Mechanisms of TOX4 deficiency-induced steatosis

The liver plays a central role in systemic glucose and lipid homeostasis. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and is strongly associated with obesity (70-90%) and T2D (80%). NAFLD is driven by complex inherited and environmental factors related to obesity, insulin resistance, and dyslipidemia. Currently, there is no approved drug for treating NAFLD due to a limited understanding of its pathological mechanisms. The PI has recently identified TOX High Mobility Group Box Family Member 4 (TOX4) as a novel insulin-regulated TF that regulates hepatic glucose production in parallel with the classic insulin receptor/FoxO1 pathway. Interestingly, acute knockdown or congenital knockout of TOX4 in hepatocytes robustly promotes lipid accumulation in the liver. Conversely, overexpression of TOX4 in primary hepatocytes suppresses lipogenesis and reduces liver lipid accumulation in vivo. Further RNA-seq analysis of liver samples from TOX4 flox and liver-specific knockout (TLKO) mice revealed that the top differentially expressed genes are enriched in lipid metabolic pathways, suggesting TOX4 as a novel negative regulator of lipid synthesis. Furthermore, complement factor D (CFD, also named adipsin) is among the top upregulated genes in the TLKO liver. Indeed, CFD is elevated in the livers of human NAFLD subjects and obese mice, distinct from its circulating levels and adipose expression. Whole-body knockout of CFD protects mice from liver steatosis. Therefore, the PI hypothesizes that hepatocyte TOX4 protects from NAFLD through transcriptional repression of lipogenic genes and CFD.

In Aim 1, the PI will first perform chromatin immunoprecipitation sequencing (ChIP-seq) to determine the genome-wide landscape of TOX4-mediated transcription regulation under normal and pathological conditions, focusing on the loci of lipogenic genes. Combining ChIP-seq and RNA-seq analyses, she will identify the direct and indirect target genes of TOX4, which will provide insights into subsequent mechanistic studies. In Aim 2, she will first establish CFD as a downstream target of TOX4 and begin to elucidate the repressive mechanisms. She will generate mice to specifically ablate CFD in TLKO hepatocytes and critically test whether TOX4 deficiency promotes steatosis through a CFD-dependent mechanism. Successful completion of the proposed studies in this application will illustrate the function and mechanism of TOX4 in regulating hepatic lipid metabolism. This proposal will identify novel genes that are critical for the development of NAFLD and therefore serve as therapeutic targets. The proposal will also help the PI to generate essential preliminary data to apply for the R01 grant.