Purification and assays for high mobility group HMG-I(Y) protein function

This chapter discusses the purification method and assays for high-mobility group (HMG)-I(Y) protein function. The HMG-I(Y) family of HMG nonhistone mammalian proteins belongs to a group of nuclear proteins collectively known as “architectural transcription factors” because of their ability to function in vivo both as components of chromatin structure and as auxiliary-gene transcription factors. In their capacity as transcription factors, HMG-I(Y) proteins have been implicated in both the positive and negative regulation of a number of human genes in vivo. The ability of HMG-I(Y) proteins to bend, straighten, unwind, and supercoil DNA substrates plays a role in gene transcriptional regulation. HMG-I(Y) proteins have also been demonstrated to be a component of the HIV-1 viral preintegration complex in human cells, and they can also be used for the efficient integration of viral DNA in vitro. HMG-I(Y) proteins, like other HMG proteins, can be isolated from nuclei or chromatin by extraction with 0.3–0.4 M NaCl. While the salt isolation method is mild and allows the efficient recovery of native, nondenatured HMG proteins, this procedure results in the extraction of a very complex mixture of nuclear proteins from which the desired HMG-I(Y) proteins must be subsequently purified. Isolation of HMG-I(Y) proteins by dilute acid extraction offers several advantages over salt extraction procedures.