and RNA-seq datasets indicate that DR1 motifs are more prevalent in the proximal promoters of TR4 direct target genes, which are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing and primary metabolic processes). In contrast, other non-DR1 repeat motifs (DR4, ER6 and IR1) are more prevalent at gene-distal TR4 binding sites. Of these, approximately 50% are also marked with epigenetic chromatin signatures (such as P300, H3K27ac, H3K4me1 and H3K27me3) associated with enhancer function. Thus, we hypothesize that TR4 regulates gene transcription via gene-proximal DR1 sites as TR4/TR2 heterodimers, while it can associate with novel nuclear receptor partners (such as RXR) to bind to distant non-DR1 consensus sites. In summary, this study reveals that the TR4 regulatory network is far more complex than previously appreciated and that TR4 regulates basic, essential biological processes during the terminal differentiation of human erythroid cells.
Nucleic Acids Research, 1992
Molecular Cell, 1997
have also been put forward in favor of a binary model that proposes that the sole function of dis... more have also been put forward in favor of a binary model that proposes that the sole function of distal elements is to generate a favorable chromatin structure that
have built an image of nuclear organization in which the molecular machines involved in transcrip... more have built an image of nuclear organization in which the molecular machines involved in transcription, RNA processing and replication assemble morphologically distinct nuclear organelles with defined functional properties. These observations indicate a very high level of structural organization for the various metabolic activities occurring within the nucleus. We discuss the possible existence of novel regulatory functions inherent to nuclear architecture itself.
Deletions at the 3 end of the human -globin locus are associated with the hereditary persistence ... more Deletions at the 3 end of the human -globin locus are associated with the hereditary persistence of fetal hemoglo- bin (HPFH) in adults, potentially through the juxtaposition of enhancer elements in the vicinity of the fetal -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 silenc- ing
Experimental Hematology, 2015
Every two years since 1978, an international group of scientists, physicians, and other researche... more Every two years since 1978, an international group of scientists, physicians, and other researchers meet to discuss the latest developments in the underlying etiology, mechanisms of action and developmental acquisition of cellular and systemic defects exhibited and elicited by the most common inherited human disorders, the hemoglobinopathies. The 19(th) Hemoglobin Switching Conference, held in September 2014 at St. John&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s College in Oxford, once again exceeded all expectations by describing cutting edge research in cellular, molecular, developmental and genomic advances focused on these diseases, comprised of about sixty short talks over three days by leading investigators in the field. This meeting report describes the highlights of the conference.
Molecular and cellular biology, Jan 13, 2015
The ordered assembly of a functional pre-initiation complex (PIC), composed of general transcript... more The ordered assembly of a functional pre-initiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cells in vivo we observed a differentiation block accompanied by deregulated GATA1 target genes, including Gata1 itself, suggesting functional crosstalk between GATA1 and TAF10. Additionally, we analyzed the composition of TFIID and SAGA complexes by mass spectrometry in mouse and human cells and found that their global integrity is maintained, wi...
Molecular and cellular biology, 2009
Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of pro... more Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of proteins. It binds with high affinity to E-box elements and, through interaction with coactivators, aids in the formation of transcription complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and beta-globin. Here, we show that the erythroid cell-specific expression of a dominant-negative mutant of USF, A-USF, in transgenic mice reduces the expression of all beta-type globin genes and leads to the diminished association of RNA polymerase II with locus control region element HS2 and with the beta-globin gene promoter. We further show that the expression of A-USF reduces the expression of several key erythroid cell-specific transcription factors, including EKLF and Tal-1. We provide evidence demonstrating that USF interacts with known regulatory DNA elements in the EKLF and Tal-1 gene loci in erythroid cells. Furthermore, A-USF-expre...
BMC bioinformatics, 2006
In the current era of high throughput genomics a major challenge is the genome-wide identificatio... more In the current era of high throughput genomics a major challenge is the genome-wide identification of target genes for specific transcription factors. Chromatin immunoprecipitation (ChIP) allows the isolation of in vivo binding sites of transcription factors and provides a powerful tool for examining gene regulation. Crosslinked chromatin is immunoprecipitated with antibodies against specific transcription factors, thus enriching for sequences bound in vivo by these factors in the immunoprecipitated DNA. Cloning and sequencing the immunoprecipitated sequences allows identification of transcription factor target genes. Routinely, thousands of such sequenced clones are used in BLAST searches to map their exact location in the genome and the genes located in the vicinity. These genes represent potential targets of the transcription factor of interest. Such bioinformatics analysis is very laborious if performed manually and for this reason there is a need for developing bioinformatic to...
Proceedings of the National Academy of Sciences of the United States of America, Jan 24, 2003
Proteomic approaches require simple and efficient protein purification methodologies that are ame... more Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method ...
Molecular cell, 2002
We have undertaken a detailed characterization of mouse globin gene expression patterns in the nu... more We have undertaken a detailed characterization of mouse globin gene expression patterns in the nucleus and cytoplasm of single erythroid cells. We demonstrate an imbalance of alpha- versus beta-globin expression in a significant proportion of cells both in nuclear transcription patterns and cytoplasmic mRNA levels. Clonal cell analysis showed these expression patterns to be clonally inherited, while analysis of a multicopy transgenic locus showed an all-or-none effect in the activation of all the genes in one locus. These data provide strong evidence for a stochastic basis of globin gene activation resulting in heritable all-or-none expression patterns.
Annals of the New York Academy of Sciences, 2005
We have described the application of a simple biotinylation tagging approach for the direct purif... more We have described the application of a simple biotinylation tagging approach for the direct purification of tagged transcription factor complexes, based on the use of artificial short peptide tags that are specifically and efficiently biotinylated by the bacterial BirA biotin ligase, which is co-expressed in cells with the tagged factor. We used this approach to initially characterize complexes formed by the hematopoietic transcription factor GATA-1 in erythroid cells. GATA-1 is essential for the erythroid differentiation, its functions encompassing upregulation of erythroid genes, repression of alternative transcription programs, and suppression of cell proliferation. However, it was not clear how all of these GATA-1 functions are mediated. Our work describes, for the first time, distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex, and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TA...
Molecular & cellular proteomics : MCP, 2007
Ring1B/Rnf2 is a RING finger protein member of the Polycomb group (PcG) of proteins, which form c... more Ring1B/Rnf2 is a RING finger protein member of the Polycomb group (PcG) of proteins, which form chromatin-modifying complexes essential for embryonic development and stem cell renewal and which are commonly deregulated in cancer. Ring1B/Rnf2 is a ubiquitin E3 ligase that catalyzes the monoubiquitylation of the histone H2A, one of the histone modifications needed for the transcriptional repression activity of the PcG of proteins. Ring1B/Rnf2 was shown to be part of two complexes, the PRC1 PcG complex and the E2F6.com-1 complex, which also contains non-PcG members, thus raising the prospect for additional Ring1B/Rnf2 partners and functions extending beyond the PcG. Here we used a high throughput proteomics approach based on the single step purification, using streptavidin beads, of in vivo biotinylated Ring1B/Rnf2 and associated proteins from a nuclear extract from erythroid cells and their identification by mass spectrometry. About 50 proteins were confidently identified of which 20 ...
Transgenic Research, 2007
One main determinant in high-density lipoprotein (HDL) metabolism is phospholipid transfer protei... more One main determinant in high-density lipoprotein (HDL) metabolism is phospholipid transfer protein (PLTP), a plasma protein that is associated with HDL. In transgenic mice overexpressing human PLTP we found that elevated plasma PLTP levels dose-dependently increased the susceptibility to dietinduced atherosclerosis. This could be mainly due to the fact that most functions of PLTP are potentially atherogenic, such as decreasing plasma HDL levels. To further elucidate the role of PLTP in lipoprotein metabolism and atherosclerosis we generated a novel transgenic mouse model that allows conditional expression of human PLTP. In this mouse model a human PLTP encoding sequence is controlled by a Tet-On system. Upon induction of PLTP expression, our mouse model showed a strongly increased PLTP activity (from 3.0 ± 0.6 to 11.4 ± 2.8 AU, p < 0.001). The increase in PLTP activity resulted in an acute decrease in plasma cholesterol of 33% and a comparable decrease in phospholipids. The decrease in total plasma cholesterol and phospholipids was caused by a 35% decrease in HDL-cholesterol level and a 41% decrease in HDL-phospholipid level. These results demonstrate the feasibility of our mouse model to induce an acute elevation of PLTP activity, which is easily reversible. As a direct consequence of an increase in PLTP activity, HDL-cholesterol and HDL-phospholipid levels strongly decrease. Using this mouse model, it will be possible to study the effects of acute elevation of PLTP activity on lipoprotein metabolism and pre-existing atherosclerosis.
Transgenic Research, 2005
The remarkable high affinity (Kd $ 10 )15 M) of avidin/streptavidin for biotin has been extensive... more 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.
The EMBO Journal, 2005
GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast ce... more GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/ proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.
The EMBO Journal, 2006
The passage from proliferation to terminal differentiation is critical for normal development and... more The passage from proliferation to terminal differentiation is critical for normal development and is often perturbed in malignancies. To define the molecular mechanisms that govern this process during erythropoiesis, we have used tagging/proteomics approaches and characterized protein complexes nucleated by TAL-1/SCL, a basic helix-loophelix transcription factor that specifies the erythrocytic lineage. In addition to known TAL-1 partners, GATA-1, E2A, HEB, LMO2 and Ldb1, we identify the ETO2 repressor as a novel component recruited to TAL-1 complexes through interaction with E2A/HEB. Ectopic expression and siRNA knockdown experiments in hematopoietic progenitor cells show that ETO2 actively represses erythroid TAL-1 target genes and governs the expansion of erythroid progenitors. At the onset of erythroid differentiation, a change in the stoichiometry of ETO2 within the TAL-1 complex activates the expression of known erythroidspecific TAL-1 target genes and of Gfi-1b and p21 Cip , encoding two essential regulators of erythroid cell proliferation. These results suggest that the dynamics of ETO2 recruitment within nuclear complexes couple cell proliferation to cell differentiation and determine the onset of terminal erythroid maturation.
Protein Expression and Purification, 2013
We describe here a simple procedure for greatly reducing contamination of nuclear extracts by nat... more We describe here a simple procedure for greatly reducing contamination of nuclear extracts by naturally biotinylated cytoplasmic carboxylases, which represent a major source of non-specific background when employing BirA-mediated biotinylation tagging for the purification and characterization of nuclear protein complexes by mass spectrometry. We show that the use of 0.5% of the non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is sufficient to practically eliminate contamination of nuclear extracts by carboxylases and to greatly reduce background signals in downstream mass spectrometric analyses.
PLoS Genetics, 2014
We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) eleme... more We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) elements in the eand cglobin promoters, and act as molecular anchors for the recruitment of epigenetic corepressors of the multifaceted DRED complex, thereby leading to eand c-globin transcriptional repression during definitive erythropoiesis. Other than the eand c-globin and the GATA1 genes, TR4-regulated target genes in human erythroid cells remain unknown. Here, we identified TR4 binding sites genome-wide using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) as human primary CD34 + hematopoietic progenitors differentiated progressively to late erythroid precursors. We also performed whole transcriptome analyses by RNA-seq to identify TR4 downstream targets after lentiviral-mediated TR4 shRNA knockdown in erythroid cells. Analyses from combined ChIP-seq and RNA-seq datasets indicate that DR1 motifs are more prevalent in the proximal promoters of TR4 direct target genes, which are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing and primary metabolic processes). In contrast, other non-DR1 repeat motifs (DR4, ER6 and IR1) are more prevalent at gene-distal TR4 binding sites. Of these, approximately 50% are also marked with epigenetic chromatin signatures (such as P300, H3K27ac, H3K4me1 and H3K27me3) associated with enhancer function. Thus, we hypothesize that TR4 regulates gene transcription via gene-proximal DR1 sites as TR4/TR2 heterodimers, while it can associate with novel nuclear receptor partners (such as RXR) to bind to distant non-DR1 consensus sites. In summary, this study reveals that the TR4 regulatory network is far more complex than previously appreciated and that TR4 regulates basic, essential biological processes during the terminal differentiation of human erythroid cells.
Nucleic Acids Research, 1992
Molecular Cell, 1997
have also been put forward in favor of a binary model that proposes that the sole function of dis... more have also been put forward in favor of a binary model that proposes that the sole function of distal elements is to generate a favorable chromatin structure that