The aim of this study is to identify the domain that is important to maintain the stability of PHF8. PHF8 (Plant Homeodomain Finger protein 8), which is a JmjC domain containing histone demethylase, it removes the histone methylation marks such as mono-methylated histone H4 lysine 20 and histone 3 lysine 9 demethylase around the transcription start sites. PHF8 positively regulates gene expression, which mostly depends on H3K4me-3 binding PHD and the JmjC catalytic domains. The mutations of PHF8 have been found in XLMR (X Linked Mental Retardation) patients, who also have craniofacial deformity such as clefts of lip and palate. In the ongoing projects being done by the Qi lab, hypoxia was found to induce PHF8 degradation, thus, identifying the key domain or amino acid in PHF8 protein will help us gain a deeper understanding of the regulatory pathways of PHF8. I carried out PCR and cloning to establish 8 truncated PHF8 forms at both N- and C- termini. These truncated PHF8, which were tagged with HA at their N-terminus, and then transfected into HeLa cells. Interestingly, at steady states, the truncated forms of PHF8 show instability and generate specific patterns of protein degradation, suggesting that the amino acids 500 to 600 might be the region that is targeted by cleavage or proteasome dependent protein degradation. With these constructs, we are further investigating which truncated PHF8 will not respond to hypoxia induced proteasome dependent PHF8 degradation. Essentially, we will generate deletion truncations or point mutations targeting the 500-600 amino acids of PHF8. The significance of this project is to identify the key residues in PHF8 that play an important role in regulating PHF8 through various signaling pathways and cellular events. Identification of the regulatory mechanism of PHF8 will provide better understanding for the cellular signaling pathways that are important for craniofacial and neural development. "