The focal distribution of regulatory macromolecules within the nucleus can effectively support the integration of regulatory networks and establish threshold levels of factors for positive and negative control in a broad spectrum of biological contexts that include development and tissue remodeling. Equally important, changes in the composition and organization of regulatory machinery in nuclear microenvironments provides insight into perturbed mechanisms that relate to human disease which is strikingly illustrated by, but not restricted to, skeletal disorders and tumorigenesis (1-5)
. Examples are modifications in the size, number, and composition of intranuclear sites that support transcription, replication, repair and altered regulatory domains that are causally associated with cleidocranial dysplasia and competency for metastatic breast cancer cells to form osteolytic lesions in bone.
Our understanding of the location of regulatory machinery for gene expression, replication, and repair and its role in functional outcome of various biological processes is increasingly evident. However, it is important to define and develop techniques that provide both specific and quantitative insight into various parameters of nuclear architecture. Development and deployment of such approaches is essential for establishing the biological relevance of subnuclear organization as well as necessary for diagnosing disease or providing a platform for development of targeted therapies.
Traditionally, compartmentalization of regulatory machinery has been identified and characterized by subnuclear fractionation followed by biochemical and molecular analyses. These are informative approaches, but with limitations. During the past several years, advances in microscopy, together with the development of highly specific antibodies and epitope tags have allowed to examine the assembly and activities of regulatory machinery at single cell level in both the fixed as well as live cell preparations. Thus, the combined use of high resolution cellular, biochemical and molecular approaches maximizes the extent to which regulatory mechanisms can be defined.
We will focus on visualization of nuclear microenvironment using Runx transcription factors as an example for compartmentalization of regulatory machinery within nuclei of osteoblastic cells. We will present approaches for imaging of focally localized regulatory complexes in interphase nuclei as well as throughout mitosis. Specificity and quantitation of regulatory complexes that are visualized by microscopy are required to informatively relate cell morphology with regulatory mechanisms. In addition, we will describe a recently developed approach in our laboratory, designated “Intranuclear Informatics”, that quantitatively assimilates multiple parameters of regulatory protein localization within the nucleus into contributions towards skeletal gene expression from a temporal/spatial perspective (6)