HIST1H1D (Ab-106) Antibody

What is the HIST1H1D protein and what role does it play in chromatin structure?

HIST1H1D is a member of the histone H1 family, specifically the H1.3 variant, which plays a key role in chromatin compaction and gene expression regulation. As a linker histone, it binds to nucleosomes and facilitates higher-order chromatin structures. Unlike core histones, the H1 histone family is more evolutionarily diverse, with multiple variants present in human somatic cells, including H1.1 to H1.5, H1.0, and H1X. HIST1H1D belongs to the replication-dependent class of histones (H1.1 to H1.5) that are expressed primarily during DNA synthesis .

How does the HIST1H1D (Ab-106) Antibody differ from antibodies targeting other H1 variants?

The HIST1H1D (Ab-106) Antibody (PACO60594) is specifically designed to target the peptide sequence around the lysine residue at position 106 in the Human Histone H1.3 protein. This specificity allows for precise detection of the HIST1H1D variant without cross-reactivity with other H1 family members. While many H1 variants share structural similarities, particularly in their globular domains, the HIST1H1D antibody targets unique epitopes that distinguish this variant from others like H1.0, H1.2, or H1X, which have been shown to have different genomic distributions and potentially distinct functions in gene regulation .

What are the optimal protocols for using HIST1H1D (Ab-106) Antibody in Western blotting experiments?

For Western blotting applications with the HIST1H1D (Ab-106) Antibody:

• Prepare cell lysates from your sample of interest (validated in HeLa and K562 cell lines)

• Run protein samples on SDS-PAGE gel and transfer to appropriate membrane

• Block membrane with standard blocking buffer

• Dilute the HIST1H1D antibody at 1:100-1:1000 in appropriate buffer (optimal working dilution may require titration)

• Incubate membrane with primary antibody solution overnight at 4°C

• Wash membrane thoroughly with PBST or TBST

• Incubate with secondary antibody (goat polyclonal to rabbit IgG recommended at 1/50000 dilution)

• Develop using standard detection methods

The antibody should detect a band at approximately 23 kDa, which corresponds to the predicted molecular weight of HIST1H1D. Positive controls include HeLa and K562 whole cell lysates, which have been validated to express detectable levels of this protein .

How can researchers optimize immunohistochemistry protocols using the HIST1H1D (Ab-106) Antibody?

For optimal IHC results with the HIST1H1D (Ab-106) Antibody:

• Deparaffinize and rehydrate tissue sections (if using FFPE samples)

• Perform antigen retrieval using high-pressure citrate buffer (pH 6.0)

• Block endogenous peroxidase activity and non-specific binding

• Dilute antibody at 1:10-1:100 (a starting dilution of 1:20 has been validated)

• Incubate sections with diluted antibody at 4°C overnight

• Wash thoroughly with PBS

• Apply appropriate detection system (e.g., polymer-based detection system)

• Counterstain, dehydrate, and mount

For enhanced specificity, researchers should include appropriate blocking steps and validate staining patterns using positive control tissues. Human cervical cancer tissue has been successfully used for validation of this antibody in IHC applications .

What methodologies can be employed to study HIST1H1D genomic distribution patterns using ChIP-seq?

To investigate the genomic distribution of HIST1H1D using ChIP-seq:

• Cross-linking and chromatin preparation:

  • Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 0.125 M glycine

  • Lyse cells and sonicate chromatin to fragments of ~200-500 bp

• Immunoprecipitation with HIST1H1D antibody:

  • Pre-clear chromatin with protein A/G beads

  • Incubate pre-cleared chromatin with HIST1H1D (Ab-106) Antibody (3-5 μg per reaction)

  • Include appropriate controls (IgG and input samples)

• DNA purification and library preparation:

  • Reverse cross-links and purify DNA

  • Prepare sequencing libraries following standard protocols

• Data analysis considerations:

  • Map reads to reference genome

  • Normalize to input samples

  • Analyze enrichment patterns at promoters, gene bodies, and intergenic regions

  • Compare distribution to other H1 variants to identify unique patterns

Research has shown that different H1 variants have distinct genomic distributions. For example, H1.2 has been found to be enriched in chromosomal domains with low GC content and associated with lamina-associated domains, while H1.0 and H1X are enriched in gene-rich chromosomes .

How does HIST1H1D interact with other chromatin modifiers to regulate gene expression?

HIST1H1D likely interacts with various chromatin modifiers similar to other H1 variants, though specific interactions may differ. Based on studies of H1 variants:

• Histone deacetylases (HDACs): H1 variants have been shown to interact with HDACs, which remove acetyl groups from histones leading to chromatin compaction and gene repression. The HIST1H1D antibody can be used in co-immunoprecipitation experiments to identify specific HDAC interactions, similar to studies showing that HDA-1 (an HDAC) opposes certain H1-associated factors in gene regulation .

• Histone methyltransferases: Some H1 variants interact with methyltransferases that modify histones. Research has shown interactions between H1 variants and SET domain-containing proteins like SET-26, which affect gene expression patterns .

• DNA methyltransferases: H1 variants may recruit or stabilize DNA methyltransferases at specific genomic loci, contributing to DNA methylation patterns.

Methodologically, researchers can study these interactions using:

• Co-immunoprecipitation with HIST1H1D antibody followed by mass spectrometry

• Proximity ligation assays to detect in situ protein-protein interactions

• Sequential ChIP (ChIP-reChIP) to identify co-occupancy of genomic regions

These approaches can reveal how HIST1H1D participates in multi-protein complexes to regulate chromatin structure and gene expression .

What experimental approaches can determine how HIST1H1D contributes to DNA replication-coupled histone gene expression?

To investigate HIST1H1D's role in DNA replication-coupled histone gene expression:

• Cell cycle synchronization and analysis:

  • Synchronize cells at different cell cycle stages using methods like double thymidine block

  • Analyze HIST1H1D levels across cell cycle using the antibody in Western blot and immunofluorescence

  • Compare expression patterns to markers of DNA replication

• HIST1H1D depletion studies:

  • Use siRNA or CRISPR-Cas9 to deplete HIST1H1D

  • Examine effects on expression of replication-dependent histone genes using RT-qPCR

  • Perform RNA-seq to identify genome-wide transcriptional changes

• Chromatin association dynamics:

  • Use HIST1H1D antibody in ChIP experiments at histone gene loci during different cell cycle phases

  • Analyze association with regulatory elements like NEG (negative regulatory elements) regions

  • Investigate co-occupancy with factors like HIR repressor complex, Asf1, and Rtt106

• Interaction with histone chaperones:

  • Investigate interactions between HIST1H1D and histone chaperones using co-immunoprecipitation

  • Focus on chaperones known to be involved in replication-coupled histone expression like Asf1

Research has shown that histone H1 variants can influence gene expression through interactions with various chromatin proteins and that their expression is tightly regulated during the cell cycle, particularly in S phase when large amounts of histones are required for chromatin assembly on newly synthesized DNA .

What are the common pitfalls when using HIST1H1D (Ab-106) Antibody in chromatin immunoprecipitation, and how can they be addressed?

Common challenges in ChIP with HIST1H1D antibody include:

• Cross-reactivity with other H1 variants:

  • Problem: H1 variants share sequence similarity, potentially causing non-specific binding

  • Solution: Validate antibody specificity through Western blot analysis of recombinant H1 variants

  • Control: Include ChIP experiments in cells where HIST1H1D has been depleted

• Poor chromatin fragmentation:

  • Problem: Inadequate sonication leads to large chromatin fragments and non-specific precipitation

  • Solution: Optimize sonication conditions (time, amplitude, pulse duration) for consistent fragmentation

  • Control: Verify fragment size distribution (200-500 bp ideal) by agarose gel electrophoresis

• Low signal-to-noise ratio:

  • Problem: High background signal masking specific binding

  • Solution: Increase blocking stringency, add competitor DNA, optimize antibody concentration

  • Control: Include IgG control and analyze signal ratios

• Epitope masking:

  • Problem: Formaldehyde cross-linking may mask the epitope recognized by the antibody

  • Solution: Test different cross-linkers or cross-linking conditions

  • Alternative: Consider native ChIP for histone proteins if appropriate

• Data analysis challenges:

  • Problem: Difficulties distinguishing specific binding from background

  • Solution: Use appropriate peak calling algorithms specific for histone modifications

  • Control: Compare enrichment patterns with published datasets of H1 variant distributions

How can researchers accurately differentiate between HIST1H1D and other H1 histone variants in their experiments?

To differentiate HIST1H1D from other H1 variants:

• Antibody validation:

  • Perform Western blot analysis with recombinant H1 variants to confirm specificity

  • Test antibody reactivity in cells where HIST1H1D has been depleted using siRNA or CRISPR-Cas9

  • Compare staining patterns with other validated H1 variant-specific antibodies

• Mass spectrometry approaches:

  • Use tandem mass spectrometry to identify unique peptides specific to HIST1H1D

  • Employ targeted proteomics approaches focusing on variant-specific peptides

  • Quantify relative abundances of different H1 variants in your samples

• Genomic distribution analysis:

  • Perform ChIP-seq with antibodies against different H1 variants

  • Compare distribution patterns, as research has shown H1 variants have distinct genomic localizations

  • Focus on regions known to preferentially bind specific variants (e.g., H1.2 associates with low GC content regions)

• Expression analysis in different cell types:

  • Different cell types and tissues express varying levels of H1 variants

  • Compare expression across cell types using RT-qPCR and Western blot

  • Consider using cells with known expression profiles of H1 variants as references

• Tagged protein approaches:

  • If antibody cross-reactivity is a concern, consider using tagged versions of HIST1H1D

  • Express HA-tagged HIST1H1D and use HA antibodies for detection

  • This approach has been successfully used in genome-wide distribution studies of H1 variants

How can researchers use the HIST1H1D (Ab-106) Antibody to investigate the role of this histone variant in cancer progression?

To investigate HIST1H1D in cancer progression:

• Expression profiling across cancer types:

  • Use the HIST1H1D antibody in tissue microarray analysis of various cancer types

  • Compare expression levels between tumor and matched normal tissues

  • Correlate expression with clinical parameters and survival data

• Functional genomics approaches:

  • Deplete or overexpress HIST1H1D in cancer cell lines

  • Assess phenotypic changes in proliferation, migration, and invasion

  • Perform RNA-seq to identify genes regulated by HIST1H1D

• Chromatin landscape analysis:

  • Conduct ChIP-seq in cancer vs. normal cells to identify differential binding patterns

  • Integrate with other epigenetic marks (DNA methylation, histone modifications)

  • Analyze enrichment at cancer-relevant genes and regulatory elements

• Combination with cancer treatment:

  • Study how HIST1H1D levels change in response to chemotherapy or radiotherapy

  • Investigate if HIST1H1D depletion sensitizes cells to treatment

  • Consider combination with epigenetic drugs like HDAC inhibitors

Research has shown that H1 variants have been differentially related to cancer processes, and their expression is regulated during differentiation and development. For example, studies have identified specific associations between certain H1 variants and neuroendocrine tumors, suggesting potential roles in cancer biology .

What methodological approaches can be used to study the role of HIST1H1D in epigenetic regulation of recombinant protein expression in CHO cells?

To investigate HIST1H1D's role in epigenetic regulation of recombinant protein expression:

• Expression analysis in production cell lines:

  • Compare HIST1H1D levels in high vs. low producer CHO cell lines using the antibody

  • Correlate expression with productivity metrics and epigenetic marks

  • Examine changes during production process and cell line development

• Genetic modification approaches:

  • Overexpress or deplete HIST1H1D in CHO cells expressing a recombinant protein

  • Quantify effects on productivity, product quality, and cell growth

  • Analyze changes in chromatin structure at the transgene locus

• Integration with epigenetic modulators:

  • Treat cells with epigenetic regulators like HDAC/LSD1 inhibitors

  • Monitor HIST1H1D occupancy changes at transgene and endogenous loci

  • Assess combinatorial effects on productivity and cellular phenotypes

• Multi-omics integration:

  • Combine ChIP-seq data with transcriptomics, proteomics, and metabolomics

  • Identify regulatory networks involving HIST1H1D

  • Develop predictive models for cell line engineering

Recent research has shown that epigenetic regulation plays a central role in cellular processes relevant to recombinant protein production, and epigenetic modulators like HDAC/LSD1 inhibitors can enhance monoclonal antibody production in CHO cells. Understanding how HIST1H1D participates in these processes could provide insights for cell line engineering and bioprocess development .

Table 1: HIST1H1D (Ab-106) Antibody Technical Specifications

Table 2: Comparative Distribution of H1 Variants in the Human Genome

*Note: Specific genomic distribution patterns for HIST1H1D require further investigation using the HIST1H1D (Ab-106) Antibody in ChIP-seq experiments.