Recombinant Drosophila melanogaster Protein charlatan (chn), partial

What is the molecular function of charlatan (chn) in Drosophila melanogaster?

Charlatan (chn) is a zinc-finger protein that functions to maintain chromatin structure compatible with stem cell properties, including proliferation in Drosophila melanogaster . The protein does not simply act as an anti-differentiation factor as previously thought, but instead plays a crucial role in maintaining a chromatin environment that supports stem cell maintenance . Research using RNAi and mutational analysis has demonstrated that chn is essential for intestinal stem cell (ISC) division and normal differentiation of enteroblasts (EBs) . When chn is knocked down, Delta-positive ISCs do not divide efficiently, resulting in smaller cell nests, while enteroblasts exhibit abnormal differentiation with increased cell size .

How is charlatan related to mammalian transcription factors?

Charlatan is considered a Drosophila REST-like molecule, showing functional similarity to the mammalian Neuron-restrictive silencing factor (NRSF), also known as RE-1 silencing transcription factor (REST) . Like REST, which regulates many neuron-specific genes in mammals, charlatan appears to have pivotal functions in neuronal development in Drosophila. The similarity extends to the regulation mechanism - both charlatan and REST undergo neuron-specific alternative splicing that produces divergent protein variants, providing regulatory complexity in the nervous system .

What is known about charlatan splice variants and their subcellular localization?

Neuron-specific alternative splicing of charlatan produces six divergent variants of Chn proteins . Recent research by Yamasaki et al. (2021) revealed that one of these variants preferentially localizes to axons . This axonal localization is determined by a small specific sequence within this variant, suggesting specialized roles beyond transcriptional regulation . This differential localization pattern may explain some of the diverse functions of charlatan in neuronal development and function.

What phenotypes are associated with charlatan mutations in Drosophila?

Mutations in the charlatan gene lead to several observable phenotypes in Drosophila. When analyzed using the MARCM (mosaic analysis with a repressible cell marker) technique, chn⁹ mutant intestinal stem cells marked by GFP expression remained as single cells over an 8-day period, while wild-type clones showed increased cell numbers over the same timeframe . Additionally, chn mutant cells remain small, and Delta staining becomes undetectable within 2 days of clone induction . These observations indicate that charlatan is essential for stem cell maintenance and division in the Drosophila intestine.

What methodologies are most effective for studying charlatan protein-DNA interactions?

For analyzing charlatan protein-DNA interactions, ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) offers the most comprehensive approach. When implementing ChIP-seq for charlatan:

• Use a GFP-tagged charlatan protein expressed at endogenous levels, which can be achieved using the fosmid-based tagging approach described by Sarov et al.

• Optimize crosslinking conditions specifically for zinc-finger proteins (1-2% formaldehyde for 10-15 minutes)

• Implement a two-step immunoprecipitation using anti-GFP antibodies for higher specificity

• Include appropriate controls such as input DNA and IgG pulldowns

• Perform peak calling using algorithms specifically designed for transcription factors (such as MACS2)

For validation of binding sites, employ electrophoretic mobility shift assays (EMSAs) using recombinant charlatan protein domains and candidate DNA sequences identified from ChIP-seq analysis.

How can recombinant charlatan protein be effectively expressed and purified for functional studies?

Expressing and purifying functional recombinant charlatan protein requires specific considerations due to its zinc-finger domains and chromatin-interacting properties:

Expression system recommendations:

• Use bacterial expression systems (E. coli BL21(DE3)) for isolated zinc-finger domains

• For full-length protein, insect cell expression systems (Sf9 or High Five) provide better folding and post-translational modifications

• Express at lower temperatures (16-18°C) to improve solubility

• Include zinc supplements (100 μM ZnCl₂) in the growth medium to ensure proper folding of zinc-finger domains

Purification protocol:

• Use a two-step chromatography approach: affinity chromatography (His-tag or GST-tag) followed by ion exchange chromatography

• Include reducing agents (1-5 mM DTT or β-mercaptoethanol) throughout purification

• Maintain zinc concentration (10-50 μM ZnCl₂) in all buffers

• Verify protein functionality through DNA-binding assays before experimental use

What approaches can be used to identify and validate novel interaction partners of charlatan?

The identification of charlatan protein interactors requires a combination of complementary techniques:

For interactome discovery:

• Implement BioID or TurboID proximity labeling using charlatan as bait

• Perform co-immunoprecipitation followed by mass spectrometry (Co-IP-MS) using GFP-tagged charlatan from transgenic flies, similar to the approach described for other Drosophila proteins by Sarov et al.

• Yeast two-hybrid screening with specific domains of charlatan as bait

For validation of interactions:

• Reciprocal co-immunoprecipitation experiments

• Bimolecular Fluorescence Complementation (BiFC) in Drosophila S2 cells

• Proximity Ligation Assay (PLA) in intact tissues

• Genetic interaction tests using available mutants or RNAi lines

How can CRISPR-Cas9 gene editing be optimized for studying charlatan function?

CRISPR-Cas9 provides powerful approaches for studying charlatan function in vivo:

For gene knockout studies:

• Design at least 3 gRNAs targeting early exons shared among all splice variants

• Include gRNAs targeting the zinc-finger domains for specific disruption of DNA binding

• Implement inducible CRISPR systems (e.g., Gal4/UAS-driven Cas9) for tissue-specific or temporal control

For generating tagged variants:

• Use homology-directed repair to introduce fluorescent tags (e.g., sGFP-V5-BLRP) similar to the approach used in the fly TransgeneOme library

• Design repair templates with at least 1kb homology arms on each side

• Include flexible linkers between charlatan and the tag to minimize functional interference

• Verify tag insertion does not disrupt function through genetic complementation tests

For creating specific splice variant knockouts:

• Target splice sites or exons unique to specific variants

• Validate splice variant disruption using RT-PCR and sequencing

• Monitor changes in protein isoform expression using isoform-specific antibodies

What computational approaches can be used to predict and analyze charlatan target genes?

Several computational methods can be employed to predict charlatan target genes:

• Motif analysis:

  • Identify enriched DNA motifs from ChIP-seq data using MEME, HOMER, or similar tools

  • Scan the Drosophila genome for occurrences of these motifs near transcription start sites

• Integrative genomics:

  • Combine ChIP-seq binding data with RNA-seq expression data from charlatan mutants

  • Implement Gene Set Enrichment Analysis (GSEA) to identify biological pathways affected

• Network analysis:

  • Construct gene regulatory networks centered on charlatan

  • Identify hub genes and key regulatory modules using algorithms like WGCNA

• Comparative genomics:

  • Compare charlatan binding sites with conserved regions across Drosophila species

  • Identify evolutionarily conserved target genes, which are more likely to be functionally important

• Machine learning approaches:

  • Train models using validated charlatan targets to predict new targets

  • Implement deep learning algorithms that integrate multiple data types (sequence, chromatin accessibility, etc.)

How can the role of charlatan in chromatin modification be experimentally assessed?

To investigate charlatan's role in chromatin modification:

• ChIP-seq for histone modifications:

  • Perform ChIP-seq for activating (H3K4me3, H3K27ac) and repressive (H3K27me3, H3K9me3) histone marks in wild-type versus charlatan mutant tissues

  • Analyze changes in modification patterns at charlatan binding sites

• ATAC-seq for chromatin accessibility:

  • Compare chromatin accessibility profiles between control and charlatan-depleted cells

  • Focus analysis on regions showing differential accessibility

• CUT&RUN or CUT&Tag:

  • These techniques provide higher resolution mapping of charlatan binding sites and associated histone modifications

  • Require fewer cells than traditional ChIP, allowing analysis from small tissue samples

• Hi-C or HiChIP:

  • Map 3D chromatin interactions influenced by charlatan

  • Identify long-range regulatory connections between charlatan binding sites and target genes

• Single-cell approaches:

  • Implement scATAC-seq or scCUT&Tag to examine cell-type-specific effects of charlatan on chromatin

  • Particularly valuable for studying developmental contexts where cell populations are heterogeneous