Nuclear microenvironments modulate transcription from low-affinity enhancers
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- European Molecular Biology Laboratory, Germany
- Albert Einstein College of Medicine, United States
Figures
Figure 1 with 7 supplements
Ubx is present in microenvironments with varying local concentrations.
(A) Stage 15 embryos stained for Ubx protein with a bounding box indicating a ventral region of abdominal segment one (A1). (B) Higher magnification, Airyscan image of the region indicated in panel …
Figure 1—figure supplement 1
Quantification of Ubx microenvironments in single nuclei.
(A) 3D projection of a single nucleus stained for Ubx protein. (B) Surface plot of contiguous Ubx regions containing a minimum of four pixels with signal intensity greater than 25% of the maximum …
Figure 1—figure supplement 2
Ubx distribution compared to other transcription factors.
(A) Stage 15 embryo stained with an antibody against Engrailed (En) and Ubx protein. (B) Same image as (A) with only En shown. (C) A higher magnification of the area in the bounding box of panel (A).…
Figure 1—figure supplement 3
Ubx distribution compared to general markers for transcriptional activity.
(A) Stage 15 embryo stained with an antibody against transcriptionally active RNA Polymerase II RPB1 (Ser5 phosphorylated) and Ubx protein, with only active Pol II shown. (B) Same image as (A) with …
Figure 1—figure supplement 4
Control experiments for HaloTag-Ubx.
(A) Schematic of the N-terminal HaloTag construct with HaloTag-Ubx under the control of 20x UAS (responsive to GAL4) and the hsp70 prometer. Ubx is inserted at the MCS. (B) Stage 5 embryo resulting …
Figure 1—figure supplement 5
Additional nuclei from live imaging of Halo-Ubx.
(A, D, G, J) Projections of summed pixel intensities over 100 s of nos::GAL4, UAS::HaloTag-Ubx nuclei imaged with JF635 dye. (B, E, H, K) Individual 100 millisecond video frames of the nuclei in …
Figure 1—figure supplement 6
Live imaging at higher concentrations of HaloTag-Ubx in late stage 6 embryos.
(A) One hundred millisecond video frame from video of nos::GAL4, UAS::HaloTag-Ubx in late stage 6 embryos with higher overall nuclear concentrations of HaloTag-Ubx molecules compared to stage 5 …
Figure 1—figure supplement 7
DNA-binding deficient Ubx is stable and localized in the nucleus.
(A, B, C) Projections of summed pixel intensity over 100 or 50 seconds (as indicated in the top right corner of the panels) from videos of (A) HaloTag-Ubx, (B) HaloTag-Ubx(R3A, N51A), and (C) …
Figure 2 with 1 supplement
Transcriptionally active svb loci reside in regions of high Ubx concentration.
(A) Embryos co-stained for both Ubx protein (magenta) and shavenbaby (svb) intronic mRNA (green). Bright spots of svb intronic nascent mRNA mark actively transcribed svb loci. Regions with high …
Figure 2—figure supplement 1
Transcription sites of minimal svb enhancers and the endogenous svb locus localize close to each other.
(A) 3D surface plot of a nuclear region showing two transcriptionally active svb loci close by and possibly within the same Ubx environment. The embryo is co-stained for Ubx (magenta) and the …
Figure 3 with 1 supplement
Manipulation of Ubx-binding site number and affinity alters the level of Ubx enrichment around svb enhancers.
(A) Schematic of the synthetic TALEA transcription network driven by the Hunchback (Hb) promoter, indicating TALEA-binding sites with green circles. (B) Early stage 15 embryos carrying the TALEA …
Figure 3—figure supplement 1
Background-subtracted Ubx intensity distributions at the transcription sites for E3N and 7H enhancers.
The Ubx intensity distributions at transcription sites were plotted for the E3N and 7 hr enhancers after subtraction of background fluorescence from raw Ubx intensities. The naming convention of the …
Figure 4
Ubx and its cofactor Hth are co-enriched around transcription sites.
(A–D) Early stage 15 embryos with 7H-lacz (A–B) or E3N-lacZ reporter constructs (C–D) stained with an antibody against ß-Galactosidase in either wild-type (WT) (A and C) or hthP2 mutant embryos (B …
Additional files
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Source code 1
Matlab script to average radially averaged intensity distributions from individual transcription sites and offset background fluorescence.
- https://doi.org/10.7554/eLife.28975.015
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Transparent reporting form
- https://doi.org/10.7554/eLife.28975.016
