Dr. Michael Goldman

goldman@sfsu.edu

http://userwww.sfsu.edu/~mgoldman

http://cistron.sfsu.edu

Early in mammalian development, all but one of the X chromosomes in each cell of an embryonic female is inactivated. X-chromosome inactivation (XCI) is one of the earliest events in developmental gene regulation, and provides an important model for gene regulation in general. Errors in gene regulation are responsible for many genetic disorders and for some changes that characterize cancerous cells. Further, abnormalities of the X chromosome occur frequently in humans, and sporadic reactivation of all or part of the inactive X chromosome is one of the few gene regulatory changes clearly associated with aging.


The existence of a nucleation point for the process of X-chromosome inactivation (XCI) has been inferred from a variety of studies, and the characterization of the X inactivation center (XIC) has been a Holy Grail for those working in X-chromosome inactivation. Quite recently, Willard, Ballabio and their colleagues demonstrated a transcript (the X-inactive -specific transcript, or XIST) that was transcribed exclusively from the inactive X chromosome, and mapped in the immediate vicinity of the X inactivation center. Subsequently, these workers confirmed the existence of a similar locus, Xist, in mouse.


The aim of this project is to characterize the chromatin domain of the newly-discovered Xist locus. Characterization of a chromatin domain involves precisely defining the size and end points of the loops or chromatin domains in which specific genes are found. The approach I have been using involves defining the extent of the domain using two methods of recognizing the end points, and one methods for determining the extent of the functional domain itself.

The DNA of a single chromatin domain shares a particular level of sensitivity to enzymes that cleave DNA (nucleases). Such nuclease sensitivity can be readily assayed by techniques we have already used on a much smaller scale to characterize the genes themselves. We plan to define length of DNA at a particular level of nuclease sensitivity for the Xist gene, in order to map the probable extent of the domain. These experiments involve isolation of nuclei from cultured cells or from fresh liver cells, treatment of these nuclei with controlled concentrations of the nuclease, DNase I, gel fractionation of the resulting DNA fragments, and characterization of those fragments using cloned molecular probes.




Last modified July 10, 2002