Integrative analysis of transcriptome dynamics during human craniofacial development identifies candidate disease genes.

Craniofacial disorders arise in early pregnancy and are one of the most common congenital defects. To fully understand how craniofacial disorders arise, it is essential to characterize gene expression during the patterning of the craniofacial region. To address this, we performed bulk and single-cell RNA-seq on human craniofacial tissue from 4-8 weeks post conception. Comparisons to dozens of other human tissues revealed 239 genes most strongly expressed during craniofacial development. Craniofacial-biased developmental enhancers were enriched +/- 400 kb surrounding these craniofacial-biased genes. Gene co-expression analysis revealed that regulatory hubs are enriched for known disease causing genes and are resistant to mutation in the normal healthy population. Combining transcriptomic and epigenomic data we identified 539 genes likely to contribute to craniofacial disorders. While most have not been previously implicated in craniofacial disorders, we demonstrate this set of genes has increased levels of de novo mutations in orofacial clefting patients warranting further study.

PHF20 is crucial for epigenetic control of starvation-induced autophagy through enhancer activation.

Autophagy is a catabolic pathway that maintains cellular homeostasis under various stress conditions, including conditions of nutrient deprivation. To elevate autophagic flux to a sufficient level under stress conditions, transcriptional activation of autophagy genes occurs to replenish autophagy components. Thus, the transcriptional and epigenetic control of the genes regulating autophagy is essential for cellular homeostasis. Here, we applied integrated transcriptomic and epigenomic profiling to reveal the roles of plant homeodomain finger protein 20 (PHF20), which is an epigenetic reader possessing methyl binding activity, in controlling the expression of autophagy genes. Phf20 deficiency led to impaired autophagic flux and autophagy gene expression under glucose starvation. Interestingly, the genome-wide characterization of chromatin states by Assay for Transposase-Accessible Chromatin (ATAC)-sequencing revealed that the PHF20-dependent chromatin remodelling occurs in enhancers that are co-occupied by dimethylated lysine 36 on histone H3 (H3K36me2). Importantly, the recognition of H3K36me2 by PHF20 was found to be highly correlated with increased levels of H3K4me1/2 at the enhancer regions. Collectively, these results indicate that PHF20 regulates autophagy genes through enhancer activation via H3K36me2 recognition as an epigenetic reader. Our findings emphasize the importance of nuclear events in the regulation of autophagy.

Estrogen exposure causes the progressive growth of SK-Hep1-derived tumor in ovariectomized mice.

Liver cancer, one of the leading death causes, has different incidence and mortality rates in men and women. The influencing factor is considered to estrogen. However, the role of estrogen in liver cancer remains controversial. In this study, we investigated the effects of estrogen on tumor progression. Total RNA sequencing was analyzed in SK-Hep1-derived tumor tissues, and 15 genes were expressed only in female mice. Among the differentially expressed genes, matrix metalloprotease 7 (MMP7), germ cell associated 1 (GSG1), and chromosome 6 open reading frame 15 (C6orf15) were associated with significantly different overall survival rates based on their expression level in liver cancer patients. Interestingly, exogenous estrogen aggravated SK-Hep1-derived tumor growth in ovariectomized (OVX) mice. When OVX mice were treated with exogenous estrogen, SK-Hep1-derived tumor tissues exhibited high MMP7 expression levels and low GSG1 and C6orf15 expression levels. These expression patterns were consistent with those of liver cancer patients with low overall survival rates. These results suggest that these genes are expected to be prognostic biomarkers of liver cancer. In conclusion, our results suggest that continuous estrogen exposure may promote tumor growth in OVX mice.

Advanced Xenograft Model with Cotransplantation of Patient-Derived Organoids and Endothelial Colony-Forming Cells for Precision Medicine.

Preclinical evaluation models have been developed for precision medicine, with patient-derived xenograft models (PDXs) and patient-derived organoids (PDOs) attracting increasing attention. However, each of these models has application limitations. In this study, an advanced xenograft model was established and used for drug screening. PDO and endothelial colony-forming cells (ECFCs) were cotransplanted in NRGA mice (PDOXwE) to prepare the model, which could also be subcultured in Balb/c nude mice. Our DNA sequencing analysis and immunohistochemistry results indicated that PDOXwE maintained patient genetic information and tumor heterogeneity. Moreover, the model enhanced tumor growth more than the PDO-bearing xenograft model (PDOX). The PDO, PDOXwE, and clinical data were also compared in the liver metastasis of a colorectal cancer patient, demonstrating that the chemosensitivity of PDO and PDOXwE coincided with the clinical data. These results suggest that PDOXwE is an improvement of PDOX and is suitable as an evaluation model for precision medicine.

Sex-dependent liver cancer xenograft models for predicting clinical data in the evaluation of anticancer drugs.

BACKGROUND: The incidence and mortality of liver cancer show a great difference between the sexes. We established sex-dependent liver cancer xenograft models and investigated whether such sex-dependent models could be used to simultaneously evaluate the therapeutic and adverse effects of anticancer drugs for drug screening. RESULTS: In the in-vitro test, the cytotoxicity of anticancer drugs (cisplatin, 5-fluorouracil, and doxorubicin) was compared between male- and female-derived liver cancer cell lines. Cisplatin and 5-fluorouracil exhibited cytotoxicity without sex-difference, but doxorubicin showed dose-dependently significant cytotoxicity only in male-derived cells. Our results showed a strong correlation between preclinical and clinical data with the use of sex-dependent liver cancer xenograft models. Moreover, the male-derived Hep3B-derived xenograft model was more sensitive than the female-derived SNU-387-derived xenograft model against doxorubicin treatment. Doxorubicin showed more severe cardiotoxicity in the male xenograft model than in the female model. We investigated the occurrence frequency of doxorubicin-related cardiotoxicity using data obtained from the Korea Institute of Drug Safety & Risk Management Database, but no significant difference was observed between the sexes. CONCLUSIONS: Our results suggest that sex-dependent xenograft models are useful tools for evaluating the therapeutic and adverse effects of anticancer drugs, because sex is an important consideration in drug development.

The chromatin-binding protein PHF6 functions as an E3 ubiquitin ligase of H2BK120 via H2BK12Ac recognition for activation of trophectodermal genes.

Epigenetic regulation is important for establishing lineage-specific gene expression during early development. Although signaling pathways have been well-studied for regulation of trophectoderm reprogramming, epigenetic regulation of trophectodermal genes with histone modification dynamics have been poorly understood. Here, we identify that plant homeodomain finger protein 6 (PHF6) is a key epigenetic regulator for activation of trophectodermal genes using RNA-sequencing and ChIP assays. PHF6 acts as an E3 ubiquitin ligase for ubiquitination of H2BK120 (H2BK120ub) via its extended plant homeodomain 1 (PHD1), while the extended PHD2 of PHF6 recognizes acetylation of H2BK12 (H2BK12Ac). Intriguingly, the recognition of H2BK12Ac by PHF6 is important for exerting its E3 ubiquitin ligase activity for H2BK120ub. Together, our data provide evidence that PHF6 is crucial for epigenetic regulation of trophectodermal gene expression by linking H2BK12Ac to H2BK120ub modification.

Sex-dependent effects of estrogen pellets in human liver cancer xenograft models.

Liver cancer shows noticeable differences in the incidence rate and mortality between genders. To investigate the estrogen effect on tumor progression in liver cancer, we developed a xenograft model using estrogen pellets. SK-Hep1 cells (human male liver carcinoma) were inoculated into male or female nude mice. Subsequently, estrogen pellets were subcutaneously implanted into these xenograft models. Interestingly, the marked adverse effect of estrogen pellets (0.5 mg/21 days) were observed in the male-derived xenograft model, with increased ulcerative dermatitis in male mice than in female mice. Additionally, necrosis was observed in male mice with SK-Hep1-derived tumors. However, the estrogen pellet (0.5 mg/60 days) did not exhibit these adverse effects. Tumor growth in female mice was significantly suppressed by estrogen (0.5 mg/60 days). Tumor growth was also suppressed in male mice implanted with estrogen (0.5 mg/60 days), but the suppression was not significant. We found that estrogen-induced skin damage was more severe in male mice than female mice. The tumor suppression of estrogen was effective in female mice compared to male mice bearing liver cancer. The results suggest that the sex difference affects estrogen activity and thus should be considered in the preclinical assessment.

Sex-Dependent Adverse Drug Reactions to 5-Fluorouracil in Colorectal Cancer.

Sex-related incidence and outcomes were reported in various cancers, including colorectal cancer. 5-Fluorouracil (5-FU) is widely used as an essential chemotherapeutic agent for colorectal cancer. However, sex-based differences in 5-FU toxicity have yet to be reported in human cancer cell lines and xenograft mouse models to date. Here, we investigated, for the first time, sex-based differences in 5-FU toxicity using human colon cancer cell lines, xenograft mouse models, and Korean patients' data. Female-derived colon cancer cell lines exhibited greater 5-FU-induced cytotoxicity than male-derived colon cancer cell lines. We established two xenograft mouse models: one with a male-derived human colon cancer cell line injected into male mice (a male-xenograft model) and another involving a female-derived human colon cancer cell line injected into female mice (a female xenograft model). Treatment with 5-FU inhibited tumor growth and led to hematological toxicity in a female xenograft model more potently than in a male xenograft model. We analyzed the data obtained from Korean patients with colorectal cancer to examine sex differences in adverse drug reactions caused by 5-FU. Korean female patients with colorectal cancer who received 5-FU chemotherapy experienced more frequent adverse drug reactions including alopecia and leukopenia than male patients. Taken together, we demonstrated that female may be associated with increased risk of toxicity to 5-FU treatment in colorectal cancer based on in vitro and in vivo investigations and clinical data analysis. Our study suggests sex as an important clinical factor, which predicts induction of toxicity related to 5-FU treatment.

AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of autophagy.

Autophagy is a highly conserved self-digestion process, which is essential for maintaining homeostasis and viability in response to nutrient starvation. Although the components of autophagy in the cytoplasm have been well studied, the molecular basis for the transcriptional and epigenetic regulation of autophagy is poorly understood. Here we identify co-activator-associated arginine methyltransferase 1 (CARM1) as a crucial component of autophagy in mammals. Notably, CARM1 stability is regulated by the SKP2-containing SCF (SKP1-cullin1-F-box protein) E3 ubiquitin ligase in the nucleus, but not in the cytoplasm, under nutrient-rich conditions. Furthermore, we show that nutrient starvation results in AMP-activated protein kinase (AMPK)-dependent phosphorylation of FOXO3a in the nucleus, which in turn transcriptionally represses SKP2. This repression leads to increased levels of CARM1 protein and subsequent increases in histone H3 Arg17 dimethylation. Genome-wide analyses reveal that CARM1 exerts transcriptional co-activator function on autophagy-related and lysosomal genes through transcription factor EB (TFEB). Our findings demonstrate that CARM1-dependent histone arginine methylation is a crucial nuclear event in autophagy, and identify a new signalling axis of AMPK-SKP2-CARM1 in the regulation of autophagy induction after nutrient starvation.