Recombinant Mouse PHD finger protein 20-like protein 1 (Phf20l1), partial

What is PHF20L1 and what is its primary function in cellular processes?

PHF20L1 functions as a H3K27me2 reader protein that coordinates with transcriptional repressor complexes to regulate gene expression. Specifically, PHF20L1 interacts with Polycomb Repressive Complex 2 (PRC2) and the Nucleosome Remodeling and Deacetylase (NuRD) complex to maintain the repressed state of target genes. This interaction leads to increased H3K27me2/me3 levels and decreased H3K27ac at target gene promoters, resulting in transcriptional silencing of tumor suppressor genes (TSGs) .

To study PHF20L1's primary function, researchers commonly employ chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify genome-wide binding sites and quantitative ChIP (qChIP) to validate specific target regions. These approaches have revealed that PHF20L1 preferentially binds to H3K27me2-enriched regions and recruits epigenetic modifiers to maintain repressive chromatin states.

How does PHF20L1 differ from its homolog PHF20?

Despite structural similarities, PHF20L1 and PHF20 have distinct interacting partners and cellular functions:

Co-immunoprecipitation (Co-IP) experiments have demonstrated that PHF20L1 specifically interacts with PRC2/NuRD components, while PHF20 associates with MOF . Notably, the interaction between PHF20 and PRC2 disappears in the presence of DNase, indicating an indirect binding mediated by DNA fragments, whereas PHF20L1's interaction with PRC2/NuRD remains stable .

What are the key structural domains of PHF20L1 and their functions?

PHF20L1 contains several crucial domains that mediate its function:

• TUDOR domain: Specifically recognizes and binds to H3K27me2 histone marks. The E92K mutation in this domain abolishes its ability to recognize H3K27me2 .

• NID (NuRD Interaction Domain): Essential for interaction with the NuRD complex components, particularly MTA1/2 through their SANT domains .

• PID (PRC2 Interaction Domain): Mediates binding to the PRC2 complex, specifically interacting with the D1 domain of EZH2 .

Rescue experiments with wild-type and mutant PHF20L1 (E92K, ΔNID, ΔPID) in knockdown cells demonstrate that all three domains are indispensable for proper function. None of the mutants can fully restore PRC2 or NuRD binding and the modification status of target genes, indicating that the intact complex is required for proper function .

What genes are directly regulated by PHF20L1 and how?

PHF20L1 primarily represses tumor suppressor genes (TSGs) including:

• BRCA1 (Breast Cancer Type 1 Susceptibility Protein)

• HIC1 (Hypermethylated in Cancer 1)

• KISS1 (Kisspeptin-1)

• GATA2 (GATA Binding Protein 2)

Mechanistically, PHF20L1 recognizes H3K27me2 marks at these gene promoters through its TUDOR domain and subsequently recruits PRC2 (which adds H3K27me3) and the NuRD complex (which removes acetylation), resulting in a condensed chromatin structure and transcriptional repression . Knockdown of PHF20L1 significantly reduces the enrichment of EZH2, MTA1, H3K27me2, and H3K27me3 on target genes while increasing H3K27ac levels, leading to transcriptional activation .

What experimental approaches are recommended for studying PHF20L1's interaction with chromatin and protein complexes?

Several complementary approaches have proven effective:

• Affinity Purification-Mass Spectrometry: Identify interacting proteins by expressing FLAG-tagged PHF20L1 in cells (such as HEK293T and MDA-MB-231), performing affinity purification with anti-FLAG beads, and analyzing eluates by mass spectrometry. This approach has successfully identified PRC2 and NuRD components as PHF20L1 interactors .

• Co-Immunoprecipitation (Co-IP): Confirm interactions in different cell types. Include DNase treatment to distinguish direct from DNA-mediated interactions .

• Protein Fractionation by Fast Protein Liquid Chromatography (FPLC): Use Superose 6 gel filtration chromatography to analyze elution patterns of PHF20L1 and its potential interactors, confirming whether they exist in the same protein complexes .

• In Vitro Pull-Down Assays: Use His-fused PHF20L1 with in vitro-transcribed/translated components or GST-fused complex components with in vitro-transcribed/translated PHF20L1 to map direct interactions and specific binding domains .

• ChIP-seq and qChIP: Determine the genome-wide and locus-specific chromatin occupancy of PHF20L1 and associated proteins/modifications .

How can researchers generate and validate effective PHF20L1 knockout or knockdown models?

Both in vitro and in vivo models can be effectively generated:

In Vitro Models:

• shRNA-mediated knockdown in breast cancer cell lines (e.g., MDA-MB-231, Hs 578T)

• Validate knockdown efficiency by qRT-PCR and Western blotting

In Vivo Models:

• Global Knockout: Use CRISPR/Cas9-mediated genome editing targeting the PHF20L1 gene. Validate by genotyping and phenotypic analysis. Note that global knockout mice exhibit growth retardation but remain viable .

• Conditional Knockout: Cross mice bearing floxed PHF20L1 with tissue-specific Cre lines (e.g., MMTV-Cre for mammary gland-specific deletion). This approach avoids systemic effects while allowing tissue-specific analysis .

• Validation Methods:

  • Genotyping PCR

  • Tissue-specific qRT-PCR to confirm deletion

  • Immunohistochemistry for downstream markers

  • Phenotypic analysis (growth curves, tissue development)

Importantly, phenotypic analysis should consider:

• Body weight measurements over time

• Reproductive development timing

• Mammary gland whole-mount analysis

• Proliferation markers (cyclin D1, Ki67) by IHC

What methodological approaches can be used to study PHF20L1's role in tumorigenesis?

Multiple complementary approaches have been employed:

• Cell-Based Assays:

  • Colony formation assays with PHF20L1 knockdown or overexpression

  • Cell migration and invasion assays

  • Cell cycle analysis by flow cytometry

  • BrdU incorporation assays to measure proliferation

  • Annexin V-FITC apoptosis detection

• In Vivo Tumor Models:

  • Orthotopic xenograft models: Inject manipulated breast cancer cells (with PHF20L1 knockdown or overexpression) into mammary fat pads of immunocompromised mice and measure tumor growth

  • Metastasis models: Intravenously inject cells stably expressing luciferase and quantify metastatic burden using bioluminescence imaging

  • Genetically engineered mouse models: Cross Phf20l1 conditional knockout mice with tumor-prone mouse models to assess effects on spontaneous tumorigenesis

• Molecular Mechanistic Studies:

  • RNA-seq to identify genome-wide transcriptional changes

  • ChIP-seq to map chromatin occupancy and histone modifications

  • Rescue experiments with wild-type and mutant PHF20L1 to determine functional domains

Studies using these approaches have demonstrated that PHF20L1 deficiency significantly reduces breast cancer cell lung metastasis in vivo, while PHF20L1 overexpression promotes lung metastasis .

How does PHF20L1 coordinate the cross-talk between histone methylation and deacetylation?

PHF20L1 serves as a critical bridge between histone methylation and deacetylation at H3K27:

• Recognition: PHF20L1's TUDOR domain specifically recognizes H3K27me2 marks .

• Recruitment: Through its PID domain, PHF20L1 recruits PRC2 complex (containing EZH2, which catalyzes H3K27 trimethylation), while its NID domain recruits the NuRD complex (containing HDACs, which remove acetylation from H3K27) .

• Coordinated Action: The simultaneous recruitment of these complexes ensures both increased methylation and decreased acetylation at H3K27, reinforcing the repressive chromatin state.

• Experimental Validation:

  • ChIP-seq analysis shows that PHF20L1 loss leads to reduced EZH2, MTA1, H3K27me2, and H3K27me3 occupancy while increasing H3K27ac levels .

  • Rescue experiments demonstrate that wild-type PHF20L1, but not domain mutants, can restore the epigenetic modifications at target genes .

This mechanism provides insights into how reader proteins can coordinate the activities of different epigenetic complexes to maintain specific chromatin states.

What are the detailed phenotypic characteristics of Phf20l1 knockout mice, and how can they be quantitatively assessed?

Phf20l1 knockout mice exhibit several distinct phenotypes that can be quantitatively characterized:

For detailed mammary gland analysis, researchers should:

• Harvest the #4 inguinal mammary gland

• Perform whole-mount staining with carmine alum

• Image using stereomicroscopy

• Quantify ductal outgrowth (distance from lymph node to terminal end buds)

• Perform IHC for proliferation markers in tissue sections

• Quantify the percentage of positive cells

These phenotypes align with PHF20L1's role in promoting growth-related gene expression and suggest its importance in normal development and tumorigenesis .