Supplementary Figure Legends 1-3 from Cancer Resistance in Transgenic Mice Expressing the SAC Module of Par-4

Zinc fingers and homeoboxes 2 (Zhx2) are transcriptional regulators of liver gene expression with key functions in embryonic development as well as tissue regeneration in response to damage and disease, presumably through its control of target genes. Previous microarray data suggested that elongation of very long chain fatty acids-3 (Elovl3), a member of the ELOVL family of enzymes that synthesize very long chain fatty acids (VLCFAs), is a putative Zhx2 target gene. VLCFAs are core component of ceramides and other bioactive sphingolipids that are often dysregulated in diseases and regulate key cellular processes including proliferation. Since several previously identified Zhx2 targets become dysregulated in liver damage, we investigated the relationship between Zhx2 and Elovl3 in liver development, damage, and regeneration. Here, using mouse and cell models, we demonstrate that Zhx2 positively regulates Elovl3 expression in the liver and that male-biased hepatic Elovl3 expression is established between 4 and 8 wk of age in mice. Elovl3 is dramatically repressed in mouse models of liver regeneration, and the reduced Elovl3 levels in the regenerating liver are associated with changes in hepatic VLCFAs. Human hepatoma cell lines with forced Elovl3 expression have lower rates of cell growth; analysis of synchronized cells indicates that this reduced proliferation correlates with cells stalling in S-phase and lower mRNA levels of cell cyclins. Taken together, these data indicate that Elovl3 expression helps regulate cellular proliferation during liver development and regeneration, possibly through control of VLCFAs.NEW & NOTEWORTHY Numerous targets of the transcription factor Zhx2 are dysregulated in liver disease. We show that the elongase Elovl3 is a novel Zhx2 target. Elovl3 and Zhx2 expression change during liver regeneration, which is associated with changes in very long chain fatty acids. Forced Elovl3 expression reduces cell growth and blocks cell cycle progression. This suggests that Elovl3 may account, at least in part, for the relationship between Zhx2 and proliferation during liver development and disease.

Liver cancer, comprised primarily of hepatocellular carcinoma (HCC), is the third leading cause of cancer deaths worldwide and increasing in Western countries. We previously identified the transcription factor zinc fingers and homeoboxes 2 (Zhx2) as a regulator of hepatic gene expression, and many Zhx2 target genes are dysregulated in HCC. Here, we investigate HCC in Zhx2-deficient mice using the diethylnitrosamine (DEN)-induced liver tumor model. Our study using whole-body Zhx2 knockout (Zhx2(KO)) mice revealed the complete absence of liver tumors 9 and 10 months after DEN exposure. Analysis soon after DEN treatment showed no differences in expression of the DEN bioactivating enzyme cytochrome P450 2E1 (CYP2E1) and DNA polymerase delta 2, or in the numbers of phosphorylated histone variant H2AX foci between Zhx2(KO) and wild-type (Zhx2(wt)) mice. The absence of Zhx2, therefore, did not alter DEN bioactivation or DNA damage. Zhx2(KO) livers showed fewer positive foci for Ki67 staining and reduced interleukin-6 and AKT serine/threonine kinase 2 expression compared with Zhx2(wt) livers, suggesting that Zhx2 loss reduces liver cell proliferation and may account for reduced tumor formation. Tumors were reduced but not absent in DEN-treated liver-specific Zhx2 knockout mice, suggesting that Zhx2 acts in both hepatocytes and nonparenchymal cells to inhibit tumor formation. Analysis of data from the Cancer Genome Atlas and Clinical Proteomic Tumor Consortium indicated that ZHX2 messenger RNA and protein levels were significantly higher in patients with HCC and associated with clinical pathological parameters. Conclusion: In contrast to previous studies in human hepatoma cell lines and other HCC mouse models showing that Zhx2 acts as a tumor suppressor, our data indicate that Zhx2 acts as an oncogene in the DEN-induced HCC model and is consistent with the higher ZHX2 expression in patients with HCC.

Hepatic PRR and its soluble form, sPRR, contribute to triglyceride and cholesterol homeostasis and hepatic inflammation. Deletion of hepatic PRR decreased triglyceride levels through a PRR-PPARγ-dependent mechanism but increased hepatic cholesterol synthesis through sPRR-medicated upregulation of SREBP-2. Our study highlighted a new paradigm of cross talk between the liver and the adipose tissue involving cholesterol and sPRR.

The mammalian Cytochrome P450 (Cyp) gene superfamily encodes enzymes involved in numerous metabolic pathways and are frequently expressed in the liver. Despite the remarkably high sequence similarity of Cyp2a4 and Cyp2a5 genes and their surrounding genomic regions, they exhibit differences in expression in the adult mouse liver. For example, Cyp2a4 is highly female-biased whereas Cyp2a5 is only moderately female-biased and Cyp2a4, but not Cyp2a5, is activated in liver cancer. We hypothesized that the limited sequence differences may help us identify the basis for this differential expression. An antisense expressed sequence tag had been uniquely annotated to the Cyp2a4 gene which led us to investigate this transcript as a possible regulator of this gene. We characterized the full-length antisense transcript and also discovered a similar transcript in the Cyp2a5 gene. These transcripts are nuclear long noncoding RNAs that are expressed similarly to their sense mRNA counterparts. This includes the sex-biased and liver tumor differences seen between the Cyp2a4 and Cyp2a5 genes, but we also find that these two genes and their antisense transcripts are expressed within different zones of the liver structure. Interestingly, while the differences in sex-biased expression of the mRNAs are established 1-2 months after birth, the antisense transcripts exhibit these expression differences earlier, at 3-4 weeks after birth. By analyzing published genomic data, we have identified candidate transcription factor binding sites that could account for differences in Cyp2a4/Cyp2a5 expression. Taken together, these studies characterize the first antisense RNAs within the Cyp supergene family and identify potential transcriptional and post-transcriptional mechanisms governing different Cyp2a4 and Cyp2a5 expression patterns in mouse liver.

The overarching goalsof the University of Kentucky (UK) IMERS workshops, supported by an IPERT grant, are to empower faculty at minority-serving institutions (MSIs) to develop and submit competitive research proposals through intensive grant-writing skills training; to build research-related individual and institutional capacity through training on mentorship of student researchers; and to sustain workshop momentum by embedding multiple levels of mentored proposal development support during and after each workshop. We hypothesize that this targeted training will enhance the research environment at MSI's, increase the diversity of NIH-funded investigators and improve the training of underrepresented students in biomedical research, thus providing a feed-forward mechanism to expand the diversity of the US biomedical workforce in future years. A hallmark of IMERS is to engage participants in hands-on, active-learning style grant-writing training for faculty who are poised to submit NIH proposals ranging from development/exploratory to R01s. This training is provided by UK faculty and experienced research development professionals. The IMERS model incorporates guided writing, participatory training, and active learning. Training is comprised of several modalities. First, IMERS offers two 3-day grant-writing retreats/year on the UK campus. These workshops are designed for highly motivated investigators who have submitted proposals to NIH without success and those who have been actively planning NIH submission; ~25 faculty are selected for each workshop via an on-line application. On-site workshops include consultation with actively funded UK investigators and staff from the UK Proposal Development Office; we strive to maintain post-workshop interactions between participants and UK faculty/staff. Three 2-day off-site workshops/year are also offered. The IMERS staff will work with off-site institutions to tailor the workshops to specific needs. In-person workshops have been postponed due to COVID-19 restrictions, but will resume when safe to do so. However, we have developed an active series of virtual workshops and seminars during this time, and we anticipate that these and additional on-line activities will continue as a third arm of training even after restriction are lifted. Travel and lodging expenses for faculty to attend UK workshops, for the IMERS team to travel, are covered by the grant. Workshop sessions cover numerous topics, including: using NIH resources for program/funding information, budget issues, the proposal review process, writing a high-impact specific aims page and an effective research strategy, rigor and reproducibility, responsible conduct of research, NIH Biosketch, navigating the NIH resubmission process, and mentoring student researchers. Program evaluation, based in part on participant survey data, communication email and an active ListServ and subsequent submission rate and success, validates that program participants have increased confidence in their grant-writing abilities and are succeeding in obtaining NIH funding.

Liver fibrosis is a consequence of persistent liver injury that can result from many insults, including exposure to natural and man‐made environmental hepatotoxins, viral infection, excessive alcohol consumption, and non‐alcoholic fatty liver disease. Carbon tetrachloride (CCl4) is an environmental toxin that can be metabolized into highly reactive toxic radicals by hepatic enzymes, primarily cytochrome P‐450 2E1 (Cyp2E1), that can damage proteins, lipids, DNA and other macromolecules. Chronic CCl4 exposure to mice injures hepatocytes and is a well‐established experimental model for human liver fibrosis. Our understanding of environmental factors influencing liver fibrosis far exceeds our knowledge of genetic factors. However, human studies provide strong evidence the fibrosis has a robust heritable component, and several genes associated with fibrosis and other liver diseases have been identified. Studies in genetically tractable organisms, including mice, complement human studies and continue to identify genes that contribute to liver disease. Moreover, mice provide experimental systems to elucidate disease mechanisms. Previously, a Quantitative Trait Locus (QTL) analysis identified a region on Chromosome 15 called hFib1 that contributed to the high liver fibrosis phenotype observed in BALB/cJ mice when given CCl4. The region of this QTL contains the gene encoding the transcription factor Zhx2, which we showed previously to be mutated in BALB/cJ mice (called Zhx2Afr1), but not in other BALB/c substrain. Furthermore, our studies indicate that Zhx2 controls the expression of hepatic genes and influences the extent of liver damage in mice maintained on a high‐fat diet. These findings led us to hypothesize that the increased liver fibrosis phenotype seen in BALB/cJ mice treated with CCl4 was due to reduced Zhx2 expression. To test this, BALB/cJ mice were crossed with C57BL/6 mice that are heterozygous for a null Zhx2 allele (Zhx2WT/KO) generated by gene targeting to obtain male and female mice that expressed (Zhx2WT/Afr1) or did not express (Zhx2KO/Afr1) Zhx2. Mice at 8 weeks of age were treated with 0.5mL/kg of 1:10 CCl4 in mineral oil or mineral oil alone twice weekly for six weeks. Forty‐eight hours after the last injection, plasma and liver were harvested for further analysis. Formalin‐fixed liver sections stained for Hematoxylin and Eosin, Masson Trichrome, and Sirius Red showed increased damage and fibrosis in the livers of the CCl4 –treated male and female Zhx2KO/Afr1 mice compared to the CCl4 –treated Zhx2WT/Afr1 mice. RT‐qPCR analysis indicated that fibrosis and inflammatory markers were altered in these mice in comparison to the control. These findings support our hypothesis that Zhx2 is the causative gene for the hFib1 phenotype. Future studies will test whether Zhx2 overexpression can block or possibly reverse liver fibrosis and identify pathways and cell types (hepatocytes, Kupffer cells, stellate cells) that are impacted by Zhx2.Support or Funding InformationR01DK074816P30 GM127211

The Zinc Fingers and Homeoboxes (Zhx) proteins, Zhx1, Zhx2, and Zhx3, comprise a small family of proteins containing two amino-terminal C-2-H-2 zinc fingers and four or five carboxy-terminal homeodomains. These multiple homeodomains make Zhx proteins unusual because the majority of homeodomain-containing proteins contain a single homeodomain. Studies in cultured cells and mice suggest that Zhx proteins can function as positive or negative transcriptional regulators. Zhx2 regulates numerous hepatic genes, and all three Zhx proteins have been implicated in different cancers. Because Zhx proteins contain multiple predicted homeodomains, are associated with interesting physiological traits, and seem to be only present in the vertebrate lineage, we investigated the evolutionary history of this small family by comparing Zhx homologs from a wide range of chordates. This analysis indicates that the zinc finger motifs and homeodomains are highly similar among all Zhx proteins and also identifies additional Zhx-specific conserved regions, including a 13 amino acid amino-terminal motif that is nearly identical among all gnathostome Zhx proteins. We found single Zhx proteins in the sea lamprey (Petromyzon marinus) and in the nonvertebrate chordates sea squirt (Ciona intestinalis) and lancelet (Branchiostoma floridae); these Zhx proteins are most similar to gnathostome Zhx3. Based on our analyses, we propose that a duplication of the primordial Zhx gene gave rise to Zhx3 and the precursor to Zhx1 and Zhx2. A subsequent tandem duplication of this precursor generated Zhx1 and Zhx2 found in gnathostomes.

The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target β-catenin in this pathway. We now report that several "Wnt inhibitors" that allegedly target β-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known "Wnt inhibitor", FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M), was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.

BALB/cJ mice exhibit considerable phenotypic differences with other BALB/c substrains. Some of these traits involve the liver, including persistent postnatal expression of genes that are normally expressed only in the fetal liver and reduced expression of major urinary proteins. These traits are due to a mutation that dramatically reduces expression of the gene encoding the transcription factor Zinc fingers and homeoboxes 2 ( Zhx2 ). BALB/cJ mice also exhibit reduced serum lipid levels and resistance to atherosclerosis compared to other mouse strains when placed on a high-fat diet. This trait is also due, at least in part, to the Zhx2 mutation. Microarray analysis identified many genes affecting lipid homeostasis, including Lipoprotein lipase , that are dysregulated in BALB/cJ liver. This led us to investigate whether hepatic lipid levels would be different between BALB/cJ and BALB/c mice when placed on a normal chow or a high-fat chow diet. On the high-fat chow, BALB/cJ mice had increased weight gain, increased liver:body weight ratio, elevated hepatic lipid accumulation and markers of liver damage when compared to BALB/c mice. These traits in BALB/cJ mice were only partially reversed by a hepatocyte-specific Zhx2 transgene. These data indicate that Zhx2 reduces liver lipid levels and is hepatoprotective in mice on a high-fat diet, but the partial rescue by the Zhx2 transgene suggests a contribution by both parenchymal and non-parenchymal cells. A model to account for the cardiovascular and liver phenotype in mice with reduced Zhx2 levels is provided.

Autophagy targets cellular components for lysosomal-dependent degradation in which the products of degradation may be recycled for protein synthesis and utilized for energy production. Autophagy also plays a critical role in cell homeostasis and the regulation of many physiological and pathological processes and prompts this investigation of new agents to effect abnormal autophagy in hepatocellular carcinoma (HCC). 2,5-Dichloro-N-(2-methyl-4-nitrophenyl) benzenesulfonamide (FH535) is a synthetic inhibitor of the Wnt/β-catenin pathway that exhibits anti-proliferative and anti-angiogenic effects on different types of cancer cells. The combination of FH535 with sorafenib promotes a synergistic inhibition of HCC and liver cancer stem cell proliferation, mediated in part by the simultaneous disruption of mitochondrial respiration and glycolysis. We demonstrated that FH535 decreased HCC tumor progression in a mouse xenograft model. For the first time, we showed the inhibitory effect of an FH535 derivative, FH535-N, alone and in combination with sorafenib on HCC cell proliferation. Our study revealed the contributing effect of Wnt/β-catenin pathway inhibition by FH535 and its derivative (FH535-N) through disruption of the autophagic flux in HCC cells.

Initiation of hepatocellular carcinoma (HCC) by chronic hepatitis B virus (HBV) infection is a complex process that includes both oncogene activation and tumor suppressor inhibition. The HBV X (HBx) protein has an important and complex role in processes leading to HCC. We previously identified the mammalian Zinc fingers and homeoboxes 2 (ZHX2) gene as an HCC‐associated tumor suppressor gene. In the present study, we investigated whether the oncogenic properties of HBV and, more specifically, HBx, involved ZHX2 silencing. Our data indicates that ZHX2 expression is significantly decreased in tumor tissues from HBV‐positive HCC patients and livers from HBV transgenic mice. In vitro and in vivo studies confirmed that HBV‐encoded proteins, particularly HBx, inhibits both the expression and tumor suppression properties of ZHX2. Further analyses identified miR‐155, a well‐known oncomiR in various cancers, as an important link between HBx and ZHX2 inhibition. Increased miR‐155 levels were found in HBV‐positive tumors, livers of HBV transgenic mice and HBx‐overexpressing hepatoma cell lines. MiR‐155 overexpression reduced ZHX2 levels via miR‐155 seed sites in the ZHX2 3′UTR, whereas blocking miR‐155 levels led to increased ZHX2 levels. Taken together, our data indicate that HCC‐promoting properties of HBV may include ZHX2 silencing via a miR‐155 dependent pathway and suggests a novel therapy for HBV‐related HCC.

In humans, liver cancer is the 5 th leading cause of cancer death in men, and the 9 th in women. Alpha‐fetoprotein (AFP) is a fetal protein that is active during liver development and hepatocellular differentiation. After the development stage, AFP is transcriptionally silenced due to the action of alpha‐fetoprotein regulator 1 ( Afr1 ). However, during liver regeneration and tumorigenesis, AFP expression is reactivated by the action of α‐fetoprotein regulator 2 ( Afr2 ). It is this observation that has led to the use of AFP levels as a diagnostic marker for liver cancers. Two strains of mice differ in their ability to develop liver tumors and reactivate expression of AFP. C3H/HeJ mice develop liver tumors and express high AFP levels in the regenerating liver while C57/BL6 do neither. Heterozygote mice express an intermediate level of AFP and show reduced tumorigenesis. Recombination mapping showed that Afr2 is located on chromosome 2. The purpose of this study was to use a refined genetic map and existing gene variation and expression data to identify Afr2 candidate genes. First, genetic variation for the region of interest was compared using genomic data from both the Mouse Genome International and the Sanger Institute. Gene ontology and domain identification databases were used to gain information about the genes that showed variation between strains. Several genes with the potential to regulate gene expression were identified. Second, microarray studies of control and reactivated liver in both strains were compared for genes that were upregulated in C3H/HeJ reactivated liver and found in the select region of chromosome 2. Through the combinatorial analysis of these lists, four genes were identified as candidates. Finally, studies are underway to investigate whether any of these candidate genes is able to activate transcription of a reporter gene cloned near the reported AFR2‐binding region. Identification of Afr2 may lead to better therapeutics for treatment of liver cancer due to its pivotal role in directing liver‐specific gene expression.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is the third leading cause of cancer death overall. In addition, HCC rates are much higher in males than in females. Our lab primarily focuses on liver genetic regulation and how dysregulated genes contribute to liver development and disease, including HCC. The cytochrome p450 (Cyp) family of enzymes are expressed mainly in the liver and have a critical role in the metabolism and biotransformation of endogenous and xenobiotic compounds. Many Cyps are expressed zonally in the livers of both mice and humans and, in addition, have been found to exhibit sex‐biased expression. Our lab has developed a novel model to study zonal hepatocyte populations within the liver. We have placed the H‐2D d reporter gene under control of pericentrally active alpha‐fetoprotein E3 enhancer. Using antibodies against the D d protein, which is expressed on the cell surface, we can separate pericentral and periportal hepatocytes using flow cytometry. This system enables us to analyze previously identified and novel zonally regulated genes. Using this system, we have identified Cyps that are both sex‐biased and zonally restricted within the mouse liver. Interestingly, the highly related Cyp2a4 and Cyp2a5 are female‐biased genes expressed periportally and pericentrally, respectively. These two genes have 97% genomic sequence identity over a 30 Kb region, providing a model to study sex‐biased and zonal regulation in the liver. Our central hypothesis is that sex‐biased zonally regulated Cyp enzymes contribute to sex‐biased HCC development. Understanding the roles of sex‐biased pericentral and periportal genes may lead to new insights into the basis for HCC predominance in males.

The mouse major urinary proteins (Mups) are encoded by a large family of highly related genes clustered on chromosome 4. Mups, synthesized primarily and abundantly in the liver and secreted through the kidneys, exhibit male-biased expression. Mups bind a variety of volatile ligands; these ligands, and Mup proteins themselves, influence numerous behavioral traits. Although urinary Mup protein levels vary between inbred mouse strains, this difference is most pronounced in BALB/cJ mice, which have dramatically low urinary Mup levels; this BALB/cJ trait had been mapped to a locus on chromosome 15. We previously identified Zhx2 (zinc fingers and homeoboxes 2) as a regulator of numerous liver-enriched genes. Zhx2 is located on chromosome 15, and a natural hypomorphic mutation in the BALB/cJ Zhx2 allele dramatically reduces Zhx2 expression. Based on these data, we hypothesized that reduced Zhx2 levels are responsible for lower Mup expression in BALB/cJ mice. Using both transgenic and knock-out mice along with in vitro assays, our data show that Zhx2 binds Mup promoters and is required for high levels of Mup expression in the adult liver. In contrast to previously identified Zhx2 targets that appear to be repressed by Zhx2, Mup genes are positively regulated by Zhx2. These data identify Zhx2 as a novel regulator of Mup expression and indicate that Zhx2 activates as well as represses expression of target genes.

Objectives We have previously found that AFP, H19, Glypican 3 (Gpc3) and Lipoprotein lipase (Lpl) are targets of Zinc Finger and Hemeoboxes (Zhx) 2 based on their elevated expression in the adult liver of BALB/cJ mice compared to other strains. Since mouse major urinary proteins (MUP) mRNA are expressed at lower levels in the adult liver of BALB/cJ mice compared to other mouse strains, we tested whether this trait is due to Zhx2. Methods At first, by using Real‐Time Polymerase Chain Reaction (PCR) techniques, we tested hepatic MUP mRNA levels in different mouse strains including BALB/c, BALB/cJ mice, BALB/cJ mice with Zhx2 liver transgene, and recently developed Zhx2 knock‐out mice. Next we performed liver regeneration and Afr2 analysis, C3H/HeJ ( Afr2 a ) and C57BL/6 ( Afr2 b ) mice (2–3 months old) were given intraperitoneal injections of 50 mL of mineral oil (MO) containing 10% (vol/vol) carbon tetrachloride (CCl 4 ); control animals were given intraperitoneal injections of 50 mL of MO alone (22). Three days later, the animals were killed, and their livers were removed for RNA preparation and analysis. Lastly, we studied how Zhx2 regulates MUP using in‐vitro cell based assays. Results Our data indicate that MUP genes are target of Zhx2 regulation. In contrast to previous Zhx2 targets, which are elevated when Zhx2 levels are reduced, MUP expression decreases when Zhx2 levels are lower. AFP, H19, Gpc3 and Lpl are reactivated in regenerating liver and HCC. With all four genes, the level of induction is significantly less in C57BL/6 mice than in other mouse strains; this trait is governed by a single locus on mouse chromosome 2 called alpha‐fetoprotein regulator 2 (Afr2). The gene for the Afr2 phenotype has not yet been identified. Since all known Zhx2 targets are also regulated by Afr2, we tested whether MUP expression was influenced by this locus in regenerating liver. We found that MUP mRNA levels decreased in regenerating liver (in contrast to AFP, H19, Gpc3 and Lpl, which are increased in regenerating liver) and that this decrease is influenced by Afr2. Additionally, data from in vitro cell‐based assays demonstrated that Zhx2 could activate MUP24 promoter and increase MUP expression in AML cells. Conclusion Taken together, MUP is a new target of Zhx2 and it is positively regulated by Zhx2.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is the third leading cause of cancer death overall. In addition, males are known to be more susceptible to HCC. Our lab primarily focuses on genetic regulation within the liver and how dysregulation of particular genes contribute to liver development and disease. We identified a novel regulator of gene expression, Zinc Fingers and Homeoboxes 2 (Zhx2), that has been shown to have potential transcriptional and post‐transcriptional mechanisms of regulation. All previously identified Zhx2 targets, including alpha‐fetoprotein (AFP), H19, and Glypican 3 (Gpc3), are dysregulated in HCC. In order to understand the contribution of Zhx2 to global gene regulation during liver development and disease, our lab developed hepatocyte‐specific and whole body Zhx2 knockout (Zhx2 ΔHep and Zhx2 − ) mice. Using these mouse models, we identified dysregulation of a subset of cytochrome p450 enzymes (CYPs) and sex‐limited protein (C4‐ Slp ), genes that are well‐known to be expressed in a gender‐biased pattern within the liver. Specifically, the absence of Zhx2 in hepatocytes leads to increased expression of female‐specific CYPs and a decreased expression of C4‐ Slp in male mice. Curiously, the majority of female‐specific CYPs that we have identified to be upregulated in the absence of Zhx2, are also upregulated in Stat5b knockout male mice, suggesting there may be a connection between Zhx2 and a previously characterized gender‐specific gene expression control pathway. My central hypothesis is that Zhx2 contributes to sexual‐dimorphic patterns of gene expression in the liver. Currently, we are designing experiments to test how Zhx2 regulates sex‐biased genes in the liver, and whether Zhx2 acts in coordination with previously known male‐biased transcriptional regulators such as Bcl6 and Stat5b. Understanding the role of Zhx2 in gender‐biased gene expression may lead to new insights into the basis for HCC predominance in males.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, ranking the sixth most common cancer and third most common cause of cancer mortality worldwide. We have been studying mouse liver gene regulation to better understand mechanisms by which changes in gene expression contribute to liver development, homeostasis and disease. We identified Zinc Fingers and Homeoboxes 2 (Zhx2) as a regulator of alpha‐fetoprotein (AFP), a plasma protein that is highly expressed in the fetal liver but is shut off after birth. AFP expression is elevated in regenerating adult liver and hepatocellular carcinoma (HCC) and has been used extensively as a diagnostic marker of liver cancer. Interestingly, all genes that are misregulated in the absence of Zhx2 are also misregulated in HCC. Thus, to better understand gene regulation during HCC, we have been studying of the mechanism by which Zhx2 regulates gene expression. While studying AFP mRNA regulation by Zhx2, we identified previously unannotated antisense transcripts (asAFP) that partially overlap the 3′ half of the mouse AFP gene. asAFP RNAs are ~5kb alternatively spliced, mainly cytoplasmic, transcripts containing 2–4 exons that are likely to be non‐coding RNAs. These antisense transcripts were also detected in mouse liver RNA‐seq data. The abundance of asAFP RNA inversely correlates with AFP mRNA levels during postnatal development. Normally, asAFP RNA levels are high and AFP mRNA levels are low in the adult mouse liver. However, in the absence of Zhx2, AFP mRNA levels are higher and asAFP RNA levels are reduced. When portions of the asAFP RNA are over‐expressed in a liver cell line, endogenous AFP mRNA was reduced, suggesting asAFP transcripts repress AFP mRNA expression in trans. We will also knock‐down asAFP RNA and predict that AFP mRNA levels will increase. My central hypothesis is that asAFP RNA contributes to the post‐transcriptional regulation of AFP mRNA through an RNA‐RNA interaction. We are designing experiments to test this hypothesis and to determine at what level the regulation may occur.Support or Funding InformationMCB‐1158234 from the National Science Foundation, R01‐DK59866 from the National Institutes of Health