DOI: 10.5061/dryad.q96g6
title: Data from: Nuclear microenvironments modulate transcription from low-affinity enhancers

description: Transcription factors bind low-affinity DNA sequences for only short
 durations. It is not clear how brief, low-affinity interactions can drive
 efficient transcription. Here we report that the transcription factor
 Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila
 melanogaster shavenbaby (svb) locus and related enhancers in nuclear
 microenvironments of high Ubx concentrations. Related enhancers colocalize
 to the same microenvironments independently of their chromosomal location,
 suggesting that microenvironments are highly differentiated transcription
 domains. Manipulating the affinity of svb enhancers revealed an inverse
 relationship between enhancer affinity and Ubx concentration required for
 transcriptional activation. The Ubx cofactor, Homothorax (Hth), was
 co-enriched with Ubx near enhancers that require Hth, even though Ubx and
 Hth did not co-localize throughout the nucleus. Thus, microenvironments of
 high local transcription factor and cofactor concentrations could help
 low-affinity sites overcome their kinetic inefficiency. Mechanisms that
 generate these microenvironments could be a general feature of eukaryotic
 transcriptional regulation.
description: Airyscan Alexa568-Ubx
 Alexa488-svb DAPI Embryo 01_01Embryo 01 imaging area
 01. Airyscan imaging in the a1 segment of a fixed stage 15 Drosophila
 melanogaster embryo. Channel 1: Ubx labeled with Alexa 568 using IF.
 Channel 2: Transcription sites of svb labeled with Alexa 488 using IF
 against DIG-RNA probes targeting the first intronic region of svb. Channel
 3: DAPI.Alexa568 Ubx Alexa488 svb DAPI
 Embryo 01_01.tifAiryscan Alexa568-Ubx
 Alexa488-svb DAPI Embryo 02_01Embryo 02 imaging area
 01. Airyscan imaging in the a1 segment of a fixed stage 15 Drosophila
 melanogaster embryo. Channel 1: Ubx labeled with Alexa 568 using IF.
 Channel 2: Transcription sites of svb labeled with Alexa 488 using IF
 against DIG-RNA probes targeting the first intronic region of svb. Channel
 3: DAPI.Alexa568 Ubx Alexa488 svb DAPI
 Embryo 02_01.tifAiryscan Alexa568-Ubx
 Alexa488-svb DAPI Embryo 02_02Embryo 02 imaging area
 02. Airyscan imaging in the a1 segment of a fixed stage 15 Drosophila
 melanogaster embryo. Channel 1: Ubx labeled with Alexa 568 using IF.
 Channel 2: Transcription sites of svb labeled with Alexa 488 using IF
 against DIG-RNA probes targeting the first intronic region of svb. Channel
 3: DAPI.Alexa568 Ubx Alexa488 svb DAPI
 Embryo 02_02.tifAiryscan Alexa568-Ubx
 Alexa488-svb DAPI Embryo 03_01Embryo 03 imaging area
 01. Airyscan imaging in the a1 segment of a fixed stage 15 Drosophila
 melanogaster embryo. Channel 1: Ubx labeled with Alexa 568 using IF.
 Channel 2: Transcription sites of svb labeled with Alexa 488 using IF
 against DIG-RNA probes targeting the first intronic region of svb. Channel
 3: DAPI.Alexa568 Ubx Alexa488 svb DAPI
 Embryo 03_01.tifAiryscan Alexa568-Ubx
 Alexa488-svb DAPI Embryo 03_02Embryo 03 imaging area
 02. Airyscan imaging in the a1 segment of a fixed stage 15 Drosophila
 melanogaster embryo. Channel 1: Ubx labeled with Alexa 568 using IF.
 Channel 2: Transcription sites of svb labeled with Alexa 488 using IF
 against DIG-RNA probes targeting the first intronic region of svb. Channel
 3: DAPI.Alexa568 Ubx Alexa488 svb DAPI
 Embryo 03_02.tifAiryscan Alexa568-Ubx
 Alexa488-svb DAPI Embryo 04_01Embryo 04 imaging area
 01. Airyscan imaging in the a1 segment of a fixed stage 15 Drosophila
 melanogaster embryo. Channel 1: Ubx labeled with Alexa 568 using IF.
 Channel 2: Transcription sites of svb labeled with Alexa 488 using IF
 against DIG-RNA probes targeting the first intronic region of svb. Channel
 3: DAPI.Alexa568 Ubx Alexa488 svb DAPI
 Embryo 04_01.tifAiryscan Alexa568-Ubx
 Alexa488-svb DAPI Embryo 04_02Embryo 04 imaging area
 02. Airyscan imaging in the a1 segment of a fixed stage 15 Drosophila
 melanogaster embryo. Channel 1: Ubx labeled with Alexa 568 using IF.
 Channel 2: Transcription sites of svb labeled with Alexa 488 using IF
 against DIG-RNA probes targeting the first intronic region of svb. Channel
 3: DAPI.Alexa568 Ubx Alexa488 svb DAPI
 Embryo 04_02.tifLive widefield
 JF635-HaloTag-NLS stage 5 embryoWide field imaging in
 the anterior region of a live stage 5 Drosophila melanogaster embryo
 expressing HaloTag-NLS. The HaloTag-NLS protein is unstable and degraded
 after translation. The embryo is injected with JF635 linked to a HaloTag
 ligand 2 hours after egg-laying. The embryo is epi-illuminated with a 633
 nm laser and videos are captured with an exposure time of 100
 ms.HaloTag-NLS.tifLive widefield JF635-HaloTag-Ubx(R3A N51A) stage 5 embryoWide field imaging in the anterior region of a live stage 5 Drosophila melanogaster embryo expressing HaloTag-Ubx(R3A N51A). This Ubx is DNA-binding deficient. The embryo is injected with JF635 linked to a HaloTag ligand 2 hours after egg-laying. The embryo is epi-illuminated with a 633 nm laser and videos are captured with an exposure time of 100 ms.HaloTag-binding deficient Ubx.tifLive widefield JF635-HaloTag-Ubx late stage 6 embryoWide field imaging in the anterior region of a live late stage 6 Drosophila melanogaster embryo expressing HaloTag-Ubx. The embryo is injected with JF635 linked to a HaloTag ligand 2 hours after egg-laying. The embryo is epi-illuminated with a 633 nm laser and videos are captured with an exposure time of 100 ms.HaloTag-Ubx old embryo.tifLive widefield JF635-HaloTag-Ubx stage 5 embryoWide field imaging in the anterior region of a live stage 5 Drosophila melanogaster embryo expressing HaloTag-Ubx. This Ubx is the wildtype protein. The embryo is injected with JF635 linked to a HaloTag ligand 2 hours after egg-laying. The embryo is epi-illuminated with a 633 nm laser and videos are captured with an exposure time of 100 ms.HaloTag-Ubx.tif