Liver-specific Transcription Factors Research Articles

Manganese inhibits HBV transcription and promotes HBsAg degradation at non-toxic levels

Chronic hepatitis B virus (HBV) infection continues to pose a significant global health challenge. However, therapeutic measures for a cure are lacking in clinical practice. Manganese, an essential trace element, has garnered attention due to its potential to activate innate immune pathways and its significant role in antiviral and antitumor immunity. Yet, the specific impact of manganese on chronic hepatitis B has been largely unexplored. Our research reveals that manganese substantially inhibits HBV replication in hepatocellular carcinoma cells at non-toxic levels. This suppression occurs independently of well-known anti-HBV innate immune pathways, such as the cGAS-STING pathway. Mechanistically, manganese decreases HBV transcription by diminishing the levels of liver-specific transcription factors. Furthermore, it activates the mTOR pathway, enhancing HBsAg ubiquitination through the upregulation of the ubiquitin ligase β-TrCP and increasing proteasome activity via the augmentation of its subunits, leading to a ubiquitin-dependent degradation of HBsAg. Significantly, our study also uncovers a notable clinical correlation between manganese levels and chronic hepatitis B infection. These findings position manganese as a critical element in diminishing HBV replication, offering a new direction in the management of chronic hepatitis B.

The long noncoding RNA HNF1A-AS1 with dual functions in the regulation of cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4) is the most important and abundant drug-metabolizing enzyme in the human liver. Inter-individual differences in the expression and activity of CYP3A4 affect clinical and precision medicine. Increasing evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in the regulation of CYP3A4 expression. Here, we showed that lncRNA hepatocyte nuclear factor 1 alpha–antisense 1 (HNF1A-AS1) exerted dual functions in regulating CYP3A4 expression in Huh7 and HepG2 cells. Mechanistically, HNF1A-AS1 served as an RNA scaffold to interact with both protein arginine methyltransferase 1 and pregnane X receptor (PXR), thereby facilitating their protein interactions and resulting in the transactivation of PXR and transcriptional alteration of CYP3A4 via histone modifications. Furthermore, HNF1A-AS1 bound to the HNF1A protein, a liver-specific transcription factor, thereby blocking its interaction with the E3 ubiquitin ligase tripartite motif containing 25, ultimately preventing HNF1A ubiquitination and protein degradation, further regulating the expression of CYP3A4. In summary, these results reveal the novel functions of HNF1A-AS1 as the transcriptional and post-translational regulator of CYP3A4; thus, HNF1A-AS1 may serve as a new indicator for establishing or predicting individual differences in CYP3A4 expression.

Chromatin accessibility dynamics in colorectal cancer liver metastasis: Uncovering the liver tropism at single cell resolution

Tumor metastasis causes over 90% of cancer related death and no currently available therapies target it. However, there is limited understanding regarding the epigenetic regulation of genes during this complex process. Here by integrating single-cell ATAC-seq (scATAC-seq), single-cell RNA-seq (scRNA-seq), microarray, bulk RNA-seq, immunohistochemistry (IHC) staining, as well as proteomics datasets from paired primary and liver metastatic colorectal cancer (CRC) patient-derived xenograft (PDX) model and patients, we discovered that liver metastatic CRC cells lose their colon-specific chromatin accessible sites yet gain liver-specific ones. Importantly, we observed elevated accessibility of HNF4A, a liver-specific transcription factor, in liver metastatic CRC cells. Subsequently, we performed clustering analysis of liver metastatic CRC cells together with cells involved in liver development, revealing significant heterogeneity among the liver metastatic CRC cells. Over 50% of the liver metastatic CRC cells exhibited characteristics similar to those of erythroid progenitors and hepatocytes, showing increased expression of genes involved in oxidative phosphorylation and glycolysis. Moreover, our discovery further revealed that the MHC and IFN response genes in these cells exhibit moderate epigenetic activity, which is significantly associated with the low objective response rates in checkpoint blockade immunotherapy. Our findings uncovered the critical roles of HNF4A and the cell populations within liver metastatic CRC cells might serve as crucial therapeutic targets for addressing liver metastasis and improving the immunotherapy response in patients with CRC.

Transcriptional regulation of Glis2 in hepatic fibrosis

The role of Gli-similar 2 (Glis2) in hepatic fibrosis (HF) is controversial. In this study, we focused on the functional and molecular mechanisms involved in the Glis2-mediated activation of hepatic stellate cells (HSCs)—a milestone event leading to HF. The expression levels of Glis2 mRNA and protein were significantly decreased in the liver tissues of patients with severe HF and in mouse fibrotic liver tissues as well as HSCs activated by TGFβ1. Functional studies indicated that upregulated Glis2 significantly inhibited HSC activation and alleviated BDL-induced HF in mice. Downregulation of Glis2 was found to correlate significantly with DNA methylation of the Glis2 promoter mediated by methyltransferase 1 (DNMT1), which restricted the binding of hepatic nuclear factor 1-α (HNF1-α), a liver-specific transcription factor, to Glis2 promoters. In addition, the enrichment of DNMT1 in the Glis2 promoter region was mediated by metastasis-associated lung adenocarcinoma transcriptor-1 (MALAT1) lncRNA, leading to transcriptional silencing of Glis2 and activation of HSCs. In conclusion, our findings reveal that the upregulation of Glis2 can maintain the resting state of HSCs. The decreased expression of Glis2 under pathological conditions may lead to the occurrence and development of HF with the expression silencing of DNA methylation mediated by MALAT1 and DNMT1.

Hepatocyte differentiation from mouse liver ductal organoids by transducing 4 liver-specific transcription factors.

Hepatocyte sources that are expandable in vitro are required for liver regenerative medicine and to elucidate the mechanisms underlying the physiological functions of the liver. Liver ductal organoids (LDOs) comprise liver tissue stem cells with a bipotential capacity to differentiate into hepatocyte and cholangiocyte lineages and can thus serve as a hepatocyte source. However, using current differentiation methods, LDOs differentiate into immature hepatocytes while retaining strong cholangiocyte characteristics. We thus investigated an alternative differentiation method for LDOs to achieve hepatocyte maturation. We extracted 12 candidate transcription factors to induce hepatocyte differentiation by comparing their gene expression in LDOs and liver tissues. After evaluating the effects of these transcription factors on LDOs, we analyzed the comprehensive gene expression profile, protein expression, and hepatic function in the transduced organoids. We identified a combination of 4 transcription factors, Hnf4a, Foxa1, Prox1, and Hlf, which upregulated hepatic lineage markers and downregulated cholangiocyte markers. Differentiation-induced LDOs showed more hepatocyte-specific characteristics than those with the conventional method, enhancing the transition from cholangiocyte to hepatocyte lineage and hepatic functions, such as liver-specific protein synthesis, lipid droplet deposition, and ammonia detoxification. Transduction of the 4 transcription factors (Hnf4a, Foxa1, Prox1, and Hlf) is a promising strategy to promote the differentiation of LDOs to obtain mature hepatocyte-like cells with better functionality.

Hepatic multiple hyperintense cystic lesions: a rare caroli disease

Caroli’s disease is a rare malformation of the biliary tract as its incidence rate is one per million of people. Most cases of hepatic cystic lesions are asymptomatic and are discovered incidentally during abdominal ultrasonography. It is reported that Caroli’s disease is an autosomal-recessive disorder with multifocal segmental non-obstructive dilation features of intrahepatic bile ducts that may involve a segment, a lobe, or the whole liver. Several complications might be developed in some cases, such as cholangiocarcinoma. Therefore, early diagnosis is vital for proper management. There is a lack of literature discussing Caroli’s disease among Africans compared to Asians. Females are more affected than males. Moreover, most of Caroli’s disease cases were about 22 years old of age. This study describes the existing case’s novelty in that we discuss a rare Caroli’s disease case of an African male patient; moreover, the patient age in mid-adulthood. In addition, the severity of the current case is that both hepatic lobes showed severe multiple cystic lesions. The embryonic justification of the case is the large cranial portion of the hepatic diverticulum gives rise to the cords of hepatocytes and intrahepatic biliary networks. On the other hand, the small caudal portion forms the extrahepatic biliary tract. The hepatocytes as well as cholangiocytes, are differentiated from the hepatoblasts under the influence of typical hepatic genes, α-fetoprotein, liver-specific transcription factors, and albumin. Based on the above, Caroli disease is defined by biliary tract malformation, whereas Caroli syndrome is determined by the presence of associated congenital hepatic fibrosis.

Hepatic multiple hyperintense cystic lesions: a rare caroli disease.

Caroli’s disease is a rare malformation of the biliary tract as its incidence rate is one per million of people. Most cases of hepatic cystic lesions are asymptomatic and are discovered incidentally during abdominal ultrasonography. It is reported that Caroli’s disease is an autosomal-recessive disorder with multifocal segmental non-obstructive dilation features of intrahepatic bile ducts that may involve a segment, a lobe, or the whole liver. Several complications might be developed in some cases, such as cholangiocarcinoma. Therefore, early diagnosis is vital for proper management. There is a lack of literature discussing Caroli’s disease among Africans compared to Asians. Females are more affected than males. Moreover, most of Caroli’s disease cases were about 22 years old of age. This study describes the existing case’s novelty in that we discuss a rare Caroli’s disease case of an African male patient; moreover, the patient age in mid-adulthood. In addition, the severity of the current case is that both hepatic lobes showed severe multiple cystic lesions. The embryonic justification of the case is the large cranial portion of the hepatic diverticulum gives rise to the cords of hepatocytes and intrahepatic biliary networks. On the other hand, the small caudal portion forms the extrahepatic biliary tract. The hepatocytes as well as cholangiocytes, are differentiated from the hepatoblasts under the influence of typical hepatic genes, α-fetoprotein, liver-specific transcription factors, and albumin. Based on the above, Caroli disease is defined by biliary tract malformation, whereas Caroli syndrome is determined by the presence of associated congenital hepatic fibrosis.

CREBH alleviates mitochondrial oxidative stress through SIRT3 mediating deacetylation of MnSOD and suppression of Nlrp3 inflammasome in NASH.

Lipotoxicity and unresolved oxidative stress are key drivers of metabolic inflammation in nonalcoholic steatohepatitis (NASH). cAMP-response element binding protein H(CREBH) is a liver-specific transcription factor and regulates the glucose and lipid metabolism of NASH. However, its role in mitochondrial oxidative stress and its association with sirtuin 3 (SIRT3), a master regulator of deacetylation for mitochondrial proteins, remains elusive. In this study, AML-12cells were treated with palmitic acid to imitate the pathological changes of NASH in vitro and 8-week-old male C57BL/6J mice were fed with a high-fat (HF) diet or a methionine-choline-deficient (MCD) diet to build the widely accepted in vivo model of NASH. We found that lipid overload induced mitochondrial oxidative stress and stimulated the expression of CREBH and SIRT3. CREBH overexpression alleviated the mitochondrial oxidative stress. Moreover, CREBH promoted SIRT3 expression, which regulated the deacetylation of manganese superoxide dismutase (MnSOD) and inhibited NOD-Like Receptor Pyrin Domain Containing 3 (Nlrp3) inflammasome activation whereas suppression of SIRT3 damaged the protecting ability of CREBH in mitochondrial oxidative stress. CREBH knockout mice were highly susceptible to HF and MCD diet-induced NASH with more severe oxidative stress. Collectively, our results firstly provided the support that CREBH could serve as a protective factor in the progression of NASH by regulating the acetylation of MnSOD and the activation of Nlrp3 inflammasome through SIRT3. These results suggest that CREBH might be a valuable therapeutic candidate for NASH.

Strategies to Inhibit Hepatitis B Virus at the Transcript Level.

Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.

A novel epigenetic mechanism unravels hsa-miR-148a-3p-mediated CYP2B6 downregulation in alcoholic hepatitis disease

Cytochrome P450 (CYP) enzymes play critical roles in drug transformation, and the total CYPs are markedly decreased in alcoholic hepatitis (AH), a fatal alcoholic liver disease. miRNAs are endogenous small noncoding RNAs that regulate many essential biological processes. Knowledge concerning miRNA regulation of CYPs in AH disease is limited. Here we presented the changes of key CYPs in liver samples of AH patients retrieved from GEO database, performed in silico prediction of miRNAs potentially targeting the dysregulated CYP transcripts, and deciphered a novel mechanism underlying miRNA mediated CYPs expression in liver cells. Nine miRNAs were predicted to regulate CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2J2, and CYP3A4, among which hsa-miR-148a-3p was selected as a case study. Biochemical and molecular evidences demonstrated that miR-148a promoted CYP2B6 expression by increasing mRNA stability via directly binding to the 3′UTR sequence, and that this positive posttranscriptional regulation was AGO1/2-dependent. Further, luciferase reporter gene assay and RNA secondary structure analysis illustrated that the seedless target site, not the seed target site, controlled miR-148a-mediated CYP2B6 upregulation. Moreover, we identified HNF4A as a liver-specific transcription factor of MIR-148A through EMSA and chromatin immunoprecipitation experiments. In conclusion, ethanol downregulated miR-148a in hepatocytes through HNF4A regulation, which eventually decreased CYP2B6 expression. Our finding will benefit the understanding of dysregulated drug metabolism in AH patients and highlight an unconventional mechanism for epigenetic regulation of CYP gene expression.

Long-term correction of ornithine transcarbamylase deficiency in Spf-Ash mice with a translationally optimized AAV vector

Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human OTC cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTCSpf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic.

Small molecule supplementation potentiates miRNA mediated direct reprogramming of human fibroblasts into functional hepatocytes

BackgroundHepatic transcription factors such as Hnf4α, Foxa1, Foxa2 or Foxa3 and Gata4 have successfully been used for directly reprogram the fibroblasts into hepatic lineages (iHeps). Although it has several advantages such as devoid of teratoma formation, robust conversion rate and potential in vivo reprograming to repair injured tissues, majority of the direct reprograming that employed genetic manipulations to modify the expression of tissue‐specific transcription factors have limitation in downstream clinical applications. Thus, it is critical to develop a new method for reprograming fibroblasts to hepatocytes.HypothesisWe hypothesized that the combination of small molecules such as epigenetic modifiers and GSK‐inhibitor, and miRNAs could successfully be used in direct differentiation of fibroblasts to iHeps.AimThe aim of this study was to directly reprogram the human fibroblasts into functional iHeps using small molecules that regulate reprogramming of somatic cells and miRNAs.MethodsHepatocyte specific miRNAs were selected from liver fibrosis, regulation of mesenchymal to epithelial transition (MET) and regulation of liver specific transcription factors HNF4α and FOXa2 miRNA datasets. Primary human fibroblasts grown to 80% confluent were transfected with either miRNA cocktail and/or small molecules. Transfected cells were cultured in hepatocyte growth media to generate iHeps. Albumin, E‐cad, HNF4α and FOXa2 expressions were detected by flow cytometry, immunostaining and qPCR analyses to establish iHeps. The iHeps mixed with human vascular endothelial cells (HUVEC) in collagen were implanted in partial‐hepatectomized immune‐compromised mice. Explants collected were characterized 4 weeks following implantation. Human albumin level was estimated by ELISA in iHeps implanted mouse serum.ResultsmiRNA cocktail alone significantly downregulated Snail1 and vimentin, and upregulation E‐cad specific mRNA abundance. Although small molecules do not exert any effect, its supplementation along with miRNA cocktail drastically downregulated Snail1 and vimentin, and upregulated E‐cad abundance. Albumin, E‐cad, HNF4α and FOXa2 expression established iHeps lineage. Integration of iHeps with vascular structures were present in liver (shown by arrows in Figure ), while human albumin (control vs iHeps: 0.78 ± 0.01 vs 63.8 ± 0.2 ng/ml) was detected in serum of mice implanted with iHeps.ConclusionsWe conclude that: 1) the down‐regulation of Snail1 and vimentin, and upregulation E‐cad establish MET; 2) the small molecule supplementation potentiate the miRNA cocktail induced MET; and 3) the direct reprograming of human fibroblast using miRNA along with small molecule supplementation generate a viable and functional iHeps.SpeculationWe speculate that small molecules along with miRNA cocktail could potentially be used to treat liver fibrosis in vivo.Support or Funding InformationFunding was provided by the Institute of Bioengineering and Nanotechnology(Biomedical Research Council, Agency for Science, Technology and Research, Singapore). VMR was supported by NIH grants DK112085 and DK104791.H&E staining of sections of explants harvested from partially hepatectomized mouse modelFigure 1

Potentiation of cancerous progression by LISCH7 via direct stimulation of TGFB1 transcription in triple-negative breast cancer.

As an aggressive breast cancer (BCa) subtype, triple-negative breast cancer (TNBC) responses poorly to chemotherapy and endocrine therapy, and usually has a worse prognosis. This is largely due to the lack of specific therapeutic targets, laying claim to an imperious demand to clarify the key signaling pathways potentiating TNBC progression. Herein, we report that expression levels of the liver-specific bHLH-Zip transcription factor (LISCH7), a recently identified key player in cancerous progression, preferentially enriched in TNBC in comparison with other BCa subtypes, and this upregulation was observed to be correlated to a poor survival outcome in patients with TNBC. Ablation of LISCH7 in TNBC cells impaired cell proliferation, reduced cell invasiveness, and enhanced sensitivity to the first-line chemotherapeutic drug docetaxel at both in vitro and in vivo levels. Importantly, concurrent induction of TGFB1, the gene encoding transforming growth factor-β1 (TGF-β1), an essential multipluripotent regulator of TNBC, was accompanied with these alterations in cancerous properties. We further showed that LISCH7 could directly bind to the TGFB1 promoter and stimulate TGFB1 transcription in TNBC cells. The recruitment of LISCH7 onto the TGFB1 chromatin and transactivation of TGFB1 were substantially augmented by treatment with the exogenous TGF-β1 in a time- and dose-dependent manner. Collectively, these findings suggest that LISCH7 and TGF-β1 form a reciprocal positive regulatory loop and cooperatively regulate cancerous progression in TNBC cells. Thus, simultaneous inhibition of both LISCH7 and TGF-β1 signaling may represent a more effective approach to counteract advanced TNBC.

Tissue-specific transcription reprogramming promotes liver metastasis of colorectal cancer.

Metastasis, the development of secondary malignant growths at a distance from a primary tumor, is the cause of death for 90% of cancer patients, but little is known about how metastatic cancer cells adapt to and colonize new tissue environments. Here, using clinical samples, patient-derived xenograft (PDX) samples, PDX cells, and primary/metastatic cell lines, we discovered that liver metastatic colorectal cancer (CRC) cells lose their colon-specific gene transcription program yet gain a liver-specific gene transcription program. We showed that this transcription reprogramming is driven by a reshaped epigenetic landscape of both typical enhancers and super-enhancers. Further, we identified that the liver-specific transcription factors FOXA2 and HNF1A can bind to the gained enhancers and activate the liver-specific gene transcription, thereby driving CRC liver metastasis. Importantly, similar transcription reprogramming can be observed in multiple cancer types. Our data suggest that reprogrammed tissue-specific transcription promotes metastasis and should be targeted therapeutically.

Hepatic Stress Response in HCV Infection Promotes STAT3-Mediated Inhibition of HNF4A-miR-122 Feedback Loop in Liver Fibrosis and Cancer Progression.

Hepatitis C virus (HCV) infection compromises the natural defense mechanisms of the liver leading to a progressive end stage disease such as cirrhosis and hepatocellular carcinoma (HCC). The hepatic stress response generated due to viral replication in the endoplasmic reticulum (ER) undergoes a stepwise transition from adaptive to pro-survival signaling to improve host cell survival and liver disease progression. The minute details of hepatic pro-survival unfolded protein response (UPR) signaling that contribute to HCC development in cirrhosis are unknown. This study shows that the UPR sensor, the protein kinase RNA-like ER kinase (PERK), mediates the pro-survival signaling through nuclear factor erythroid 2-related factor 2 (NRF2)-mediated signal transducer and activator of transcription 3 (STAT3) activation in a persistent HCV infection model of Huh-7.5 liver cells. The NRF2-mediated STAT3 activation in persistently infected HCV cell culture model resulted in the decreased expression of hepatocyte nuclear factor 4 alpha (HNF4A), a major liver-specific transcription factor. The stress-induced inhibition of HNF4A expression resulted in a significant reduction of liver-specific microRNA-122 (miR-122) transcription. It was found that the reversal of hepatic adaptive pro-survival signaling and restoration of miR-122 level was more efficient by interferon (IFN)-based antiviral treatment than direct-acting antivirals (DAAs). To test whether miR-122 levels could be utilized as a biomarker of hepatic adaptive stress response in HCV infection, serum miR-122 level was measured among healthy controls, and chronic HCV patients with or without cirrhosis. Our data show that serum miR-122 expression level remained undetectable in most of the patients with cirrhosis (stage IV fibrosis), suggesting that the pro-survival UPR signaling increases the risk of HCC through STAT3-mediated suppression of miR-122. In conclusion, our data indicate that hepatic pro-survival UPR signaling suppresses the liver-specific HNF4A and its downstream target miR-122 in cirrhosis. These results provide an explanation as to why cirrhosis is a risk factor for the development of HCC in chronic HCV infection.

Liver biopsy derived induced pluripotent stem cells provide unlimited supply for the generation of hepatocyte-like cells.

Background & aimsHepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells (iPSCs) have emerged as a promising cell culture model to study metabolism, biotransformation, viral infections and inherited liver diseases. iPSCs provide an unlimited supply for the generation of HLCs, but incomplete HLC differentiation remains a major challenge. iPSC may carry-on a tissue of origin dependent expression memory influencing iPSC differentiation into different cell types. Whether liver derived iPSCs (Li-iPSCs) would allow the generation of more fully differentiated HLCs is not known.MethodsIn the current study, we used primary liver cells (PLCs) expanded from liver needle biopsies and reprogrammed them into Li-iPSCs using a non-integrative Sendai virus-based system. Li-iPSCs were differentiated into HLCs using established differentiation protocols. The HLC phenotype was characterized at the protein, functional and transcriptional level. RNA sequencing data were generated from the originating liver biopsies, the Li-iPSCs, fibroblast derived iPSCs, and differentiated HLCs, and used to characterize and compare their transcriptome profiles.ResultsLi-iPSCs indeed retain a liver specific transcriptional footprint. Li-iPSCs can be propagated to provide an unlimited supply of cells for differentiation into Li-HLCs. Similar to HLCs derived from fibroblasts, Li-HLCs could not be fully differentiated into hepatocytes. Relative to the originating liver, Li-HLCs showed lower expression of liver specific transcription factors and increased expression of genes involved in the differentiation of other tissues.ConclusionsPLCs and Li-iPSCs obtained from small pieces of human needle liver biopsies constitute a novel unlimited source for the production of HLCs. Despite the preservation of a liver specific gene expression footprint in Li-iPSCs, the generation of fully differentiated hepatocytes cannot be achieved with the current differentiation protocols.

Abstract 4334: Epigenetic reprogramming of tissue-specific transcription promotes metastasis

Abstract Metastasis is the cause of death for 90% of cancer patients, but little is known about how cancer cells adapt to and colonize new tissue environments. Colorectal cancer (CRC) is the third most common cancer and the fourth most common cancer cause of death globally. Here, we investigated how metastatic cancer cells, such as CRC cells, with original-tissue specificity adapt to the environment of a distant tissue, like liver. By analyzing transcriptome data of multiple cohorts of clinical samples and primary/metastatic cell lines, we found metastatic CRC cells lose their colon-specific gene transcription program and gain a liver-specific gene transcription program as they metastasize in the liver. The elevated expression of a number of liver-specific genes was confirmed in human liver metastatic tumors by immunohistochemistry. Through epigenomic profiling, we revealed this transcription reprogramming is driven by a reshaped epigenetic landscape of both typical and super-enhancers, and chemical inhibition of enhancer activity disrupts the ability of cells to execute this altered transcription program and consequently inhibits metastasis. Further we identified liver-specific transcription factors, FOXA2 and HNF1A, bind to the gained enhancers in liver metastatic cells and activate the expression of liver-specific genes. We also found both FOXA2 and HNF1A are highly expressed in human liver metastatic tumors compared to primary CRC tumors. Loss of function of FOXA2 or HNF1A inhibits liver-specific transcription and impairs the ability of metastatic CRC cells to adapt to liver. Consistently, the gain of function of HNF1A activated liver-specific transcription and enhanced CRC liver metastasis. Our findings indicate the direct contribution of a reprogrammed tissue-specific transcription program, which is induced by reshaped epigenetic landscape as well as distant-tissue-specific transcription factors, to the adaption and colonization of metastatic CRC cells to a distal organ. Notably, in addition to CRC liver metastasis, this tissue-specific transcription reprogramming is also observed in other multiple distant organs and/or cancer types. In summary, our data suggest that epigenetically reprogrammed tissue-specific transcription promotes metastasis and might have implications for targeted anti-metastasis therapies. Citation Format: Shuaishuai Teng, Yang Li, Ming Yang, Rui Qi, Qianyu Wang, Zhi Lu, Dong Wang. Epigenetic reprogramming of tissue-specific transcription promotes metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4334.

Transcriptional regulation of protein S gene

Background: Protein S (PS) is a known Vitamin K dependent plasma glycoprotein that is produced in many human tissues including the liver. Furthermore, it has an important function in the coagulation cascade as an anticoagulant cofactor when it binds active protein C. The deficiency of PS may lead to the development of deep vein thrombosis. There are many transcription factors which play an important role in the regulation of the transcription of PS. One of these regulatory factors is the liver specific transcription factor hepatocyte nuclear factor 1 (HNF1), a binding site which is located in the 638 base pair promoter area at the 5' end of the PS gene. HNF1 and PS is not well understood and to confirm the importance of HNF1 in regulating expression of PS small interfering ribonucleic acid (siRNA) which will knockout the HNF1 gene expression can be used, and the effect on PS expression monitored. The levels of expression of HNF1 and PS can be determined by real time polymerase chain reaction. Materials and Methods: Preparation of total ribonucleic acid for complementary DNA synthesis, Evaluation the quality of HepG2 ribonucleic acid by agarose gel electrophores is, Real time polymerase chain reaction, Acrylamide gel electrophoresis. Results: HepG2 rRNA bands shows a good quality of RNA expression, Analysis of the total RNA quantity can be determined by spectrophotometric analysis, The best housekeeping genes were UBC and YWHAZ therefore, there has been used as a reference for the primers samples in efficiency test. HNF1 and PROS1 shows a good expression but with low efficiency which is not required to carry further gene knockdown experiment, SYBR green dye is less sensitive than TaqMan because it presents a non specific detection (primer dimers) were all dsDNA products are detected including primer dimers, contaminating DNA, and PCR product from mis annealed primer. Conclusion: The primer pairs are not efficient to carry further gene knockdown experiment and required to design another HNF1 and PROS1 primers. The efficiency of the PCR should be 90%-100% meaning doubling of the amplicon at each cycle. This corresponds to a slope of -3.1 to -3.6 in the Ct vs log template amount standard curve. To obtain accurate and reproducible results, reactions should have efficiency as close to 100% as possible.

Ancestral transcriptome inference based on RNA-Seq and ChIP-seq data.

With the help of high-throughput NGS (next-generation sequencing) technologies, ancestral transcriptome reconstruction is helpful to understand the complexity of transcriptional regulatory systems that underlies the evolution of multiple cellular metazoans with sophisticated functions and distinctive morphologies. To this end, we report a new method of ancestral state inference. The new method used Ornstein-Uhlenbeck (OU) model, which is more biologically realistic, to replace the Brownian motion (BM) model and is suitable for multi-transcriptome data. Implemented in the free R package, AnceTran is specially designed for RNA-seq and ChIP-seq data, which is feasible. It should be noticed that our work will be integrated to a unified, statistically-sound phylogenetic framework to study the evolution of many other molecular phenomes such as proteomics, chromatin accessibility, methylation status, and metabolomics. We exemplify our method by a case study, using the ChIP-seq binding data of three liver-specific transcription factors and the RNA-seq liver expression data in four closely related mice species, and some technical issues are discussed.

HRD1-ERAD controls production of the hepatokine FGF21 through CREBH polyubiquitination.

The endoplasmic reticulum-associated protein degradation (ERAD) is responsible for recognizing and retro-translocating protein substrates, misfolded or not, from the ER for cytosolic proteasomal degradation. HMG-CoA Reductase (HMGCR) Degradation protein-HRD1-was initially identified as an E3 ligase critical for ERAD. However, its physiological functions remain largely undefined. Herein, we discovered that hepatic HRD1 expression is induced in the postprandial condition upon mouse refeeding. Mice with liver-specific HRD1 deletion failed to repress FGF21 production in serum and liver even in the refeeding condition and phenocopy the FGF21 gain-of-function mice showing growth retardation, female infertility, and diurnal circadian behavior disruption. HRD1-ERAD facilitates the degradation of the liver-specific ER-tethered transcription factor CREBH to downregulate FGF21 expression. HRD1-ERAD catalyzes polyubiquitin conjugation onto CREBH at lysine 294 for its proteasomal degradation, bridging a multi-organ crosstalk in regulating growth, circadian behavior, and female fertility through regulating the CREBH-FGF21 regulatory axis.