Base-unpairing regions (BURs) contain a specialized DNA context with an exceptionally high unwinding propensity, and are typically identified within various matrix attachment regions. A BUR affinity column was used to purify a doublet of Mr 20,000 proteins from human breast carcinoma cells. These proteins were identified as the high-mobility group (HMG) protein, HMG-I, and its splicing variant, HMG-Y. We show that HMG-I(Y) specifically binds BURs. Mutating BURs so as to abrogate their unwinding property greatly reduced their binding affinity to HMG-I(Y). Numerous studies have indicated that elevated HMG-I(Y) expression is correlated with more advanced cancers and with increased metastatic potential. We studied whether the expression of HMG-I(Y) responds to signaling through the heregulin (HRG)-erbB pathway and the extracellular matrix. HMG-I(Y) expression was increased in MCF-7 cells after stable transfection with an HRG expression construct that led cells to acquire estrogen independence and metastasizing ability. A high level of HMG-I(Y) expression was detected in metastatic MDA-MB-231 cells, but the expression was virtually diminished, and the metastasizing ability was lost after cells were stably transfected with an antisense HRG cDNA construct. HMG-I(Y) was also decreased in MDA-MB-231 cells when treated with a chemical inhibitor for matrix metalloproteinase-9 that led to a reduction of invasive capability in vitro. The level of HMG-I(Y) expression, therefore, is dynamically regulated in human breast cancer cells in response to varying types of signaling that affect metastatic ability, including the HRG-erbB pathway and those from the extracellular matrix.