ABSTRACT
KAP1/TIF1β
is proposed to be a universal corepressor protein for the KRAB zinc
finger protein (KRAB-zfp) superfamily of transcriptional repressors. To
characterize the role of KAP1 and KAP1-interacting proteins in
transcriptional repression, we investigated the regulation of stably
integrated reporter transgenes by hormone-responsive KRAB and KAP1
repressor proteins. Here, we demonstrate that depletion of endogenous
KAP1 levels by small interfering RNA (siRNA) significantly inhibited
KRAB-mediated transcriptional repression of a chromatin template.
Similarly, reduction in cellular levels of
HP1α/β/γ and SETDB1 by siRNA attenuated
KRAB-KAP1 repression. We also found that direct tethering of KAP1 to
DNA was sufficient to repress transcription of an integrated transgene.
This activity is absolutely dependent upon the interaction of KAP1 with
HP1 and on an intact PHD finger and bromodomain of KAP1, suggesting
that these domains function cooperatively in transcriptional
corepression. The achievement of the repressed state by wild-type KAP1
involves decreased recruitment of RNA polymerase II, reduced levels of
histone H3 K9 acetylation and H3K4 methylation, an increase in histone
occupancy, enrichment of trimethyl histone H3K9, H3K36, and histone
H4K20, and HP1 deposition at proximal regulatory sequences of the
transgene. A KAP1 protein containing a mutation of the HP1 binding
domain failed to induce any change in the histone modifications
associated with DNA sequences of the transgene, implying that
HP1-directed nuclear compartmentalization is required for
transcriptional repression by the KRAB/KAP1 repression complex. The
combination of these data suggests that KAP1 functions to coordinate
activities that dynamically regulate changes in histone modifications
and deposition of HP1 to establish a de novo microenvironment of
heterochromatin, which is required for repression of gene transcription
by
KRAB-zfps.
Tetrameric Oligomerization Mediates Transcriptional Repression by the BRCA1-dependent Kruppel-associated Box-Zinc Finger Protein ZBRK1