The Barr body has long been recognized as the cytological manifestation of the 
inactive X chromosome (Xi) in interphase nuclei. Despite being known for over 50 
years, relatively few components of the Barr body have been identified. In this 
study, we have screened over 30 histone variants, modified histones and 
non-histone proteins for their association with or exclusion from the Barr body. 
We demonstrate that, similar to the histone variant macroH2A, heterochromatin 
protein-1 (HP1), histone H1 and the high mobility group protein HMG-I/Y are 
elevated at the territory of the Xi in interphase in human cell lines, but only 
when the Xi chromatin is heteropycnotic, implicating each as a component of the 
Barr body. Surprisingly, however, virtually all other candidate proteins 
involved in establishing heterochromatin and gene silencing are notably absent 
from the Barr body despite being localized generally elsewhere throughout the 
nucleus, indicating that the Barr body represents a discrete subnuclear 
compartment that is not freely accessible to most chromatin proteins. A similar 
dichotomous pattern of association or exclusion describes the spatial 
relationship of a number of specific histone methylation patterns in relation to 
the Barr body. Notably, though, several methylated forms of histone H3 that are 
deficient in Xi chromatin generally are present at a region near the 
macrosatellite repeat DXZ4, as are the chromatin proteins CTCF and SAP30, 
indicating a distinctive chromatin state in this region of the Xi. Taken 
together, our data imply that the Xi adopts a distinct chromatin configuration 
in interphase nuclei and are consistent with a mechanism by which HP1, through 
histone H3 lysine-9 methylation, recognizes and assists in maintaining 
heterochromatin and gene silencing at the human Xi.