Benoit de Crombrugghe - US grants

collagen; protein biosynthesis; genetic transcription; transcription factor; fibroblasts; extracellular matrix; hormone regulation /control mechanism; DNA binding protein; oncogenes; genetic enhancer element; cell differentiation; site directed mutagenesis; transforming growth factors; genetic promoter element; tissue /cell culture; laboratory mouse; laboratory rat; molecular cloning; chemical binding; genetically modified animals; transfection;

protein structure function; collagenase; model design /development; disease /disorder model; collagen disorder; genetically modified animals; autosomal dominant trait; osteoarthritis; genetic recombination; gene expression; genetic regulatory element; chondrodystrophy; posttranslational modifications; Ehlers Danlos syndrome; tissue /cell culture; polymerase chain reaction; laboratory mouse; site directed mutagenesis;

The purpose of this shared instrument is to provide access to the most effective and modern DNA sequencing methodology for a number of different laboratories at The University of Texas M.D. Anderson Cancer Center. Laboratories in eight different departments have requested to participate in this grant proposal. They represent a total in excess of 1.9 million dollars in NIH supported research. Determination of DNA sequences has become an essential method in a large number of Cell Biology and Molecular Biology projects. A growing number of laboratories at The University of Texas M.D. Anderson Cancer Center are utilizing manual DNA sequencing techniques as part of their efforts to characterize cDNA and genomic clones. These projects include clones for transcription factors, oncoproteins, proteins related to development, differentiation cancer, membrance receptors etc. Other projects study the function of gene regulatory segments and other biologically important molecules by site specific mutagenesis. Still other projects seek to define the mutations occuring in the retinoblastoma gene of patients with various forms of neoplasms or in the Wilms' tumor gene in patients with such tumors. The availability. of an automated DNA sequencer will greatly facilitate and improve the research of many NIH supported laboratories at this Institution. It is estimated that during the first year of operation of this instrument the requests will be in excess of 500,000 nucleotide sequences. We anticipate an increasing demand on this type of instrument by funded investigators and by new faculty members. Mr. Dave Toman, A Senior Research Assistant in the laboratory of the P.I., with extensive experience in manual DNA sequencing, will perform all DNA sequencing reaction and perform all operations with the instrument. An Internal Committee will be established, the composition of which will be broadly representative of the different users of the DNA sequencing facility. The Committee will decide about priorities and evaluate the quality of the DNA sequences, and review the fee structure. We anticipate that initially, a fee of about $30 per sequencing run will be charged to the users.

The genes for type I collagen are activated in a precise spatial and temporal pattern during embryonic development. This proposal seeks to identify and characterize the lineage specific transcriptional mechanisms which activate these genes in fibroblasts, osteoblasts and odontoblasts. One can postulate that the same genetic programs might also activate these genes in fibrotic diseases, maybe in response to similar cues from the extracellular environment. Previous results have established that defined segments of the 5' flanking sequences of the type I collagen genes confer tissue-specific expression to reporter genes in transgenic mice, that largely mimics expression of the endogenous genes. The following Specific Aims are proposed: (1) Determine by mutagenesis which precise sequences within a short reconstructed alpha2(I) collagen promoter are responsible for this tissue-specificity. This reconstructed promoter contains a segment between -40 and +54 and 5' to this sequence the -315 to -284 segment which is tandemly repeated. (2) Demonstrate in a in vitro transcription system that activation of a reconstructed alpha2(I) collagen promoter is mediated by the sequences which are needed for tissue-specificity in animals. (3) Identify and clone the transcription factor(s) which determine the cell specificity of a minimal alpha2(I) collagen promoter. Approaches to clone such cDNAs include: (a) a Southwestern method to identify and clone cell-specific DNA-binding proteins which bind to a alpha2(I) collagen promoter sequence, which is needed for cell specificity; (b) establishment of a mammalian expression system or a yeast expression system to clone cDNAs which activate a reconstructed alpha2(I) collagen promoter through the sequence that is needed for cell-specificity in transgenic mice (c) test whether the products of two homeobox genes, which show a pattern of expression in embryos that is similar to that of the type I collagen genes, can activate these genes. (4) Identify within the 350 bp promoter the cis-acting elements that are needed for activity in osteoblasts. (5) Identify additional far-upstream, or intragenic or 3' flanking cis-acting enhancer elements that confer high level expression to the alpha2(I) collagen gene. (6) Use transgenic mice which harbor various alpha2(I) collagen promoter constructions to determine at which time during bleomycin induced lung fibrosis, the fibroblasts acquire a more active alpha2(I) collagen promoter and which sequences in the alpha2(I) collagen promoter are needed for this bleomycin-induced activation.

Type 1 collagen is a major structural protein which is abundantly expressed in bones, tendons and skin and is present in lower concentrations in connective tissues of other organs. The genes for the two chains of type I collagen are coordinately expressed during embryonic development and also in most physiological and pathological situations in adult animals. The expression of these genes is likely to be the result of complex combinatorial control mechanisms involving both tissue-specific and ubiquitous transcription factors. The exaggerated synthesis of type I collagen in a number of fibrotic diseases, probably results from the abnormal activation of some of these transcription factors. Our laboratory has focused its efforts on a ubiquitous transcription factor, CBF, which is an important physiological activator of both the alpha1(I) and alpha2(I) collagen genes. CBF binds to a CCAAT motif in each promoter within 100 bp upstream of the start of transcription, as a heteromeric protein which needs three different subunits, CBF-A, CBF-B and CBF-C to bind to DNA. CBF-A forms a tight complex with CBF-C and this complex binds to CBF-B; the complex of three different polypeptides then interacts with DNA. Based on the hypothesis that CBF is a key transcriptional activator of the two type I collagen genes, the experimental approach outlined in this application focuses on an extensive molecular characterization of this protein in order to better understand the mechanisms whereby CBF activates these genes. These studies will examine how the subunits of CBF interact with each other, how they interact with a specific DNA sequence, and how they interact with other transcription factors in order to activate the type I collagen promoters. We believe that these studies will constitute an important step in the elucidation of the complex transcription controls of the type I collagen genes.

DESCRIPTION: (Adapted From Investigator's Abstract) This application is based on the hypothesis that the determination and differentiation of chondrocytes during embryonic development is controlled by a multistep developmental pathway in which specific transcription factors play a critical role. This application proposes to study two different steps in this pathway. The applicants have recently identified the DNA for a novel homeodomain containing protein, Cartl, which is selectively expressed in prechondrocytes and chondrocytes and have speculated, based on the tissue distribution of Cartl transcripts, that Cartl plays a role as a transcription factor in the pathway of chondrocytes determination and differentiation, probably at an early step. The first part of this proposal is to characterize the function of Cartl in intact mice and embryos by generating mice that are deficient in Cartl and to study in transgenic mice the transcriptional mechanisms which control the expression of the Cartl gene in prechondrocytes and chondrocytes. The second part of the application is based on the hypothesis that another chondrocyte-specific transcription factor controls the activation of the gene for proa1(II) collagen, a typical marker of terminal differentiation of mature chondrocytes. The investigators propose to define the minimal sequence in intron 1 of the COL2A1 gene which confers chondrocyte-restricted expression to a B-galactosidase reporter gene in transgenic mice and embryos, to characterize chondrocyte-specific proteins which bind to this minimal functionally active sequence and to clone their cDNAs. This application is a two- pronged approach to studying two different steps in the pathway of chondrocyte determination and differentiation.

A meeting will be organized in Bethesda, M.D., September 28-30, 1995 under the auspices of the New York Academy of Science which will focus on the genetics and developmental biology of cartilages. This is an emerging area of research to which a large group of scientists from different disciplines are making important contributions. Organizers of this meeting are Benoit de Crombrugghe, Francesco Ramirez, Bjorn Olsen and William Horton. Speakers will be human and mouse geneticists, developmental biologists, molecular biologists and biochemists. Our program includes women and junior scientists. The meeting will be subdivided in five sessions which strongly complement each other. In each session we have left one slot open to be filled at a later date by scientists who will report new results relevant to the theme of the conference which have not yet been published to date. A majority of the presentations will discuss the effects of mouse mutations in transcription factors, cytokines and extra cellular matrix proteins on the development of cartilages and the formation of the skeleton. Human mutations which affect the formation and role of cartilage will also be discussed. A poster session will be organized to present additional results and to further stimulate interactions among participants. We believe that this multi disciplinary meeting should greatly stimulate research on the molecular bases of cartilage development and cartilage function. The meeting should be very beneficial for an improved understanding of diseases of cartilages in humans.

This Program Project proposes a multidisciplinary approach to examine the function of several extracellular matrix (ECM) components of cartilage and bone by characterizing mice in which the genes for these components will have been disrupted by homologous recombination of embryonic stem cell. The program also proposes to use transgenic mice in order to study the mechanisms which control the tissue-specific expression of the genes for these components. Our goal is to capitalize on the strengths of the investigators, the complementary nature of the different projects and the powerful genetic approaches that are proposed to develop strongly interactive, collaborative projects. This program should help us gain considerable new knowledge about the mechanisms controlling the lineages and phenotypes of cells of skeletal tissues and about the function of individual cartilage and bone ECM components within larger multiprotein structures. These advances, which could not be effectively achieved by individual projects, should be very beneficial for our understanding of cartilage and bone diseases.

DESCRIPTION: The hypothesis behind this application is that type I collagen genes are activated during embryonic development in a discrete number of cell types by some of the same transcriptional mechanisms which also control the differentiated phenotypes of these cells. The objective of the proposal is to elucidate the mechanisms that control expression of these genes in fibroblasts and osteoblasts and, consequently, broader genetic programs in these cell types. Previous work has indicated that separate cis-acting elements in the type I collagen genes control expression of reporter genes in different type I collagen producing cells. One of these elements in the mouse proa1(I) gene controls osteoblast expression; another one is a potent far-upstream enhancer in the proa2(I) gene which controls expression mainly in fibroblasts. This application is divided into two parts. One proposes to delineate the minimal elements in the proa2(I) far-upstream enhancer which control expression of a reporter gene in fibroblastic cells of transgenic mice. It is expected that this minimal segment can then be used to identify fibroblast-specific or fibroblast-enriched DNA binding transcription factors which can activate the fibroblast elements in the proa2(I) gene. Subsequently, cDNAs for these DNA binding proteins will be cloned and characterized. The second part of the project will further define the DNA sequence in the proa1(I) gene critical for osteoblast expression in transgenic mice. It is expected that this sequence can be used to identify osteoblast-specific or osteoblast-enriched DNA binding proteins which control expression of the proa1(I) gene in osteoblasts. Also in this case, cDNAs for these polypeptides will then be cloned and characterized. Together, these two studies will enhance our understanding of the molecular determinants underlying the differentiation of mesenchymal cell precursors.

TGFbeta increases the levels of type I collagen in fibroblasts and at least part of this stimulation is mediated by transcriptional mechanisms. One of the hallmarks of fibrotic diseases including Systemic Sclerosis is an abnormal accumulation of type I collagen and other extracellular matrix (ECM) components in the skin and some internal organs, but the mechanisms of this abnormal accumulation are poorly understood. It has been postulated that excess TGFbeta might mediate the exaggerated accumulation of ECM components. A similar abnormal accumulation of type I collagen and other ECM components is found in the autosomal dominant Tight Skin mutant mice (tsk1) in which a partial in- frame duplication of the fibrillin 1 gene has been directly linked to the abnormal phenotype. Our laboratory has recently identified a potent far-upstream enhancer in the mouse pro alpha2(I) collagen gene which directs high levels of expression of reporter genes in transgenic mice specifically in fibroblasts of the skin and visceral organs. Since a spatial and temporal correlation exists between the presence of extracellular TGFbeta and the activation of type I collagen genes in these tissues during embryonic development, we postulate that the Colla2 upstream enhancer may contain elements that are responsive to TGFbeta. To further explore the mechanisms by which TGFbeta increases type I collagen synthesis and those leading to the abnormal accumulation of type I collagen in tsk1 mutant mice, we propose the following Specific Aims. (a) Delineate sequences in the Colla2 far-upstream enhancer that are responsive to TGFbeta in vivo. (b) Delineate sequences within the Colla2 far-upstream enhancer and/or proximal promoter which are responsive to signals that cause increased type I collagen synthesis in tsk1 mutant mice. (c) Generate and characterize transgenic mice in which the activity of the TGFbeta signaling pathway in fibroblasts is increased or decreased. (d) Examine the effects on the activity of the Colla2 upstream enhancer of dominant negative and constitutively active mutants of signaling components of the TGFbeta pathway in fibroblasts in culture. (f) Measure the levels of activity of the TGFbeta signaling pathway in fibroblasts of normal and tsk1 mice.

This application is based on the hypothesis that specific transcription factors play a critical role in the formation of specific parts of the mammalian skeleton and in the determination and differentiation of chondrocytes during embryonic development. This application is subdivided in two different parts. We have recently identified the DNA for a novel homeodomain containing protein, Cart1, which is selectively expressed in specific mesenchymal cells, in prechondrocytes and chondrocytes. The first part of this proposal is to examine the function of Cart1 in intact mice and embryos by an extensive characterization of the phenotype of mice that are deficient in Cart1 and to study in transgenic mice the transcriptional mechanisms which control the expression of the Cart1 gene in prechondrocytes and chondrocytes. Cart-1 deficient mice show a phenotype that is similar to a human acrania syndrome. The second part of this application is based on the hypothesis that other chondrocyte-specific transcription factors(s) control the activation of the gene for proalpha1(II) collagen, a typical marker of terminal differentiation of mature chondrocytes. We propose to define the minimal sequence in intron 1 of the COL2A1 gene which confers chondrocyte- restricted expression to a beta-galactosidase reporter gene in transgenic mice and embryos, to characterize chondrocyte-specific proteins which bind to this minimal functionally active sequence and to clone their cDNAs. This application proposes to study transcription factors that play a critical role in two different aspects of skeletogenesis.

The Macromolecular Analysis Facility consists of three separate but related entities: the DNA Sequencing Facility (DSF), which performs automated DNA sequencing, automated microsatellite analysis, and single- strand conformation polymorphism (SSCP) analysis; the Computational Analysis Facility (CAF), which helps investigators with computational analysis of macromolecular structures; and the Nucleic Acid Core Facility (NACF), a centralized laboratory for standardized DNA extraction from various sources and genotyping of transgenic mice. These resources prepared DNA from blood, tumor samples, and paraffin-embedded or frozen sections (in the NACF); determine DNA sequences and perform microsattelite and SSCP analyses (in the DSF). DNA sequences are analyzed by the CAF. The DSF provides help to a wide range of investigators in basic, clinical and population-based research involved in studies of growth control, growth inhibition, commitment to specific cell lineages, cell adhesion and mobility, metastasis, angiogenesis, and other fields, as well as supports studies on the mapping and cloning of new cancer susceptibility genes and screening for tumor and constitutional mutations in known cancer susceptibility genes and screening for tumor and constitutional mutations in known cancer susceptibility genes and screening for tumor and constitutional mutations in known cancer susceptibility genes and oncogenes. The DSF provides fast and reliable sequence information to investigators in an efficient and cost-effective manner. The number of DNA sequences determined by the DSF increased 400% from about 1,500 in 1992 to over 6,000 between July 1, 1996 and June 1, 1997, and the length of readable sequences increased from about 300-350 to more than 650 bases per sample. To generate the DS sequences, the DSF currently uses on ABI 373 DNA sequencer equipped with stretch configuration and one recently acquired ABI 377 DNA sequencer. The resource provides investigators with the polymerase chain reaction (PCR)-generated DNAs, to produce the most accurate DNA sequences and expertise in primer design and maintain rigorous quality control to ensure consistency of service. A substantial effort of the facility is dedicated to determining optimal conditions for DNA sequencings with new and improved fluorescent dyes and more efficient enzymes. The facility recently began to use the ABI 377 DNA sequencer for automated analysis of the sizes and amounts of PCR products. The analysis detects SSCPs in PCR products and allows genetic linkage analysis by determining the sizes and amounts of specific microsatellite-containing PCR products. The 377 ABI DNA sequencer is also used to perform microsatellite analysis to evaluate constitutional and tumor-specific genomic instability.

This Program Project proposes to examine the functional of critical regulatory molecules in the formation of cartilage and bone and the role of a specific class of extracellular matrix (ECM) proteins in the function of cartilage and bone and the role of a specific class of extracellular matrix (ECM) proteins in the function of cartilage and bone. The program will provide important and novel information about the roles of the transcription factors Sox9 and Cbfa2 in chondrogenesis, about the role of Bone Morphogenetic Proteins in Somite, Cartilage and Bone development and about the function of Small Leucine-Rich Proteoglycans and their interactions with other ECM components in cartilage and bone. A large portion of the application proposes to utilize gene-targeting and transgenic approaches to better understand the function of these proteins in intact mice and embryos. The strengths of the Program are based on the productivity and expertise of the participating investigators and their interactions during the period of the current P011, on the interactive nature of the individual projects, on the essential support provided by the cores, and on other complementary projects ongoing in the laboratories of the project leaders. The anticipated advances in the molecular and cellular biology of skeletal development and function could not be achieved by individual projects. This Program Project should provide a more rational basis for the understanding of both genetic and acquired diseases of cartilage and bone.

Chondrogenesis is a multistep process by which cartilages are formed. Cartilage formation begins with condensations of mesenchymal cells followed by their differentiation into chondrocytes. Our recent studies have identified Sox9 as the first master transcription factor that is required for differentiation of all chondrocytes. Indeed, homozygous Sox9-/- mutant cells are unable to differentiate into chondrocytes and to express a series of chondrocyte-specific marker genes such as Col2a1, Collla2, Col9a2 and Aggrecan. Heterozygous mutations in the SOX9 gene cause the human skeletal malformation syndrome Campomelic Dysplasia. This application is centered on the study of the function of Sox9 during chondrogenesis. It proposes to further characterize the cellular phenotype of Sox9-/- mutant mesenchymal like cells that are blocked from differentiation into chondrocytes and to isolate these cells from chimeric embryos for functional studies. It will also characterize the abnormal skeletal phenotype of heterozygous Sox9+/- mice that present an almost perfect phenocopy of the human disease Campomelic Dysplasia. It will examine the role of cyclic AMP on the activity of Sox9 and that of the FGF pathway on the level of expression of Sox9 during chondrogenesis in intact mice. Signaling pathways controlled by these molecules are known to be active in growth plate cartilages. In addition, this application proposes to identify other transcription factors that interact with Sox9 and with two other Sox family members that are active in chondrocytes.

The overall objective of this application is to establish new mouse models of fibrotic diseases. To achieve this objective we propose two approaches, one is to disrupt in fibroblasts of transgene mice the TGF- beta signalling pathway, the other is to alter the balance between metalloproteinases and their tissue inhibitors in the extracellular matrix of fibroblasts. Our application is anchored on our previous identification and characterization of a promoter/enhancer in the mouse gene for the proalpha2 chain of mous4e type I collagen (Co11alpha2) that has the ability to direct expression of reporter genes specifically to fibroblasts of transgene mice. To avoid undesirable effects during embryonic development we use a dual system. This system, based on Cre mediated recombination, allows to switch-on the DNAs that we propose to activate in fibroblasts at a specific time after birth. The system used throughout this proposal is based on a Cre recombinase, developed by others, which becomes activated only after administration of Tamoxifen. In this proposal, expression of the DNA for this recombinase is directed by the Co11alpha2 promoter/enhancer which restricts its expression to fibroblastic cells. The genes that we propose to express in fibroblasts postnatally are cloned downstream of the potent and ubiquitously active ROSA26 promoter. Interposed between this promoter and the DNAs that we propose to express we placed a transcription stop cassette, flanked by two Lox P sites, which are substrates for Cre recombinase. Thus expression of the DNAs is only possible after Cre-mediated removal of the STOP cassette. Hence in this dual system, Tamoxifen controls expression of these DNAs which is restricted to fibroblasts because Cre is only expressed in fibroblasts. We hypothesize that the expression of DNA for a constitutively active TGF-beta1 receptor in fibroblasts as well as the over-expression of TIMP1, in fibroblasts will produce fibrotic phenotypes. To attempt to correct the postulated fibrotic phenotype caused by expression of the constitutively active TGF-beta type I receptor, we propose to over- expression in fibroblasts a DNA for a dominant-negative Smad3. In each case of the phenotype of transgene mice and that of their fibroblasts in culture will be extensively characterized with the use of specific molecular markers and gene expression arrays. The results of such gene profiling of fibroblasts from fibrotic animals will also be compared to those performed with fibroblasts of scleroderma patients performed by Dr. Frank Arnett.

DESCRIPTION (provided by applicant): The extracellular matrix (ECM) strongly influences cell behavior and tissue homeostasis, both in normal physiology and disease. Components of the ECM have critical roles in a variety of diseases including diseases of craniofacial development, diseases of the muscular-skeletal system, tumor progression and tumor cell metastasis, diseases of the vascular system and infectious diseases. The ECM also plays a pivotal role in many developmental processes in the embryo. The recent founding of the American Society for Matrix Biology (ASMB) demonstrates the growth and new importance of the field of ECM biology and cell matrix interactions. The first meeting of the ASMB is scheduled to take place at the Marriott Galleria Hotel, Houston, TX, Nov 6-9, 2002. The target audience is composed of physicians, academic scientists and industrial researchers, as well as graduate students and postdoctoral fellows. The Keynote address will discuss the role of key secreted molecules, which control many developmental decisions including craniofacial development, skeletal development, and tooth formation. Lectures in the plenary sessions will discuss broad concepts of the mechanisms by which the ECM controls critical aspects of cell behavior during embryo development in the normal physiological functions of organs and in diseases. These sessions will include discussions on the assembly and remodeling of the ECM, the complex mechanisms by which the ECM signals to cells, how the ECM controls cell migration and motility and influences cell differentiation and fate. The concepts discussed in these sessions will be based on specific examples including studies of muscular-skeletal and skin disease, diseases of vasculogenesis, cancer progression, neuronal cell development, and embryo development. Breakout afternoon sessions will expand on these concepts and discuss progress in structural biology of ECM components, the role of the ECM in infection and host defense, in cell adhesion, in the control of blood vessel formation, in skeletal development and in chronic degenerative diseases. The program has broad implications that are closely related to the missions of several NIH institutes, including NIDCR, NIAMS, NHLBI, NCI, NICHDI, NINDS, and NIA. We anticipate that the ASMB meeting will occur on a biennial basis and the society in the near future will be able to financially support these symposia. However, for the initial meeting, we rely entirely on outside support. We are requesting $40,000 in NIH support for the 1st meeting.

[unreadable] DESCRIPTION (provided by applicant): Similarly to the formation of other organs, skeletogenesis involves two broad classes of regulatory factors. Patterning factors control the shape, size and number of skeletal element, as well as initial decisions regarding the body plan of the embryo, whereas differentiation factors control the fate of the constituent cells of the skeleton. Previous cell biological experiments suggested that osteoblast differentiation occurs along a multistep pathway. More recently, the transcription factor Cbfa1/Runx2 was shown to be needed for osteoblast differentiation. Very recently, we have discovered that a novel member of the Kruppel family of transcription factors, called Osterix (Osx), is required for bone formation and osteoblast differentiation. Osx null mice have no membranous, and no endochondral bones, although chondrocyte differentiation and cartilage formation occur normally. Furthermore, our experiments indicate that Osx acts downstream of Cbfa1/Runx2. This application proposes to characterize the mechanisms by which Osx controls osteoblast differentiation. We plan to examine the extent of the osteoblast-genetic program that is controlled by Osx and identify sequences in target genes that directly mediate the transcriptional activation by Osx of these genes in osteoblasts in vivo. Our studies will also identify the proteins that either physically or functionally interact with Osx, or control its activity. Finally, we will determine whether Osx is a negative regulator of Sox9 expression and of the chondrocyte differentiation program. Overall, these experiments should greatly improve our understanding of the molecular mechanisms of osteoblast differentiation.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): Our long term goal is to understand the transcriptional mechanisms of chondrocyte differentiation. Our previous work has demonstrated that Sox9 has a central role in chondrogenesis and is needed at multiple steps in the pathway of chondrocyte differentiation. Sox9 is initially needed to establish an osteochondroprogenitor;subsequently it is required for chondrogenic mesenchymal condensations. Sox9 is then needed for overt differentiation of chondrocytes, in part because Sox9 is required for expression of Sox5 and Sox6, which are needed for overt chondrocyte differentiation. Later in the pathway Sox9 still has another important role because it participates in the physiological inhibition of the maturation of chondrocytes into hypertrophic chondrocytes. In addition to its roles in the chondrocyte differentiation pathway, Sox9 is also needed for the differentiation of a small number of other cell lineages. Four specific aims are proposed to gain new insights in the mechanisms by which Sox9 and L-Sox5 control chondrocyte differentiation. The repertoire of genes controlled by Sox9 at two major steps in the chondrocyte differentiation program will first be identified. The hypothesis will also be tested whether Sox9 controls the expression of specific cell surface associated proteins or other proteins, needed for chondrogenic mesenchymal condensations. The role of TIP60 as a coactivator of Sox9 and L-Sox5 during chondrogenesis will be further characterized and new polypeptides that are part of transcriptional complexes, which interact with Sox9 will be identified. The patterns of Sox9 and L-Sox5 binding sites and those of polypeptide complexes, which interact with chondrocyte-specific promoters and enhancers, in intact chondrocytes will also be determined. In vitro reconstituted nucleosomal templates of the Col2a1 regulatory segments will be used in order to dissect the function of Sox9, L-Sox5 and other transcriptionally active polypeptides in chromatin disruption and transcription. These experiments should greatly enhance our understanding of the mechanisms whereby Sox9 controls chondrocyte differentiation and may suggest new therapeutic approaches for cartilage diseases.

laboratory mouse; model

Scleroderma or systemic sclerosis (SSc) is a disorder of the connective tissues affecting various organ[unreadable] systems. The disease is complex and is characterized by excessive accumulation of collagen and other[unreadable] extracellular matrix components in the skin and internal organs. Because increased signaling by TGF-beta has[unreadable] been implicated in this disease we recently established a novel mouse model in which the TGF-beta Receptor1[unreadable] is constitutively activated in fibroblasts post-natally (TBR1CA; Col1a2-CreER). These mice recapitulated the[unreadable] major features of human SSc, showing pronounced and generalized fibrosis of the dermis, thinner epidermis,[unreadable] loss of hair follicles, and fibrotic thickening of small blood vessel walls in lung and kidney. Primary skin[unreadable] fibroblasts of these mice showed elevated expression of downstream TGF-beta targets, reproducing the[unreadable] hallmark biochemical phenotype of explanted SSc dermal fibroblasts. In particular there was a marked[unreadable] increase in connective tissue growth factor (CTGF) expression. Since increased expression of CTGF has[unreadable] been implicated to play a key role in the disease process, we generated transgenic mice that over-express[unreadable] CTGF in fibroblasts (Col1a2-CTGF) by using a fibroblast-specific promoter/enhancer from the pro-D2(l)[unreadable] collagen gene. The animals exhibit a severe loss of hair. Initial histological examination of skin biopsies[unreadable] showed pronounced and generalized fibrosis of the dermis, thicker epidermis and inflammatory infiltrates in[unreadable] the area of the skin fibrosis. Preliminary analysis of mouse embryonic fibroblasts derived from these[unreadable] transgenic mice showed elevated expression of collagen type I and Timp-3.[unreadable] We propose to characterize the Col1a2-CTGF mice as well as their explanted skin fibroblasts. To[unreadable] understand the mechanisms by which increased expression of CTGF in fibroblasts causes a fibrotic disease,[unreadable] we will also perform a comparison of the molecular phenotypes of the skin fibroblasts of these mice with[unreadable] those of TBR1CA; Col1a2-CreER mice and with those of specific human scleroderma patients. To further[unreadable] examine these mechanisms we propose to attenuate the fibrotic phenotypes of the skin fibroblasts of the two[unreadable] transgenic mouse models of scleroderma by pharmacological inhibitors of specific signaling pathways or[unreadable] siRNAs. We also will attempt to inhibit the fibrotic phenotypes of these transgenic mice in vivo by crossing[unreadable] null mutations in either SmadS or integrin D6 in these mice. Finally, we will examine the mechanisms that[unreadable] cause increased sensitivity to bleomycin-induced lung fibrosis in TBR1CA; Col1a2-CreER mice and test[unreadable] whether a similar sensitivity occurs in Col1a2-CTGF mice. These studies should give information about the[unreadable] relative importance of TGF-beta and CTGF in causing fibrotic diseases.

[unreadable] DESCRIPTION (provided by applicant): The DNA Analysis Core Facility (DAF) at the University of Texas M. D. Anderson Cancer Center requests funds for the purchase of a 3730 DNA Genetic Analyzer (48capillary instrument) to perform DNA sequencing, resequencing, fluorescent fragment analysis and SNPlex analysis (for identifying polymorphisms and sequence variants). The DAF is a CCSG supported centralized Core facility that provides services to 187 of principal investigators in 44 departments at M.D. Anderson; 74% hold peer-reviewed grants. When NIH investigators are not using these services, they are made available to other members of the institution. The DAF which opened in 1992 has become an integral part of the research laboratories at M.D. Anderson. Members of the institution have come to rely heavily on the facility to provide fast and accurate DNA services, allowing researchers to progress more rapidly with their research goals. The facility is currently supervised by Ms. Erika Thompson M.S. Ms. Thompson has 11 years experience in DNA analysis and has been managing the facility since 2003. The requested instrument will: 1) increase sequencing throughput, processing 48 samples in 2.5hour for DNA sequencing, versus only 16 samples on the 3100 instrument which the 3730 Analyzer will replace, 2) provide significant cost savings e.g. running costs on 3100, $0.60 per sample versus $0.10 on 3730, 3) permit the use of the latest SNPlex technology to rapidly identify up to 48 SNP genotypes per sample in one run. This is ability to multiplex 48 SNPs per sample is especially important in the analysis of small amounts of DNA from cancer patients as SNPlex requires less than 0.8ng of genomic DNA per genotype. The 3730 genetic analyzer will allow the facility to meet the growing demands for existing services and also allow the latest technology in the form of VariantSEQr and SNPlex to be made available to NIH supported investigators at M.D Anderson. At this time the facility cannot provide these additional services as the 3730 XL Genetic analyzer is being used solely for DNA sequencing and the remaining 3100 for genotyping human and mouse samples using fluorescent fragment analysis technology. The DAF has supported the completion of several hundred publications including studies reported in Nature Medicine, Nature Genetics, and Cell. The purchase of a 3730, would allow the facility to increase throughput, reduce turnaround time, provide PIs access to the latest technology (SNPlex and VariantSEQr) and reduce reagent cost. This instrument would also serve as a back up to the 3730XL instrument (the 96 capillary instrument). The 3730 XL, which was purchased by M.D. Anderson in 2002, is currently the workhorse of the facility, and is now reaching capacity. It is critical that the DAF obtain a state of the art, 3730 Genetic Analyzer, so that the core can continue to provide NIH funded investigators with the latest technology to support cancer research. [unreadable] [unreadable] [unreadable]

Since its inception in 1991, the goal of the DNA Analysis Facility (DAF) has been to provide MDACC investigators with reliable nucleic acid analysis using state-of-the-art technology to provide a high level of technical expertise in a centralized facility. Today, 7 highly-skilled technical staff operate 3 robotic workstations: a QiagenSOOO Biorobot, a Qiagen 8000 Biorobot, and a Beckman Biomek 2000. The staff also operate a Transgenomic WAVE mutation detection system, an Applied Biosystems 3730 XL genetic analyzer, an Applied Biosystems 3130 genetic analyzer, an Applied Biosystems 3100 genetic analyzer and 4 Applied Biosystems real-time PCR instruments; the 7000, 7300, 7500 and 7900 with low density array. The DAF performs automated DNA sequencing, gene resequencing, fluorescent fragment analysis, highthroughput SNP analysis, murine DNA extraction and genotyping by real-time PCR, quantitative gene expression analysis and real-time PCR assay design. Provision of these essential services has freed investigators to focus their efforts on characterizing the molecular and biological functions of their target genes. A shared resource director, co-director, and a 5-member oversight committee direct the development of the DAF. This committee meets annually to evaluate the scientific and financial performance of the facility. The co-director manages the daily activities of the DAF. In the current grant period, the institution invested $569,500 in the DAF for the purchase of new equipment, in addition to providing a new 1,600 sq ft lab at a cost of $876,000. Funding for the DAF is provided by the CCSG (38%) and user fees (62%). The DAF successfully processed 560,413 samples in the last 5 years, representing 280 investigators from 20 programs. The total number of samples processed increased 198% compared to the previous grant period. All services had more than 75% use by peer-reviewed investigators, with more than 90% of samples processed for peer-reviewed investigators. Output in DNA sequencing during the present grant period compared to the last showed an increase of 130%, while Fragment analysis increased 158%. Newer services have also shown significant growth. Murine genotyping increased 103%, gene resequencing increased 718% (2 years) and 14,600 SNPs were determined using the SNPlex service. The DAF recently introduced a quantitative gene expression analysis service. This service includes RNA quantification, RNA QC, reverse transcription and assay set-up. The facility plans to expand this service to include microRNA assays.