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.
Airyscan Alexa568-Ubx Alexa488-svb DAPI Embryo 01_01
Embryo 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.tif
Airyscan Alexa568-Ubx Alexa488-svb DAPI Embryo 02_01
Embryo 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.tif
Airyscan Alexa568-Ubx Alexa488-svb DAPI Embryo 02_02
Embryo 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.tif
Airyscan Alexa568-Ubx Alexa488-svb DAPI Embryo 03_01
Embryo 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.tif
Airyscan Alexa568-Ubx Alexa488-svb DAPI Embryo 03_02
Embryo 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.tif
Airyscan Alexa568-Ubx Alexa488-svb DAPI Embryo 04_01
Embryo 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.tif
Airyscan Alexa568-Ubx Alexa488-svb DAPI Embryo 04_02
Embryo 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.tif
Live widefield JF635-HaloTag-NLS stage 5 embryo
Wide 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.tif
Live widefield JF635-HaloTag-Ubx(R3A N51A) stage 5 embryo
Wide 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.tif
Live widefield JF635-HaloTag-Ubx late stage 6 embryo
Wide 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.tif
Live widefield JF635-HaloTag-Ubx stage 5 embryo
Wide 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