Abstract: For humans to develop properly, the fertilized egg must divide in a manner that generates a body that has the correct organs and appendages in the correct places. Much of this is determined by the proper temporal and spatial regulation of master regulatory genes that guide formation of the differentiated cells that make up organs and appendages. A key aspect of this developmental process is the ability to maintain master regulatory genes in a repressed state in cells where their expression might cause inappropriate cell behavior. Mis-expression of even a single master regulatory gene, such as those encoded in the HOX loci, can result in a cell behaving in a manner incompatible with its body location and tissue type. The Polycomb- Group (PcG) group of genes is largely responsible for maintaining this repression, based upon intensive study over the past 70 years. Maintaining active expression patterns for genes is equally critical for development. A set of genes that maintains activation, called the trithorax- Group (trxG), was isolated via their ability to suppress PcG mutations. This application describes the continuation of our work on understanding the function of the proteins encoded by the PcG and trxG genes. These proteins form several complexes. The PcG complexes PRC1 and PRC2 are each large complexes that contain several PcG gene products. PRC1 is the main `engine' of repression in the Polycomb-Group and is known to interact with chromatin, the structure that packages genes in the nucleus of cells. Chromatin structure can render the enclosed DNA inaccessible to activating factors. A prominent hypothesis is that repression can be generated by generating highly packaged DNA that is no longer able to be transcribed and expressed. We describe approaches to investigate the various mechanisms used by PRC1 to generate packaged chromatin. PRC1 works together with PRC2 to generate repression. PRC2 methylates nucleosomes, the primary packaging unit of chromatin, on residue H3K27, a topic of intensive study in numerous laboratories and companies. We intend to provide novel information by exploring repressive activities of PRC2 that are unrelated to methylation. We will also characterize methylation by PRC2 of a separate protein involved in gene regulation, ElonginA. Finally, we will characterize two separate nucleosome remodeling activities encoded by trxG genes, the mammalian SWI/SNF (BAF) complex and mammalian CHD7. We will explore interactions between SWI/SNF subunits and cohesin, a chromatin organizing activity, and determine how those interactions impact long range interactions in chromatin. We will examine the role for a lncRNA called HERVH in targeting CHD7 activity.

To develop properly, mammalian cells must express master regulatory genes only in the proper cell lineages. This requires that both repressive states and active states of these genes be ‘remembered’ throughout the lifetime of the organism, functions carried out by gene products from the Polycomb-Group and trithorax-Group. This application continues our longstanding characterization of the functional mechanisms of these complexes.