6. HNF4α isoforms and their specific interactome
Despite the above findings, few studies have assessed the landscape of
HNF4α isoforms’ specific interactome and their impact on gene
expression. Only three studies have addressed these aspects [31, 32,
65]. The pioneering study performed by Daigo et al. used two
independent HNF4α antibodies, one raised against the F domain and
another against the A/B domain, to pulldown native endogenous HNF4α
isoforms from the human hepatic HepG2 cell line. [65].
Semi-quantitative proteomic analysis revealed that HNF4α could interact
with a wide variety of protein complexes, some associated with the
DNA-dependent protein kinase catalytic subunit, histone
acetyltransferase complexes, alternative splicing, chromatin remodeling,
and nucleosome remodeling. These authors also identified the formation
of heterodimers between HNF4α and HNF4γ and further explored the impact
of this novel interaction on the transcriptional regulation of genes
involved in liver metabolism [65]. With the use of large-scale
approaches, two additional studies indicated that each of these HNF4α
isoforms could influence a variety of genes with different patterns of
expression and that for most tested isoforms, a positive modulatory
effect was observed on their target genes with some exceptions. For
example, the study from Ko et al . [32] reported that
non-canonical isoforms of the P1b group negatively modulate gene
expression. In contrast, Lambert et al. [31] showed that only
the P2a-α9 isoform acted likewise. These discrepancies may result from
the nature of these analyses, where Lambert et al . investigated
the entire transcriptome in their cellular system. In contrast, Koet al. focused on a cluster of genes restricted to inflammation
and immune response. Nevertheless, the study from Lambert et al.raised the possibility that the P1b subgroup of isoforms may act as
negative regulators of the expression of several genes through
interaction with other transcription factors [31]. Another
significant difference between these two studies is the cellular system.
The different or contradictory results reported could be related to the
biological models and the overall design used in each study.
Lambert et al. chose to stably single-insert a
doxycycline-inducible copy of each HNF4α isoform into an intestinal
epithelial cell line (HCT116) devoid of endogenous HNF4α expression.
This approach presented the advantage of comparing the transcriptome in
the absence and presence of any given isoform in an endogenous range
level that would be expected under this biological context. On the other
hand, Ko et al . used a hepatocyte cell line (HuH7) that
endogenously expressed HNF4α and transiently transfected each of the
isoforms in different combinatory setups. This approach presents the
disadvantage of producing possible supraphysiological levels of each of
these isoforms and possible interference of endogenous HNF4α isoforms
naturally produced in this cell line. However, both studies recognize a
complex variability of isoforms in the N- and C-terminal regions and
agree that this could be why the non-canonical isoforms of the P1b-α4 to
α6 subgroup harbor different affinity with the DR1 motif, the classical
and functional response element for HNF4α. Additionally, Ko et
al. noted that canonical P1b group isoforms could bind and activate
transcription, even when the binding sites in their DR1 motif 1 and 2
are mutated [32].
The study by Lambert et al. used powerful and complementary
quantitative proteomic approaches to precisely identify the interactome
of each HNF4α isoform in the context of HCT116 cells [31]. The
authors observed that HNF4α isoforms interacted with a joint group of 69
proteins, for which some were previously identified and discussed above.
For example, interactions with transcriptional activating complexes
CBP/p300 and repressing complexes NCOR1 and 2 were observed (Figure 2).
Other and newly identified large complexes were identified to interact
with most individual HNF4α isoforms [31]. For example, the
Switch/Sucrose-Nonfermentable (SWI/SNF) family of chromatin remodeling
complexes, including the subunits AT-rich interaction domain (ARID)1A,
ARID1B, ARID2, bromodomain containing 7 (BRD7), double PHD fingers 2
(DPF2), polybromo 1 (PBRM1), SWI/SNF related, matrix associated, actin
dependent regulator of chromatin (SMARC)A4, SMARCC1, SMARCC2, SMARCD2,
and SMARCE1 were all identified in this context (Figure 2). These
complexes can activate or inhibit transcription in the presence of
activators or repressors, and alterations in some of their units have
been associated with different types of cancer and neurological
disorders [66-68]. In addition, interactions between several HNF4α
isoforms and subunits of the mediator complex, including mediator
complex (MED)1, MED14, and MED15, were identified (Figure 2) [31].
Interestingly, Daigo et al. previously identified MED16 and MED24
as interactors of HNF4α [65], and some of these interactions were
also observed to take place using GST-pulldown and
co-immunoprecipitations assays [69, 70]. Given this complex’s
importance in regulating the expression of many genes, mutations in some
of these subunits are often associated with different diseases,
including cancer [71, 72]. Interactions between HNF4α isoforms and
enhancer of polycomb homolog 1 (EPC1), BRD8, and E1A binding protein
P400 (EP400), all subunits of the histone H4/ nucleosome
acetyltransferase complex (NuA4/Tip60), were also observed [31].
This complex involves transcriptional regulation, chromatin
modification, cell migration and invasion, mitosis, and genomic
instability (10). In addition, some of these subunits are also
associated with colorectal cancer progression [73].
Finally, additional interactions involving some of the HNF4α P1 and or
P2 isoforms with members of the nucleosome remodeling and deacetylase
(NuRD) complex, including metastasis associated (MTA)1, MTA2, GATA zinc
finger domain containing (GATAD)2A, and GATAD2B have also been observed
(Figure 2) [31]. Likewise to SWI/SNF complexes, NuRD is involved in
regulating transcriptional events, genome integrity, and cell cycle
progression. Defects in the activity of the complex have been linked to
defects in embryonic development, premature aging, oncogenesis, and
cancer progression [74]. Given the importance of the complex in
tumor diseases, some of the members of this complex, such as MTA1, have
been proposed as potential therapeutic targets [75].