TRANSCRIPTION FACTOR BINDING SITE ENRICHMENT ANALYSIS PREDICTS\nDRIVERS OF ALTERED GENE EXPRESSION IN NONALCOHOLIC\nSTEATOHEPATITIS
The molecular mechanisms behind the transition from simple steatosis to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD) are not clearly understood. This hinders development of effective therapies for treatment and prevention of NASH. In this study expression profiling data from normal, steatosis, and NASH human livers was used to predict transcription factors that are misregulated as mechanistic features of NAFLD progression. Previously-published human NAFLD gene expression profiling data from normal, steatosis, and NASH livers was subjected to transcription factor binding site enrichment analysis. Selected transcription factors that bind enriched transcription factor binding sites were analyzed for changes in expression. Distinct transcription factor binding sites were enriched in genes significantly up-or down-regulated in NASH livers. Those enriched in up-regulated genes were bound by transcription factors such as FOXA, CEBP, and HNF1 family members, while those enriched in down-regulated genes were bound by nuclear receptors involved in xenobiotic sensing and lipid metabolism. Levels of mRNA and protein for selected transcription factors were significantly changed during disease progression. The study indicates that NAFLD progression involves changes in activity or expression of transcription factors that regulate genes involved in hepatic processes known to be altered in NASH. Transcription factors such as PPAR receptors, FoxA family members, and HNF4A might be targeted therapeutically to prevent NAFLD progression.
1. Introduction<!>2.1 Human Liver Samples<!>2.2 Human NAFLD Microarray Data<!>2.3 Transcription Factor Binding Site Analysis<!>2.4 Immunoblot Analysis<!>3.1 TFBS Enrichment Analysis of Human NAFLD GEP Data<!>3.2 TFs that bind TFBS enriched in genes up-regulated in human NASH\nlivers<!>3.3 TFs that bind TFBS enriched in genes down-regulated in human NASH\nlivers<!>4. Discussion
<p>Approximately 30–40% of the U.S. population has nonalcoholic
fatty liver disease (NAFLD) [1] and 5–17% are estimated to have progressed to
nonalcoholic steatohepatitis (NASH) [2]. NAFLD occurs when altered processing of fatty acids and
triglycerides leads to abnormal hepatic lipid accumulation resulting in steatosis.
Increased inflammation and oxidative stress can cause progression to NASH, which is
characterized by fibrosis and higher risk of cirrhosis and hepatocellular carcinoma
[2]. While the
pathological progression and characteristics of NAFLD are defined, a clear
understanding of the underlying mechanisms is lacking.</p><p>Gene expression profiling (GEP) has been used to identify changes in
regulatory mechanisms and signaling that drive disease progression [3;4]. However, this remains challenging due to the complexity of the
resulting data and the poorly-understood functions of many genes. Cell signaling
pathways ultimately target transcription factors (TFs) to alter gene expression.
Thus, identifying TFs that drive changes in gene expression during disease
progression may reveal key signaling pathways that are misregulated and could be
targeted therapeutically. To regulate transcription TFs bind specific DNA sequences,
generally referred to as transcription factor binding sites (TFBS). TFBS enrichment
analysis has gained utility in elucidating the mechanistic basis for altered gene
expression [4]. In this
method, potential TFBS within regulatory regions of genes that show significant
changes in expression between distinct conditions are identified. Statistical
analysis is then performed to identify TFBS enriched around genes that are either
up-or down-regulated. The enrichment of particular TFBS in the promoters of
co-expressed genes can predict which TFs drive changes in their expression and
ultimately reveal underlying regulatory mechanisms.</p><p>Previously, a GEP data set representing individual microarrays for multiple
normal, steatosis, and NASH liver samples was generated to study mechanisms
underlying NAFLD progression [5]. In the current study, TFBS enrichment analysis was performed to
identify those that are over-represented in the promoter regions of genes either
significantly up-regulated or down-regulated in NASH livers compared to normal or
steatosis livers. The results revealed enriched TFBS bound by TFs that regulate key
liver functions. In some cases their expression was found to be altered during NAFLD
progression. Altogether the results indicate changes in the activity and/or
expression of specific TF families during NAFLD progression that could be targeted
therapeutically to prevent NASH or limit its severity.</p><!><p>Human liver samples representing the complete spectrum of NAFLD were
acquired from the Liver Tissue Cell Distribution System. Post mortem and liver
biopsy samples were scored and diagnosed using the Kleiner NAFLD activity
scoring method [6], as
previously described [5].
Demographics and other information have been previously published [7].</p><!><p>GEP data from individual Affymetrix Human GeneChip 1.0 ST arrays of
normal (n=19), steatosis (n=10), NASH (n=16) liver
samples were utilized in the analyses of TFBS enrichment and TF mRNA levels. A
total of 33,252 genes were analyzed and are accessible at the ArrayExpress
public repository under the accession number E-MEXP-3291 (http://www.webcitation.org/5zyojNu7T). All microarray data,
archiving, and analysis were generated as previously described [5]. Analysis of gene expression was
done as follows. In most cases, expression data for each gene examined was
derived from GEP for each biopsy sample. These data were then converted to Log2
transformed values (+/− the standard deviation) normalized to
the median of the normal or steatosis groups (see Fig. 2 as an example). In some cases Log2 transformed values for a
gene were derived through normalization to the median expression of a related
gene in the normal or NASH groups (see Fig.
1 for an example) Statistical analysis of the Log2 transformed values
was performed using 1-way ANOVA with post hoc Tukey testing.</p><!><p>Genes that were significantly up-or down-regulated between normal and
NASH microarray data sets were subjected to TFBS enrichment analysis using the
TRANSFAC (Biobase) TFBS database and software package. Predicted TFBS were
identified in gene regions 5000 bp upstream and downstream of the transcription
start site (TSS) for all genes contained in the microarrays. Statistical
analysis was then performed to identify TFBS enriched in the significantly up-or
down-regulated genes relative to all genes in the expression profiling set. This
yielded two significance scores for each TFBS: the Fisher score, which measured
its occurrence in each gene, and the Z score, which measured the number of its
occurrences in the promoter of each gene. In the TRANSFAC analysis Z scores and
Fisher of ≤ 0.05 were considered significant. An additional analysis was
done using genes that were significantly up-or down-regulated between steatosis
and NASH microarray data sets. In this case, the JASPAR TFBS database and the
oPOSSOM platform were used for enrichment analysis. Z scores and Fisher scores
were generated but, in this case, Z scores were expressed differently and scores
>1.6 were considered significant.</p><!><p>Protein from human liver whole cell lysates (80 Î¼g/sample) was
separated in 10% SDS-PAGE gels and transferred to polyvinylidene
difluoride membranes. Membrane blocking and antibody application was
accomplished using 5% non-fat dry milk dissolved in phosphate-buffered
saline with 0.1% Tween-20. Primary antibodies for HNF3A/B (sc-377033)
and HNF1A (sc-10791) were acquired from Santa Cruz Biotechnology (Santa Cruz,
CA) while those for LXRÎ±/Î² (ab24532) and Pan-Cadherin (ab16505)
were purchased from Abcam (Cambridge, MA). Immunoblots were imaged using
Supersignal West Femto chemiluminescent substrate (Fisher Scientific, Rockford,
IL). Densitometry was performed using ImageJ software (NIH, Bethesda, MD).</p><!><p>Previously-obtained GEP data were used to identify TFBS
significantly-enriched in the extended promoter regions (+/−
5000 bp relative to the TSS) of genes up-regulated or down-regulated in NASH.
Significantly-enriched TFBS from the normal versus NASH comparison are listed in
Tables 1 (up-regulated genes) and
2 (down-regulated genes) while those
from the steatosis versus NASH comparison are listed in Table 3 (upregulated genes, columns 1–3 and
down-regulated genes, columns 4–6). For both comparisons, the TFBS
enriched in the up-regulated genes were mostly distinct from those enriched in
the down-regulated genes. While the two comparisons did not yield identical
results, many enriched TFBS were common (Table
4). The liver-specific actions of multiple TFs listed, such as NKX
and PAX families, are poorly understood, so further analysis was focused on
those with known hepatic functions. HNF4A, and members of the C/EBP, HNF1, HNF3
(FOXA), PPAR, LXR, and COUP-TF families regulate lipid
metabolism, xenobiotic sensing/metabolism, and lipotoxic stress, all of which
are impacted in NASH [8–11].
Changes in TF activity or expression during NAFLD progression may alter their
ability to regulate target genes and thereby contribute to NAFLD progression and
NASH pathology.</p><!><p>TFs that bind TFBS enriched in NASH up-regulated genes include members
of the C/EBP, FOXA, and HNF1 families. Their enrichment in up-regulated genes
implies that their transcription-promoting activity is enhanced or that their
transcription-repressing activity is diminished.</p><p>C/EBP family members bind similar DNA sequences as homo-or heterodimers
[12]. Further
analysis was focused on those with known hepatic functions, namely C/EBPalpha
(CEBPA), C/EBPbeta (CEBPB), C/EBPdelta
(CEBPD), and C/EBPgamma (CEBPG). These TFs
regulate hepatic energy metabolism, liver regeneration, and the inflammatory
acute phase response [8].
The GEP data set was analyzed to reveal that CEBPA,
CEBPB, and CEBPD mRNAs were expressed at
similar levels in normal (not shown) and NASH livers while
CEBPG mRNA levels were significantly lower (Fig. 1A). Furthermore, C/EBP mRNA levels do not
significantly change between normal, steatosis, and NASH livers (Fig. 2A–D), indicating that
potentially-increased transcriptional activity is not due to elevated
expression.</p><p>HNF3 TFs are essential regulators of liver-specific gene expression
[9] and include three
isoforms, now referred to as FOXA1, FOXA2, and FOXA3. FOXA1 and FOXA2 mRNAs were
expressed at significantly higher levels than FOXA3 mRNA in normal and NASH
livers (Fig. 1B,C), but all FOXA mRNAs were
significantly down-regulated in NASH (Fig.
2E–G). These TFs act primarily as transcriptional activators,
so down-regulated mRNA expression is not consistent with enrichment of their
binding sites in NASH up-regulated genes. Western blotting shows that, in
contrast to mRNA, the combined level of the FOXA1 and FOXA2 proteins was
significantly elevated in NASH livers (Fig.
3A,B), which is consistent with increased expression of their target
genes.</p><p>The HNF1 family consists of HNF1A and HNF1B, which can form homo-or
heterodimers to bind DNA [9]. Only HNF1A is robustly expressed in adult liver
[13;14], consistent with results from the GEP
data, which shows that HNF1B mRNA levels are significantly lower than those of
HNF1A in normal liver (Fig. 1D). GEP data
analysis shows that HNF1A mRNA is significantly down-regulated upon progression
to NASH (Fig. 2H). Because HNF1A functions
predominantly as a transcriptional activator and its binding site was enriched
in NASH-upregulated genes, HNF1A protein levels were measured by Western blot
(Fig. 3A,C). The results showed a
significant increase of HNF1A protein in NASH livers (Fig. 3B). HNF1B mRNA levels are significantly
up-regulated in NASH (Fig. 2I) to the
extent that they become significantly higher than those of HNF1A (Fig. 1D). Due to the very limited amount of liver
extracts, measurement of HNF1B protein was not feasible. However, the results
suggest that combined HNF1 expression is elevated in NASH livers and drives
increased expression of target genes.</p><!><p>TFBS enriched in NASH-downregulated genes include those bound by members
of the nuclear receptor superfamily that have hepatic functions perturbed in
NASH, including xenobiotic sensing/metabolism, lipid metabolism, and
inflammation. Their enrichment in down-regulated genes implies that their
transcription-repressing activity is enhanced or that their
transcription-promoting activity is diminished.</p><p>As heterodimers with RXR, both LXR isoforms bind the same DNA element
[15]. LXRalpha
(NR2H3) is the primary regulator of cholesterol and bile
acid metabolism in adult liver [16]. Accordingly, LXRalpha mRNA is expressed at higher
levels relative to LXRbeta mRNA (NR2H2) in normal livers (Fig. 4A). However, progression to NASH
results in a significant drop in both mRNAs (Fig.
4B,C). Western blot analysis of liver extracts with an antibody
recognizing both LXR isoforms shows that, in contrast to the decline in mRNA,
LXR protein levels trend upward in NASH livers (Fig. 4D). The increase did not reach significance but the median
level of LXR protein is clearly higher in NASH relative to normal and
steatosis.</p><p>HNF4A is a nuclear receptor that regulates many liver-specific genes.
The GEP data shows that its mRNA is significantly decreased in NASH livers
(Fig. 5A). The same was observed for
GABPA (Fig. 5B) which has multiple
interactions with HNF4A. HNF4A binds to the GABPA promoter,
suggesting a positive role in its expression [17] and consistent with the coordinate
down-regulation of both mRNAs in NASH. A recent study showed significant
co-occupancy of GABPA and HNF4A near the TSS of selected HNF4A target genes and
established that HNF4A and GABPA interact by co-immunoprecipitation
[18]. Thus, decreased
expression of HNF4A and GABPA may cause down-regulation of their target genes in
NASH.</p><p>The PPAR family of nuclear receptors has three isoforms: PPARalpha
(PPARA), PPARdelta (PPARD), and PPARgamma
(PPARG). Various fatty acid derivatives bind PPARs and
increase or inhibit transcription at target genes [19]. PPARs can also repress transcription in
the unliganded state [20;21]. In normal livers,
PPARA mRNA is expressed at significantly higher levels than
mRNAs for PPARD or PPARG (Fig. 5C). During NAFLD progression,
PPARA and PPARG mRNA levels do not change
significantly (Fig. 5D,F) while
PPARD mRNA is significantly up-regulated in NASH livers
(Fig. 5E).</p><p>The nuclear receptors COUP-TFI (NR2F1) and COUP-TFII
(NR2F2) are transcriptional repressors [22]. GEP data analysis shows that
COUP-TFII is expressed at significantly higher levels in normal and NASH livers
(Fig. 7A). mRNAs for both COUP-TFs increase significantly in NASH relative to
steatosis (Fig. 7B,C) but the magnitude of the change is greater for COUP-TFII.
Thus, the increased expression of COUP-TFs may cause down-regulation of genes in
NASH.</p><!><p>The mechanisms underlying NAFLD progression to NASH are not well-understood
resulting in a dearth of effective therapeutic options. The current study utilizes
bioinformatics to predict TFs that drive changes in gene expression which occur upon
progression to NASH. These TFs and the signaling pathways that regulate their
activity and/or expression provide potential targets for therapeutic intervention.
TFBS enrichment analyses performed revealed a potential role for hepatic nuclear
factors and nuclear receptors in the altered expression profile that characterizes
NASH.</p><p>Distinct groups of TFBS were enriched in the genes repressed or activated
upon progression to NASH. The activated genes were enriched in sequences bound by
members of the C/EBP, FOXA, and HNF1 families, suggesting that the
transcription-promoting function of these proteins is increased. Levels of C/EBP
proteins were not measured in this study but their activity can be regulated by
other mechanisms. C/EBPalpha and C/EBPbeta are expressed as multiple isoforms
through alternative translation start sites [8;12]. C/EBPbeta isoforms
include LAP and LAP*, that activate transcription, and LIP, that represses
transcription when hetero-dimerized with LAP or LAP*. The relative
expression of these isoforms may alter transcription of target genes. In addition,
post-translational modification regulates C/EBP DNA binding and transcriptional
activity [12]. C/EBP proteins
are targets of cytokine signaling, which is activated in NASH due to chronic
inflammation. Expression of C/EBP isoforms and their post-translational
modifications in NAFLD have not been investigated.</p><p>FoxA proteins are critical for proper hepatic function. FoxA1 and A2 mRNAs
were significantly decreased upon progression to NASH. In contrast, combined
expression of these proteins is significantly increased in NASH livers. Consistent
with the current findings, FOXA2 overexpression in transgenic mouse liver caused
significant down-regulation of mRNAs from the endogenous FOXA genes [23;24], indicating the existence of a feedback mechanism for regulation
of FOXA expression that is perturbed in NASH. Elevated FOXA1 and A2 levels could
increase expression of their target genes.</p><p>Hepatic FOXA2 overexpression was also associated with increased
lipid-containing vesicles, elevated serum bile acids, decreased expression of
sinusoidal bile uptake transporters, mitochondrial membrane damage/swelling, influx
of immune cells, and fibrosis [23;24]. Since many of
these features are also observed in NASH, dysregulated FOXA2 expression and activity
may contribute to NAFLD progression, and thus highlight its potential as a
therapeutic target. Deacetylation by SIRT1 leads to reduced levels of FOXA2 protein
through increased degradation [25]. Small molecule sirtuin activators are under development, and
their potential use in NAFLD treatment has been suggested [26].</p><p>While HNF1A mRNA levels decline upon NAFLD progression, protein levels
increase in NASH livers along with levels of HNF1B mRNA. While HNF1B protein levels
were not measured, the combined results raise the possibility that total HNF1
expression is elevated in NASH. Because normal hepatocytes express only HNF1A
[13;14], it is not clear whether HNF1B up-regulation
occurs in hepatocytes or in another cellular compartment that expands in NASH, such
as stellate or inflammatory immune cells. Because HNF1A regulates genes involved
with carbohydrate and lipid metabolism, drug metabolism and transport, co-expression
of both HNF1 proteins in NASH hepatocytes could have a significant impact. It is
unclear whether target gene specificity would be altered upon heterodimer formation
or whether heterodimers would be cooperative or antagonistic at target genes.</p><p>Genes down-regulated in NASH were enriched in sequences bound by several
nuclear receptors. While both LXR mRNAs were down-regulated upon progression to
NASH, combined protein levels trend upward. In support, Ahn et al. demonstrated that
LXRalpha staining was more intense in NASH livers and correlated with the extent of
hepatic inflammation and fibrosis [27]. The enrichment of LXR binding sites in NASH-downregulated genes
could be explained in several ways. First, LXR agonist treatment in mouse liver
caused both activation and repression of genes [15]. Interestingly, LXR-repressed genes are
enriched in those associated with PPAR signaling [15]; PPAR binding sites were also enriched in
NASH down-regulated genes. The gene repressive activity of LXR may predominate in
NASH. Second, the endogenous ligands for LXR nuclear receptors are oxysterol
cholesterol derivatives [28].
Because cholesterol and bile acid metabolism is altered during NAFLD progression
[29–31], the mix of endogenous LXR ligands, both
agonists and antagonists, could influence the balance of transcription-activating
versus –repressing LXR activity.</p><p>Consistent with the enrichment of its binding site in NASH down-regulated
genes, HNF4A mRNA was significantly decreased in NASH livers. Metabolomic analysis
of NAFLD liver samples revealed a shift from classical biIe acid synthesis toward
the alternative pathway in NASH [31]. Chenodeoxycholic acid, a product of the alternative pathway,
negatively affects HNF4A expression and activity [32]. Down-regulation of HNF4A mRNA in NASH is
also consistent with elevated IL-1beta signaling, which occurs in human NASH livers
[33]. Treatment of mice
with IL-1beta downregulated HNF4A mRNA and protein [34].</p><p>While HNF4A protein expression was not measured in the current study,
several lines of evidence suggest reduced expression or activity. First, HNF4A binds
and activates the HNF1A [17;35] and CYP8B1 [36] promoters; mRNA levels for both are
significantly decreased in NASH (Fig. 2H and
[31]). Second, the
effects of hepatic HNF4A knockout in mice share key characteristics with human
NAFLD, including hepatic lipid accumulation and increased serum bile acids
[37]. The latter was
associated with downregulation of mRNAs for the NTCP and Oatp1a1 bile acid uptake
transporters. While NTCP mRNA was unchanged during human NAFLD progression,
SLCO1B3 (OATP1B3) mRNA is significantly down-regulated
[5]. HNF4A binds linoleic
acid [38], and one synthetic
ligand has been reported [39]. Accumulated evidence suggests that impaired expression or
activity of HNF4A contributes to NAFLD progression or severity, indicating its
potential as a therapeutic target.</p><p>While levels of PPAR mRNAs were not dramatically altered during NAFLD
progression, the enrichment of PPAR binding sites in NASH down-regulated genes
suggests that PPAR activity is impaired. In support, multiple PPAR agonists have
been shown to slow NAFLD progression and/or ameliorate its severity in animal models
[40–42]. While fibrates, which are weak PPARalpha
agonists, have variable effects on NAFLD in humans, newer, more potent agonists such
as the dual PPARalpha/delta agonist, GFT505, and the PPARalpha-selective agonist,
K-877, have shown promise in clinical trials [40–42].</p><p>COUP-TFI and COUP-TFII, generally considered to be transcriptional
repressors, can antagonize the activating function of nuclear receptors such as
PPARs and HNF4A [43].
Although COUP-TF protein levels were not measured, their mRNAs were significantly
up-regulated in NASH suggesting the possibility they down-regulate gene expression
in NASH through increased expression. COUP-TF expression has not been measured in
NAFLD biopsies but other evidence suggests a potential role for COUP-TFII in NAFLD
progression. Mice heterozygous for the COUP-TFII gene are lean and protected from
the effects of high fat diet, including excess hepatic lipid storage [44]. COUP-TFII mRNA levels are low in
human hepatocytes but high in hepatic endothelial and Kupffer cells [45]. The latter are activated by
lipotoxic stress and secrete IL-1β and TNF-α [46], which activate stellate cells to
produce collagen that contributes to fibrosis. Thus, activated Kupffer cells may
contribute to increased COUP-TFII expression in NASH.</p><p>This study combined TFBS enrichment with analysis of TF expression to reveal
potential drivers of altered gene expression in NASH. Among hepatic TFs, the current
study indicates that FOXA2, PPARs, LXRs, and HNF4 are misregulated in NASH and
represent promising therapeutic targets. Finally, this study revealed a disconnect
between the pattern of mRNA and protein expression for multiple TFs during NAFLD
progression. This is consistent with several studies that measured the correlation
between protein and RNA abundance in cell populations. The results showed a limited
correlation, implying that only about 40% of variation in protein abundance
can be explained by abundance of the corresponding mRNA and that
post-transcriptional mechanisms generally make a strong contribution to protein
expression levels [47–50]. Altogether these observations
suggest that quantitative proteomic analysis of patient biopsies would add
significantly to knowledge of NAFLD pathology by providing a more accurate readout
of protein expression than can be predicted from GEP analysis.</p>