Browser does not support script.
logo
Image of Fred Sablitzky
Professor of Genetics, Faculty of Medicine & Health Sciences
Diplom in Biology, University of Cologne (1981) - PhD Genetics/Immunology, University of Cologne, Fellowship 'Fonds der Chemischen Industrie' (1985) - Habilitation (venia legendi) in Genetics, University of Cologne (1996) - Research Fellow in Immunology, University of Cologne (1985-1987) - Research Fellow in Haematopoiesis, Hospital For Sick Children, University of Toronto, Fellowship 'Deutsche Forschungsgemeinschaft' (1987-1989) - Head of Developmental Haematology Unit, Max-Delbrück-Laboratory, Max-Planck Society, Cologne, 'Nachwuchsförderung, Budesministerium für Bildung und Forschung', scientific award 'Bennigsen-Foerder-Preis des Landes NRW' (1989-1995) - Wellcome Trust European Senior Research Fellow, Deptartment of Medicine, University College London (UCL), London (1995-1999) - Reader in Molecular Genetics, Deptartment of Medicine, UCL, London (1998-1999) - Professor of Genetics and Scientific Director of the Gene Targeting & Transgenic Unit, School of Biology, Molecular Cell & Developmental Biology, The University of Nottingham (1999-Current)
Molecular regulators of cell fate. Utilising molecular, cell biology and biochemical techniques including gain- and loss-of-function analysis in vertebrate model systems such as mice and zebrafish,… read more
Current:
Senior Scientific Officer, Gene Targeting & Transgenic Unit (GTTU)
Dr Annemarie Kelly
PhD PGs:
Tamilvendhan Dhanaseelan
Xiaoou Xu
MRes PGs:
Former:
Postdocs:
Dr Yee Heng (The University of Nottingham, UK)
Dr Jing Jing Zhang (Pennington Biomedical Research Center, Baton Rouge, USA)
Dr Yu-Jung (Sarah) Yeh (National Health Research Institute, Taiwan)
PGs:
Maria Mirotsou (PhD 2001; post-doc Harvard, Boston, USA)
Lynn Bedford (PhD 2002; post-doc The University of Nottingham, UK)
Emerson King (PhD 2002; post-doc; Columbia, New York, USA)
Emma Heath (PhD 2003; post-doc Genetics, Cambridge, UK)
Konstantinos (Dino) Mavrakis (PhD 2003; post-doc Imperial College London, UK)
Robert Walker (PhD 2004; post-doc Developmental Biology, Sheffield, UK)
Joëlle Alcock (PhD 2005; post-doc The University of Nottingham, UK)
Paul Martin (PhD 2007; gap year)
Santosh Patlola (MRes 2009; Research Centre Borstel, Germany)
Katerina Goudevenou (PhD 2010; post-doc Imperial College London, UK)
Shabana Bashir (MRes 2010; gap year)
Wai Ho (Timothy) Shuen (MRes 2010 with distinction; PhD The University of Hong Kong, China)
Taina Theodore (MRes 2011; gap year)
Fiona Hey (PhD 2011; post-doc University of Leicester, UK)
Selam Aman (MRes submitted)
Tamilvendhan Dhanaseelan (MRes 2011; PhD The University of Nottingham, UK)
Nathan Czyzewicz (MRes 2011 with merit; PhD The University of Nottingham, UK)
Technicians:
Paul Roach
Molecular regulators of cell fate. Utilising molecular, cell biology and biochemical techniques including gain- and loss-of-function analysis in vertebrate model systems such as mice and zebrafish, we are analysing several genes to determine their functional role in cell fate.
We have shown that the dominant negative HLH protein Id4 plays a crucial role in neural and glial progenitor cell proliferation and timing of differentiation. In addition, analysis of Id4/lacZ knockin mice demonstrated that Id4 promotes osteoblast differentiation and acts as tumour suppressor gene in chronic lymphocytic leukemia (CLL). Recently it was shown that Id4 regulates mammary gland development by suppressing p38MAPK activity.
Utilising lyl-1 /lacZ knockin mice, transplantation experiments have shown that lyl-1-deficient bone marrow cells have a reduced capacity to repopulate lymphoid and myeloid lineages. Importantly, either lyl1 or scl/tal1, a related bHLH transcription factor, is required for survival of adult haematopoietic stem and progenitor cells. In addition, lyl-1 activity is required for the maturation of newly formed blood vessels in adult mice. In addition, lyl-1-dificiency induces a stress erythropoiesis.
We showed that def-3/rbm6 is a novel, evolutionary conserved RNA-binding protein that is targeted to splicing speckles and nascent transcripts and we discovered the def6 gene and demonstrated that def6 protein functions as an up-stream activator of Rho GTPases and as a filamentous actin binding protein. We have shown that def6 is required for convergent extension cell movements during zebrafish gastrulation downstream of Wnt5b signaling.
Id4 function in neural stem cell fate: Complex intrinsic and extrinsic mechanisms determine neural cell fate during development of the nervous system. Using Id4-deficient mice, we show that Id4 is required for normal development of the central nervous system (CNS), timing neural differentiation in the developing forebrain.
Id4-lacZ expression (blue) during mouse embryogenesis
In the absence of Id4, the ventricular zone of the neocortex, future hippocampus as well as lateral and medial ganglionic eminences exhibited a 20-30% reduction in mitotic neural precursor cells (NPCs). Although the number of apoptotic cells was significantly increased, the neocortex of Id4-deficient embryos was consistently thicker due to premature neuronal differentiation, which resulted in an increase in early-born neurons in the adult Id4-null cortex. Late-born cortical neurons and astrocytes in the cortex, septum, hippocampus and caudate putamen of Id4-deficient adult brains were decreased, however, likely due to the depletion of the NPC pool. Consequently, adult Id4-deficient brains were smaller and exhibited enlarged ventricles. A similar phenotype was observed when Id4 was ectopically over-expressed through Cre-mediated transgene activation in the developing brain.
Adult brains from wild type (left) and mutant Id4-deficient (top right) and Id4-NSE Cre transgenic mice (bottom right)
In vitro analysis of neurosphere cultures revealed that proliferation of Id4-deficient NPCs was impaired and that BMP2-mediated astrocyte differentiation was accelerated in the absence of Id4.
EMBO J cover Vol 23 (21) 2004
Myelination in the central nervous system is a complex process requiring the integration of oligodendrocyte progenitor differentiation and the coordinate expression of myelin genes. Overexpression of Id4 in oligodendrocyte progenitors prevents differentiation and consequently decreases the endogenous expression of all myelin genes. Conversely, progenitors lacking Id4 display precocious differentiation both in vitro and in vivo, and this phenotype is partially compensated by increased apoptosis. Besides this role, Id4 also has the ability to decrease the activity of specific myelin promoters, since Id4 overexpression decreases the activity of luciferase reporter genes driven by the ceramide galactosyltransferase (CGT) or myelin basic protein (MBP) promoter, but not by a myelin proteolipid protein (PLP) promoter. Consistent with these results, the expression levels of MBP and CGT are greater in neonatal Id4 null mice when compared with wild-type siblings and correlate with the early detection of MBP immunoreactive myelinated fibers. In contrast, the levels of other myelin proteins, such as PLP and myelin associated glycoprotein (MAG) are decreased in the Id4 null mice. MAG expression is localized to the soma rather than the fibers of immunoreactive cells in the neonatal brain and compensated at later developmental stages. These data support the role of Id4 as oligodendrocyte differentiation inhibitor with the ability to differentially regulate the expression and subcellular distribution of myelin gene products.
Together, these data demonstrate a crucial role for Id4 in regulating neuronal and glial stem and progenitor cell expansion and timing of differentiation.
Id4-lacZ expression (blue) in the adult cerebellum
Id4 promotes osteoblast differentiation: Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Pparc2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Pparc2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.
Id4 acts as a tumour suppressor: ID4 promoter methylation has been reported in acute myeloid leukemia and chronic lymphocytic leukemia (CLL), although the expression, function and clinical relevance of this gene have not been characterized in either disease. We demonstrate that the promoter of ID4 is consistently methylated to varying degrees in CLL cells, and increased promoter methylation in a univariable analysis correlates with shortened patient survival. However, ID4 mRNA and protein expression is uniformly silenced in CLL cells irrespective of the degree of promoter methylation. Crossing of ID4+/- mice with Eμ-TCL1 mice triggers a more aggressive murine CLL as measured by lymphocyte count and inferior survival. Hemizygous loss of ID4 in non-transformed TCL1-positive B cells enhances cell proliferation triggered by CpG oligonucleotides and decreases sensitivity to dexamethasone-mediated apoptosis. Collectively, this study confirms the importance of the silencing of ID4 in murine and human CLL pathogenesis.
ID4 regulates mammary gland development by suppressing p38MAPK activity: The ID family of helix-loop-helix proteins regulates cell proliferation and differentiation in many different developmental pathways, but the functions of ID4 in mammary development are unknown. We report that mouse Id4 is expressed in cap cells, basal cells and in a subset of luminal epithelial cells, and that its targeted deletion impairs ductal expansion and branching morphogenesis as well as cell proliferation induced by estrogen and/or progesterone. We discover that p38MAPK is activated in Id4-null mammary cells. p38MAPK is also activated following siRNA-mediated Id4 knockdown in transformed mammary cells. This p38MAPK activation is required for the reduced proliferation and increased apoptosis in Id4-ablated mammary glands. Therefore, ID4 promotes mammary gland development by suppressing p38MAPK activity.
Dong J., Huang S., Caikovski M., Ji S., McGrath A., Custorio M.G., Creighton C.J., Maliakkal P., Bogoslovskaia E., Du Z., Zhang X., Lewis M.T., Sablitzky F., Brisken C. & Li Y. (2011) ID4 regulates mammary gland development by suppressing p38MAPK activity. Development 138: 5247-5256
CHEN, S.-S., CLAUS, R., LUCAS, D.M., YU, L., QIAN, J., RUPPERT, A.S., WEST, D.A., WILLIAMS, K.E., JOHNSON, A.J., SABLITZKY, F., PLASS, C. AND BYRD, J.C., 2010. Silencing of the inhibitor of DNA binding protein 4 (ID4) contributes to the pathogenesis of mouse and human CLL. Blood 117(3), 862-871
TOKUZAWA, Y., YAGI, K., YAMASHITA, Y., NAKACHI, Y., NIKAIDO, I., BONO, H., NINOMIYA, Y., KANESAKI-YATSUKA, Y., AKITA, M., MOTEGI, H., WAKANA, S., NODA, T., SABLITZKY, F., ARAI, S., KUROKAWA, R., FUKUDA, T., KATAGIRI, T., SCHöNBACH, C., SUDA, T., MIZUNO, Y. AND OKAZAKI, Y., 2010. Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation. PLoS Genetics 6(7), e1001019.
Sablitzky, F. Id4. AfCS-Nature Molecule Pages [online], Nature Publishing Group, London. (2006) Available at: <http://dx.doi.org/10.1038/mp.a001159.01>
Marin-Husstege, M., He, Y., Li, J., Kondo, T., Sablitzky, F. and Casaccia-Bonnefil, P. Multiple roles of id4 in developmental myelination: predicted outcomes and unexpected findings. Glia 54, 285-296 (2006)
Bedford, L., Walker, R., Kondo, T., van Cruechten, I., King, R. E. and Sablitzky, F. Id4 is required for the correct timing of neural differentiation. Developmental Biology 280, 386-395 (2005)
Norton, J. D., Deed, R. W., Craggs, G. and Sablitzky, F. Id helix-loop-helix proteins in cell growth and differentiation. Trends Cell Biol. 8, 58-65 (1998)
Sablitzky, F., Moore, A., Bromley, M., Deed, R. W., Joshua S. Newton, J. S. and Norton, J. D. Stage- and subcellular-specific expression of Id proteins in male germ and sertoli cells implicates distinctive regulatory roles for Id proteins during meiosis, spermatogenesis and sertoli cell function. Cell Growth Differ . 9, 1015-1024 (1998)
van Crüchten, I., Cinato, E., Fox, M., King, E. R., Newton, J. S., Riechmann, V. and Sablitzky, F. Structure, chromosomal localisation and expression of the murine dominant negative helix-loop-helix Id4 gene. Biochim Biophys Acta. 1443, 55-64 (1998)
Riechmann, V. and Sablitzky, F. Mutually exclusive expression of two dominant-negative helix-loop-helix (dnHLH) Genes, Id4 and Id3, in the developing nervous system of the mouse suggests distinct regulatory roles of theses dnHLH proteins during cellular proliferation and differentiation of the nervous system. Cell Growth Differ. 6, 837-843 (1995)
Riechmann, V. van Crüchten, I. and Sablitzky, F. The expression pattern of Id4, a novel dominant negative helix-loop-helix protein, is distinct from Id1, Id2 and Id3. Nucl. Acids Res. 22, 749-755 (1994)
Lyl-1 function in haematopoietic stem cell fate
Haematopoietic stem cells (HSCs) arise self-renew, or give rise to all hematopoietic lineages through the effects of transcription factors activated by signaling cascades. Lyl-1 encodes a transcription factor containing a basic helix-hoop-helix (bHLH) motif closely related to scl/tal-1, which controls numerous decisions in embryonic and adult haematopoiesis. During ontogeny, scl/tal-1is required for haematopoietic cell generation, both in the yolk sac, where erythro-myeloid cells are first produced, then in the intra-embryonic compartment, where hematopoietic stem cells independently arise. Similar to scl/tal-1, lyl-1 mRNA expression occurs in the developing cardiovascular and hematopoietic systems.
Lyl-1-lacZ expression during mouse embryogenesis
In lyl-1-lacZ knock-in heterozygous and homozygous embryos, lyl-1/lacZ expression completely correlates with lyl-1 mRNA expression in the intra-embryonic compartment and is present: (i) in the developing hematopoietic system, precisely where hematopoietic stem cells emerge, and thereafter in the fetal liver, (ii) in the developing vascular system, and (iii) in the endocardium. In contrast, whereas lyl-1 mRNA is expressed in yolk sac-derived endothelial and hematopoietic cells, lyl-1/lacZ expression is either absent or very weak in these cell types, thus differing from scl/tal-1, which is highly expressed there at both mRNA and protein levels.
Lyl-1 null mice are viable and display normal blood cell counts, except for a reduced number of B cells resulting from a partial block after the pro-B stage. Nevertheless, the deletion of lyl-1 results in a diminution in the frequency of immature progenitors (LSK), and LSK-side population (LSK-SP) and in S12 colony forming unit (CFU-S12) and long-term culture-initiating cell (LTC-IC) content in embryonic day 14 fetal liver (E14 FL) and adult bone marrow (BM). More important, lyl-1-deficient E14 FL cells and BM are severely impaired in their competitive reconstituting abilities, especially with respect to B and T lineage reconstitution. Thus, ablation of lyl-1 quantitatively and functionally affects HSCs, a cell population that transcribes lyl-1 more actively than their differentiated progenies. Our results demonstrate for the first time that lyl-1 functions are important for HSC properties and B-cell differentiation and that they are largely distinct from scl/tal-1 functions.
Moreover, ectopic expression of lyl1 did not rescue haematopoietic differentiation in scl/tal-1-deficient ES cells thus providing a molecular explanation for the vastly different phenotypes of scl/tal-1- and lyl1-deficient mouse embryos.
Adult Hematopoietic Stem and Progenitor Cells Require Either Lyl1 or Scl for Survival: Single-knockout mice have not revealed an essential function for Scl or Lyl1 in adult hematopoietic stem cells (HSCs). To determine if maintenance of HSCs in single-knockout mice is due to functional redundancy, we generated Lyl1; Scl-conditional double- knockout mice. We revealed a striking genetic interaction between the two genes, with a clear dose dependence for the presence of Scl or Lyl1 alleles for HSC function. Bone marrow repopulation assays and analyses demonstrated rapid loss of hematopoietic progenitors due to apoptosis. The function of HSCs could be rescued by a single allele of Lyl1 but not Scl. These results show that expression of at least one of these factors is essential for maintenance of adult HSC function.
LYL activity is required for the maturation of newly formed blood vessels in adulthood: We revealed a role for Lyl as a major regulator of adult neovascularization. When compared to their wild type or heterozygous littermates, tumors implanted into Lyl-deficient mice grew significantly faster, although they showed similar angiogenic response measured by global CD31 staining of blood vessels. On the other hand, tumor blood vessels from Lyl-deficient mice displayed an immature phenotype, as demonstrated by diameter enlargement, reduced pericyte coverage, increased permeability and endothelial-specific Tal-1 upregulation. This phenomenon was not restricted to tumors as blood vessels forming in Matrigel implanted in Lyl-deficient mice also showed excessive dilation. Notably though, LYL was not required for early morphogenetic events as demonstrated by siRNA-mediated LYL depletion in HUVECs, but was a key element in later events directing the expression of molecules important for the assembly and stabilization of endothelial junctions. Thus, LYL is dispensable to initiate angiogenesis but essential to control blood vessel maturation.
LYL-1 deficiency induces a stress erythropoiesis
LYL-1 is a transcription factor containing a basic helix-loop-helix motif closely related to SCL/TAL-1, a regulator of erythroid differentiation. Because LYL-1 is expressed in erythroid cell populations, we addressed its role in erythropoiesis using knockin mice. MaterialsandMethods. ErythropoiesisofLYL-1 -/- micewasstudiedbyprogenitorassays,flow cytometry, reconstitution assays, and functional tests. Expression of LYL-1, SCL, and GATA-1 was assessed at messenger RNA level by quantitative reverse transcription polymerase chain reaction. LYL-1 -/- mice displayed decreased erythropoiesis with a partial arrest in differenti- ation, and enhanced apoptosis associated with decreased Bcl-xL expression in the bone marrow (BM). In addition, LYL-1 -/- BM cells were severely impaired in their abilities to reconstitute the erythroid lineage in competitive assays, suggesting a cell autonomous abnor- mality of erythropoiesis. In parallel, erythroid progenitor and precursor cells were signifi- cantly increased in the spleen of LYL-1 -/- mice. Expression of LYL-1 was differentially regulated during maturation of erythroblasts and strikingly different between spleen- and BM-derived erythroblasts. Expression of LYL-1 decreased during erythroid differentiation in the spleen whereas it increased in the BM to reach the same level in mature erythroblasts as in the soleen. Loss of Lyl-1 expression was accompanied with an increase of SCL/TAL-1 and GATA-1 transcripts in spleen but not in BM-derived erythroblasts. Furthermore, phenylhydrazine-induced stress erythropoiesis was elevated in LYL-1 -/- mice and mutant BM and spleen erythroid progenitors were hypersensitive to erythropoietin. Taken together, these results suggest that LYL-1 plays a definite role in erythro- poiesis, albeit with different effects in BM specifically regulating basal erythropoiesis, and spleen, controlling stress-induced erythropoiesis.
CAPRON C, LACOUT C, LÉCLUSE Y, WAGNER-BALLON O, KAUSHIK A.L, CRAMER-BORDÉ E, SABLITZKY F and DUMÉNIL D. AND VAINCHENKER W., 2011. (External) LYL-1 deficiency induces a stress erythropoiesis. Experimental Hematology 39(6), 629-642
PIROT, N., DELEUZE, V., EL-HAJJ, R., DOHET, C., SABLITZKY, F., COUTTET, P., MATHIEU, D. AND PINET, V., 2010. LYL activity is required for the maturation of newly formed blood vessels in adulthood. Blood 115(25), 5270-5279.
SOUROULLAS, G.P., SALMON, J.M., SABLITZKY, F., CURTIS, D.J. AND GOODELL, M.A., 2009. Adult Hematopoietic Stem and Progenitor Cells Require Either Lyl1 or Scl for Survival. Cell Stem Cell 4(2), 180-186.
Giroux, S., Kaushik, A.-L., Capron, C., Jalil, A., Kelaidi, C., Sablitzky, F., Duménil, D., Albagli, O., Godin, I. Lyl-1 and tal-1/scl, two genes encoding closely related bHLH transcription factors, display highly overlapping expression patterns during cardiovascular and hematopoietic ontogeny. Gene Expression Patterns 7, 215-226 (2007)
Chan, W.Y.I., Follows, G. A., Lacaud, G., Pimanda, J. E., Landry, J.-R., Kinston, S., Knezevic, K., Piltz, S., Donaldson, I. J., Gambardella, L., Sablitzky, F., Green, A. R., Kouskoff, V. and Göttgens, B. The paralogous haemopoietic regulators Lyl1 and SCL are co-regulated by Ets and GATA factors yet Lyl1 cannot rescue the early SCL-/- phenotype. Blood 109(5), 1908-1916. (2007)
Capron, C., Lécluse, Y., Koushik, A. L., Foudi, A., Lacout, C., Sekkai, D., Godin, I., Albagli, O., Poullion, I., Svinartchouk, F., Schanze, E., Vainchenker, W., Sablitzky, F., Bennaceur-Griscelli, A. and Duménil, D. The SCL relative LYL-1 is required for fetal and adult hematopoietic stem cell functions and B-cell differentiation. Blood 107, 4678-4686 (2006).
DEF6, a novel PH-DH-like domain protein, is an up-stream activator of the Rho GTPases Rac1, Cdc42 and RhoA.
Utilising a gene trap approach, we isolated genes (def-1 to -8) which were differentially expressed in the haematopoietic system. Def6 was down-regulated upon differentiation of FDCP-mixA4 progenitor cells into myeloid as well as erythroid lineages. By Northern blot analysis, high expression of the human DEF6 gene was observed in lymph node, thymus and peripheral leukocytes. In mice, def6 expression was highest in spleen.
Sequence analysis of DEF6 revealed a predicted protein (ac no. NP_071330) of about 74 kDa with a modular organisation of four domains very similar to SWAP-70, that was originally identified as part of a B cell specific DNA recombination complex which preferentially recombines, or 'swaps', sequences derived from immunoglobulin heavy chain switch regions in vitro (2). The N-terminal end of DEF6 contains two potential EF-hands showing 63% and 68% similarity with an EF-hand calcium-binding domain consensus sequence. A region between amino acids 73 to 216 is a strongly conserved in DEF6 and SWAP-70 and we refer to this novel domain as DSH (DEF6-SWAP-70 homology) domain. A central PH domain (amino acids 217-312) is followed by an amino acid sequence that shows similarities to a DH domain. However, like SWAP-70, the similarities to the DH domain are rather limited and we therefore refer to this region as DH-like (DHL) domain.
InNIH-3T3cells, stimulationofthePI3-kinasesignallingpathwaywitheitherH2O2orPDGFresultedinthetranslocationofanover-expressedDEF6-GFPfusionproteintothecellmembraneandinducedtheformationoffilopodiaandlamellipodia. Incontrasttofull-lengthDEF6, expressionoftheDH-likedomainasaGFPfusionproteinpotentlyinducedactinpolymerisation, includingstressfibreformationinCOS-7cells, intheabsenceofPI3kinasesignalling, indicatingthatitwasconstitutivelyactive. TheGTP-loadingofCdc42wasstronglyenhancedinNIH3T3cellsexpressingtheDH-likedomainwhilefilopodiaformation, membranerufflingandstressfibreformationcouldbeinhibitedbytheco-expressionoftheDH-likedomainwithdominantnegativemutantsofeitherN17Rac1, N17Cdc42orN19RhoA, respectively.
This indicated that DEF6 acts up-stream of the Rho GTPases resulting in the activation of the Cdc42, Rac1 and RhoA signalling pathways. In vitro , DEF6 specifically interacted with Rac1, Rac2, Cdc42 and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerisation and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity and movement.
GFP-DEF6 expression in COS-7 cells stained with phalloidin (red) and DAPI (blue)
Def6 Is Required for Convergent Extension Movements during Zebrafish Gastrulation Downstream of Wnt5b Signaling During gastrulation, convergent extension (CE) cell movements are regulated through the non-canonical Wnt signaling pathway. Wnt signaling results in downstream activation of Rho GTPases that in turn regulate actin cytoskeleton rearrangements essential for co-ordinated CE cell movement. Rho GTPases are bi-molecular switches that are inactive in their GDP-bound stage but can be activated to bind GTP through guanine nucleotide exchange factors (GEFs). Here we show that def6, a novel GEF, regulates CE cell movement during zebrafish gastrulation. Def6 morphants exhibit broadened and shortened body axis with normal cell fate specification, reminiscent of the zebrafish mutants silberblick and pipetail that lack Wnt11 or Wnt5b, respectively. Indeed, def6 morphants phenocopy Wnt5b mutants and ectopic overexpression of def6 essentially rescues Wnt5b morphants, indicating a novel role for def6 as a central GEF downstream of Wnt5b signaling. In addition, by knocking down both def6 and Wnt11, we show that def6 synergises with the Wnt11 signaling pathway.
Def6 expression (blue) is ubiquitous during early development (A-F) but becomes restricted to the developing nervous system (K, K',L), somite boundaries (L'), otic vesicle (L), pectorial and medial fins (M) and neuromasts of the anterior and posterior lateral lines as well as pharyngeal arches (M, M'). Control hybridisation with the corresponding sense probe performed in parallel did not give rise to any detectable signal (examples shown in I, J N). A and A' etc. depicts the same embryo from different view points.
Goudevenou, K., Martin, P., Yeh, Y-J, Jones, P. and Sablitzky, F. Def6 is required for convergent extension movement during zebrafish gastrulation downstream of Wnt5b. Plos ONE 6(10): e26548 (2011)
Mavrakis, K. J., McKinlay, K. J., Jones, P. and Sablitzky, F. DEF6, a novel PH-DH-like domain protein, is an up-stream activator of the Rho GTPases Rac1, Cdc42 and RhoA. Exp. Cell Res. 294, 335-344 (2004)
Hotfilder, M., Baxendale, S., Cross, M. A. and Sablitzky, F. Def-2, -3, -6 and -8, novel mouse genes differentially expressed in the haematopoietic system. Brit J Haematology 106, 335-344 (1999)
Def-3/rbm6 defines a novel family of RNA-binding proteins
Def-3/rbm6, a novel RNA-binding protein was isolated as a differentially expressed gene during myelopoiesis and in parallel by positional cloning from the SCLC homozygous deletion region on human chromosome 3p21.3(1). The cDNA sequence of def-3 (also referred to as g16, NY-LU-12), predicted a hydrophilic nuclear protein of 127kDa, containing a unique combination of functional domains implicated in both RNA-binding and protein-protein interactions. Two RNA recognition motifs (RRMs) are located centrally and surround a C4 type Zn-finger. The C-terminal end contains a second, C2H2, Zn-finger domain as well a glycine-rich region. A database search identified related proteins in human (LUCA15 or RBM5), rat (S1-1), Drosophila, C. elegans and S. pombe. Although the RRMs of def-3/rbm6 and LUCA15 exhibit homology to those found in Sex-lethal, elav and Hu proteins, in vitro experiments indicated that def-3/rbm6 and LUCA 15 bind distinct RNA targets. In contrast to elav and Hu proteins which interact with AU rich target sequences, recombinant def-3/rbm6 and/or LUCA15 comprising the RRMs and the central C4 Zn-finger bound specifically poly (G) RNA homopolymers. Specific binding of poly (G) RNA was also observed using the full-length def-3/rbm6 protein. Whereas the N-terminal end of def-3/rbm6 did not bind any RNA targets in vitro, the glycine-rich C-terminal region as well as the C2H2 Zn-finger domain on its own also bound specifically poly (G) RNA homopolymers(2).
By Northern blot analysis, def-3/rbm6 expression was ubiquitous during mouse embryogenesis and in adult organs, while in human haematopoietic tissues def-3/rbm6 showed differential expression and alternative splicing. However, we have recently shown that the mouse genome contains two def-3/rbm6 pseudogenes in addition to the functional def-3/rbm6 locus. Whereas both pseudogenes map on chromosome #1, the def-3/rbm6 gene maps on chromosome #9 F1-F2, a region of conserved synteny with the SCLC deletion region on human chromosome 3p21.3.
Sub-nuclear targeting of the RNA Binding Motif protein def-3/rbm6 to splicing speckles and nascent transcripts: Using antibodies raised against mouse def-3/rbm6 to immmunostain mammalian cell lines we found that the endogenous protein was both distributed diffusely in the nucleus and concentrated in a small number of nuclear foci that corresponded to splicing speckles/interchromatin granule clusters (IGCs). Tagged def-3/rbm6 was also targeted to IGCs, although it accumulated in large bodies confined to the IGC periphery. The basis of this distribution pattern was suggested by the targeting of tagged def-3/rbm6 in the giant nuclei (or germinal vesicles, GVs) of Xenopus oocytes. In spread preparations of GV contents def-3/rbm6 was localised both to lampbrush chromosomes and to the surface of many oocyte IGCs, where it was confined to up to 50 discrete patches. Each patch of def-3/rbm6 labelling corresponded to a bead-like structure of 0.5-1 μm diameter that assembled de novo on the IGC surface. Assembly of these novel structures depended on the repetitive N-terminal region of def-3/rbm6, which acts as a multimerization domain. Without this domain def-3/rbm6 was no longer excluded from the IGC interior but accumulated homogeneously within it. Assembly of IGC-surface structures in mammalian cell lines also depended on the oligomerization domain of def-3/rbm6. Oligomerization of def-3/rbm6 also had morphological effects on its other major target in GVs, namely the arrays of nascent transcripts visible in lampbrush chromosome transcription units. The presence of oligomerized def-3/rbm6 on many lampbrush loops caused them to appear as dense structures with a spiral morphology that appeared quite unlike normal, extended loops. This distribution pattern suggests a new role for def-3/rbm6 in the co-transcriptional packaging or processing of most nascent transcripts.
HEATH, E., SABLITZKY, F. AND MORGAN, G.T., 2010. Subnuclear targeting of the RNA-binding motif protein RBM6 to splicing speckles and nascent transcripts. Chromosome Research 18(8), 851-872
Drabkin, H. A., West, J. D., Hotfilder, M., Heng, Y. M., Erickson, P., Calvo, R., Dalmau, J., Gemmill, R. M. and Sablitzky, F. Def-3 (g16/NY-LU-12), an RNA binding protein from the 3p21.3 homozygous deletion region in SCLC. Oncogene 18, 2589-2597 (1999)
Heng, Y. M., Fox, M. and Sablitzky, F. Assignment of the murine def-3 (Rbm6) gene to chromosome 9 F1-F2 and its pseudogenes Rbm6-ps1 and Rbm6-ps2 to chromosome 1 by in situ hybridisation. Cytogenet and Cell Genet. 89, 238-239 (2000).
Id2 protein function in spermatogenesis
We discovered that Id proteins displayed a unique temporal and spatial expression pattern during spermatogenesis. In addition, each Id protein displayed a distinctive, dynamic pattern of subcellular distribution. The observed expression of Id proteins in post-proliferative Sertoli cells and spermatids and during specific stages of meiosis implies novel functional roles for Id proteins during meiotic cell division as well as sperm maturation. To elucidate the role of Id2 protein in spermatogenesis we have analyse Id2-deficient mice (in collaboration with Y. Yokota).
Sablitzky, F., Moore, A., Bromley, M., Deed, R. W., Joshua S. Newton, J. S. and Norton, J. D. Stage- and subcellular-specific expression of Id proteins in male germ and sertoli cells implicates distinctive regulatory roles for Id proteins during meiosis, spermatogenesis and sertoli cell function. Cell Growth Differ. 9, 1015-1024 (1998)
Last updated 25.11.2011
University of Nottingham University Park Nottingham, NG7 2RD
telephone: +44 (0)115 9513300 (Undergraduate Enquiries) +44 (0)115 8230311 (Postgraduate Enquiries) fax: +44 (0)115 8230338 email: biology@nottingham.ac.uk
Change Text Only Settings
Graphic version of this page