Our research is focused on elucidating molecular mechanisms
of gene regulation, with particular emphasis on the function
of co-activators and co-repressors in mediating the action
of transcription regulators involved in tumor suppression
and carcinogenesis, as well as cholesterol and lipid
homeostasis.
Activator-Recruited Cofactor/Mediator Family of
Transcription Co-Activators
We have identified a novel family of mammalian co-activators,
referred to as Activator-Recruited Co-factor
(ARC) that is required for full transcriptional activation
by several transcriptional activators in vitro.
Cloning of many of the ARC subunits indicated that ARC
is related to the previously identified yeast Mediator
co-activator. Recent biochemical studies together with
investigations employing electron microscopy revealed
that the human ARC/Mediator co-activator functions at
least in part by bridging transcriptional activators
and RNA Polymerase II. A substantial portion of the research
in the Näär laboratory is currently centered
on the identification and characterization of ARC/Mediator
subunits that are directly targeted by gene activators,
such as the SMAD family of tumor suppressors, the SREBP
family of cholesterol and lipid regulators, and the inflammatory
and stress-response activator NF-kappaB, to help elucidate
their functional role in mediating activator-signaling
controlling cell proliferation/cancers and cholesterol/lipid
homeostasis.
The ARC/Mediator subunit ARC105 is required for
SREBP gene activation and regulation of cholesterol
and lipid homeostasis
Cholesterol and fatty acids play important functional
roles in metazoans, such as modulating membrane fluidity,
serving as signaling molecules, and providing energy
storage in the form of triacylglycerides. Abnormal cholesterol
and fat levels have been linked to prevalent diseases,
such as atherosclerosis, obesity, type II diabetes, hypertriglyceridemia,
hepatic steatosis, and hypertension (all associated with
Metabolic Syndrome), as well as Alzheimer’s disease
and several types of cancer, including prostate and breast
cancer, underscoring the importance of understanding
fully how cholesterol and lipid homeostasis are modulated
and maintained.
The mammalian SREBP family of bHLH-Zip transcription factors
are critical regulators of cholesterol and fatty acid
homeostasis by controlling the expression of cholesterogenic
and lipogenic genes, as well as promoting differentiation
of adipocytes. Investigations of the molecular mechanism
of SREBP gene regulation by others and us revealed that
human SREBPs can activate target genes by recruiting
the chromatin-targeting CBP/p300 acetyltransferases and
the Pol II-interacting ARC/Mediator co-activators. The
activation domain of SREBPs can interact with the CBP/p300
KIX domain, a sequence bound by many activators, however
the molecular target of SREBPs in the ARC/Mediator has
been unclear. We have now demonstrated that the 105 kDa
subunit of ARC/Mediator (ARC105, also known as MED15),
is a functionally important and evolutionarily conserved
mediator of SREBP gene activation. ARC105 interacts with
SREBPs in vitro and in vivo, and RNAi-mediated ablation
of ARC105 in human cells inhibits transcription of SREBP
target genes. Collaborative NMR-based structural analysis
with the group of Gerhard Wagner at Harvard Medical School revealed
that the SREBP-binding domain in ARC105 folds into a
three-helix bundle structure with striking similarity
to the CBP/p300 KIX domain. This finding may explain,
at least in part, the ability of SREBPs to recruit both
CBP/p300 and ARC/Mediator co-activators.
The Caenorhabditis elegans SREBP homologue sbp-1 plays
a key role in regulating fat storage and metabolism in
this nematode by regulating the expression of lipogenic
enzymes, indicating evolutionary conservation of the
function of SREBP in controlling lipid homeostasis in
metazoans. The ARC/Mediator co-activator is also conserved
in C. elegans and we have therefore initiated
a collaborative effort with the groups of Anne Hart and
Sander van den Heuvel at the MGH Cancer Research Center
to investigate the involvement of the ARC/Mediator and
the ARC105 subunit in SREBP-dependent gene regulation
and fat metabolism/storage in C. elegans. Remarkably,
RNAi-mediated ablation of the C. elegans ARC105
homolog mdt-15 results in the same phenotype
as ablation or mutation of SREBP, i.e. pale or clear
worms which indicates mobilization of lipids stored in
intestinal fat granules. In sharp contrast, RNAi-mediated
ablation of 20 other ARC/Mediator subunits (out of a
total of 23 conserved subunits) did not yield this phenotype,
suggesting that ARC105 is uniquely involved in SREBP
signaling and fat homeostasis in C. elegans.
By employing gas chromatography and mass spectrometry
we have also found that RNAi of mdt-15 results
in a dramatically altered fatty acid profile, similar
to that observed when depleting sbp-1. Moreover,
RNAi-mediated depletion of mdt-15 reveals a
critical role for this ARC/Mediator subunit in controlling
transcription of genes governing fatty acid homeostasis
in C. elegans. Collectively, our findings demonstrate
that ARC105 is a key effector of SREBP-dependent gene
regulation and control of lipid homeostasis in metazoans
(Yang et al., Nature 2006). Dysregulation of ARC105 in
humans could thus potentially contribute to diseases
caused by cholesterol and fatty acid imbalance, including
Metabolic Syndrome and several types of cancer.
ARC105 Mediates Gene Activation by Several Physiologically
and Developmentally Important Transcription Regulators
We have in collaboration with Xi He’s group at
Harvard Medical School found that the ARC105 is also
a critical component of the TGFß/SMAD2/3 signaling
pathway (Kato et al., Nature 2002). Our recent unpublished
findings indicate that ARC105 is also involved in mediating
signaling by the stress-response regulator NF-kappaB.
These results together suggest that ARC105 is a central
integrator of several cellular signaling pathways implicated
in human disease.
Gal11, the Yeast Homolog of ARC105, is Required
for Multidrug Resistance (MDR) in Yeast
Because ARC105 serves as a key transducer of multiple
important gene regulatory signals in metazoans and based
on the conservation of the ARC/Mediator co-activator
complex in the yeast S. cerevisiae, we have
asked whether the yeast Mediator harbors a subunit homologous
to ARC105. Using bioinformatics analyses, we have now
found that the activator-targeted yeast Mediator subunit
Gal11 contains a KIX domain, suggesting that ARC105 and
Gal11 are homologous subunits. To identify yeast
transactivators that utilize the Gal11 KIX domain as
their regulatory conduit, we have employed affinity chromatography
of yeast whole cell extract with the Gal11 KIX domain.
This led to the purification and identification of the
pleiotropic drug resistance protein 1 (PDR1) transcription
factor as a Gal11 KIX interaction partner. Our genetic
studies have confirmed the critical and specific importance
of Gal11 in PDR1 function and in multidrug resistance
(MDR) in yeast. In collaboration with the Wagner lab,
we have initiated NMR studies of the Gal11 KIX domain
in complex with the PDR1 transactivation domain to gain
further insights into mechanisms of gene regulation in
eukaryotes. This work will have direct impact on our
understanding of MDR in fungi and may ultimately yield
novel anti-fungal therapeutics. As gain-of-function mutations
in PDR1 have been shown to result in increased MDR in
yeast, our finding may also have implications for the
regulatory circuitry guiding MDR in human cancers.
Identification of novel Retinoblastoma (pRB) Tumor
Suppressor-Associated Polypeptides
Lastly, we have initiated a collaboration with the group
of Nick Dyson at the MGH Cancer Center to identify nuclear
binding partners of the human Retinoblastoma (pRB) tumor
suppressor. We have developed an effective pRB-affinity
purification procedure that has allowed us to purify
and identify by tandem mass spectrometry a number of
nuclear pRB-binding protein complexes, several of which
have previously been implicated in transcriptional repression,
consistent with the observed functional role of pRB in
inhibition of gene expression. Surprisingly, we have
additionally found strong biochemical and functional
association of pRB with the Anaphase Promoting Complex/Cyclosome
(APC/C), a key cell cycle-regulatory complex, suggesting
that pRB may also act more directly to modulate cell
cycle progression. We are currently confirming these
associations and developing functional assays to assess
their relative contribution to the activities of pRB
as a transcriptional regulator and tumor suppressor.
Center
for Cancer Research 