Recombinant Human Protein FAM138D (FAM138D)

What is FAM138D and what is its genomic location?

FAM138D (family with sequence similarity 138 member D) is primarily classified as a non-coding RNA gene located on chromosome 12 at position 36602-38133 (hg38 reference genome) on the negative strand . It belongs to the FAM138 family and is also known as F379 . This gene is categorized as a long intergenic non-coding RNA (lincRNA) gene according to Sequence Ontology classification .

Methodological consideration: When designing experiments involving FAM138D, researchers should note the discrepancy between its classification as a non-coding RNA in current databases versus potential protein-coding capacity suggested in some research contexts. This discrepancy requires careful experimental validation of any putative protein product.

How many transcript variants of FAM138D have been identified?

Four transcript variants of FAM138D have been documented with the following specifications :

Methodological consideration: When designing PCR primers or hybridization probes for FAM138D, researchers should carefully consider which transcript variants they wish to target and design experiments accordingly.

What is currently known about the function of FAM138D?

The exact function of FAM138D remains largely unknown. Current research suggests potential involvement in cellular processes related to protein interactions and signaling pathways . FAM138D has 534 functional associations with biological entities spanning 4 categories (molecular profile, chemical, cell line, cell type or tissue, gene, protein or microRNA) extracted from 14 datasets .

Methodological consideration: Given the limited functional characterization, researchers should employ multiple approaches to elucidate FAM138D function, including association studies, cellular localization experiments, and interaction analyses with predicted functional partners.

What approaches can be used to study potential protein products of FAM138D given its classification as a non-coding RNA?

Despite FAM138D being primarily classified as a non-coding RNA gene, researchers interested in potential protein products should implement a multi-faceted approach:

• In silico analysis: Use advanced computational tools to identify potential open reading frames (ORFs) and assess conservation across species

• Mass spectrometry: Employ targeted proteomics to detect low-abundance peptides that might be translated from FAM138D transcripts

• Ribosome profiling: Determine if FAM138D transcripts associate with ribosomes, suggesting potential translation

• Epitope tagging: Generate constructs with tagged putative ORFs to track expression and localization

• Custom antibody development: Develop antibodies against predicted peptide sequences to detect endogenous expression

Methodological consideration: Any protein product detection should be validated through multiple independent techniques, given the discrepancy between current classification and potential protein-coding capacity.

How might FAM138D be involved in cancer pathogenesis, particularly in the context of the 12p13.33 amplicon?

FAM138D is located within the 12p13.33 genomic region, which shows frequent copy number gains in high-grade serous carcinoma (HGSC) . This amplicon contains FOXM1 and 32 additional genes, and amplification at this locus is associated with reduced patient survival . FAM138D's presence in this amplicon raises questions about its potential role in cancer development.

Methodological consideration: To investigate FAM138D's potential role in cancer:

• Analyze FAM138D expression levels in cancer vs. normal tissues using RNA-seq or qRT-PCR

• Perform knockdown/overexpression studies to assess effects on cancer cell phenotypes

• Use CRISPR-Cas9 direct fusions for targeted genome editing to study gain or loss of function effects

• Investigate potential co-expression or functional relationships with known oncogenes in the 12p13.33 amplicon, particularly FOXM1

How can CRISPR-Cas9 technology be optimized for studying FAM138D function?

CRISPR-Cas9 technology offers powerful approaches for studying genes with unknown function like FAM138D. Recent advances involve fusing CRISPR-Cas9 to functional domains of proteins that impact DNA repair processes, enhancing genome editing capabilities .

For FAM138D research, optimization might include:

• CRISPR activation (CRISPRa): Using dCas9-VP64 fusions to upregulate FAM138D expression

• CRISPR interference (CRISPRi): Using dCas9-KRAB fusions to repress FAM138D expression

• CRISPR knock-in: Inserting reporter genes to monitor FAM138D expression in real-time

• Bidirectional promoter editing: If FAM138D shares a bidirectional promoter with adjacent genes (similar to the FOXM1/RHNO1 BDG pair discussed in result ), targeted modification of promoter elements to dissect regulatory mechanisms

Methodological consideration: When designing sgRNAs for FAM138D targeting, researchers should carefully evaluate potential off-target effects and consider the impact on all four transcript variants.

How can transcription factor binding at the FAM138D promoter be experimentally validated?

Given that FAM138D has associations with transcription factor binding site profiles , researchers can validate these interactions through:

• Chromatin Immunoprecipitation (ChIP): Using antibodies against predicted transcription factors to determine in vivo binding

• Electrophoretic Mobility Shift Assay (EMSA): Evaluating direct binding of purified transcription factors to FAM138D promoter sequences

• Luciferase reporter assays: Creating reporter constructs with wild-type and mutated binding sites to assess functional significance

• CRISPR-dCas9 recruitment assays: Targeting transcription factors to the FAM138D promoter to test sufficiency for activation

Methodological consideration: Integrating data from ChEA and ENCODE Transcription Factor Binding Site Profiles can guide hypothesis generation for which transcription factors to prioritize for experimental validation.

What are the optimal conditions for expressing recombinant FAM138D in heterologous systems?

For researchers attempting to produce recombinant FAM138D:

• Expression system selection: For a potentially human-specific gene like FAM138D, mammalian expression systems (HEK293, CHO cells) may be preferable over bacterial systems

• Vector design: Consider using vectors with strong promoters (CMV, EF1α) and appropriate tags (His, FLAG, GFP) for detection and purification

• Codon optimization: Optimize codons based on the expression system while maintaining key structural elements

• Purification strategy: For a protein of unknown function, employ mild purification conditions to preserve potential structure and function

• Quality control: Verify protein identity via mass spectrometry and assess folding via circular dichroism

Methodological consideration: Given FAM138D's current classification as a non-coding RNA, researchers should validate that any recombinant product accurately represents a biologically relevant protein.

How can researchers address the discrepancy between FAM138D's classification as a non-coding RNA and potential protein-coding capacity?

This represents a fundamental challenge in FAM138D research. A comprehensive approach includes:

• Computational re-analysis: Apply multiple gene prediction algorithms to assess protein-coding potential

• Conservation analysis: Examine evolutionary conservation patterns typical of coding vs. non-coding sequences

• RNA structural analysis: Investigate structural features characteristic of mRNAs vs. functional ncRNAs

• Polysome profiling: Determine if FAM138D transcripts associate with translating ribosomes

• Direct protein detection: Use custom antibodies or mass spectrometry to detect endogenous protein products

Methodological consideration: Researchers should explicitly address this classification discrepancy in publications and be transparent about the lines of evidence supporting either coding or non-coding status.

What approaches can be used to study FAM138D's potential role in histone modification processes?

Given FAM138D's association with histone modification site profiles from ENCODE data , researchers can investigate this connection through:

• ChIP-seq for histone marks: Map the genomic distribution of histone modifications in relation to FAM138D expression levels

• RNA immunoprecipitation (RIP): Test for physical interactions between FAM138D transcripts and histone-modifying enzymes

• Gain/loss-of-function studies: Assess changes in global histone modification patterns upon FAM138D modulation

• Co-localization studies: Use fluorescence microscopy to determine if FAM138D transcripts localize to chromatin regions undergoing active modification

Methodological consideration: When designing ChIP-seq experiments, consider examining both activating (H3K4me3, H3K27ac) and repressive (H3K27me3, H3K9me3) histone modifications to comprehensively characterize FAM138D's potential regulatory roles.

How should researchers integrate multi-omics data to understand FAM138D function?

Understanding FAM138D likely requires integrating multiple data types:

• Genomics: Analyze copy number variations and mutations in disease contexts

• Transcriptomics: Examine co-expression networks to identify functionally related genes

• Proteomics: Search for potential protein interactors if a protein product exists

• Epigenomics: Assess regulatory landscape and chromatin interactions

• Phenomics: Connect molecular findings to cellular and organismal phenotypes

Methodological consideration: Weighted gene co-expression network analysis (WGCNA) can help identify gene modules that contain FAM138D, potentially revealing functional associations that direct further experimental validation.

How can researchers interpret inconsistent results when studying a poorly characterized gene like FAM138D?

When working with poorly characterized genes like FAM138D, inconsistent results are common. A systematic approach includes:

• Methodological variation analysis: Determine if discrepancies arise from different experimental approaches

• Context-dependent function hypothesis: Test if FAM138D functions differently across cell types or conditions

• Isoform-specific effects: Examine if different transcript variants have distinct functions

• Technical artifact assessment: Rigorously evaluate potential sources of technical variability

• Independent validation: Confirm key findings using orthogonal techniques and multiple biological replicates

Methodological consideration: Maintain a comprehensive lab notebook documenting exact experimental conditions to help identify sources of variability, and consider publishing negative or inconsistent results to advance the field's understanding.

What emerging technologies could accelerate functional characterization of FAM138D?

Several cutting-edge technologies hold promise for elucidating FAM138D function:

• Single-cell multi-omics: Integrate single-cell RNA-seq with single-cell ATAC-seq to connect FAM138D expression with chromatin accessibility changes

• Spatial transcriptomics: Map FAM138D expression within tissue contexts to gain insights into its physiological relevance

• Organoid models: Study FAM138D in more physiologically relevant 3D tissue models

• High-throughput CRISPR screens: Identify genetic interactions through synthetic lethality or enhancement screens

• Nanopore direct RNA sequencing: Characterize RNA modifications that might regulate FAM138D function

Methodological consideration: Given FAM138D's unknown function, exploratory approaches that do not require prior hypotheses about its role may be particularly valuable.

How might understanding FAM138D contribute to broader knowledge about non-coding RNAs that potentially encode functional peptides?

FAM138D research could provide insights into the emerging field of non-coding RNAs with coding potential:

• Establishing detection criteria: Develop robust frameworks for identifying truly non-coding vs. peptide-producing transcripts

• Evolutionary significance: Illuminate how genes transition between coding and non-coding states during evolution

• Regulatory bifunctionality: Explore how RNA might serve both coding and non-coding functions simultaneously

• Translation regulation: Uncover mechanisms that control translation of non-canonical ORFs

• Disease relevance: Connect dysregulation of such transcripts to pathological processes

Methodological consideration: Researchers studying FAM138D should contextualize their findings within the broader conceptual framework of "pervasive translation" – the observation that many previously annotated non-coding RNAs show evidence of translation.