Recombinant Danio rerio Sterol regulatory element-binding protein 2 (srebf2)

What is srebf2 and what are its primary functions in Danio rerio?

Srebrp2 (encoded by the srebf2 gene) in Danio rerio is a transcription factor that functions as a master regulator of cholesterol metabolism. It belongs to the sterol regulatory element-binding protein (SREBP) family, with the specific role of controlling cholesterol homeostasis, which is distinct from SREBP1 that primarily regulates fatty acid metabolism . In zebrafish, srebf2 is located on chromosome 3 and encodes a protein that enables DNA-binding transcription factor activity specific to RNA polymerase II and binds to cis-regulatory regions with sequence specificity .

The protein is initially produced as a large precursor molecule anchored to the endoplasmic reticulum membrane. When cellular sterol levels are depleted, the N-terminal segment containing the basic helix-loop-helix-leucine zipper (bHLH-LZ) domain is released through sequential proteolytic cleavages, allowing it to translocate to the nucleus where it activates transcription of target genes .

What is the nuclear transport mechanism of srebf2?

The nuclear transport of srebf2 involves a distinct pathway mediated by importin beta, which differs from the classical nuclear import mechanism. When the mature form of srebf2 is released from the endoplasmic reticulum membrane following sterol depletion, it undergoes active transport into the nucleus through the following mechanism:

• Srebf2 binds directly to importin beta without requiring importin alpha as an adapter protein

• This binding is regulated by the Ran GTPase cycle, where Ran-GTP (but not Ran-GDP) causes dissociation of the srebf2-importin beta complex

• The nuclear import can be inhibited by G19VRan-GTP in living cells

• The nuclear import process requires the coordination of Ran and its interacting protein p10/NTF2

• The helix-loop-helix-leucine zipper motif contains a novel type of nuclear localization signal that enables direct binding to importin beta

This specialized nuclear transport mechanism ensures precise control over srebf2's transcriptional activity in response to cellular sterol levels.

What are the target genes regulated by srebf2 in zebrafish?

Srebf2 in zebrafish regulates multiple genes involved in cholesterol metabolism and lipid homeostasis. The primary targets include:

The srebf2-mediated regulation of these genes ensures coordinated control of cholesterol synthesis and uptake in response to cellular needs.

How does the experimental manipulation of srebf2 affect lipid metabolism in zebrafish models?

Experimental manipulation of srebf2 in zebrafish models reveals significant effects on lipid metabolism, particularly triglyceride accumulation. When investigating the functional impact of srebf2, researchers have observed:

• Knockdown of srebf2 inhibits LDLR expression upregulated by palmitic acid (PA), indicating its crucial role in LDLR regulation

• Srebf2 deficiency prevents PA-induced triglyceride accumulation in hepatocytes

• The mechanism appears to operate through both direct and indirect pathways:

  • Direct: srebf2 binds to the LDLR promoter at the conserved sequence motif (GCGGTGTGAC) to activate transcription

  • Indirect: srebf2 activates lncRNA LDLR-AS, which stabilizes LDLR mRNA by recruiting heterogeneous nuclear ribonucleoprotein R (hnRNPR) to the 5' UTR region

These findings highlight that experimental manipulation of srebf2 can significantly alter lipid metabolism in zebrafish models, making it a valuable target for studying metabolic disorders.

What are the optimal conditions for expressing and purifying functional recombinant Danio rerio srebf2?

For optimal expression and purification of functional recombinant Danio rerio srebf2, researchers should consider the following methodological approach:

• Expression System Selection:

  • E. coli expression systems may be suitable for partial domains (particularly the N-terminal transcription activation domain)

  • For full-length protein, eukaryotic expression systems (insect cells or mammalian cells) provide better post-translational modifications and folding

• Purification Strategy:

  • Affinity chromatography using tag-based systems (His-tag or GST-tag)

  • Tag selection should be determined during the production process for optimal protein stability and activity

  • Storage in Tris-based buffer with 50% glycerol as indicated in commercial preparations

• Stability Considerations:

  • Avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

  • For extended storage, conserve at -20°C or -80°C

• Functional Validation:

  • DNA binding assays to confirm interaction with SRE (Sterol Regulatory Element) sequences

  • Reporter gene assays to assess transcriptional activation capacity

Researchers should note that the full-length membrane-bound form may present additional purification challenges compared to the transcriptionally active N-terminal domain.

How does TNFα-mediated activation of srebf2 influence LDLR expression and hepatic triglyceride accumulation?

TNFα-mediated activation of srebf2 creates a signaling cascade that significantly impacts LDLR expression and hepatic triglyceride accumulation through the following mechanism:

• Palmitic acid (PA) exposure induces inflammatory responses in liver cells, increasing TNFα, IL-1β, and MMP9 expression

• TNFα activates srebf2, promoting its translocation to the nucleus

• Nuclear srebf2 binds to the LDLR promoter, enhancing transcription of LDLR

• Simultaneously, srebf2 activates transcription of lncRNA LDLR-AS by binding to its promoter at the AACACACCAT site

• LDLR-AS functions as an RNA scaffold that recruits hnRNPR to the 5' UTR of LDLR mRNA, stabilizing it post-transcriptionally

• The resulting increase in LDLR protein levels enhances LDL uptake into hepatocytes

• Increased LDL internalization leads to triglyceride accumulation within liver cells

This pathway represents a molecular link between inflammation and lipid accumulation in liver cells, with srebf2 serving as a central regulatory node.

What are the differences in srebf2 function between mammals and zebrafish models?

While srebf2 maintains its core function as a regulator of cholesterol metabolism across species, several important differences exist between mammalian and zebrafish srebf2:

Understanding these similarities and differences is crucial when using zebrafish as a model for studying human lipid metabolism disorders and when translating findings between species.

How can researchers differentiate between direct and indirect effects of srebf2 on gene expression in experimental designs?

Differentiating between direct and indirect effects of srebf2 on gene expression requires a multi-faceted experimental approach:

• Chromatin Immunoprecipitation (ChIP) Assays:

  • ChIP experiments can confirm direct binding of srebf2 to promoter regions

  • For example, ChIP assays confirmed srebf2 binding to the LDLR-AS promoter at position -1273 ~ -862 bp

  • This technique definitively identifies direct transcriptional targets

• Mutation Analysis of Binding Sites:

  • Site-directed mutagenesis of predicted srebf2 binding sites

  • Dual-luciferase reporter assays with wild-type vs. mutant promoters

  • As demonstrated with LDLR promoter, mutation of the binding site (GCGGTGTGAC) significantly reduced promoter activity

• Time-Course Expression Analysis:

  • Compare timing of srebf2 activation with target gene expression

  • Direct targets typically show more immediate response than indirect targets

• RNA-Sequencing with srebf2 Knockdown:

  • Transcriptome analysis after srebf2 silencing identifies both direct and indirect targets

  • Integration with ChIP-seq data can separate direct from indirect effects

• Intermediate Factor Inhibition:

  • Selective inhibition of potential intermediate factors (like lncRNAs)

  • For example, silencing LDLR-AS to determine its role in mediating srebf2 effects on LDLR stability

These approaches, particularly when used in combination, provide robust evidence for distinguishing direct transcriptional regulation from indirect effects mediated by intermediate factors.

What role does srebf2 play in immune regulation and how can this be investigated experimentally?

Srebf2 has emerged as an important regulator of immune function, particularly in the context of tumor microenvironments. Its role in immune regulation can be investigated through several experimental approaches:

• Identification of srebf2-dependent immune cell populations:

  • Research has shown that srebf2 is critical for the development and function of myeloid regulatory dendritic cells (mregDCs)

  • These CD63+ mregDCs exhibit reduced capacity to drive CD8+ T cell proliferation

  • They enhance regulatory T cell (Treg) differentiation and suppress T cell cross-priming by other dendritic cell subsets

• Experimental approaches to investigate srebf2 in immune regulation:

  • Conditional knockout models targeting srebf2 in specific immune cell populations

  • Flow cytometry analysis of CD63+ dendritic cells from tumor-draining lymph nodes (TDLNs)

  • Quantitative real-time PCR to measure expression of MVA pathway genes (Hmgcs1, Hmgcr, Pmvk, Mvd, and Idi1) in sorted CD63+ vs. CD63- dendritic cells

  • Functional assays measuring T cell proliferation and Treg differentiation in co-culture with srebf2-deficient dendritic cells

• Translational relevance:

  • CD63+ mregDCs have been identified in tumor-draining lymph node tissues of melanoma patients

  • These cells maintain enriched expression of genes involved in cholesterol homeostasis

  • Therapeutic targeting of srebf2 and dendritic cell lipid metabolism represents a promising approach to overcome immune tolerance in cancer

This emerging area connects lipid metabolism with immune function and offers new avenues for immunotherapy development.