SSU72 PAT45E2AT Antibody

What is SSU72 and what is its primary function in mammalian systems?

SSU72 is a highly conserved CTD (C-terminal domain) phosphatase that regulates RNA polymerase II (Pol II) activity through dephosphorylation of specific residues on Pol II's CTD. In mammalian systems, SSU72 preferentially contributes to transcriptional elongation rather than polyadenylation or RNA processing, which differs somewhat from its yeast homolog . Its primary function involves the dephosphorylation of Ser5-P and Ser7-P residues on the CTD heptapeptide repeat (YSPTSPS) . This activity is essential for gene expression regulation, as demonstrated by the lethality of SSU72 deletion in mice .

Mammalian SSU72 shows distinctive tissue-specific regulation patterns, preferentially affecting actively transcribed genes in a cell-type-specific manner. Research has demonstrated that SSU72 depletion leads to aberrant Pol II pausing and elongation defects .

What specific phosphorylation sites does SSU72 target on the RNA polymerase II CTD?

SSU72 primarily targets two phosphorylation sites on the RNA polymerase II CTD:

• Serine 5 phosphorylation (Ser5-P): SSU72 works in coordination with the prolyl-isomerase Ess1 to remove Ser5-P marks, particularly at the cleavage and polyadenylation site (CPS) .

• Serine 7 phosphorylation (Ser7-P): SSU72 serves as a major Ser7-P phosphatase, with persistent Ser7 phosphorylation or phosphomimetic substitution (S7E) being lethal to cells .

The phosphatase activity of SSU72 is substrate-specific, as it has a strong preference for substrates in the cis configuration of the Ser5-Pro6 motif within CTD . This specificity is critical for its function in transcriptional regulation.

How does the PAT45E2AT antibody recognize SSU72?

The PAT45E2AT antibody is a mouse monoclonal antibody derived from hybridization of mouse F0 myeloma cells with spleen cells from BALB/c mice that were immunized with recombinant human SSU72 (amino acids 1-194) purified from E. coli . This antibody specifically recognizes the SSU72 protein and has been validated for use in ELISA and Western blot applications .

The antibody belongs to the mouse IgG1 subclass with κ light chain and is typically purified using protein-A affinity chromatography from mouse ascitic fluids . When using this antibody, researchers should consider its specificity for human SSU72 in their experimental design and validation.

What are the optimal experimental controls when using the PAT45E2AT antibody in ChIP-Seq experiments?

When designing ChIP-Seq experiments with the PAT45E2AT antibody, the following controls are essential:

• Input DNA control: Reserve a portion of chromatin before immunoprecipitation to normalize for differences in DNA amounts and sonication efficiency .

• IgG control: Use normal mouse IgG antibodies in parallel immunoprecipitations to identify non-specific binding .

• Biological controls:

  • SSU72 knockout/knockdown samples: If available, include samples where SSU72 has been depleted using Cre-mediated deletion in conditional knockout models (Ssu72 f/f mice) or other knockdown methods .

  • SSU72 overexpression samples: Consider including samples with HA-tagged SSU72 expression to validate antibody specificity .

• Cross-validation with other antibodies: Validate findings by comparing the genomic distribution of SSU72 with that of Pol II (Rpb1) and different phosphorylated forms (pSer5, pSer2, pSer7) .

This comprehensive control strategy will help ensure the specificity and reliability of ChIP-Seq results when studying SSU72 genomic localization.

How should researchers design experiments to study the impact of SSU72 depletion on gene expression?

To effectively study SSU72 depletion effects on gene expression, researchers should consider the following experimental design strategy:

Experimental Design Framework:

• Generate appropriate cellular models:

  • Use conditional knockout systems (e.g., Ssu72 f/f mice with loxP-flanked exon 1)

  • Employ Cre recombinase delivery through adenoviral infection (Ad-Cre) or tissue-specific Cre expression (e.g., Alb-Cre for liver-specific deletion)

• Include diverse cell types:

  • Study multiple cell types with different proliferation capabilities:

    • Embryonic stem cells (highly proliferating)

    • Mouse embryonic fibroblasts (moderate proliferating)

    • Hepatocytes (almost quiescent)

• Implement comprehensive controls:

  • Use adenoviruses incorporating luciferase (Ad-Luc) as infection controls

  • Include wild-type cells (Ssu72 +/+) as baseline controls

  • Consider transgenic models expressing HA-tagged SSU72 at levels similar to endogenous expression

• Analysis methodology:

  • RNA sequencing for genome-wide expression profiling

  • ChIP sequencing to correlate changes in gene expression with Pol II occupancy and phosphorylation status

  • qRT-PCR validation of key target genes

  • Immunoblot analyses to confirm SSU72 depletion and effects on CTD phosphorylation

This experimental framework accounts for the tissue-specific effects of SSU72 and enables comprehensive analysis of its role in transcriptional regulation .

What methodology should be used to assess SSU72's phosphatase activity in vitro?

To accurately assess SSU72's phosphatase activity in vitro, researchers should follow this detailed methodology:

Materials Required:

• Recombinant GST-CTD substrate

• Purified kinases (Kin28-TAP, Ctk1-TAP, or activated MAPK)

• Recombinant GST-purified SSU72

• ATP (1 mM)

• Phosphatase buffer (50 mM Tris-HCl, pH 6.5, 10 mM MgCl₂, 20 mM KCl, 5 mM DTT)

• Gel filtration spin columns

• Antibodies against different phosphorylated forms of CTD (pSer2, pSer5, pSer7)

Protocol:

• Substrate preparation:

  • Phosphorylate GST-CTD (approximately 5 pmol) with specific kinases in the presence of 1 mM ATP for 1 hour at 30°C

  • Remove residual ATP using gel filtration spin columns

• Phosphatase reaction:

  • Incubate phosphorylated GST-CTD with increasing concentrations of recombinant SSU72 (1.25, 2.5, and 5 pmol) in phosphatase buffer

  • Maintain reaction for 1 hour at 30°C

  • Quench reactions by adding SDS loading buffer and heating at 98°C for 5 minutes

• Analysis:

  • Perform SDS-PAGE and Western blotting

  • Probe with antibodies specific for Ser5-P, Ser2-P, or Ser7-P forms of CTD

  • Quantify dephosphorylation using densitometry

• Controls:

  • Include no-enzyme controls to establish baseline phosphorylation

  • Consider using known phosphatase inhibitors as negative controls

  • Use catalytically inactive SSU72 mutants to confirm specificity

This methodology allows for precise measurement of SSU72's phosphatase activity and substrate specificity against different phosphorylated CTD residues.

How can researchers investigate the interaction between SSU72 and the P-TEFb complex?

To investigate the interaction between SSU72 and the P-TEFb complex, researchers should employ a multi-method approach:

1. Co-immunoprecipitation (Co-IP) analysis:

• Immunoprecipitate cell extracts with antibodies against Cdk9, Cyclin T1, or SSU72

• Analyze immunoprecipitates by Western blotting with antibodies against SSU72, Cdk9, Cyclin T1, and Pol II

• Include RNase treatment to determine if interactions are RNA-dependent or direct protein-protein interactions

2. Transgenic models for validation:

• Utilize inducible HA-SSU72 expression systems

• Perform immunoprecipitation with anti-HA antibodies

• Probe for endogenous P-TEFb components (Cdk9, Cyclin T1, Hexim1)

3. Proteomic analysis:

• Perform mass spectrometry (MudPIT analysis) on SSU72 immunocomplexes

• Identify associated proteins such as NcoR1, Sart1, Hexim, and DDX5

4. High-resolution microscopy:

• Conduct 3D confocal microscopy to visualize co-localization of SSU72 with P-TEFb components in cells

• Quantify co-localization coefficients for protein pair combinations

5. Functional analysis of P-TEFb activity:

• Assess P-TEFb kinase activity by immunoprecipitating the P-TEFb complex with anti-Cdk9 antibody

• Compare kinase activity under control and SSU72-depleted conditions

• Use purified GST-CTD proteins as substrates with cold ATP

• Include flavopiridol treatment as a control for P-TEFb inhibition

This comprehensive approach will provide robust evidence for the physical and functional interactions between SSU72 and the P-TEFb complex, elucidating the mechanism by which SSU72 regulates transcriptional elongation.

How can the tissue-specific effects of SSU72 be effectively studied?

To investigate the tissue-specific effects of SSU72, researchers should implement a comprehensive experimental strategy that accounts for cell-type differences in gene regulation:

1. Conditional knockout system design:

• Generate Ssu72 f/f mice with loxP sites flanking exon 1

• Employ tissue-specific Cre expression systems (e.g., Alb-Cre for liver-specific deletion)

• Develop cell-type-specific inducible knockout models

2. Multi-tissue comparative analysis:

• Isolate primary cells from different tissues with varied proliferation rates:

  • Embryonic stem cells (highly proliferating)

  • Mouse embryonic fibroblasts (moderately proliferating)

  • Hepatocytes (almost quiescent)

3. Integrative genomics approach:

• Perform RNA-Seq across multiple tissue types with and without SSU72

• Conduct ChIP-Seq for SSU72 and phosphorylated forms of Pol II CTD

• Generate genome browser views to compare SSU72 binding patterns across tissues

• Identify tissue-specific target genes and associated pathways

4. Mechanistic investigation:

• Examine CTD phosphorylation patterns in different cell types

• Analyze interaction partners of SSU72 across tissues using proteomics

• Investigate the relationship between proliferation status and SSU72 dependency

5. Validation experiments:

• Perform rescue experiments with ectopic expression of SSU72 in knockout backgrounds

• Conduct qRT-PCR validation of tissue-specific target genes

• Use immunoblotting to confirm tissue-specific effects on CTD phosphorylation patterns

The data from these experiments indicate that SSU72 displays significant tissue specificity in its genomic localization and function. For example, in ES cells, SSU72 occupancy is increased at both promoter and 3'-end regions, while in MEFs and hepatocytes, it is predominantly found at promoter regions. This methodological framework allows for comprehensive characterization of SSU72's tissue-specific roles in transcriptional regulation .

What methodology is recommended for analyzing contradictory data regarding SSU72's function in different experimental systems?

When faced with contradictory data regarding SSU72's function across different experimental systems, researchers should employ the following analytical framework:

1. Systematic comparison of experimental conditions:

• Create a comprehensive table documenting key experimental variables:

  • Cell/tissue types used

  • SSU72 depletion/knockout strategies

  • Time course of depletion (acute vs. chronic)

  • Analysis methods employed

  • Genetic background of models

  • Developmental stage of samples

2. Validation across multiple model systems:

• Repeat key experiments in both yeast and mammalian systems

• Compare results from different cell types with varying proliferation rates

• Test both in vitro biochemical assays and in vivo functional studies

3. Contextual analysis of phosphorylation dynamics:

• Examine the interplay between different CTD phosphatases (SSU72, Fcp1, Rtr1)

• Analyze the reciprocal phosphorylation patterns of different CTD residues

• Consider the impact of cell-type-specific transcriptional programs

4. Consideration of SSU72's dual roles:

• Distinguish between SSU72's catalytic phosphatase activity and its structural role in protein complexes

• Separate analysis of termination defects from elongation defects

• Investigate potential context-dependent functions

5. Resolution strategy:

• Design experiments that directly test competing hypotheses

• Implement molecular complementation studies with domain-specific mutants

• Utilize advanced techniques like NET-seq or PRO-seq to resolve transcriptional dynamics

When analyzing contradictory data, it's important to recognize that SSU72 has been shown to have both phosphatase-dependent and phosphatase-independent functions. For example, while its catalytic activity is critical for proper transcription in vitro, its essential role in 3' end processing can be independent of its catalytic function . This distinction may explain some apparent contradictions in the literature.

What are the essential controls for validating the specificity of the PAT45E2AT antibody in research applications?

To properly validate the specificity of the PAT45E2AT antibody in research applications, implement these essential controls:

1. Genetic validation controls:

• Knockout/knockdown samples: Test antibody reactivity in SSU72-depleted cells generated through:

  • Conditional knockout using Cre-mediated deletion of loxP-flanked SSU72 exon

  • siRNA or shRNA knockdown of SSU72

  • CRISPR/Cas9-mediated deletion

• Overexpression samples: Test specificity using:

  • Cells expressing HA-tagged SSU72 at levels similar to endogenous expression

  • Recombinant SSU72 protein as a positive control

2. Technical validation controls:

• Pre-absorption controls: Pre-incubate antibody with purified antigen (recombinant human SSU72 amino acids 1-194) before application

• Isotype controls: Use irrelevant mouse IgG1 antibodies of the same concentration

• Secondary antibody controls: Test secondary antibody alone to rule out non-specific binding

3. Application-specific controls:

• Western blot: Include molecular weight markers to confirm band size (SSU72 is approximately 23-24 kDa)

• Immunoprecipitation: Compare with normal mouse IgG immunoprecipitates

• ChIP/ChIP-Seq: Include IgG ChIP controls and input DNA controls

4. Cross-validation approaches:

• Test alternative SSU72 antibodies from different sources

• Correlate antibody reactivity with mRNA expression levels

• For tagged constructs, compare results between the PAT45E2AT antibody and tag-specific antibodies

These comprehensive controls will ensure the reliability and specificity of results obtained using the PAT45E2AT antibody across different experimental applications.

How should researchers interpret changes in CTD phosphorylation patterns following SSU72 depletion?

Interpreting changes in CTD phosphorylation patterns following SSU72 depletion requires careful consideration of several interconnected factors:

1. Expected primary effects:

• Increased Ser5-P and Ser7-P levels: As SSU72 directly dephosphorylates these residues, expect significant increases in their phosphorylation levels, particularly at promoter regions

• Acidic shift in isoelectric focusing: Hyperphosphorylation of Ser5 and Ser7 residues will manifest as an acidic shift of Pol II in isoelectric focusing experiments

2. Secondary effects on other phosphorylation sites:

• Reduced Ser2-P and Thr4-P levels: Despite not being direct targets of SSU72, these sites show decreased phosphorylation following SSU72 depletion, likely through altered P-TEFb recruitment and activity

• Basic shift of Ser2-P and Thr4-P: In isoelectric focusing, these residues will shift to a more basic isoelectric point in SSU72-deficient cells

3. Gene-specific interpretation framework:

• For actively transcribed genes, examine changes in phosphorylation patterns across different regions:

  • Promoter-proximal regions: Increased Ser5-P and Ser7-P

  • Gene body: Decreased Ser2-P and Thr4-P

  • 3' end: Complex patterns reflecting termination defects

4. Mechanistic considerations:

• Interpret changes in light of SSU72's interaction with P-TEFb complex

• Consider the reciprocal relationship between different phosphorylation marks

• Analyze results in context of the role of SSU72 in releasing paused Pol II

5. Tissue-specific interpretation:

• Account for cell-type-specific effects on actively transcribed genes

• Compare changes across cells with different proliferation rates

• Consider the differential impact on housekeeping versus tissue-specific genes

This interpretative framework allows researchers to distinguish direct effects of SSU72 depletion from secondary consequences and to understand the complex interplay between different CTD modifications in transcriptional regulation.

What experimental designs can effectively demonstrate the functional consequences of SSU72 phosphatase activity?

To effectively demonstrate the functional consequences of SSU72 phosphatase activity, researchers should implement the following experimental designs:

1. Catalytic mutant complementation studies:

• Generate phosphatase-dead SSU72 mutants through site-directed mutagenesis

• Express wild-type or catalytically inactive SSU72 in SSU72-depleted backgrounds

• Compare rescue efficiency for various phenotypes:

  • Transcriptional defects

  • CTD phosphorylation patterns

  • Cell viability and proliferation

  • Specific gene expression changes

2. Phosphomimetic CTD substitution experiments:

• Construct CTD variants with serine-to-glutamate (S7E) substitutions to mimic constitutive phosphorylation

• Employ plasmid shuffle strategies to test viability with mutant CTDs

• Compare phenotypes of S7E mutants with SSU72 depletion

3. In vitro transcription systems:

• Deplete SSU72 from nuclear extracts

• Test transcription efficiency with phosphorylated templates

• Rescue experiments with recombinant catalytically active SSU72

• Compare with other phosphatases (Fcp1, Rtr1) to establish specificity

4. Coupled phosphorylation-dephosphorylation assays:

• Set up sequential kinase and phosphatase treatments of CTD substrates

• Analyze the interplay between different CTD modifications

• Test the impact of prolyl isomerases (Pin1/Ess1) on SSU72 activity

5. Genome-wide occupancy and transcription analysis:

• Conduct ChIP-Seq for SSU72 and phosphorylated Pol II forms

• Perform RNA-Seq and PRO-Seq to measure transcriptional outcomes

• Map termination defects and correlate with phosphorylation changes

• Compare results between wild-type SSU72 and catalytic mutants

These experimental approaches can effectively distinguish between the phosphatase-dependent and phosphatase-independent functions of SSU72, providing insight into how its catalytic activity contributes to transcriptional regulation and cell viability.

What are common pitfalls when studying SSU72 function, and how can researchers avoid them?

When studying SSU72 function, researchers frequently encounter several challenges. Here are the common pitfalls and strategies to avoid them:

1. Lethal phenotype complications:

• Pitfall: Complete SSU72 knockout causes lethality, complicating functional studies.

• Solution: Implement inducible or tissue-specific conditional knockout systems using Cre-loxP technology. Use adenoviral delivery of Cre (Ad-Cre) for temporal control or tissue-specific Cre expression (e.g., Alb-Cre for liver) .

2. Phosphorylation site cross-reactivity:

• Pitfall: Antibodies against phosphorylated CTD residues may show epitope interference.

• Solution: Validate antibody specificity with synthetic phosphopeptides. Use multiple antibodies from different sources and complement with mass spectrometry analysis of phosphorylation sites .

3. Dual function misinterpretation:

• Pitfall: Confusing SSU72's phosphatase-dependent and structural roles.

• Solution: Use catalytically inactive mutants to distinguish between enzymatic and structural functions. Compare phenotypes between phosphatase-dead mutants and complete knockouts .

4. Insufficient temporal resolution:

• Pitfall: Missing dynamic changes in CTD phosphorylation during transcription.

• Solution: Implement time-course experiments after SSU72 depletion. Use advanced techniques like ChIP-seq with spike-in normalization to accurately track changes over time .

5. Overlooking tissue-specific effects:

• Pitfall: Generalizing findings from one cell type to all systems.

• Solution: Study multiple cell types with different proliferation rates. Compare results between embryonic stem cells, fibroblasts, and terminally differentiated cells like hepatocytes .

6. P-TEFb complex heterogeneity:

• Pitfall: Failing to account for different P-TEFb subcomplexes.

• Solution: Distinguish between active and 7SK snRNP-sequestered P-TEFb. Include RNase treatments in immunoprecipitation experiments to determine if interactions are RNA-dependent .

7. Technical artifacts in ChIP experiments:

• Pitfall: Cross-linking efficiency varies between proteins and genomic regions.

• Solution: Optimize cross-linking conditions. Include appropriate controls (input DNA, IgG) and use spike-in normalization for quantitative comparisons .

By anticipating these challenges and implementing the suggested solutions, researchers can significantly improve the reliability and interpretation of their studies on SSU72 function.

How can researchers optimize ChIP-Seq protocols specifically for studying SSU72 genomic localization?

To optimize ChIP-Seq protocols specifically for studying SSU72 genomic localization, researchers should follow these detailed recommendations:

1. Sample preparation optimization:

• Cross-linking conditions: Use 1% formaldehyde for 10-15 minutes at room temperature. For SSU72, which may have transient interactions with chromatin, consider using dual cross-linking with DSG (disuccinimidyl glutarate) followed by formaldehyde .

• Sonication parameters: Optimize sonication conditions to achieve DNA fragments of 200-300 bp, which is ideal for high-resolution mapping of SSU72 binding sites. Verify fragment size distribution by agarose gel electrophoresis .

2. Immunoprecipitation refinements:

• Antibody selection: Use the PAT45E2AT antibody at optimal concentration (typically 2-5 μg per ChIP reaction). Consider validating with HA-tagged SSU72 and anti-HA antibodies in parallel .

• Pre-clearing strategy: Implement stringent pre-clearing with protein A/G beads and non-specific IgG to reduce background.

• Washing conditions: Use increasingly stringent wash buffers to reduce non-specific binding while preserving specific SSU72 interactions .

3. Controls and normalization:

• Input controls: Reserve 5-10% of chromatin before immunoprecipitation as input control.

• Negative controls: Include IgG ChIP and SSU72-depleted samples as negative controls.

• Spike-in normalization: Add a small amount of chromatin from a different species (e.g., Drosophila) as a spike-in control for quantitative comparisons between samples .

4. Sequencing considerations:

• Library preparation: Use methods optimized for low-input samples if SSU72 ChIP yields are modest.

• Sequencing depth: Aim for at least 20-30 million uniquely mapped reads to detect SSU72 binding sites with high confidence.

• Paired-end sequencing: Consider paired-end sequencing for improved mapping specificity .

5. Bioinformatic analysis tailored for SSU72:

• Peak calling parameters: Use MACS2 with parameters optimized for transcription factors (narrow peaks) rather than histone modifications.

• Co-occupancy analysis: Compare SSU72 binding patterns with Pol II occupancy and various phosphorylated forms (pSer5, pSer2, pSer7).

• Promoter-focused analysis: Pay special attention to promoter regions where SSU72 shows strong enrichment .

6. Validation approaches:

• ChIP-qPCR validation: Confirm ChIP-Seq peaks at selected target genes using ChIP-qPCR.

• Sequential ChIP (Re-ChIP): Consider Re-ChIP to determine co-occupancy of SSU72 with other factors like TFIIB or P-TEFb components.

• Correlation with RNA-Seq: Integrate ChIP-Seq data with RNA-Seq to correlate SSU72 binding with gene expression changes upon SSU72 depletion .

These optimizations will help researchers obtain high-quality, reproducible ChIP-Seq data for SSU72, enabling precise mapping of its genomic localization and insights into its function in transcriptional regulation.