MPHOSPH8 Antibody

What is MPHOSPH8 and what are its key functional domains?

MPHOSPH8 is an epigenetic regulator containing multiple functional domains with distinct roles in chromatin regulation. The protein contains an N-terminal chromodomain capable of binding to H3K9me3 histone marks and four consecutive ankyrin-repeat domains towards its C-terminus with previously unknown function . MPHOSPH8 interacts with multiple epigenetic silencing proteins, including the H3K9 mono- and di-methyltransferase proteins GLP/G9a, DNA methyltransferase DNMT3A, histone deacetylase SIRT1, and ATF7IP (a known binding partner of H3K9 tri-methyltransferase SETDB1) . These interactions position MPHOSPH8 as a hub for coordinating various epigenetic silencing mechanisms.

The specificity of the MPHOSPH8 chromodomain for H3K9me3 is comparable to high-quality antibodies currently used in chromatin research, making it a valuable tool for studying this histone modification . Notably, the chromodomain shows minimal cross-reactivity with H3K27me3, as demonstrated by Western blot experiments with cells treated with EZH2 inhibitors .

How does MPHOSPH8 contribute to the HUSH complex?

MPHOSPH8 forms the human silencing hub (HUSH) core complex together with TASOR and PPHLN1, with SETDB1 serving as an associated catalytic subunit . This complex plays a crucial role in heterochromatin formation and maintenance. The current model suggests that the core complex recruits SETDB1, enabling the propagation of H3K9me3 from heterochromatin-adjacent regions onto target loci, leading to their silencing – a phenomenon known as position-effect variegation .

Gene expression analyses reveal significant concordance between PPHLN1- and MPP8-repressed genes, evidenced by a large number of mutually upregulated genes when either component is depleted . Interestingly, this includes numerous KZNF genes (zinc finger proteins), suggesting that the HUSH complex may regulate the expression of these transcription factors .

What is the role of MPHOSPH8 in stem cell biology?

MPHOSPH8 has been identified as an essential factor for ground-state pluripotency in mouse embryonic stem cells (mESCs). Its depletion leads to significant cellular consequences:

• Cell cycle arrest with a 3-fold increased percentage of cells in G1 phase and 2-fold reduced percentage in S phase within 72 hours of MPP8 depletion

• Spontaneous differentiation with a significant increase (>2-fold) of differentiated colonies as assessed by alkaline phosphatase staining

• Rapid destabilization of the naïve gene expression program during the early phase of mESC transition after withdrawal from 2i culture conditions

These findings demonstrate that MPHOSPH8 is critical for maintaining self-renewal capacity of naïve mESCs by preserving their proliferative state . Interestingly, its function appears partially dependent on signaling pathways, as MPP8-depleted mESCs remained viable when cultured with GSK inhibitor alone but not with MEK inhibitor or both inhibitors, indicating that MEK/ERK signaling pathway activation can overcome the requirement for MPP8 in mESCs .

How can MPHOSPH8 antibodies be used for chromatin immunoprecipitation studies?

MPHOSPH8 antibodies provide valuable tools for studying chromatin modifications, particularly H3K9me3. Research indicates that the specificity of MPHOSPH8 Chromo domain is comparable to high-quality antibodies currently used in chromatin research, making it an excellent choice for chromatin immunoprecipitation studies .

For Chromatin Interaction Domain Precipitation (CIDOP) and ChIP experiments, researchers should follow these methodological considerations:

• Validate antibody specificity by comparing CIDOP-qPCR profiles with established anti-H3K9me3 antibodies

• Include methylation-binding pocket mutants as negative controls to confirm modification-dependent precipitation

• Use approximately 100,000 cells per assay for optimal results when studying histone modifications

• For genome-wide analyses, compare MPHOSPH8 Chromo binding profiles with anti-H3K9me3 antibody profiles to confirm specificity

Studies show that MPHOSPH8 Chromo and anti-H3K9me3 antibody binding profiles produce similar results in genome-wide chromatin studies, confirming their comparable efficacy .

What are the optimal conditions for using MPHOSPH8 antibodies in CUT&Tag assays?

CUT&Tag (Cleavage Under Targets and Tagmentation) represents an advanced method for profiling chromatin-associated proteins with higher signal-to-noise ratio than traditional ChIP-seq. For MPHOSPH8 antibodies in CUT&Tag:

• Cell number considerations:

  • Use 100,000 cells per assay for optimal enrichment of target loci

  • For limited samples, as few as 20,000 cells can be used for transcription factors and cofactors

  • Up to 250,000 cells can be used without scaling up beads, antibodies, enzyme, or buffers

• Buffer optimization:

  • Ensure appropriate salt concentration in the tagmentation buffer to prevent bias toward euchromatin or heterochromatin

  • The active tethering of pAG-Tn5 to chromatin allows for tagmentation even in less accessible heterochromatin regions where MPHOSPH8 often binds

• Controls:

  • Include IgG controls to assess background signal

  • Use antibodies against known euchromatic (H3K4me3) and heterochromatic (H3K27me3) marks as positive controls to validate the assay

How should MPHOSPH8 antibody specificity be validated?

Validating MPHOSPH8 antibody specificity is critical for reliable experimental results. Multiple complementary approaches should be employed:

• Western blot validation:

  • Compare binding to histones isolated from wild-type and knockout cells (such as Suv39h1/Suv39h2 double-knockout MEF cells)

  • Treatment with specific inhibitors (e.g., EZH2 inhibitors) can help distinguish between H3K9me3 and H3K27me3 specificity

• Peptide array analysis:

  • Test binding against arrays containing modified histone peptides

  • Assess potential cross-reactivity with related modifications (e.g., H3K27me3)

• Functional validation:

  • Use methyllysine-binding pocket mutants as negative controls in precipitation experiments

  • In CUT&Tag assays, non-specific tagmentation at open chromatin regions indicates poor antibody specificity

• Genomic profile comparison:

  • Compare binding profiles with established antibodies targeting the same modification

  • Distinguish from other histone marks by comparing with H3K9me2 and H3K27me3 profiles

How does MPHOSPH8 regulate transposable elements?

MPHOSPH8 plays a critical role in repressing LINE-1 elements through the HUSH complex, providing a defense mechanism against retroelement activation. Key insights include:

• Mechanism of repression:

  • MPHOSPH8 depletion leads to overexpression of LINE-1 elements, as evidenced by increased LINE-1 ORF1-protein levels

  • RNA-sequencing data reveals that specific LINE-1 subfamilies, particularly the hominid-specific families L1PA1 (L1HS) and L1PA2, are differentially expressed in MPHOSPH8-depleted cells

  • These elements are expressed bidirectionally, producing both sense and antisense transcripts that significantly increase upon MPHOSPH8 depletion

• Domain requirements:

  • Surprisingly, LINE-1 elements are efficiently repressed by MPHOSPH8 lacking the chromodomain, indicating that H3K9me3 binding is not essential for this function

  • The previously unannotated C-terminus appears essential for MPHOSPH8's repressive function

  • SETDB1 recruits MPHOSPH8 to genomic target loci, while transcriptional repression of LINE-1 elements is maintained without retaining H3K9me3 levels

This unexpected finding challenges previous models suggesting that MPHOSPH8 represses LINE-1 elements primarily through recognition of H3K9me3 marks.

What is the relationship between MPHOSPH8 and interferon responses?

MPHOSPH8 functions as a gatekeeper of type I interferon responses through epigenetic control of LINE-1 elements. Upon MPHOSPH8 depletion:

• A robust interferon response is triggered:

  • The response is independent of cGAS/STING DNA-sensing pathways

  • The response depends on signaling through the type I interferon receptor (IFNAR)

  • Double-stranded RNA (dsRNA) becomes detectable in MPHOSPH8-depleted cells, and this signal is abolished by treatment with RNase III, suggesting dsRNA drives the response

• LINE-1 elements contribute to interferon activation:

  • The interferon response can be triggered by siRNAs against MPHOSPH8, indicating that the ligand driving the response is endogenous

  • Bidirectional transcription of LINE-1 elements (particularly L1PA1 and L1PA2) occurs in MPHOSPH8-depleted cells, potentially generating dsRNA

  • Reverse transcription is not necessary to drive the interferon response, as reverse transcriptase inhibitors that are effective against LINE-1 RNA do not prevent it

These findings establish MPHOSPH8 as a critical suppressor of interferon responses by preventing LINE-1 activation and subsequent dsRNA production.

What domain requirements exist for MPHOSPH8 function in stem cell maintenance?

Detailed structure-function analyses have revealed surprising insights about MPHOSPH8 domain requirements:

• Chromodomain independence:

  • Mutation of all three residues forming the aromatic cage of the chromodomain (MPP8 F59A;W80A;Y83A) does not impair MPHOSPH8's ability to maintain mESC self-renewal

  • This mutant can rescue the growth defect of MPHOSPH8-depleted cells to the same extent as wild-type MPHOSPH8

• Ankyrin-repeat domain dispensability:

  • Complete removal of the ankyrin-repeat domain (MPP8 ΔARD) also does not affect MPHOSPH8's function in maintaining mESC self-renewal

  • The mutant lacking this domain rescues proliferation defects as effectively as wild-type MPHOSPH8

These findings suggest that neither of the two defined domains of MPHOSPH8 is required for its ability to maintain mESC self-renewal, indicating that other regions of the protein or its interaction with partner proteins may be more critical for this function.

How can conflicting results between MPHOSPH8 chromatin binding and functional outcomes be resolved?

Researchers may encounter an apparent paradox: MPHOSPH8 functions in heterochromatin maintenance despite evidence that its chromodomain is dispensable for some functions. Consider these approaches to resolve such conflicts:

• Conduct sequential ChIP experiments to determine whether MPHOSPH8 recruitment to chromatin occurs through alternative mechanisms besides direct H3K9me3 binding

• Employ proximity ligation assays to identify protein-protein interactions that may recruit MPHOSPH8 to chromatin independently of its chromodomain

• Consider the temporal dynamics of MPHOSPH8 recruitment versus maintenance of silencing:

  • Initial recruitment may require the chromodomain

  • Maintenance of silencing may continue through other domains once established

• Design experiments to test the model where "SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE-1 elements is maintained without retaining H3K9me3 levels"

What factors affect MPHOSPH8 antibody performance in different cell types?

When working with MPHOSPH8 antibodies across different experimental systems, consider these variables:

• Epigenetic landscape variations:

  • Ground-state pluripotent stem cells have distinct chromatin configurations characterized by "distinctly open chromatin and repressed endogenous retroviruses"

  • Differentiated cells may show different MPHOSPH8 binding patterns and requirements

• Cell-type specific experimental considerations:

  • For pluripotent cells, use 100,000 cells per CUT&Tag assay for optimal results

  • When working with limited primary cell numbers, as few as 20,000 cells may be used, though with potentially reduced signal

• Culture conditions impact:

  • MPHOSPH8 function in mESCs depends on culture conditions (2i/LIF versus serum/LIF)

  • MPHOSPH8-depleted mESCs remain viable when cultured with GSK inhibitor but not with MEK inhibitor

  • Consider these variables when interpreting antibody binding patterns across different culture systems

How can researchers distinguish MPHOSPH8-specific effects from general HUSH complex functions?

Differentiating MPHOSPH8-specific roles from those of the broader HUSH complex requires careful experimental design:

• Comparative depletion studies:

  • Analyze the concordance between genes upregulated upon depletion of different HUSH components

  • Data shows significant overlap between PPHLN1- and MPP8-repressed genes, but TASOR may affect different gene sets

  • Use Venn diagrams to visualize shared and unique targets:

• Domain-specific mutations:

  • Utilize chromodomain mutants and ankyrin-repeat domain deletion mutants to separate MPHOSPH8-specific functions from general HUSH complex roles

  • Competition-based proliferation assays with these mutants can reveal domain-specific contributions

• Sequential ChIP approaches:

  • Perform ChIP first for MPHOSPH8 followed by other HUSH components (or vice versa)

  • This can identify genomic loci bound by complete versus partial HUSH complexes