Histone acetyltransferase 1 (Hat1) is responsible for the acetylation of newly synthesized histone H4 on lysines 5 and 12 during the process of chromatin assembly. To understand the broader biological role of Hat1, we have generated a conditional mouse knockout model of this enzyme. We previously reported that Hat1 is required for viability and important for mammalian development and genome stability. In this study, we show that haploinsufficiency of Hat1 results in a significant decrease in lifespan. Defects observed in Hat1+/− mice are consistent with an early‐onset aging phenotype. These include lordokyphosis (hunchback), muscle atrophy, minor growth retardation, reduced subcutaneous fat, cancer, and paralysis. In addition, the expression of Hat1 is linked to the normal aging process as Hat1 mRNA and protein becomes undetectable in many tissues in old mice. At the cellular level, fibroblasts from Hat1 haploinsufficient embryos undergo early senescence and accumulate high levels of p21. Hat1+/− mouse embryonic fibroblasts (MEFs) display modest increases in endogenous DNA damage but have significantly higher levels of reactive oxygen species (ROS). Consistently, further studies show that Hat1−/− MEFs exhibit mitochondrial defects suggesting a critical role for Hat1 in mitochondrial function. Taken together, these data show that loss of Hat1 induces multiple hallmarks of early‐onset aging.

The replisome is a protein complex on the DNA replication fork and functions in a dynamic environment at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter DNA duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the activity of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12, which accompanies replication-coupled chromatin assembly. Here, using proximity ligation assay-based chromatin assembly assays and DNA fiber analysis, we analyzed the role of murine HAT1 in replication-coupled chromatin assembly. We demonstrate that HAT1 physically associates with chromatin near DNA replication sites. We found that the association of HAT1 with newly replicated DNA is transient, but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant, as HAT1 loss decreased replication fork progression and increased replication fork stalling. Moreover, in the absence of HAT1, stalled replication forks were unstable, and newly synthesized DNA became susceptible to MRE11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.

A central component of the epigenome is the pattern of histone post-translational modifications that play a critical role in the formation of specific chromatin states. Following DNA replication, nascent chromatin is a 1:1 mixture of parental and newly synthesized histones and the transfer of modification patterns from parental histones to new histones is a fundamental step in epigenetic inheritance. Here we report that loss of HAT1, which acetylates lysines 5 and 12 of newly synthesized histone H4 during replication-coupled chromatin assembly, results in the loss of accessibility of large domains of heterochromatin, termed HAT1-dependent Accessibility Domains (HADs). HADs are mega base-scale domains that comprise ∼10% of the mouse genome. HAT1 globally represses H3 K9 me3 levels and HADs correspond to the regions of the genome that display HAT1-dependent increases in H3 K9me3 peak density. HADs display a high degree of overlap with a subset of Lamin-Associated Domains (LADs). HAT1 is required to maintain nuclear structure and integrity. These results indicate that HAT1 and the acetylation of newly synthesized histones may be critical regulators of the epigenetic inheritance of heterochromatin and suggest a new mechanism for the epigenetic regulation of nuclear lamina-heterochromatin interactions.

During S phase, eukaryotic cells must faithfully duplicate both the sequence of the genome and the regulatory information found in the epigenome. A central component of the epigenome is the pattern of histone post-translational modifications that play a critical role in the formation of specific chromatin states. During DNA replication, parental nucleosomes are disrupted and re-deposited on the nascent DNA near their original location to preserve the spatial memory of the epigenetic modifications. Newly synthesized histones must be also incorporated into the nascent chromatin to maintain nucleosome density. Transfer of modification patterns from parental histones to new histones is a fundamental step in epigenetic inheritance. Whether new histones play an active or passive role in epigenetic inheritance is unknown. Here we report that HAT1, which acetylates lysines 5 and 12 of newly synthesized histone H4 during replication-coupled chromatin assembly, regulates the epigenetic inheritance of chromatin states. HAT1 regulates the accessibility of large domains of heterochromatin termed HAT1-dependent Accessibility Domains (HADs). HADs are mega base-scale domains that comprise ~10% of the mouse genome. HAT1 functions as a global negative regulator of H3 K9me2/3 and HADs correspond to the regions of the genome that display HAT1-dependent increases in H3 K9me3 peak density. HADs display a high degree of overlap with a subset of Lamin-Associated Domains (LADs). HAT1 is required to maintain nuclear structure and integrity and physically associates with the nuclear lamina. These results indicate that HAT1 and the acetylation of newly synthesized histones are critical regulators of the epigenetic inheritance of heterochromatin and suggest a new mechanism for the epigenetic regulation of nuclear lamina-heterochromatin interactions.

Adrenocortical carcinoma (ACC) has a poor prognosis, and no new drugs have been identified in decades. The absence of drug development can partly be attributed to a lack of preclinical models. Both animal models and 2D cell cultures of ACC fail to accurately mimic the disease, as animal physiology is inherently different than humans, and 2D cultures fail to represent the crucial 3D architecture. Organoids are small 3D in vitro models of tissues or tumors, which can model certain complexities of human in vivo biology; however, this technology has yet to be applied to ACC. In this study, we describe the generation of organoids from an established ACC cell line, H295R. H295R cells were encapsulated to generate ACC organoid constructs. Organoids were assessed for biomarker expression, viability, proliferation, and cortisol production. In addition, matrix metalloproteinase (MMP) functionality was assessed directly using fluorogenic MMP-sensitive biosensors and through infusion of H295R cells into a metastasis-on-a-chip microfluidic device platform. ACC organoids showed expression of biomarkers associated with ACC, including SF-1, Melan A, and inhibin alpha. Treatment of ACC organoids with chemotherapeutics demonstrated decreased drug sensitivity compared to 2D cell culture. Since most tumor cells migrate through tissue using MMPs to break down extracellular matrix, we validated the utility of ACC organoids by integrating fluorogenic MMP-sensitive peptide biosensors within the organoids. Lastly, in our metastasis-on-a-chip device, H295R cells successfully engrafted in a downstream lung cell line-based organoid construct, but invasion into the lung organoid construct was decreased by MMP inhibition. These studies, which would not be possible using 2D cell cultures, demonstrated that H295R cells secreted active MMPs that are used for migration in 3D. This work represents the first evidence of a 3D organoid platform for ACC that can be deployed for future mechanistic studies as well as development of new targets for intervention and therapies.

Adrenocortical carcinoma (ACC) is an aggressive malignancy of the adrenal cortex. Five-year survival in Stage IV disease is 6%. Surgical resection is the mainstay of treatment; however, most patients present with unresectable disease or experience tumor recurrence after surgery. No targeted therapies are available for treatment of ACC. Thus, there is a critical need to understand molecular mechanisms driving advanced ACC. Matrix metalloproteinases (MMPs) have been associated with worse prognosis in other human cancers. Although pan-inhibition had limited clinical success, recent studies suggest select inhibition may improve patient outcomes. Here, we use The Cancer Genome Atlas (TCGA) and single nuclei RNA sequencing data (snRNAseq) to evaluate the expression of MMPs in ACC. RNA sequencing data from 77 ACC patients in TCGA was downloaded from UCSC Treehouse Public Data Tumor Compendium v11. Using the Kaplan-Meier estimator, survival was compared between the highest and lowest quartile of RNA expression for each MMP in R v.4.1. For snRNAseq, normal adrenal and adrenal adenoma samples were collected from a patient diagnosed with adrenal hypercortisolism. Seven primary and metastatic ACC samples were collected from patients diagnosed with ACC at time of surgical resection and included one primary tumor-metastasis pair. Single nuclei were isolated from frozen samples. Sequencing libraries were generated and sequenced on the Illumina NovaSeq 6000. Data were preprocessed using the 10x Genomics CellRanger software suite. Downstream analysis was performed using Seurat v.4 for R. ACC specimens in TCGA expressed all 24 MMPs with MMP14 demonstrating the highest expression. MMP14 expression was associated with significantly worse survival with a hazard ratio of 3.6 (95% CI 1.2 - 11.1). In addition, increased MMP14 expression was associated with higher AJCC tumor stage (p < 0.05). snRNAseq analysis of our ACC primary and metastatic samples identified 7 adrenal cortex clusters and one cluster each - fibroblasts, macrophages, infiltrating lymphocytes, endothelial, and adrenal medulla cells. Two cell clusters were present in primary and metastatic samples but not benign adrenal samples and were characterized by high KI67 expression or high ribosomal content. MMP14 expression was significantly higher in metastatic versus primary tumors in 2 adrenal cortex clusters with 3.6 and 2 fold increased expression, respectively (p < 0.05); and one of these clusters was defined by a 15-fold increase in expression of ZNRF3, which is involved in activation of the Wnt pathway. These data suggest that MMP14 expression is associated with worse survival and may be associated with metastatic progression. Further studies are needed to confirm whether simultaneous targeting of Wnt pathway and MMP14 may result in decreased metastatic progression. Citation Format: Liudmila V. Popova, Elaine Mardis, Katherine Miller, Priya H. Dedhia. Matrix metalloproteinase 14 in associated with poor survival in adrenocortical carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5286.