ASPSCR1 Human

What is the genomic location and protein structure of ASPSCR1?

ASPSCR1 is located on chromosome 17q25.3 in humans . The protein contains an N-terminal region involved in protein binding and a C-terminal portion (residues 313-553) that possesses the ability to disassemble VCP (Valosin-containing protein) hexamers . This C-terminal function is notably absent in the ASPSCR1::TFE3 fusion protein, which only retains the N-terminal portion (approximately the first 311 amino acids) of ASPSCR1 . For detailed structural analysis, researchers typically employ biochemical techniques including size exclusion chromatography and negative stain transmission electron microscopy (TEM), which have revealed distinct differences in how native ASPSCR1 versus the fusion protein interact with VCP hexamers .

What are the primary protein interactions of native ASPSCR1?

The most well-characterized interaction partner of ASPSCR1 is VCP (also known as p97), an AAA+ ATPase with segregase function. Full-length ASPSCR1 has been shown to interact with and disassemble VCP hexamers through its C-terminal motifs, which can be visualized using blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by Western blotting for VCP . When recombinant VCP is mixed with equimolar ASPSCR1 in vitro, the resulting complexes elute later than VCP alone during size exclusion chromatography, consistent with ASPSCR1-mediated disassembly of VCP hexamers . This interaction is functionally significant as VCP participates in multiple cellular processes including protein quality control and chromatin-associated functions.

How is ASPSCR1 expression distributed across normal human tissues?

ASPSCR1 shows a broad expression pattern across human tissues. According to The Human Protein Atlas data, ASPSCR1 protein expression has been detected in numerous tissues including the hippocampal formation, cerebral cortex, thyroid gland, lung, gastrointestinal tract, urinary system, and reproductive organs . Understanding this expression pattern is important for contextualizing the pathological role of ASPSCR1 in fusion-positive tumors. When analyzing ASPSCR1 expression, researchers should employ multiple complementary techniques such as RNA-seq, quantitative PCR, and immunohistochemistry to ensure accurate tissue distribution mapping.

What are the molecular characteristics of the ASPSCR1::TFE3 fusion variants?

The ASPSCR1::TFE3 fusion results from a t(X;17)(p11;q25) chromosomal translocation that creates a chimeric gene encoding a fusion oncoprotein . There are two primary fusion variants:

• Type-1 fusion: Contains a specific breakpoint junction between ASPSCR1 and TFE3

• Type-2 fusion: Has a different breakpoint where ASPSCR1 is joined in-frame to TFE3

Both variants retain the N-terminal region of ASPSCR1 (approximately 311 amino acids) and most of TFE3, including its DNA-binding domain . This fusion is often referred to as AT3 in the scientific literature . Detection methods include RT-PCR with variant-specific primers, RNA sequencing for precise breakpoint characterization, and fluorescence in situ hybridization (FISH) for chromosomal rearrangement confirmation .

How do the fusion variants differ functionally?

The type-1 and type-2 ASPSCR1::TFE3 fusion variants demonstrate significant functional differences that affect tumor biology:

These differences suggest that the specific fusion variant may influence therapeutic response, particularly to immune checkpoint inhibitors and VCP-targeting agents .

How does the fusion protein reprogram chromatin organization?

ASPSCR1::TFE3 functions as an aberrant transcription factor that dramatically alters the chromatin landscape. ChIP-seq analysis demonstrates that the fusion protein predominantly binds to distal regulatory regions (69-78% of binding sites) with only 16-22% of peaks located at gene promoters in both mouse and human ASPS . The consensus binding motif for the MiT/TFE family transcription factors, CACGTGAC, is highly enriched at these binding sites .

A critical mechanism of ASPSCR1::TFE3 action involves its frequent association with super-enhancers (SEs). ChIP-seq reveals that 75-85% of ASPSCR1::TFE3 binding sites in mouse and human ASPS coincide with H3K27ac, a histone mark of active enhancers . Loss of ASPSCR1::TFE3 expression significantly reduces H3K27ac signals at these regions, demonstrating that the fusion protein actively maintains enhancer activity . Furthermore, ASPSCR1::TFE3 facilitates the assembly of higher-order chromatin conformation structures as demonstrated by HiChIP analysis, orchestrating long-range interactions that drive oncogenic gene expression programs .

What experimental models exist for studying ASPSCR1::TFE3-positive cancers?

Several complementary experimental models have been developed for studying ASPSCR1::TFE3-driven cancers:

Cell Line Models:

• ASPS-1: Derived from a female patient, expresses the type-1 fusion (AT3.1)

• FU-UR-1: Derived from a male patient, expresses the type-2 fusion (AT3.2)

• HEK293T: Frequently used for ectopic expression studies of ASPSCR1::TFE3 constructs

Mouse Models:

• Conditional AT3 expression models that develop tumors recapitulating human ASPS histology and transcriptome in perivascular tissues of the brain and retina

• Ex vivo mouse model for human ASPS developed by Tanaka et al.

Advanced Experimental Systems:

• Organ-on-a-chip systems for investigating ASPSCR1::TFE3-induced angiogenesis

• Patient-derived materials, including frozen samples and tissue microarrays from repositories

When selecting an experimental model, researchers should consider factors including the specific fusion variant being studied, the need for an intact tumor microenvironment, and the specific research question being addressed.

What genomic analysis techniques are optimal for ASPSCR1::TFE3 research?

For comprehensive genomic analysis of ASPSCR1::TFE3-driven cancers, several complementary techniques are recommended:

Fusion Detection and Characterization:

• RNA Capseq panel, a targeted sequencing approach focusing on coding regions of cancer-related genes, is particularly effective for identifying ASPSCR1::TFE3 fusions

• RT-PCR with specific primers to distinguish between fusion variants

• FISH for detecting chromosomal rearrangements in archival materials

Genome-wide Binding Analysis:

• ChIP-seq using antibodies against TFE3 (C-terminal region) or ASPSCR1 (N-terminal region), or with epitope-tagged constructs

• For optimal ChIP-seq results, consider both formaldehyde crosslinking and dual crosslinking approaches to capture protein-protein interactions

• Include parallel ChIP-seq for histone marks (H3K27ac, H3K4me1) to identify active enhancers and super-enhancers

Higher-order Chromatin Analysis:

• HiChIP to identify long-range chromatin interactions mediated by ASPSCR1::TFE3

• ATAC-seq to assess chromatin accessibility at ASPSCR1::TFE3 binding sites

Transcriptomic Analysis:

• RNA-seq to identify differentially expressed genes and pathways

• Single-cell RNA-seq to capture cellular heterogeneity within tumors

When analyzing ChIP-seq data, researchers should focus on the consensus MiT/TFE family binding motif (CACGTGAC) and examine the distribution of binding sites relative to gene features, particularly super-enhancers associated with angiogenesis pathways .

How can CRISPR-based techniques be applied to ASPSCR1::TFE3 research?

CRISPR-based techniques have emerged as powerful tools for investigating ASPSCR1::TFE3 biology:

Epigenomic CRISPR Screening:

• dCas9-KRAB (CRISPRi) can be used to identify critical enhancers regulated by ASPSCR1::TFE3

• This approach has successfully identified genes associated with reduced enhancer activities due to ASPSCR1::TFE3 loss, including Pdgfb, Rab27a, Sytl2, and Vwf

• The screening specifically targeted 494 super-enhancers and active enhancers where H3K27ac signals were reduced by >40% upon perturbation

Functional Validation:

• CRISPR/Cas9 knockout of specific target genes (e.g., Gpnmb) has demonstrated their role in processes like tumor cell intravasation

• Knockdown experiments have shown that Gpnmb is required for transendothelial migration of ASPS tumor cells

Engineered Models:

• CRISPR-mediated knock-in of ASPSCR1::TFE3 fusions can generate new cellular models

• Artificial chimeric genes consisting of 5' ASPSCR1 and 3' TFE/MITF sequences have been tested, revealing that ASPSCR1-TFE3 and ASPSCR1-TFEB, but not ASPSCR1-TFEC and ASPSCR1-MITF, induce sarcoma

When designing CRISPR screens, researchers should consider both gene-level and enhancer-level targeting strategies, as ASPSCR1::TFE3 exerts much of its oncogenic effect through enhancer reprogramming rather than direct promoter regulation.

How does the VCP interaction differ between native ASPSCR1 and the fusion protein?

The interaction between ASPSCR1 and VCP undergoes a fundamental change in the context of the fusion protein:

Native ASPSCR1-VCP Interaction:

• Full-length ASPSCR1 interacts with and disassembles VCP hexamers through its C-terminal motifs (residues 313-553)

• This disassembly can be visualized by blue native polyacrylamide gel electrophoresis (BN-PAGE) and Western blotting for VCP

• In size exclusion chromatography, VCP:ASPSCR1 complexes elute later than VCP alone, consistent with disassembly of the larger hexameric structure

ASPSCR1::TFE3-VCP Interaction:

• The fusion protein, which lacks the C-terminal portion of ASPSCR1, interacts with VCP but does not disassemble VCP hexamers

• Negative staining and transmission electron microscopy (TEM) of co-immunoprecipitates reveals intact hexameric VCP with ASPSCR1::TFE3, but not with full-length ASPSCR1

• In size exclusion chromatography, VCP-ASPSCR1::TFE3 complexes elute earlier than VCP alone, suggesting preservation of the hexameric structure

This difference has profound functional implications, as VCP has emerged as "a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology" . The intact VCP hexamer appears essential for the oncogenic transcriptional program driven by ASPSCR1::TFE3.

How does ASPSCR1::TFE3 regulate tumor angiogenesis?

ASPSCR1::TFE3 orchestrates a sophisticated angiogenic program that distinguishes ASPS from many other cancers:

Angiogenic Gene Regulation:

• ASPSCR1::TFE3 directly regulates key angiogenic factors through super-enhancer modulation

• Epigenomic CRISPR screening has identified Pdgfb, Rab27a, Sytl2, and Vwf as critical targets associated with reduced enhancer activities due to ASPSCR1::TFE3 loss

• These genes play crucial roles in vascular development and stability

Vesicular Transport Mechanisms:

• Upregulation of Rab27a and Sytl2 specifically promotes angiogenic factor trafficking

• This facilitates the construction of ASPS's distinctive vascular network

• Unlike many cancers that have disorganized vasculature, ASPS induces abundant and highly integrated blood vessels with pericyte wrapping

In Vivo Requirement:

• While ASPSCR1::TFE3 expression is dispensable for in vitro tumor maintenance, it is required for in vivo tumor development via angiogenesis

• The loss of ASPSCR1::TFE3 expression induces super-enhancer distribution changes related to genes in the angiogenesis pathway

Vascular Integration:

• Mouse ASPS models show tumor intravasation with intravascular tumor cells presenting as organoid structures covered with hemangiopericytes, a feature also observed in human ASPS

• Culture supernatant from mouse ASPS cells strongly induces hemangiopericyte migration in vitro

This sophisticated angiogenic program contributes to ASPS's distinctive histology and likely plays a role in its metastatic behavior, as the high-quality vascular network facilitates tumor cell intravasation and dissemination.

What downstream transcriptional pathways are activated by different fusion variants?

The two ASPSCR1::TFE3 fusion variants activate distinct downstream transcriptional pathways:

Type-1 Fusion (AT3.1) Pathways:

• Immunogenic pathways including PD-L1/PD-1 checkpoint expression

• NK-cell-mediated cytotoxicity pathways

• Higher interferon-gamma (IFNG) and tumor inflammation signature (TIS) scores

• Strong association with HIF1A regulation in ASPS-1 cells and human ASPS tumors

Type-2 Fusion (AT3.2) Pathways:

• Autophagy-related pathways

• Apelin signaling pathway activation

• Lower inflammatory gene signatures [10