Recombinant Mouse Zinc fingers and homeoboxes protein 2 (Zhx2), partial

What is the basic structure and function of Zhx2 protein?

Zhx2 is a transcriptional regulator characterized by two amino-terminal C2-H2 zinc finger motifs and four to five carboxy-terminal homeodomains. These structural features facilitate DNA-protein and protein-protein interactions, enabling Zhx2 to function primarily as a transcriptional repressor.

The protein contains:

• Two Cys-Xaa2-Cys-Xaa12-His-Xaa4-His-type zinc finger motifs

• Five HOX-like homeodomains

• Nuclear localization signals that direct the protein to the nucleus

Functionally, Zhx2 regulates numerous genes including:

• Major urinary proteins (Mups)

• Alpha-fetoprotein (AFP) and H19

• Genes controlling lipid and cholesterol homeostasis

• Cytochrome P450 (Cyp) genes

Zhx2 can form homodimers or heterodimers with other Zhx family members and interacts with nuclear factor Y subunit alpha (NF-YA), further expanding its regulatory potential .

How does Zhx2 regulate gene expression in hepatocytes?

Zhx2 regulates hepatic gene expression through several mechanisms:

Direct promoter interaction: In transfection studies, Zhx2 can activate endogenous Mup genes in mouse hepatocyte cell lines. Research demonstrates that when a Zhx2-GFP fusion protein is transiently expressed in the AML12 mouse hepatocyte cell line, endogenous Mup20, Mup3, and class B Mup mRNA levels increase compared to control cells with empty vector .

Homeodomain-dependent activation: The homeodomain region of Zhx2, rather than the zinc-finger region, is primarily responsible for activating the Mup20 promoter. This was demonstrated through hybrid protein studies where researchers created Zhx1/Zhx2 hybrid proteins and found that the construct containing Zhx2 homeodomains significantly activated Mup20 p-luc, while Zhx1 did not .

Strain-specific differences: The BALB/cJ mouse substrain contains a natural hypomorphic mutation in Zhx2, resulting in dysregulation of various hepatic target genes including AFP and Mup genes. Transgenic BALB/cJ mice expressing Zhx2 specifically in hepatocytes (TTR-Zhx2) show restoration of Mup20 and class B Mup expression to approximately 45% and 86% of levels seen in wild-type BALB/c mice, respectively .

Multiple approaches can be employed to detect and quantify Zhx2 expression:

RT-qPCR Analysis:

• Isolate RNA from tissue of interest using standard protocols

• Perform reverse transcription to generate cDNA

• Use Zhx2-specific primers for qPCR analysis

• Normalize to appropriate housekeeping genes for the tissue being examined

• This approach has been used to show that Zhx2 is ubiquitously expressed in adult mouse tissues

Western Blotting:

• Use commercially available antibodies such as Rabbit anti-Zhx2 (Cell Signaling Technology #20937)

• Recommended dilution: 1:1000 for Western blotting

• Expected molecular weight: approximately 110 kDa

• Validated for human, mouse, and monkey samples

Immunofluorescence/Confocal Microscopy:

• Can be used to determine subcellular localization

• In most normal tissues, Zhx2 shows nuclear localization

• In podocytes, Zhx2 shows predominantly peripheral (non-nuclear) localization, forming heterodimers with Zhx1 in the cell body and with Zhx3 in the slit diaphragm

• Co-staining with markers like Aminopeptidase A (for podocyte body) or Nephrin (for slit diaphragm) can help determine precise localization

Immunoprecipitation:

• Can be performed using anti-Zhx2 antibody (recommended dilution 1:200)

• Useful for studying protein-protein interactions

• Has been used to demonstrate interactions of Zhx2 with aminopeptidase A and EPHRIN B1 in podocytes

What is the role of Zhx2 in kidney disease, particularly in podocyte function?

Zhx2 plays critical roles in kidney podocyte function and glomerular disease:

Localization and heterodimer formation:

• In healthy podocytes, Zhx2 is predominantly expressed at the cell membrane

• Forms heterodimers with different partners in distinct locations:

  • Zhx2-Zhx1 heterodimers: predominantly at the cell membrane of the podocyte cell body

  • Zhx2-Zhx3 heterodimers: predominantly at the slit diaphragm

Disease-specific nuclear translocation:

• In focal segmental glomerulosclerosis (FSGS), increased nuclear localization of Zhx3 and Zhx2 is observed

• In minimal change disease (MCD), increased nuclear localization of Zhx1 is observed

Experimental disease models:

• Zhx2-deficient mice develop worse experimental FSGS than controls

• Podocyte-specific Zhx2-overexpressing transgenic rats develop worse FSGS but less severe MCD than controls

• This differential effect appears related to the subcellular sequestration of different Zhx proteins

Protein interactions in disease:

• Zhx2 interacts with aminopeptidase A in the podocyte body cell membrane

• Zhx2 interacts with EPHRIN B1 in the slit diaphragm

• These interactions are central to disease pathogenesis:

  • Mice deficient in Enpep (aminopeptidase A gene) or Efnb1 (ephrin B1 gene) develop worse albuminuria in glomerular disease models

  • Targeting aminopeptidase A in Zhx2-deficient mice induces albuminuria and upregulates the MCD mediator angiopoietin-like 4 through nuclear entry of Zhx1

How can I design experiments to study Zhx2 transcriptional activity?

To investigate Zhx2 transcriptional activity:

Promoter-reporter assays:

• Clone the promoter region of a suspected Zhx2 target gene (e.g., Mup20) into a luciferase reporter vector

• Co-transfect with Zhx2 expression vector into appropriate cell lines (HEK293 cells have been used successfully)

• Measure luciferase activity to determine activation or repression

• Include appropriate controls (empty vector, known activators/repressors)

This approach has demonstrated that Zhx2 can activate the Mup20 promoter approximately 2.3-fold, while Zhx1 did not activate this promoter .

Domain mapping experiments:

• Create hybrid proteins containing different domains of Zhx2 fused with domains from related proteins (e.g., Zhx1)

• Co-transfect with reporter constructs to determine which domains are responsible for transcriptional effects

• This approach has shown that the homeodomain region of Zhx2, rather than the zinc-finger region, is primarily responsible for activating the Mup20 promoter

Endogenous gene expression analysis:

• Transiently express Zhx2 (or Zhx2-GFP fusion) in appropriate cell lines

• Extract RNA after 48 hours

• Perform RT-qPCR to measure expression changes in endogenous target genes

• Confirm protein expression and subcellular localization by fluorescence microscopy

This approach has demonstrated that Zhx2-GFP localizes to the nucleus in transfected cells and increases endogenous Mup20, Mup3, and class B Mup mRNA levels .

What is known about Zhx2 protein-protein interactions and how can they be studied?

Zhx2 engages in various protein-protein interactions that are crucial to its function:

Known interaction partners:

• Zhx1 and Zhx3: Forms heterodimers with other Zhx family members

• NF-YA (Nuclear Factor Y subunit A): Enhances transcriptional repression

• Aminopeptidase A: Interacts in podocyte body cell membrane

• EPHRIN B1: Interacts in the slit diaphragm of podocytes

Experimental approaches:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate with anti-Zhx2 antibody (1:200 dilution recommended)

  • Western blot for potential interacting partners

  • Has successfully identified interactions with aminopeptidase A and EPHRIN B1 in podocytes

• Yeast two-hybrid screening:

  • Has been used to identify Zhx family interactions

  • Can be performed using Zhx2 as bait to identify novel interaction partners

• Fluorescence microscopy and co-localization:

  • Perform immunofluorescence with antibodies against Zhx2 and potential partners

  • Analyze co-localization using confocal microscopy

  • This approach has shown co-localization of Zhx2 with aminopeptidase A and Nephrin in rodent glomeruli

• Proximity ligation assays:

  • Can detect protein-protein interactions in situ

  • Provides spatial information about where interactions occur within cells

• Domain mapping:

  • Create deletion constructs to identify which domains mediate specific interactions

  • Has shown that the homeodomain region of Zhx2 is important for its function

How does Zhx2 contribute to disease pathogenesis beyond kidney disorders?

Zhx2 has been implicated in multiple disease processes:

Hepatocellular carcinoma (HCC):

• Functions as a tumor suppressor in liver

• Loss of Zhx2 is associated with HCC development

• Mechanisms include dysregulation of AFP and other target genes

Hematological malignancies:

• Loss of Zhx2 is associated with lymphoma and myeloma development

• Zhx family members function in hematopoietic cell development and differentiation

Renal cell carcinoma:

• May function as an oncogene in clear cell renal carcinoma (ccRCC)

• Loss of von Hippel-Lindau protein (VHL) leads to increased Zhx2 expression and increased cellular proliferation

Atherosclerosis:

• Promotes macrophage survival and pro-inflammatory function

• Contributes to growth of atherosclerotic lesions

Neurological functions:

• Promotes maintenance of neural progenitor cells through repression of ephrin-B

• May have implications for neural development and neurological disorders

Experimental approaches to study these disease associations include:

• Mouse disease models (mentioned in FAQ #3)

• Expression analysis in patient samples

• Correlation of expression levels with disease progression and prognosis

• Mechanistic studies of downstream target genes in relevant cell types

What methodological challenges exist when working with recombinant Zhx2 protein?

Several methodological challenges must be addressed when working with recombinant Zhx2:

Protein size and complexity:

• Full-length Zhx2 is approximately 110 kDa

• Contains multiple structural domains that may fold independently

• Consider expressing partial constructs for specific applications

Expression systems:

• E. coli: Commonly used for partial Zhx2 constructs

• Mammalian cells (HEK-293): Better for full-length protein with proper folding and post-translational modifications

• Cell-free protein synthesis: Alternative approach for difficult-to-express proteins

Purification considerations:

• Use affinity tags (His, Strep) for purification

• Optimize buffer conditions (PBS, pH 7.4, containing DTT has been used)

• Consider additives for stability (0.01% SKL, 1 mM DTT, 5% Trehalose, Proclin-300)

Quality control:

• Verify purity by SDS-PAGE (>95% purity is achievable)

• Confirm identity by Western blotting with specific antibodies

• Analytical SEC (HPLC) can assess homogeneity

Storage stability:

• Store at 2-8°C for up to one month

• For longer storage, maintain at -80°C for up to one year

• Avoid repeated freeze/thaw cycles

Functional validation:

• DNA binding assays to confirm homeodomain functionality

• Protein interaction studies to verify structural integrity

• Reporter assays to confirm transcriptional activity

How can I investigate the role of Zhx2 in the context of cell-specific expression patterns?

To investigate cell-specific Zhx2 functions:

Tissue/cell isolation techniques:

• For podocyte studies: Dynabead isolation of glomeruli has been used to demonstrate 5-7 fold lower Zhx2 mRNA expression in BALB/cJ mice compared to BALB/c and C57BL/6 mice

• For hepatocytes: Primary hepatocyte isolation or use of liver-specific promoters in transgenic models

Cell-type specific expression analysis:

• Single-cell RNA sequencing:

  • Provides comprehensive expression patterns across cell types

  • Can identify co-expressed genes that may function with Zhx2

• Immunohistochemistry/Immunofluorescence:

  • Use co-staining with cell-type specific markers:

    • WT1 for podocyte nuclei

    • Nephrin for slit diaphragm

    • Aminopeptidase A for podocyte body

    • VWF for endothelial cells

• Cell-type specific transgenic models:

  • Use tissue-specific promoters:

    • NPHS2 (podocin) promoter for podocyte-specific expression

    • Albumin promoter for hepatocyte-specific expression

    • TTR (transthyretin) promoter for hepatocyte expression

Functional validation in specific cell types:

• Podocyte-specific Zhx2 transgenic rats showed:

  • Increased peripheral podocyte expression of Zhx2 protein

  • Partial overlap with Nephrin

  • No increased overlap with nuclear marker WT1

  • No overlap with endothelial marker VWF

• Three transgenic rat lines with varying Zhx2 expression levels have been generated:

  Transgenic Line	Glomerular Zhx2 mRNA Increase
  TG 14	13.7%
  TG 142	50.7%
  TG 144	309.8%

These models showed morphologically normal glomeruli by light microscopy but exhibited differential responses in experimental kidney disease models .