DRB and heat-shock treatment of fetal liver cells inhibits transcription. (A) FUrd incorporation into nascent transcripts in untreated and DRB- and heat-shock-treated fetal liver cells. (Top panel) FUrd immunostaining. (Bottom panel) Overlay of FUrd (green) and DAPI (blue; bar, 4 μm). (B) Hbb-b1 RNA FISH signals (green) in untreated and DRB- and heat-shock-treated fetal liver nuclei. DAPI is in blue (bar, 4 μm). (C) Percent transcribed Hbb-b1 alleles at 0, 10, 20, and 30 min (n > 100).

Punctate localization of RNAPII is maintained in the absence of transcription. (A) Immunofluorescence of RNAPII in untreated and DRB- and heat-shock-treated fetal liver nuclei (4H8 or H14 antibody; bar, 4 μm). (B) Average number of foci per optical section (4H8) in untreated and DRB- and heat-shock-treated nuclei (n > 45; P = 0.5828; error bars depict SEM). (C) RNAPII was detected by Western in the saponin supernatant (SS) and pellet (SP) using the ARNA-3 antibody. Coomassie staining of histones, retained in the pellet, and globin proteins, extracted by 1% saponin, shows equal loading.

Heat-shock but not DRB treatment causes gene dissociation from RNAPII foci. (A) DNA immuno-FISH, Hbb, or Eraf loci (red) and RNAPII protein (green, 4H8) in untreated and DRB- and heat-shock-treated fetal liver nuclei (bar, 4 μm). Inset shows a higher magnification of each DNA FISH signal. Representative examples of nuclei showing two alleles colocalizing with RNAPII foci in untreated and DRB-treated nuclei or two dissociated alleles in heat-shock-treated nuclei. (B) Quantification of RNAPII-associated Hbb or Eraf alleles by DNA immuno-FISH in untreated and DRB- and heat-shock-treated nuclei (n > 40; [*] P < 0.05; [***] P < 0.001).

Heat-shock but not DRB treatment causes dissociation of distal-clustered genes while Hbb locus conformation is largely maintained. (A) 3C interactions in fetal brain (1) and untreated (2), DRB-treated (3), and heat-shock-treated (4) fetal liver cells. 3C control template (5). (C) 3C interactions across the Hbb locus relative to Hbb-b1 (black bar). Chromatin digested with BglII or HindIII. Relative cross-linking frequency determined at each point for the restriction fragments indicated in gray (adjacent fragments in alternating intensities) normalized to that of the most 3′ fragment. A schematic of the Hbb locus indicates the position of the globin genes (blue sideways triangles), HS (red bars), and olfactory receptor genes (black squares). Error bars depict SEM (n = 3).

Model of gene occupancy in transcription factories in the presence and absence of transcription. (A) Expressed genes, Hbb-b1, b2, Eraf, and Uros (green arrows) dynamically associate with transcription factories (blue circle) when transcribed and often coassociate in a shared factory. The Hbb locus forms chromatin loops, HS (red bars) of the LCR are in close contact with transcribed Hbb genes (contact with Hbb-b1 depicted). HS2 and HS3 form a tighter association with the Hbb-b1 promoter during transcription initiation, possibly through transcription factor–RNAPII interactions. This conformation of the Hbb locus and gene association with transcription factories occurs during initiation and is maintained in the absence of elongation. (B) In the absence of transcription initiation, previously active genes dissociate from transcription factories. Conformation of the Hbb locus is largely maintained; however, there is a disruption in the association of HS2 and HS3 with the Hbb-b1 promoter.