BACKGROUND: The INK4b-ARF-INK4a tumour suppressor locus controls the balance 
between progenitor cell renewal and cancer. In this study, we investigated how 
higher-order chromatin structure modulates differential expression of the human 
INK4b-ARF-INK4a locus during progenitor cell differentiation, cellular ageing 
and senescence of cancer cells.
RESULTS: We found that INK4b and INK4a, but not ARF, are upregulated following 
the differentiation of haematopoietic progenitor cells, in ageing fibroblasts 
and in senescing malignant rhabdoid tumour cells. To investigate the underlying 
molecular mechanism we analysed binding of polycomb group (PcG) repressive 
complexes (PRCs) and the spatial organization of the INK4b-ARF-INK4a locus. In 
agreement with differential derepression, PcG protein binding across the locus 
is discontinuous. As we described earlier, PcG repressors bind the INK4a 
promoter, but not ARF. Here, we identified a second peak of PcG binding that is 
located approximately 3 kb upstream of the INK4b promoter. During progenitor 
cell differentiation and ageing, PcG silencer EZH2 attenuates, causing loss of 
PRC binding and transcriptional activation of INK4b and INK4a. The expression 
pattern of the locus is reflected by its organization in space. In the repressed 
state, the PRC-binding regions are in close proximity, while the intervening 
chromatin harbouring ARF loops out. Down regulation of EZH2 causes release of 
the approximately 35 kb repressive chromatin loop and induction of both INK4a 
and INK4b, whereas ARF expression remains unaltered.
CONCLUSION: PcG silencers bind and coordinately regulate INK4b and INK4a, but 
not ARF, during a variety of physiological processes. Developmentally regulated 
EZH2 levels are one of the factors that can determine the higher order chromatin 
structure and expression pattern of the INK4b-ARF-INK4a locus, coupling human 
progenitor cell differentiation to proliferation control. Our results revealed a 
chromatin looping mechanism of long-range control and argue against models 
involving homogeneous spreading of PcG silencers across the INK4b-ARF-INK4a 
locus.