BEGIN:VCALENDAR
VERSION:2.0
PRODID:-//Talks.cam//talks.cam.ac.uk//
X-WR-CALNAME:Talks.cam
BEGIN:VEVENT
SUMMARY:3D single-molecule imaging of nuclear proteins and chromatin in pl
 uripotent cells - Srinjan Basu (University of Cambridge)
DTSTART:20231018T120000Z
DTEND:20231018T130000Z
UID:TALK205168@talks.cam.ac.uk
DESCRIPTION:How pluripotent cells in the mammalian embryo decide their fat
 e is a fundamental question in developmental biology and regenerative medi
 cine. Nuclear proteins that play a key role in these fate decisions have t
 he capacity to form intra-nuclear compartments called condensates and to i
 nfluence spatiotemporal enhancer-promoter relationships. Using single-cell
  Hi-C\, we recently demonstrated multi-way enhancer-promoter relationships
  in pluripotent cells enriched for the binding sites of nuclear proteins k
 nown to influence pluripotent cell differentiation &ndash\; transcription 
 factors (SOX2/NANOG) and chromatin regulators (the nucleosome remodelling 
 and deacetylase complex\, NuRD\, and the H3K4me3 methyltransferase\, MLL2)
 . However\, little is known about the dynamics of these proteins or how th
 ey influence enhancer-promoter relationships in live cells. To address thi
 s\, we develop 3D single-molecule localisation microscopy (SMLM) approache
 s for tracking of single HaloTag-tagged nuclear proteins within live mamma
 lian nuclei. We then establish machine-learning algorithms that extract bi
 ological information from SMLM to determine (1) the chromatin binding kine
 tics of nuclear proteins\; and (2) how nuclear proteins influence chromati
 n mobility. Finally\, we integrate nuclear-scale molecular modelling into 
 our pipelines as well as spatial information from high-density 3D SMLM to 
 reveal whether proteins form condensates in live cells and to characterise
  condensate properties. Using these approaches\, we show that while NANOG 
 and SOX2 bind to enhancers for seconds (with NANOG binding ~3 times more s
 tably than SOX2)\, the NuRD remodeller binds over much longer periods. Alt
 hough in vitro studies have shown that NANOG and SOX2 can in principle for
 m condensates\, we find evidence for only NANOG forming condensates in liv
 e pluripotent cells. Moreover\, we confirm in live cells our single-cell H
 i-C observation that NANOG\, SOX2 and NuRD binding sites cluster together.
  Finally\, we show that NuRD and MLL2 influence the range that genes explo
 re within the nucleus. Using chromosome conformation capture\, we show tha
 t these changes in chromatin mobility are linked to changes in the length-
 scale over which enhancers activate transcription at nearby genes\, and pr
 opose that this influences gene activation during pluripotent cell differe
 ntiation. Our results highlight the importance of making dynamic live-cell
  measurements at single-molecule resolution to provide insight into conden
 sates. Furthermore\, we have now established our approaches within live mo
 use embryos\, which will lead to future insights into these mechanisms in 
 vivo.
LOCATION:Seminar Room 2\, Newton Institute
END:VEVENT
END:VCALENDAR