Andre Van Wijnen, Ph.D.

Academic Role: Associate Professor

Faculty Appointment(s) In:
   Cell Biology

Gene regulation/cell cycle/skeletal development/nuclear architecture

1. Runt-related transcription factors.
   

   Runt-related transcription factors are an exciting class of proteins that contain a highly conserved DNA binding module, the runt domain, which has been evolutionarily conserved in most if not all animal species. These factors are key regulators of hematopoiesis (RUNX1/AML1), osteogenesis (RUNX2/CBFA1) and gastro-intestinal development (RUNX3/PEBP2aC). RUNX proteins are components of gene regulatory master-switches that regulate transcription of groups of genes required for the formation of hematopoietic cells, mature osteoblasts or other specialized tissues. Defects in the RUNX2/AML1 gene [e.g., t(8;21) chromosomal translocation] cause leukemia. Mutation of the RUNX2/CBFA1 gene causes cranio-facial abnormalities, supernumerary teeth and/or absence of clavicles [e.g., Cleido-Cranial Dysplasia]. Genetic loss of RUNX3/PEBP2aC contributes to gastric cancer. One major goal of our studies is to map the gene regulatory pathways by which RUNX proteins determine lineage commitment, proliferative expansion and maturation of hematopoietic and osseous cells.

   One focus of our studies is analysis of the function of RUNX2 proteins in regulating osteoblast cell growth and differentiation. RUNX2 may regulate genes that are required for the expansion of osteo-progenitor cells, which has immediate implications for skeletal development, wound healing and tissue-engineering. Current experiments are focused on the role of RUNX2 in regulating cell cycle progression, and the mechanisms involved in cell growth control of RUNX2 activity. To study RUNX2 function we use both anti-sense interference strategies and stable cell lines that conditionally express RUNX2 proteins. In addition, we are interested in the identification and functional characterization of target genes of RUNX2 using gene profiling techniques (e.g., Affymetrix gene micro-arrays).

   Another focus of our studies is the transcriptional and post-transcriptional regulation of RUNX2 activity in response to osteogenic signals. The activity of RUNX2 is regulated by physiological mediators of skeletal development and bone cell differentiation (e.g., BMP2, TGFbeta, Vitamin D, glucocorticoids, FGF). Our experiments are aimed at understanding how regulatory signals are integrated at the RUNX2 promoter, or its mRNA or protein, to control the biological activity of RUNX2.

2. Cell cycle control.
   

   One of the principal advances we have made is the development of the concept that transcriptional control of the histone H4 gene at the G1/S phase transition occurs independent of the E2F class of transcription factors. This concept is based on our determination of the molecular identity of the three critical DNA binding proteins HiNF-M, -D and -P that modulate histone gene transcription through a phylogenetically conserved promoter element we refer to as the Site II cell cycle domain. None of these HiNFs are related to E2F, and hence activation of histone gene transcription factors defines a novel cell cycle transition at the onset of S phase (which we refer to as the "S-point") that is temporally and functionally distinct from the Restriction (R) point in late G1. The biomedical relevance of our findings is indicated by early evidence from our laboratory suggesting that deregulation of HiNFs, and hence S point control, may be deregulated in cancer cells.

   We propose that the putative S point occurs subsequent to the R-point and reflects the commitment of the cell to initiate DNA replication and to express its multiplicity of histone genes. The onset of DNA synthesis requires prior activation of enzymes involved in nucleotide metabolism (several of which are encoded by E2F dependent genes activated at the R-point) to ensure appropriate cellular pools of nucleotide-triphosphates, while histone proteins are required subsequently for the packaging of nascent DNA. Our current data suggests that a distinct set of regulatory decisions is being made at the S point to ensure that enhancement of histone gene transcription is coupled with the onset of DNA synthesis.

   The R-point is defined as the cell cycle transition stage when cell cycle progression becomes independent of growth factor stimulation. The key regulatory event at the R-point is activation of the CDK2/cyclin E kinase complex, which has at least two consequences for functionally distinct events that control histone H4 gene transcription at the S-point. First, activation of CDK2/cyclin E results in the hyperphosphorylation of pRB, its release from the E2F protein, and subsequent activation of E2F responsive genes, including those involved in nucleotide metabolism, by "free" E2F in late G1. The R-point related and CDK2/cyclin E dependent hyperphosphorylation of pRB precedes the upregulation of HiNF-D, which contains CDP-cut/pRB/CDK1/cyclin A, and its interaction with the Site II cell cycle element in S phase. Second, recently it was shown that CDK2/cyclin E activates NPAT. Data from our laboratory have established that NPAT functions as a co-activator for HiNF-P in control of H4 gene transcription at the S point. The mechanistic linkages between pRB and CDP-cut, as well as NPAT and HiNF-P reflect the unique cell cycle regulatory mechanisms that are operative at the S-point, and are consistent with functional and temporal distinctions in the E2F-dependent R-point and E2F-independent S-point during the cell cycle. Based on our findings, we are now experimentally addressing the hypothesis that the molecular functions of Site II transcription factors in histone gene transcription at the S point are components of a broader biological mechanism that controls cell growth and differentiation during development in vivo.

3. Subnuclear targeting of gene regulatory factors.
   

   One main goal of our studies is to understand control of gene expression in situ within the dynamic environment of the cell nucleus. Our program focuses on establishing the developmental function and mechanisms by which gene regulatory factors are targeted to distinct subnuclear foci to integrate developmental signals into the molecular instructions required for controlling the expression of phenotypic genes that are packaged as chromatin. Our projects focus on establishing (i) the physiological requirement of subnuclear targeting of transcription factors for normal development in vivo, (ii) the role of intranuclear trafficking of gene regulatory proteins in cellular differentiation and transcription of tissue-specific genes, (iii) the dynamics of targeting regulatory proteins to subnuclear foci in live cells, and (iv) the molecular mechanisms that mediate intranuclear trafficking to foci containing gene regulators to support tissue-specific transcriptional control.

Figures

Bone Cell Growth Regulation

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Cell Division

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In situ integration of cell signaling at Runx foci

Representative Publications

van der Meijden CM, Lapointe DS, Luong MX, Peric-Hupkes D, Cho B, Stein JL, Van Wijnen AJ, Stein GS. Gene Profiling of Cell Cycle Progression through S-Phase Reveals Sequential Expression of Genes Required for DNA Replication and Nucleosome Assembly. Cancer Res. 2002 Jun 1; 62(11): 3233-43.

Zaidi SK, Sullivan AJ, Van Wijnen AJ, Stein JL, Stein GS, Lian JB. Integration of Runx and Smad regulatory signals at transcriptionally active subnuclear sites. Proc Natl Acad Sci U S A. 2002 Jun 11; 99(12): 8048-53.

Lengner CJ, Drissi H, Choi JY, van Wijnen AJ, Stein JL, Stein GS, Lian JB. Activation of the bone-related Runx2/Cbfa1 promoter in mesenchymal condensations and developing chondrocytes of the axial skeleton. Mech Dev. 2002 Jun; 114(1-2): 167-70.

Xie RL, van Wijnen AJ, van der Meijden CM, Stein JL, Stein GS. Forced expression of the interferon regulatory factor 2 oncoprotein causes polyploidy and cell death in FDC-P1 myeloid hematopoietic progenitor cells. Cancer Res. 2002 May 1; 62(9): 2510-5.

Drissi H, Pouliot A, Koolloos C, Stein JL, Lian JB, Stein GS, van Wijnen AJ. 1,25-(OH)2-vitamin D3 suppresses the bone-related Runx2/Cbfa1 gene promoter. Exp Cell Res. 2002 Apr 1; 274(2): 323-33.

Shen J, Montecino M, Lian JB, Stein GS, Van Wijnen AJ, Stein JL. Histone Acetylation in Vivo at the Osteocalcin Locus Is Functionally Linked to Vitamin D-dependent, Bone Tissue-specific Transcription. J Biol Chem. 2002 Jun 7; 277(23): 20284-92.

Luong MX, van der Meijden CM, Xing D, Hesselton R, Monuki ES, Jones SN, Lian JB, Stein JL, Stein GS, Neufeld EJ, van Wijnen AJ. Genetic ablation of the CDP/Cux protein C terminus results in hair cycle defects and reduced male fertility. Mol Cell Biol. 2002 Mar; 22(5): 1424-37.

Mitra P, Vaughan PS, Stein JL, Stein GS, van Wijnen AJ. Purification and functional analysis of a novel leucine-zipper/nucleotide-fold protein, BZAP45, stimulating cell cycle regulated histone H4 gene transcription. Biochemistry. 2001 Sep 4; 40(35): 10693-9.

Choi JY, Pratap J, Javed A, Zaidi SK, Xing L, Balint E, Dalamangas S, Boyce B, van Wijnen AJ, Lian JB, Stein JL, Jones SN, Stein GS.  Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development. Proc Natl Acad Sci U S A. 2001 Jul 17; 98(15): 8650-5.

Staal A, Enserink JM, Stein JL, Stein GS, van Wijnen AJ. Molecular characterization of celtix-1, a bromodomain protein interacting with the transcription factor interferon regulatory factor 2. J Cell Physiol. 2000 Nov; 185(2): 269-79.

Drissi H, Luc Q, Shakoori R, Chuva De Sousa Lopes S, Choi JY, Terry A, Hu M, Jones S, Neil JC, Lian JB, Stein JL, Van Wijnen AJ, Stein GS. Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene. J Cell Physiol. 2000 Sep; 184(3): 341-50.

Javed A, Guo B, Hiebert S, Choi JY, Green J, Zhao SC, Osborne MA, Stifani S, Stein JL, Lian JB, van Wijnen AJ, Stein GS. Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX/ CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription. J Cell Sci. 2000 Jun; 113 (Pt 12): 2221-31.

van Gurp MF, Pratap J, Luong M, Javed A, Hoffmann H, Giordano A, Stein JL, Neufeld EJ, Lian JB, Stein GS, van Wijnen AJ. The CCAAT displacement protein/cut homeodomain protein represses osteocalcin gene transcription and forms complexes with the retinoblastoma protein-related protein p107 and cyclin A. Cancer Res. 1999 Dec 1; 59(23): 5980-8.

van der Meijden CM, Vaughan PS, Staal A, Albig W, Doenecke D, Stein JL, Stein GS, van Wijnen AJ. Selective expression of specific histone H4 genes reflects distinctions in transcription factor interactions with divergent H4 promoter elements. Biochim Biophys Acta. 1998 Oct 23; 1442(1): 82-100.

Runt-related transcription factors.

Runt-related transcription factors are an exciting class of proteins that contain a highly conserved DNA binding module, the runt domain, which has been evolutionarily conserved in most if not all animal species. These factors are key regulators of hematopoiesis (RUNX1/AML1), osteogenesis (RUNX2/CBFA1) and gastro-intestinal development (RUNX3/PEBP2aC). RUNX proteins are components of gene regulatory master-switches that regulate transcription of groups of genes required for the formation of hematopoietic cells, mature osteoblasts or other specialized tissues. Defects in the RUNX2/AML1 gene [e.g., t(8;21) chromosomal translocation] cause leukemia. Mutation of the RUNX2/CBFA1 gene causes cranio-facial abnormalities, supernumerary teeth and/or absence of clavicles [e.g., Cleido-Cranial Dysplasia]. One major goal of our studies is to map the gene regulatory pathways by which RUNX proteins determine lineage commitment, proliferative expansion and maturation of hematopoietic and osseous cells.

Potential Rotation Projects

Project #1: Analysis of the function of RUNX2 proteins in regulating osteoblast cell growth and differentiation. RUNX2 may regulate genes that are required for the expansion of osteo-progenitor cells, which has immediate implications for skeletal development, wound healing and tissue-engineering. The student will perform experiments focused on the identification and functional characterization of target genes of RUNX2. This project will use gene profiling techniques (e.g., Affymetrix gene micro-arrays) and stable cell lines conditionally expressing RUNX2 proteins.

Project #2: Transcriptional and post-transcriptional regulation of RUNX2 activity in response to osteogenic signals. The activity of RUNX2 is regulated by physiological mediators of skeletal development and bone cell differentiation (e.g., BMP2, TGFbeta, Vitamin D, glucocorticoids, FGF). The student will perform experiments aimed at understanding how regulatory signals are integrated at the RUNX2 promoter, or its mRNA or protein, to control the biological activity of RUNX2. Techniques include ligation-mediated PCR of genomic protein/DNA interactions, transient transfections of bone cells, and/or metabolic labeling studies.

Office: S3-326
Phone: 508-856-5942
E-mail: Andre.VanWijnen@umassmed.edu
Keywords: Signal Transduction, Developmental Biology

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