Characterizing the Mechanisms of Transcription Regulation of RGMc/Hemojuvelin

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SPACE PROVIDED. Iron-related metabolic and hematologic disorders affect millions of individuals worldwide. Repulsive guidance molecule c (RGMc), or Hemojuvelin (HJV), is a recently discovered gene shown to be critical for iron regulation. Mutations in RGMc/HJV cause the severe systemic iron overload disorder, juvenile hemochromatosis. The molecular mechanisms responsible for the regulation of RGMc under physiological and pathological conditions are unknown. In addition, understanding the regulation of RGMc expression has been complicated by its unique tissue distribution. To date there are no other genes whose expression is restricted to striated muscle and liver, placing RGMc in a unique position in that understanding its regulatory mechanisms will provide insight into systemic iron regulation and tissue-specific gene expression. The primary focus of this application will be to define the mechanisms responsible for the tissue-specific expression of RGMc. I hypothesize that the RGMc locus goes from a 'closed' to an 'open' chromatin state in differentiating muscle cells, and is 'open' in the liver. In order to define the molecular mechanisms that regulate the expression of RGMc, I propose a series of experiments with the following specific aims: (1) To determine the transcriptional regulatory mechanisms of RGMc in muscle. (2) To define the mechanisms of regulation of RGMc gene transcription in the liver. Regulatory mechanisms will be characterized using DNA- protein interactions and functional mutagenesis studies to define transcription factor binding sites in each type of tissue, restriction enzyme accessibility assays to query the state of chromatin condensation under different conditions of RGMc expression, and ChiP to determine the kinetics of histone modifications associated with repressed and active gene transcription, as well as the in vivo analysis of transcriptional machinery (including chromatin-remodeling complexes) at the RGMc locus in tissues that express RGMc. The long range goals are to understand the mechanisms of tissue-specific regulation of RGMc/HJV in response to developmental or physiological cues and how this expression influences iron metabolism in normal and diseased states. This proposal will focus on the expression of RGMc in two developmentally and physiologically distinct tissues, and establish a foundation for regulation of the gene with the long term goal of understanding tissue-specific response of RGMc expression and its role in systemic iron homeostasis. This work is of particular importance to public health in that it is becoming increasingly apparent that disorders of iron metaboilsm show different tissue-specific responses during iron overload or anemia; treatment will require novel insight into the molecular mechanisms of tissue-specific expression.