David G. Skalnik - US grants

This proposal examines the molecular mechanisms that regulate tissue-specific gene expression and development in myelomonocytic cells (phagocytes). The phagocyte-specific gp9l-phox gene, which when defective causes chronic granulomatous disease, encodes a component of the respiratory burst NADPH-oxidase complex. We have previously shown that the proximal gp9l-phox promoter will direct the expression of linked reporter genes in a subset of monocyte/macrophages in, transgenic mice. In addition, macrophage tumors arise at 6-12 months of age in animals carrying a gp9l-phox promoter/SV40 T-antigen transgene. A major goal of the proposed work is to identify the cis-elements within the gp9l-phox promoter, and the DNA-binding proteins interacting with these sequences, that mediate monocyte/macrophage-specific transcription. Transgene constructs consisting of various portions of the gp9l-phox promoter linked to reporter genes will be introduced into mice, and the tissue-distribution of transgene expression determined by Northern blot analysis and immunohistochemical detection of the reporter protein. In addition, efforts will be made to develop a tissue culture system as a more convenient method to assay gp9l-phox promoter function. Nuclear extracts will be used in in vitro DNA-binding protein assays to identify DNA-binding activities that interact with the proximal gp9l-phox promoter. DNA-binding protein recognition sites will then be ablated by site-directed mutagenesis, and mutant promoter constructs will be introduced into transgenic mice (or suitable tissue culture cells) to evaluate the effect that loss of protein binding has on transgene expression. DNA-binding proteins found to be functionally relevant to the regulation of gp9l-phox expression will be molecularly cloned by ligand screening of a lambda-GT11 expression library, or by library screening with antibody or degenerate oligonucleotide probes following biochemical (DNA affinity chromatography) purification. Isolation of transcription factor cDNA clones will allow a detailed study of the regulation of their activity, with the ultimate goal of understanding how a progenitor cell becomes committed to differentiate along the myelomonocytic lineage. The time lag prior to the appearance of macrophage malignancies in gp9l-phox promoter/SV40 T-antigen transgenic mice suggests that somatic events, in addition to T-antigen expression, are required for tumorigenesis. Genetic loci that contribute to tumor progression in these cells will be identified by proviral "tagging" with Moloney murine leukemia virus, followed by recovery of proviral integration sites from tumors that arise at unusually early times.

Molecular dissection of the complex and overlapping network of interactions between GTPases is currently a major challenge. Dozens of GTPases have been identified, many of which contain highly related amino acid sequences. Each species of GTPase much interact with regulatory and effector molecules to appropriately transduce specific external stimuli, leading to specific cellular responses. The long term goal of this Program Project is to gain insight into this regulatory network by studying one sub-family of GTPases, the RAC proteins. Rac1 and Rac2 proteins are highly similar (92% identical), yet disruption of the Rac2 gene, which is specifically expressed in hematopoietic cells, leads to multiple defects of blood cell development and function of phenotypes has recently been identified in a human patient carrying a dominant-mutant Rac2 allele. The specific goals of this Program Project include elucidation of the networks of upstream signaling molecules and downstream effector molecules that are utilized by Rac2 in hematopoietic cells, as well as gaining insight into the molecular controls responsible for coordinate expression of Rac GTPases. Each project within the questions of Rac2 function, including cross-talk between expression of Rac proteins; the molecular basis of tissue restricted expression of the Rac2 gene; the functional intersection of Rac2 with proteins such as BCR-ABL and Nf-1 that are involved in the control of hematopoietic cell proliferation; the unique role of Rac2 in the control of phagocyte function; the role of Rac2 in transducing integrin- mediated signals associated with inflammation and angiogenesis; and the role of Rac2 in the control of cytoskeletal structure and function. On a broader level, completion of these highly integrated studies will not only provide significant new information on the functional relationships and regulation of Rac1 and Rac2 GTPases in hematopoietic cells, but will also likely provide insights into GTPase regulation that will be relevant to other small molecular weight GTPases.

GTPases of the Rac family are important regulators of cytoskeletal remodeling in response to signals received via growth factor responses. The expression patterns of these GTPases are tightly regulated in response to cytochalasin B exposure. In contrast, Rac2 expression is restricted to hematopoietic cells, and is further induced upon terminal myeloid cell differentiation and T cell stimulation. Furthermore, there appears to be cross-talk between the expression of the Rac1 and Rac2 genes, as Rac1 expression is dysregulated in Rac2-null cells. The specific aims of this proposal are: (AIM #1) Identify genetic elements and cognate transcription factors responsible for restriction of Rac2 expression to hematopoietic cells. Cytosine methylation patterns and DNase hypersensitive sites surrounding the Rac2 locus will be determined. Bacterial artificial chromosome clones carrying the human Rac2 gene locus will be introduced into mice and murine cell lines, and transgene expression assessed by RNase protection assays in order to identify cis-elements necessary and sufficient to direct lineage-specific expression. In vitro DNA-binding protein assays will be performed to detect and identify transcription factors that interact with critical cis- elements, and novel factors will be molecularly cloned by either ligand screening or biochemical purification approaches. (AIM #2) Analyze cross-talk between the Rac1 and Rac2 genes. Biochemical studies will be conducted to determine the level at which expression of the Rac1 gene is modulated in response to Rac2 levels. The Rac1 promoter will be cloned and functionally characterized, and critical cis-elements and cognate trans-factors will be identified. The molecular basis for dysregulated Rac1 expression in response to Rac 2 levels will be determined by introducing Rac1 promoter/luciferase reporter genes into primary murine blood cell cultures derived from Rac2-null mice.

Cytosine and histone methylation are important epigenetic modifications involved in the regulation of chromatin structure and gene expression. Little is known about how DNA and histone methyltransferase enzymes are controlled and targeted to appropriate genomic sites for action. This laboratory cloned CpG binding protein (CGBP), a novel factor that binds DNA containing unmethylated CpG dinucleotides, the site of cytosine methylation in mammals. This factor is also a component of the Set1 histone H3-Lys4 methyltransferase complex. Disruption of the CGBP gene leads to embryonic death in mice, and embryonic stem (ES) cells lacking CGBP are unable to differentiate in vitro and carry altered patterns of epigenetic modifications such as reduced cytosine methylation. They also contain elevated levels of histone H3-Lys4 methylation, correlated with inappropriate drifting of the Set1 protein into heterochromatin. Thus, CGBP is a regulator of both major classes of epigenetic modifications. The CGBP -/- mutant phenotype is ""rescued"" upon introduction of a CGBP-expression vector, thus offering an experimental system with which to probe structure/function relationships of CGBP. The specific aims are: (1) Determine domains of CGBP that are necessary and sufficient to rescue epigenetic modifications in CGBP -/- ES cells. Various truncated or mutated versions of CBGP will be introduced into CGBP-/- ES cells. Clones expressing physiologic levels of these CGBP variants will be examined for genomic cytosine methylation, DNA methyltransferase activity, sub-nuclear targeting of the Set1 histone methyltransferase complex, and histone H3-Lys4 and Lys9 methylation. (2) Determine domains of CGBP that are necessary and sufficient to rescue developmental potential of CGBP -/- ES cells. Transfected ES cell clones isolated in Specific Aim #1 will be assessed for developmental capacity. ES clones will be induced to differentiate in vitro by the removal of leukemia inhibitory factor from the growth media, and differentiation will be assessed by morphological examination of subsequent embryoid body formation, and by histochemical detection of alkaline phosphatase activity, a marker of stem cells. RT-PCR will also be performed to assess the expression of a panel of stem cell and lineage-restricted molecular markers. In vivo developmental potential of ""rescued"" ES cell lines will be assessed by isolating cells expressing enhanced green fluorescent protein (EGFP), injecting these cells into blastocysts, and introduction into pseudo-pregnant mice to produce chimeric animals. The contribution of ES cells to each tissue will be examined by fluorescence microscopy.


Intellectual Merit.These studies are targeted at gaining an understanding of the molecular mechanism of CGBP function. These studies will provide novel information regarding the molecular mechanisms that control the epigenetic regulation of chromatin structure. This is a fundamental question that relates to many aspects of normal development and disease.

Broader Impacts. This laboratory has a long history of integrating graduate student education with its research program. Nine doctoral students have been trained over 15 years. Ten undergraduate students, including minority students, have participated in the research as well. A similar level of student participation is anticipated for these studies.

DESCRIPTION (provided by applicant): The Science Animal Resource Center (SARC) provides animal housing and use facilities for the School of Science at Indiana University Purdue University Indianapolis (IUPUI). In recent years the School of Science has enjoyed a rapid expansion of its research program, including a doubling of external research support. Recent inspections by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) noted deficiencies in the SARC facilities that were structural in nature and not easily remedied. These include the inability to effectively separate clean and dirty animal caging, insufficient HVAC capacity, and inadequate storage capacity. In response to these deficiencies and the need to provide appropriate support for its growing research program, the School of Science (in collaboration with the School of Engineering and Technology) has embarked on the construction of a new $25 million Science and Engineering Laboratory Building (SELB). Construction began in March 2012, and is expected to be completed by early 2014. Approximately one-third of this new facility (~10,400 assignable square feet) will be devoted to the housing and experimental use of animals, including twenty-four animal housing rooms. It is estimated that the cost of equipment for the new SARC vivarium within SELB will be approximately $1.3 million (this is in addition to the above-mentioned construction costs). The Specific Aim of this proposal is to purchase twenty-seven Maxi-Miser ventilated mouse housing cage rack systems and four changing stations from Thoren Caging Systems, Inc., to be installed in four animal housing rooms within the new SELB vivarium. These units will permit the individual housing of 3,360 mice. Single housing of rodents is essential for faculty users from the Psychology Department, who are the major users of mice within the facility and whose research focuses on the neuroscience of drug addiction. Thus, this proposal is responsive to the major goal of the Developing and Improving Institutional Animal Resources program at the NIH, which is to upgrade animal facilities to support the conduct of biomedical and/or behavioral research. The remaining ~$800,000 needed to equip the new vivarium will be provided by the School of Science. Compared to the current open caging systems utilized by the existing SARC facility, these new mouse caging units and changing stations will provide state-of-the-art rodent housing and will significantly minimize the expulsion of allergens and potential zoonotic agents into the environment. The proposed caging systems will also permit the housing of mice in a controlled microenvironment with superior protection from exposure to pathogens. The proposed caging systems will maximize housing capacity of the available space.