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Reposted by Apratim (Appy) Mukherjee

Mir Lab @mirlab.bsky.social · 09/10/2026

1/ Our paper, “Enhancer binding kinetics explain transcription factor hub formation,” is now published! Here we set out to understand why transcription factors form bright clusters or “hubs” in the nucleus, and whether those hubs actually control gene activity. tinyurl.com/38uf8348

tinyurl.com

Enhancer binding kinetics explain transcription factor hub formation

Transcription factors (TFs) form dynamic, high-concentration clusters, condensates, or hubs, proposed to increase TF binding frequency at target enhancers. However, how enhancer sequence shapes hub pr...

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Reposted by Apratim (Appy) Mukherjee

Mir Lab @mirlab.bsky.social · 07/06/2026

1/🧵 Can transcription factor condensate formation be explained without phase separation? Our new preprint introduces SPARK, a simulation tool that reproduces condensate behavior (clustering, fusion, FRAP) from diffusion & binding kinetics alone. Movie: 60 sec FRAP sim www.biorxiv.org/content/10.6...

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 20/03/2026

Excited that our work on RNAPII clusters during development is finally in print today! Thanks to everyone who worked incredibly hard on this project and the community for their engaging (and fun!) discussion during each iteration. Please see the post below for details!

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Reposted by Apratim (Appy) Mukherjee

Anders Sejr Hansen @andersshansen.bsky.social · 11/02/2026

Come on join us! We are searching for the next technical associate to join our lab ~Summer 2026. This could be a great position for someone who is graduating this spring and is looking for 2 more years of research experience before starting their PhD: careers.peopleclick.com/careerscp/cl...

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 16/01/2026

Are you asking how TFs might form clusters if they are diffusing so rapidly? They can form clusters via multiple mechanisms: repetitive binding on the DNA (high k_on), potential trapping because of the local chromatin environment, multivalent interactions, etc (sorry if I misunderstood your ques.!)

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Reposted by Apratim (Appy) Mukherjee

Wendy Bickmore @wbickmor.bsky.social · 16/01/2026

Delighted to have a contribution in this issue together with @eliasfriman.bsky.social

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 16/01/2026

Very cool review! Skimmed through it but printed it out for my weekend read.

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 16/01/2026

(2/2) Also, we ran a number of controls: Made sure that alpha-injected embryos would not gastrulate properly. Also used MS2/MCP to mark specific genes and confirmed signal disappears after alpha-injection. Finally, looked at vehicle injection to ensure injection itself wasn't killing the embryo.

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 16/01/2026

(1/2) Always glad to have a discussion! We could of course use ActoD or DRB which more selective. However, the timescale of degradation with alpha (~hour) is much longer than timescale of these nuclear cycles in Drosophila (~mins). So we were ok with using alpha for these experiments.

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 15/01/2026

Thank you for the thoughtful discussion! Always happy to discuss further :)

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 15/01/2026

Thanks! 1. Yes, we show that perturbing elongation using alpha-amanitin leads to increased cluster lifetimes (Fig. 3). 2. Release of Pol2 molecules into elongation (latter nuclear cycles) drives the decrease in cluster lifetime. This does not require additional mechanisms (eg. RNA feedback, etc)

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 15/01/2026

Yes, TF molecules may help modify the chromatin environment and recruit relevant cofactors/Pol II. But like Pol II clusters, TF clusters also might emerge due to repetitive protein-protein/DNA binding.

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 15/01/2026

@msuskiewicz.bsky.social Yes exactly! Our model suggests that enough polymerase molecules at a single gene is sufficient to give rise to a visible cluster above the nuclear background.

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 15/01/2026

(2/2): We are also currently looking into the temporal sequence of upstream transcription machinery such as Mediator, TFIIH, etc at loci tagged with ParS/ParB system, so stay tuned!

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 15/01/2026

(1/2): Work from Sarah Bray’s lab, O’Farrel lab, and our lab (in prep.) have suggested that upstream TFs can form clusters/hubs at their genomic targets prior to the gene turning on so that could definitely be a factor.

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 15/01/2026

(3/3) Finally, our simulations (Fig 6h,i) indicate there could be instances (low burst duration/slow loading rate) which prevent a detectable cluster (over the background) suggesting that # of engaged polymerases at the gene is the key driver of transcription, not the formation of a visible cluster.

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 15/01/2026

(2/3) Furthermore, our Triptolide injections (Fig. 3d) show that disrupting initiation results in the abolishment of all visible clusters (other than HLBs) suggesting that transcription initiation is what drives the formation of visible clusters (as eloquently described the concert analogy!).

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 15/01/2026

(1/3) Thanks for the question! If cluster formation drove upregulated transcription, we would expect a time-lag between the cluster intensity and the MCP intensity (marking the gene). However, our cross-correlation across multiple nuclei and genes consistently shows a time-lag of zero (Fig 5d).

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 18/09/2025

Huge props to our grad student @manyakapoor.bsky.social for developing the simulations and the new MS2 analysis amongst other things! We also thank the transcription community for their invaluable feedback and always welcome further discussion😊

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 18/09/2025

(3/3) We also include new simulations showing cluster visibility over background is mediated by # of engaged polymerases at a locus. Our data + sim. show that RNAPII clusters do not require higher-order assembly mechanisms and reflect local accumulations of transcriptionally engaged polymerases.

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 18/09/2025

(2/3) Previously, we had only looked at one gene (hunchback). Here, we extend our study to 3 additional genes (eve, snail, and sog), sister chromatids, and include multiple controls (non-transcribing loci and control spots) to more definitively claim single-cluster association with individual genes.

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Apratim (Appy) Mukherjee @mukherja.bsky.social · 18/09/2025

(1/3) Excited to post our updated pre-print! We show RNAPII clusters are simply collections of polymerase molecules stably engaged at single genes & cluster intensity is strongly correlated with txn output during Drosophila embryogenesis. www.biorxiv.org/content/10.1...

biorxiv.org

A single cluster of RNA Polymerase II molecules is stably associated with active genes

In eukaryotic nuclei, transcription is associated with the clustering of RNA Polymerase II (RNAPII) molecules. The mechanisms underlying cluster formation, their interactions with genes, and their imp...

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Reposted by Apratim (Appy) Mukherjee

Mir Lab @mirlab.bsky.social · 09/04/2025

1/n: Excited to share our new preprint. We find that Dorsal transcription factor hubs exhibit gene-specific biophysical properties that tune transcriptional output. Read here: www.biorxiv.org/content/10.1.... Project was led by amazing graduate student Samantha Fallacaro (not on bsky)

biorxiv.org

Transcription factor hubs exhibit gene-specific properties that tune expression

The spatial and temporal control of gene expression relies on transcription factors binding to and occupying their target sites. Transcription factor hubs - localized, high-concentration microenvironm...

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Reposted by Apratim (Appy) Mukherjee

Mir Lab @mirlab.bsky.social · 12/02/2025

1/n Excited to share our new preprint! Using live imaging in Drosophila embryos, we show that RNA Pol II clusters switch from sites of initiation to elongation during zygotic genome activation and that they are stably associated with an active gene: www.biorxiv.org/content/10.1...

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