CHRAC1 Human

What is CHRAC1 and what is its primary function?

CHRAC1 is a histone-fold protein encoded by the CHRAC1 gene located on human chromosome 8. It functions by interacting with other histone-fold proteins to bind DNA in a sequence-independent manner . The primary function of CHRAC1 involves chromatin remodeling, playing a crucial role in altering chromatin structure and controlling the accessibility of transcription factors to DNA . This activity is essential for dynamic regulation of gene expression, particularly in contexts like cancer progression.

CHRAC1 has several aliases including CHARC1, CHARC15, CHRAC-1, CHRAC-15, CHRAC15, YCL1, and is also referred to as chromatin accessibility complex 15 kDa protein or DNA polymerase epsilon subunit p15 . The protein combines with other histone-fold protein dimers within larger enzymatic complexes to facilitate DNA transcription, replication, and packaging .

How is CHRAC1 expression regulated in normal versus disease states?

The elevated expression of CHRAC1 in cancer tissues suggests that its overexpression may contribute to oncogenic processes. Notably, CHRAC1 expression has been found to be statistically associated with YAP (Yes-associated protein) expression in breast and cervical cancer biopsies, indicating a potential co-regulatory mechanism in cancer progression .

What are the most effective methods for studying CHRAC1 expression and function?

When investigating CHRAC1 expression and function, researchers should consider multiple complementary approaches:

Protein Expression Analysis:

• Western blotting remains a gold standard for CHRAC1 protein detection. Use specific antibodies (e.g., ABclonal rabbit CHRAC1: A14896, 1:500 dilution) for reliable detection .

• Immunohistochemistry is valuable for assessing CHRAC1 expression in tissue samples and correlating with clinical parameters.

Functional Studies:

• RNA interference using shRNA or siRNA offers effective means to knock down CHRAC1 expression. Validated sequences include 5′-ACTCCACTGTCTCTAAGTAAA-3′ .

• Cell proliferation assays (CCK-8), colony formation assays, and wound-healing assays provide functional readouts of CHRAC1 modulation .

• For in vivo validation, xenograft models in immunocompromised mice (e.g., BALB/c nude mice) can assess tumor growth characteristics after CHRAC1 modulation .

Interaction Studies:

• Co-immunoprecipitation (Co-IP) is effective for identifying protein-protein interactions with CHRAC1. This method has successfully demonstrated interaction between CHRAC1 and YAP .

• The Bio-ID method has proven valuable for identifying the wider interactome of CHRAC1-associated proteins .

How should RNA-seq experiments be designed to study CHRAC1-mediated transcriptional changes?

When designing RNA-seq experiments to investigate CHRAC1-mediated transcriptional changes, researchers should follow these methodological guidelines:

• Experimental Design:

  • Include appropriate biological replicates (minimum three per condition)

  • Implement CHRAC1 knockdown using validated shRNAs with appropriate controls (shNC)

  • Consider including both short-term and long-term knockdown timepoints to distinguish between direct and indirect effects

• Sample Preparation:

  • Extract high-quality total RNA using validated methods (e.g., Vazyme RNA extraction Kit)

  • Verify RNA concentration and purity using spectrophotometry

  • Perform quality control using RNA integrity number (RIN) assessment

• Data Analysis Pipeline:

  • Analyze differential gene expression comparing CHRAC1-knockdown to control samples

  • Implement Gene Ontology (GO) annotation to identify enriched biological processes

  • Conduct pathway analysis (e.g., KEGG) to identify affected signaling networks

  • Use Gene Set Enrichment Analysis (GSEA) to detect subtle but coordinated changes in gene expression

• Validation:

  • Confirm key findings using RT-qPCR for selected genes

  • Prioritize validation of YAP target genes and cancer-related pathways

This approach revealed that CHRAC1 knockdown affects 2,595 genes (1,300 upregulated, 1,295 downregulated) with enrichment in pathways related to transcription, cell proliferation, apoptosis, migration, and the Hippo signaling pathway .

What in vivo models are most appropriate for studying CHRAC1 in cancer?

Based on existing research, the following in vivo models have proven effective for studying CHRAC1 in cancer:

Xenograft Models:

• BALB/c nude mice (5-week-old) have been successfully used for CHRAC1 studies in both breast and cervical cancer

• Typical protocol involves subcutaneous injection of 5 × 10^6 cancer cells (control vs. CHRAC1-knockdown) into the dorsal side of nude mice

• Tumor volume should be monitored every other day using the formula V = (L × W^2) / 2 (where L is length and W is width)

• The experimental endpoint typically occurs at 35-40 days post-injection, with tumors generally not exceeding 2000 mm^3

Assessment Methods:

• Tumor weight comparison between control and CHRAC1-modulated groups

• Immunohistochemistry of tumor sections for proliferation markers (e.g., Ki67)

• RNA extraction from tumor samples for gene expression analysis

• Protein extraction for western blot analysis of CHRAC1 and related pathways

The statistical power of this experimental design can be determined through preliminary experiments, with a minimum of three mice per group recommended . Housing conditions should include specific-pathogen-free environments (22 ± 2°C, 60 ± 10% relative humidity, 12-hour light/dark cycle) .

How does CHRAC1 regulate YAP-mediated transcriptional activity?

CHRAC1 has been identified as a crucial regulator of YAP-mediated transcriptional activity through several mechanisms:

• Direct Protein Interaction:

  • CHRAC1 was identified as a potential YAP interactor using the Bio-ID method

  • Co-immunoprecipitation confirmed physical interaction between CHRAC1 and YAP

  • This interaction appears to be functionally significant in promoting YAP's transcriptional activity

• Transcriptional Program Regulation:

  • CHRAC1 silencing leads to significant downregulation of YAP target genes

  • Gene Set Enrichment Analysis (GSEA) demonstrates that YAP target gene signatures are enriched in control cells but not in CHRAC1-silenced cells

  • RT-qPCR validation confirms that depletion of CHRAC1 reduces mRNA levels of classical YAP target genes

• Chromatin Remodeling:

  • As a component of chromatin remodeling complexes, CHRAC1 likely facilitates YAP's access to target gene promoters

  • This allows YAP to recruit transcription factors like TEAD to activate oncogenic gene expression programs

  • The ATP-dependent chromatin remodeling function of CHRAC1 may be essential for this process

This regulatory relationship appears particularly important in cancer contexts, as CHRAC1 expression is statistically associated with YAP expression in breast and cervical cancer biopsies . This suggests a potential co-regulatory network that drives oncogenic transcription programs in these cancer types.

What is the role of CHRAC1 in cancer progression and metastasis?

CHRAC1 plays a multifaceted role in cancer progression and metastasis:

Proliferation and Tumor Growth:

• CHRAC1 silencing significantly suppresses cancer cell proliferation in vitro, as demonstrated by CCK-8 assays and colony formation assays

• In vivo, CHRAC1 knockdown reduces tumor growth in xenograft models, with the average weight of CHRAC1-silenced Hela xenografts being approximately one-third of control tumors

• Ki67 staining of tumor sections confirms reduced proliferative capacity in CHRAC1-silenced tumors

Cell Migration and Invasion:

• Wound-healing assays demonstrate that CHRAC1 downregulation restrains cancer cell migration

• Previous studies have shown that CHRAC1 disruption inhibits migration and invasion in cisplatin-resistant ovarian cancer cells and H1299 lung cancer cells

Transcriptional Regulation:

• RNA-seq analysis reveals that CHRAC1 regulates genes involved in cell proliferation, apoptosis, and migration

• CHRAC1 ablation suppresses expression of cancer hallmark genes related to DNA repair, G2M checkpoint, and the P53 pathway

• The protein may contribute to tumor progression by promoting oncogenic transcription through interaction with YAP

Clinical Correlation:

• CHRAC1 is elevated in cervical and breast cancer biopsies

• High CHRAC1 expression correlates with shorter survival, poor pathological stages, and increased metastasis in cancer patients

• These clinical associations support CHRAC1's role as a potential oncogenic driver and therapeutic target

How does CHRAC1 influence DNA repair mechanisms in cancer cells?

While the search results don't provide extensive details specifically about CHRAC1's influence on DNA repair mechanisms, several key insights can be inferred:

RNA-seq data from CHRAC1-silenced cells shows that CHRAC1 knockdown suppresses the expression of representative cancer hallmarks, including genes involved in DNA repair processes . This suggests that CHRAC1 may positively regulate DNA repair mechanisms in cancer cells, potentially contributing to therapy resistance.

As a chromatin remodeler, CHRAC1 likely plays a role in controlling chromatin accessibility during DNA repair processes. Chromatin remodeling is essential for allowing repair proteins to access damaged DNA sites. Given that CHRAC1 interacts with other histone-fold proteins to bind DNA in a sequence-independent manner , it may facilitate this access in response to DNA damage.

Research has shown that CHRAC1 expression is increased in cisplatin-resistant ovarian cancer cell lines , suggesting a potential role in developing resistance to DNA-damaging therapies. Cisplatin primarily acts by forming DNA-platinum adducts that disrupt DNA structure, so increased CHRAC1 may help cancer cells repair or tolerate this damage.

What are the potential therapeutic approaches targeting CHRAC1 in cancer?

Based on the research findings, several therapeutic approaches targeting CHRAC1 show promise:

RNA Interference:

• Short hairpin RNA (shRNA) targeting CHRAC1 has demonstrated efficacy in reducing tumor growth in both in vitro and in vivo models

• This approach could be developed into therapeutic RNAi strategies targeting CHRAC1 expression

Small Molecule Inhibitors:

• Development of small molecules that disrupt the interaction between CHRAC1 and YAP could inhibit the oncogenic transcription program

• Compounds targeting the histone-fold domain of CHRAC1 might interfere with its DNA-binding capacity

Combination Therapies:

• Given CHRAC1's role in cisplatin resistance , combining CHRAC1 inhibition with conventional chemotherapeutics might overcome resistance mechanisms

• Dual targeting of CHRAC1 and YAP pathways could provide synergistic anti-cancer effects

Patient Stratification:

• CHRAC1 expression levels could serve as a biomarker for patient stratification, identifying those who might benefit most from targeted therapies

• The correlation between CHRAC1 expression and poor prognosis suggests its value as a prognostic marker

The development of these therapeutic approaches is supported by the observation that CHRAC1 depletion significantly reduces cancer cell proliferation and tumor growth . Furthermore, the specific role of CHRAC1 in promoting YAP-mediated oncogenic transcription provides a rational basis for targeted intervention in multiple cancer types.

What technical challenges exist in CHRAC1 research, and how can they be addressed?

Researchers studying CHRAC1 face several technical challenges that require careful methodological considerations:

Protein Complex Analysis:

• Challenge: CHRAC1 functions within large protein complexes, making it difficult to distinguish its individual contribution

• Solution: Implement BioID or proximity labeling approaches to identify context-specific interaction partners

• Utilize CRISPR-based genetic screens to identify synthetic lethal interactions with CHRAC1

Specificity in Chromatin Remodeling Studies:

• Challenge: Determining which chromatin regions are specifically affected by CHRAC1 activity

• Solution: Combine CHRAC1 modulation with ATAC-seq or DNase-seq to identify differential chromatin accessibility regions

• Perform ChIP-seq for CHRAC1 and its interaction partners (e.g., YAP) to map genomic binding sites

Translation to Clinical Applications:

• Challenge: Translating basic CHRAC1 research findings into clinically relevant applications

• Solution: Validate findings across diverse patient-derived xenograft models and clinical samples

• Develop tissue-specific CHRAC1 modulation approaches to minimize off-target effects

Distinguishing Direct vs. Indirect Effects:

• Challenge: Separating direct CHRAC1-mediated effects from downstream consequences

• Solution: Implement time-course experiments after CHRAC1 modulation

• Use rapid protein degradation systems (e.g., auxin-inducible degron) for acute CHRAC1 depletion

By addressing these challenges with innovative methodological approaches, researchers can advance our understanding of CHRAC1 biology and exploit its therapeutic potential in cancer treatment.