EZH2 antibody and chromatin enzyme reagents: measure the mark for the activity

Designing chromatin work

1. Measure the mark to measure the activity. The enzyme level reports capacity. The abundance of the mark it deposits, and its distribution across the genome, report what the enzyme actually did. Both belong in a chromatin figure.
2. Treat the complex as the unit. Catalytic subunits are inactive alone and their targeting depends on partners. Where the claim concerns targeting, immunoprecipitation of the complex is the informative experiment rather than a blot of the subunit.
3. Use histone variants as their own controls. Variant specific reagents have to distinguish sequences that differ by a few residues. Demand the supplier's discrimination data and, where a conclusion depends on it, confirm with an orthogonal method.
4. Normalise chromatin experiments to total histone. Modification levels are reported relative to the histone carrying them, not to a cytoplasmic loading control. Blot the total histone on the same membrane.
5. Validate chromatin immunoprecipitation reagents separately. An antibody that works on a blot may not work in chromatin immunoprecipitation, where the epitope is in cross linked, native chromatin. Application specific validation is required rather than assumed.

Writers, readers, erasers and the substrate

Four classes of reagent appear in this area and answer four questions: which enzymes are present, which marks exist, which proteins are bound to those marks, and what the substrate itself looks like. Confusing them produces claims the data does not support.

State the class in the figure legend. It costs a phrase and makes the logic of the experiment visible.

Chromatin protocols are their own discipline

Cross linking time, sonication, chromatin concentration and antibody amount dominate the outcome of a chromatin immunoprecipitation, and small changes shift results substantially. Reagent quality is necessary and not sufficient.

Fix the protocol, include a positive and a negative locus in every run, and report both. Those two controls make a chromatin result comparable across time.

A bmi1 antibody and the other polycomb complex

Polycomb repression runs through two complexes, and the page's own antigen sits in one while BMI1 sits in the other, so a bmi1 antibody is what shows the second is present. The two complexes leave different marks, so the honest figure carries a mark antibody beside each enzyme. BMI1 is chromatin associated, which means the fraction and a histone loading reference decide whether a faint band is real.

An fto antibody and the mark it removes

FTO erases methylation on messenger RNA, so an fto antibody shows the eraser is present while an antibody against the modified base or a sequencing method shows the mark changed. Both belong in a figure that claims an effect on the mark. The protein shuttles between nucleus and cytoplasm, so a fractionation is often the more informative experiment than a level.

A ythdc1 antibody and a reader rather than a writer

Readers bind the modified base and carry its consequences, so a ythdc1 antibody reports the machinery that acts on the mark rather than the mark itself. It concentrates in nuclear speckles, which is a pattern distinctive enough to act as its own control in an image. The family shares a binding domain, so specificity against the cytoplasmic readers has to be stated.

A brd2 antibody and the bromodomain family

The BET proteins share tandem bromodomains and are often co-expressed, so a brd2 antibody raised on that region reports BRD3 and BRD4 too, and the relatives run at different masses which is how a cross reacting clone is spotted. They are chromatin bound, so the fraction matters, and a degrader or inhibitor treated lane is the control that shows the reagent is reading the right protein.

A tet2 antibody and a large enzyme read for loss

TET2 is frequently mutated in blood malignancies and the mutations are often truncating, so a tet2 antibody has to say where its epitope lies: a clone against the carboxy terminal region misses a truncated protein that is present. The protein is very large, so transfer is the usual failure, and the functional readout is the level of the oxidised base rather than the enzyme.

An hdac1 antibody and the paralogue it partners with

HDAC1 and HDAC2 are close paralogues that sit in the same complexes and compensate for each other, so an hdac1 antibody needs cross reactivity data and a double knockdown is often the only experiment that shows a phenotype. Activity belongs to an enzyme assay, since the protein level rarely changes. Complex integrity is read by immunoprecipitation with the partner subunits.

A kdm6a antibody and an X linked demethylase

KDM6A escapes X inactivation and has a Y encoded relative, so expression differs between sexes and a kdm6a antibody has to be specific against that relative for a comparison to mean anything. It is frequently mutated with loss of protein, so the same proof of absence applies as for any suppressor: an internal positive population, or a positive control line on the same blot.

Common questions

Does more enzyme mean more modification?

Not reliably. Activity depends on complex assembly, targeting and substrate availability. Measuring the mark itself is what reports the outcome, and both measurements together are what support a mechanistic claim.

Can a BRD4 antibody blot reagent be used for chromatin immunoprecipitation?

Only if it has been validated for it. The epitope in cross linked native chromatin is presented very differently from a denatured band, and many reagents work in one and not the other.

How should modification blots be normalised?

To the total amount of the histone carrying the mark, on the same membrane. Normalising to a cytoplasmic protein confounds the modification with the amount of chromatin loaded.

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