Development of Site-specific O-GlcNAc Antibodies for Epigenetic Research

Project SummaryO glycosylation of nuclear and cytoplasmic proteins by a singleN acetyl D glucosamine moietyOGlcNAcis a common post translational modification that is highly dynamic and fluctuates in response to cellular stimuliThis type of glycosylation has been found on approximately a thousand human proteins to dateand is thought to be nearly as wide spread and abundant as protein phosphorylationIn factO GlcNAc often competes with protein phosphorylationand these two modifications have extensive crosstalk in the regulation of signalingtranscriptionand the functions of oncogenes and tumor suppressorsThe modification appears to play a major role in key pathophysiological conditions including cancerAlzheimer s diseaseand diabetesMany of the first proteins identified carrying this modification were transcription factorsand it has become clear in the last several years that O GlcNAc plays a major role in chromatin remodeling and gene expressionThe focus of this proposal is to develop site specific antibodies that can be used as tools in the elucidation of the role that O GlcNAc plays in epigeneticsIn the predecessor Phase I grant we focused on evaluating synthetic immunogensthe production of polyclonal antibodiesPAbsto five sites of O GlcNAc modification on the four core histonesHistoneABandand characterizing these antibodiesWe were successful with each Aim of this previous projectwhich leads to this Phase II proposalHerewe propose to utilize our proprietary immunization strategy to significantly expand our repertoire of site specific O GlcNAc antibodies to include the majority of proteins currently known to be modified in this manner that are also involved in gene expressionConsequentlyif we are successfulresearchers will have access to a wide range of site specific Abs developed for epigenetic researchand thus we feel that this study will have an immediate impact on epigenetic research and could have far reaching implications in disease research Project NarrativeModification of proteins involved in gene regulation and epigenetics by a single sugar termed O GlcNAc is now under intense scrutiny for its role in cell biology and human diseases such as diabetescancer and Alzheimer sThere are no protein specific reagents to study O GlcNAc s role in epigeneticsWe propose to develop site specific O GlcNAc antibodies to a number of epigenetic modifiersincluding the histone proteins and proteins involved in the histone complexthereby providing valuable tools for the study of O GlcNAc s role in epigenetics and possible causative links to a variety of diseases