COMET Histone Tails

Noah Gordon

Rotation Student

Gilbert Lab Meeting
Feb 26, 2026

Good morning, everyone!

My name is Noah; I am a rotation student in the lab.

I’m going to take you all through what I’ve been working on over the last eight weeks.

So I’ll start with some background just to introduce the idea of histone tails.

I found these beautiful simulations done on supercomputers at riken in japan that show the basic units of chromatin very nicely.

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So in this lab we’re interested in editing the epigenome

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• CRISPRoff

DNMT3 methyltransferases are regulated by an autoinhibitory mechanism (48), which CRISPRoff bypasses by only using the catalytically active methyltransferase domain D3A (32). The D3A domain on its own can have detrimental effects when overexpressed in target cells; indeed, a ZFPoff construct transiently overexpressed in HEK293T cells exhibited substantial cytotoxicity, whereas cells transfected by the same ZFP fusion without the D3A catalytic domain recovered quickly

• CHARM
• Coupled Histone tail for Autoinhibition Release of Methyltransferase

here we see the first example of a technique we’ll see a few times, which is where we can use point amino acid changes to survey their effects. later we’ll see examples where we rationally mutate an amino acid to mimic, basically only on the level of charge, a type of epigenetic modification.

here, they show mutate the lysine on histone 3 at position 4 to an alanine, and show that there isn’t stimulation there like there is when position 4 is an unmethylated lysine

• COMET
• COMbinatorial Effector Targeting

allowing us to systematically investigate more than 110,000 combinations of effector proteins at endogenous human loci for their influence on transcription

• COMET
• Large library (>110,000) combinations of effector proteins
• Includes
  • Writers and erasers of histone methyl and acetyl marks
  • Chromatin remodelers
  • DNA methyltransferases
  • KRAB and VPR

Library Design

• Positive control

• Single cloning

• H3.1
  • MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPG
• H3K4R
  • MARTRQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPG
• H3K4Q
  • MARTQQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPG
• H3K27R
  • MARTKQTARKSTGGKAPRKQLATKAARRSAPATGGVKKPHRYRPG
• H3K27Q
  • MARTKQTARKSTGGKAPRKQLATKAARQSAPATGGVKKPHRYRPG

• Kurdistani, Tavazoie, & Grunstein (2004)
• “K12R (a lysine to arginine mutation mimics the unacetylated state)”
• “K16Q (a lysine to glutamine mutation mimics the acetylated state)” -Acetylation neutralizes the positive charge on lysine by adding an acetyl group to the ε-amino group. Glutamine mimics this by being uncharged, while arginine maintains the positive charge like unacetylated lysine.

-Yes, arginine is constitutively positive! Arginine has a guanidinium group in its side chain with a pKa of 12.5, which means it remains protonated and positively charged at physiological pH (7.4). Unlike lysine’s ε-amino group which can be acetylated to neutralize its charge, arginine’s side chain cannot be acetylated in this way, so it maintains its positive charge. - since methylation doesn’t change charge, none of these mimicked methylation, rather acetylation or non acetylation

H3.3

H2A

H2AX

H2AY

H2AZ

H2B

H2B.E

Experiment workflow

Gating

• a lysine to arginine mutation mimics the unacetylated state
• a lysine to glutamine mutation mimics the acetylated state -Acetylation neutralizes the positive charge on lysine by adding an acetyl group to the ε-amino group. Glutamine mimics this by being uncharged, while arginine maintains the positive charge like unacetylated lysine.

-Yes, arginine is constitutively positive! Arginine has a guanidinium group in its side chain with a pKa of 12.5, which means it remains protonated and positively charged at physiological pH (7.4). Unlike lysine’s ε-amino group which can be acetylated to neutralize its charge, arginine’s side chain cannot be acetylated in this way, so it maintains its positive charge. - since methylation doesn’t change charge, none of these mimicked methylation, rather acetylation or non acetylation

• show H31 vs positive control
• show H3K4R which mimicks the charge at position 4 of unacetylated lysine, it has higher activity than H31 on its own which is interesting because methylated lysine maintains its positive charge
• show H33 which is a variant we thought might have activation activity, and in fact it was a stronger repressor than H31
  • Activation: Tethering H3.3 tail will recruit HIRA complex and cause transcriptional activation, as H3.3 is enriched at active genes and regulatory elements and associated with open chromatin (Szenker et al., 2011, Nature Reviews Molecular Cell Biology)

OK and then the histone 2 tails were surprising! We talked about the importance of modifications to especially lysines on Histoine 3, but many of these histone 2 variants arent single amino acid changes in the same way, they’re known variants that are correlated with changes in expression–this is NOT causal though, as no one has ever put a histone tail on dCas9

• the strongest effects of our tails came from histone 2a/2b modifications
• the first two are just h2a and h2b regular
• the rest are variants

Future work

• Future work

• CRSIPR cut-and-tag/pulldown

• Combinatorial effector screening of strong candidates

Acknowledgments

• thank luke
• thank chris hsiung
• Special note for caroline
  • credit for the design of this project goes all to her
  • scope was perfect for a rotation project
  • i learned so much! thank you thank you
• thank ashir
• thank izabella
• thank gary and tyler and mandy
• thank michael sarah and kiwa and ishwa

Thank you!

• PC vs CHARM

Overlays

Negative Controls

RC433, Negative Control, Rep 1

RC433, Negative Control, Rep 2

Positive Controls

H3_DNMT3L, charm-like positive control,Rep 1

H3_DNMT3L, charm-like positive control, Rep 2

H3 vs H3 + 3L

H3_DNMT3L, charm-like positive control, Rep 1

H31_HUMAN, Histone H3 Tail, Rep 1

Negative Controls

RC433, Negative Control, Rep 1

RC433, Negative Control, Rep 2

H33

H33_HUMAN, Histone, H3.3 Variant Tail, Rep 1

H33_HUMAN Histone, H3.3 Variant Tail, Rep 2

H3 Other Variants

H31_HUMAN_H3K27R, H3K27R, Rep 1

H31_HUMAN_H3K4Q H3K4Q, Rep 1

H31_HUMAN_H3K27Q, H3K27Q, Rep 1

H31_HUMAN_H3K4R, H3K4R, Rep 1

H3K4R

H31_HUMAN_H3K4R, H3K4R, Rep 1

H31_HUMAN_H3K4R, H3K4R, Rep 2

H2 STRONG 💪

H2AX_HUMAN_A, H2A.X N term tail, Rep 1

H2B1B_HUMAN, Histone H2B N term tail, Rep 1

H2A1_HUMAN_B, Histone H2A C term tail, Rep 1

H2B2E_HUMAN Histone, H2B.E N term tail, Rep 1

H2 variants weaker than H2A and H2B

H2AX_HUMAN_B, H2A.X C term tail

H2AB1_HUMAN Histone H2A.B, N term tail

H2AY_HUMAN, Center macro-H2A.1 linker

H2A N vs C

H2A1_HUMAN_A, Histone H2A N term tail, Rep 1

H2A1_HUMAN_B, Histone H2A C term tail, Rep 1

H2 Weak

H2A1_HUMAN_A, Histone H2A N term tail

H2AZ_HUMAN, Histone H2AZ.1

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