Recombinant Human Lysine-specific demethylase 3B (KDM3B), partial

What is KDM3B and what is its primary function?

KDM3B (Lysine-specific demethylase 3B) is an epigenetic regulator that specifically demethylates 'Lys-9' of histone H3, playing a central role in histone code regulation and chromatin modification . The protein belongs to the Jumonji domain-containing family of histone demethylases and functions as a dioxygenase. During the demethylation process, KDM3B removes methyl groups from lysine residues, generating formaldehyde and succinate as byproducts . This enzymatic activity is crucial for regulating gene expression through modifying chromatin accessibility and transcriptional activation or repression of target genes.

What are the known alternative names and identifiers for KDM3B?

KDM3B is known by several alternative names in scientific literature and databases:

• JmjC domain-containing histone demethylation protein 2B

• Jumonji domain-containing protein 1B (JMJD1B)

• Nuclear protein 5qNCA

• Gene symbols: KDM3B, C5orf7, JHDM2B, JMJD1B, KIAA1082

• UniProt Primary Accession: Q7LBC6

• NCBI GeneID: 51780

• NCBI Accession: NP_057688.2

Understanding these alternative identifiers is essential when conducting literature searches or database queries to ensure comprehensive coverage of relevant research.

What are the key structural domains of KDM3B protein?

KDM3B contains several functional domains that are critical for its demethylase activity:

• JmjC (Jumonji C) catalytic domain - responsible for the demethylase enzymatic activity

• Zinc finger domain - required for proper catalytic activity

• Nuclear localization sequences - direct the protein to the nucleus where it functions

The protein structure reveals that KDM3B is divergent from its paralog KDM3A at the protein level outside of these conserved domains . The JmjC domain contains a catalytic center with specific residues that coordinate metal ions essential for demethylase activity. Understanding these structural features is critical for designing experiments targeting specific functional domains.

How can recombinant KDM3B be efficiently expressed and purified for in vitro studies?

Recombinant KDM3B can be expressed using bacterial expression systems, particularly E. coli BL21(DE3) strains with modified expression vectors. Based on protocols for related demethylases, the following methodology is recommended:

• Clone the KDM3B sequence (full-length or partial) into an expression vector (such as modified pET28b) with appropriate affinity tags (6×His-SUMO fusion tags improve solubility)

• Transform into E. coli BL21(DE3) cells and induce expression with IPTG

• Lyse cells and purify using affinity chromatography (Ni-NTA columns for His-tagged proteins)

• Remove fusion tags using specific proteases (e.g., ULP1 for SUMO tags)

• Further purify using size-exclusion chromatography

This method typically yields 1-2 mg of purified protein per liter of bacterial culture. Addition of FAD cofactor (0.5 mM) during purification may improve stability and activity of the recombinant protein. Verification of protein purity can be performed using SDS-PAGE and Western blotting with specific antibodies against KDM3B or the affinity tags.

What assays can be used to measure KDM3B demethylase activity in vitro?

Several approaches can be employed to assess KDM3B demethylase activity:

• Radioactive assay: Using core histone substrates methylated with 3H-SAM (S-adenosyl methionine), measure the release of 3H-formaldehyde following demethylation by KDM3B. The released formaldehyde can be detected using the modified NASH method, with measurement by scintillation counting .

• Mass spectrometry-based assay: Analyze peptide samples before and after incubation with KDM3B to detect changes in methylation status. This approach can be coupled with proteome analysis for identification of demethylated substrates.

• Immunological detection: Western blotting using antibodies specific for H3K9me1/2 (mono- or di-methylated lysine 9 on histone H3) can be used to monitor demethylation activity.

For optimal activity, the reaction buffer should contain glycine (pH 9.0), DTT, KCl, and potentially metal cofactors. The choice of assay depends on the specific research question, with radioactive assays offering high sensitivity while mass spectrometry provides more comprehensive analysis of methylation states.

What are the functional differences between KDM3B and other histone demethylases?

KDM3B belongs to the KDM3 subfamily of Jumonji domain-containing histone demethylases but displays both overlapping and distinct functions compared to other demethylases:

• Substrate specificity: KDM3B specifically demethylates mono- and di-methylated lysine 9 on histone H3 (H3K9me1/2) but not tri-methylated H3K9 or other methylated residues. This differs from KDM4 family members that can demethylate H3K9me3.

• Redundancy with KDM3A: KDM3A and KDM3B have identical catalytic activity and partially redundant functions in several model systems. In mouse embryonic stem cells (ESCs), deletion of both Kdm3a and Kdm3b decreases cell number within 4 days, suggesting functional overlap .

• Unique biological roles: Despite similarities in catalytic activity, KDM3B has unique functions, including potential tumor suppressor activity and regulation of alternative splicing in maintenance of naïve pluripotency .

Understanding these functional differences is essential for designing targeted studies and interpreting phenotypes in knockout or knockdown experiments.

How can researcher generate cell lines with controlled KDM3B expression for functional studies?

To generate cell lines with regulated KDM3B expression, researchers can employ several approaches:

• Inducible expression system: Create cell lines with FLAG-epitope tagged KDM3B under a doxycycline-inducible promoter. This allows for controlled expression of KDM3B at approximately 1.5-2 fold over endogenous levels after induction . The doxycycline-inducible system permits temporal control of expression and can minimize potential toxicity from constitutive overexpression.

• CRISPR/Cas9 gene editing: For loss-of-function studies, CRISPR/Cas9 can be used to generate knockout cell lines or to introduce specific mutations in KDM3B. This approach allows for studying the effects of complete loss of KDM3B function or the consequences of specific domain disruptions.

• shRNA-mediated knockdown: For partial reduction of KDM3B expression, short hairpin RNAs targeting different regions of KDM3B mRNA can be used.

Validation of expression levels should be performed using Western blotting, qRT-PCR, and functional assays to confirm altered demethylase activity. For interaction studies, immunoprecipitation followed by mass spectrometry can be performed using nuclear extracts with benzonase digestion to identify KDM3B-interacting proteins .

What proteomic approaches are most effective for studying KDM3B interactome?

To comprehensively analyze the KDM3B interactome, the following proteomic workflow is recommended:

• Generate cell lines expressing epitope-tagged KDM3B (e.g., FLAG-tagged) under inducible control to avoid artifacts from constitutive overexpression.

• Perform immunoprecipitation from nuclear extracts using anti-FLAG antibodies. Include benzonase digestion to remove DNA-mediated interactions and isolate direct protein-protein interactions .

• Analyze immunoprecipitated proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using high-resolution instrumentation.

• Filter data against common contaminants using databases such as the Crapome database with appropriate statistical thresholds (e.g., Saint score 0.7) .

• Perform network analysis using tools like STRING to identify functional protein clusters.

• Validate key interactions using reciprocal co-immunoprecipitation, proximity ligation assays, or functional studies.

This approach has successfully identified KDM3B-specific interaction partners distinct from those of KDM3A, despite their similar catalytic domains . When analyzing results, nuclear compartment filtering using DAVID GO analysis can help focus on relevant interactions.

What is the evidence for KDM3B involvement in human diseases?

KDM3B has been implicated in several human diseases and pathological conditions:

• Neurodevelopmental disorders: De novo and inherited pathogenic variants in KDM3B have been associated with intellectual disability syndromes. Nonsense variants in KDM3B have been identified in affected individuals, suggesting haploinsufficiency as a disease mechanism .

• Cancer: KDM3B may have tumor suppressor activity, with high expression of KDM3B associated with better prognosis in breast cancer patients (no recurrence after mastectomy and response to docetaxel treatment) .

• Developmental abnormalities: Given its role in stem cell maintenance and pluripotency, disruption of KDM3B function may contribute to developmental abnormalities through dysregulation of cell differentiation programs .

The high probability of loss-of-function intolerance (pLI score of 1.00) and selection against missense variants (Z score of 4.99) in population databases further support the clinical significance of KDM3B disruption . Researchers investigating disease mechanisms should consider both loss-of-function and dominant-negative effects of KDM3B variants.

How can researchers assess the pathogenicity of KDM3B variants identified in clinical settings?

To evaluate the potential pathogenicity of KDM3B variants identified in clinical sequencing, researchers should implement a systematic approach:

• Population frequency analysis: Check variant frequency in population databases like gnomAD. KDM3B has a high pLI score of 1.00, indicating strong selection against loss-of-function variants .

• In silico prediction tools: Utilize computational algorithms that predict the functional impact of variants on protein structure and function.

• Functional assays: Develop cell-based assays to measure:

  • Demethylase activity using H3K9me1/2 substrates

  • Protein stability and localization

  • Impact on gene expression of known KDM3B target genes

  • Effects on alternative splicing regulation

• Model systems: Generate cellular or animal models expressing the variant of interest to assess phenotypic consequences.

• Clustering analysis: Compare phenotypes of individuals with similar variants using approaches like clustering improvement factors (CIFs) to distinguish KDM3B-related conditions from other intellectual disability syndromes .

This multifaceted approach helps establish causality and mechanism for KDM3B variants, which is particularly important given its diverse cellular functions.

How can ChIP-seq be optimized for studying KDM3B genomic targets?

Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) for KDM3B requires specific optimization due to the enzymatic nature of the protein and its interaction with chromatin:

• Antibody selection: Use well-validated antibodies against endogenous KDM3B or epitope-tagged versions (FLAG, HA) if using engineered cell lines. Perform antibody validation using Western blot and immunoprecipitation followed by mass spectrometry.

• Crosslinking optimization: Test both formaldehyde (1%) and dual crosslinking approaches (DSG followed by formaldehyde) to capture potentially transient interactions of KDM3B with chromatin.

• Sonication conditions: Optimize chromatin fragmentation to obtain 200-500 bp fragments while preserving epitope recognition.

• Controls: Include input DNA, IgG controls, and ideally KDM3B knockout cells as negative controls.

• Bioinformatic analysis: When analyzing data, focus on:

  • H3K9me1/2 levels at KDM3B binding sites

  • Integration with RNA-seq data to correlate binding with expression changes

  • Motif discovery to identify potential DNA binding preferences

  • Comparison with datasets for interacting partners identified in proteomic studies

Additionally, performing parallel ChIP-seq for H3K9me1/2 provides valuable information about the relationship between KDM3B binding and its enzymatic activity at specific genomic loci.

What strategies can be employed to study the role of KDM3B in alternative splicing regulation?

To investigate KDM3B's function in alternative splicing regulation as identified in embryonic stem cells , researchers should consider the following approaches:

• RNA-seq with splice junction analysis:

  • Compare transcriptomes of wild-type and KDM3B-depleted cells using RNA-seq

  • Analyze alternative splicing events using tools such as rMATS, VAST-TOOLS, or MAJIQ

  • Focus on exon skipping, alternative 5'/3' splice sites, and intron retention events

• Direct RNA-protein interaction studies:

  • Perform RNA immunoprecipitation (RIP) or CLIP-seq to identify RNAs directly bound by KDM3B

  • Analyze binding motifs and RNA structural features at binding sites

• Splicing reporter assays:

  • Design minigene constructs containing alternatively spliced exons identified as KDM3B targets

  • Test the effect of KDM3B depletion or overexpression on splicing patterns

• Interactome analysis:

  • Identify interactions between KDM3B and known splicing factors using immunoprecipitation followed by mass spectrometry

  • Validate key interactions using co-immunoprecipitation or proximity ligation assays

• Mechanistic dissection:

  • Determine whether KDM3B's effect on splicing is dependent on its demethylase activity using catalytically inactive mutants

  • Investigate whether KDM3B affects splicing through histone modification or through direct interaction with the splicing machinery

This multi-faceted approach can help clarify whether KDM3B's role in alternative splicing is direct or indirect and identify the specific splicing events most sensitive to KDM3B function.

What are common technical challenges when working with recombinant KDM3B and how can they be addressed?

Researchers working with recombinant KDM3B often encounter several technical challenges:

• Protein solubility issues:

  • Express KDM3B as fusion proteins with solubility tags (SUMO, GST, MBP)

  • Consider expressing only the catalytic domain rather than full-length protein

  • Optimize expression conditions: lower temperature (16-18°C), reduced IPTG concentration

• Low enzymatic activity:

  • Ensure proper cofactor inclusion (Fe²⁺ and α-ketoglutarate) in buffers

  • Verify protein folding using circular dichroism or limited proteolysis

  • Test activity with different substrates (peptides vs. nucleosomes)

• Protein stability concerns:

  • Add glycerol (10%) and reducing agents (DTT or βME) to storage buffers

  • Aliquot and flash-freeze protein to avoid freeze-thaw cycles

  • Include protease inhibitors in all buffers

• Assay interference:

  • When using colorimetric or fluorescence-based assays, test compounds for potential interference

  • Include appropriate controls to detect false positives or negatives

• Specificity verification:

  • Confirm substrate specificity using mass spectrometry

  • Include catalytically inactive mutants as negative controls

  • Validate key findings with orthogonal assays

These technical considerations are based on protocols established for related histone demethylases and should be optimized specifically for KDM3B in each experimental system.

How can researchers differentiate between KDM3B-specific effects and redundant functions shared with KDM3A?

Distinguishing KDM3B-specific functions from those redundantly shared with KDM3A requires careful experimental design:

• Single vs. double knockout/knockdown approaches:

  • Generate KDM3B-specific, KDM3A-specific, and double knockout/knockdown models

  • Compare phenotypes to identify unique vs. shared functions

  • In mouse embryonic stem cells, both single and double knockouts should be characterized, as previous research has shown deletion of both genes decreases cell number within 4 days

• Rescue experiments:

  • Perform rescue experiments with KDM3B or KDM3A expression in double knockout backgrounds

  • Create chimeric proteins exchanging domains between KDM3A and KDM3B to identify regions responsible for specific functions

• Interactome analysis:

  • Compare protein interaction partners of KDM3A and KDM3B using immunoprecipitation followed by mass spectrometry

  • Focus on unique interactors that might explain KDM3B-specific functions

• Genome-wide binding profiles:

  • Perform ChIP-seq for both KDM3A and KDM3B to identify shared and unique genomic targets

  • Correlate binding sites with gene expression changes and histone modification patterns

• Domain-specific function analysis:

  • Generate truncation or point mutants affecting specific domains that differ between KDM3A and KDM3B

  • Test these mutants for their ability to complement specific functions

This systematic approach can help delineate the unique biological roles of KDM3B despite its catalytic similarity to KDM3A, particularly in contexts such as alternative splicing regulation and tumor suppression .