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. 2013 Aug 22;51(4):480-92.
doi: 10.1016/j.molcel.2013.08.009.

Gene-specific transcriptional mechanisms at the histone gene cluster revealed by single-cell imaging

Benjamin Guglielmi  1 , Natalie La Rochelle, Robert Tjian
Affiliations

Gene-specific transcriptional mechanisms at the histone gene cluster revealed by single-cell imaging

Benjamin Guglielmi et al. Mol Cell. .

Abstract

To bridge the gap between in vivo and in vitro molecular mechanisms, we dissected the transcriptional control of the endogenous histone gene cluster (His-C) by single-cell imaging. A combination of quantitative immunofluorescence, RNA FISH, and FRAP measurements revealed atypical promoter recognition complexes and differential transcription kinetics directing histone H1 versus core histone gene expression. While H1 is transcribed throughout S phase, core histones are only transcribed in a short pulse during early S phase. Surprisingly, no TFIIB or TFIID was detectable or functionally required at the initiation complexes of these promoters. Instead, a highly stable, preloaded TBP/TFIIA "pioneer" complex primes the rapid initiation of His-C transcription during early S phase. These results provide mechanistic insights for the role of gene-specific core promoter factors and implications for cell cycle-regulated gene expression.

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Figures

Figure 1
Figure 1
Visualizing RNA polymerase II transcription at the histone gene locus. (A) Left panel, Co-staining of S2 cells with MPM2 antibody (red) and Pol II P-Ser5 antibody in green. Middle panel, fraction of cells showing MPM-2 staining (light Red) and MPM-2/Pol II co-localization (light green). Right panel, fraction of cells showing Pol II His-C staining (light green) and MPM-2/Pol II co-localization (light red) (N=336). Bar = 5μm. (B) A stable cell line expressing GFP-RPII215 (Pol II-green) stained with P-Ser5 antibody (red); and showing co-localization. (C) A stable cell line expressing GFP-RPII215 is stained with Lsm11 (red) antibody, showing co-localization. (D) A stable cell line expressing both GFP-RPII215 (green) and RFP-Lsm11 (red); and showing co-localization. (E) FRAP experiment tracking GFP-RPII215 at the histone locus. Left panel: Black line shows recovery of signal from untreated cells. The red line shows recovery of signal from cells treated with DRB. Average of 10-15 FRAP experiments, standard error <15%. Right panel: Example of of GFP-RPII215 FRAP at the histone locus. Bar = 5μm.
Figure 2
Figure 2
Visualizing nascent transcription of the histone genes by FISH in single S2 cells. (A) Co-staining of MPM2 antibody (green) with H1 or H2A mRNA FISH (red), bar=5μm. Middle panel shows fraction of MPM-2 signal co-localized with H1 or H2A. Right panel shows fraction of H1 and H2A foci colocalized with MPM-2 staining (N=60-116) (B) Co-staining of Pol II P-Ser5 (green) with H1 or H2A mRNA FISH (red), bar=5μm. Middle panel shows fraction of Pol II His-C signal co-localized with H1 or H2A. Right panel shows fraction of H1 and H2A foci colocalized with Pol II His-C staining (N=40-93) (C) Co-staining through FISH of H1 (green) and H2A (red). Middle panel shows fraction of co-localized signals of H1 with H2A, bar=5μm. Right panel shows fraction of co-localized signals of H2A with H1.
Figure 3
Figure 3
Differential Expression of H1 and H2A during S-phase. (A) Co-staining of H1 or H2A mRNA through FISH (Red) with EdU incorporation (green), marking S-phase. (B) Schematic of the double pulse chase experiment for S-phase timing. (C) Co-staining of BrdU (green) with EdU (red) when using a 90 minutes chase between BrdU and EdU incubations. (D) Upper panel, Association of EdU staining patterns with BrdU staining as a function of chase time. Each colored graph (A to F) represents one of 6 distinct EdU staining patterns. Lower panel, Repartition of EdU staining patterns across S-phase. Each colored bar represents one independently detectable EdU staining pattern (N=80-166). Lower Panel, The length of the bar on the arrow represents approximately when an EdU pattern is occurring the most during S-phase. (E) Fraction of cells that are stained by Edu and BrdU both in function of chase time. (F) Fraction of cells with nascent H2A mRNA for cells within each different EdU staining pattern (N=68-103). Each colored bar corresponds to a EdU staining pattern with the same color code from panel (D).
Figure 4
Figure 4
TBP and TFIIA accumulation at the histone locus in interphase. (A) Left panel, co-staining of Lsm11 antibody (red) with TBP antibody (green). Right panel, distribution of Lsm11 and TBP signal at His-C among the total number of cells, using Lsm11 signal as reference. (B) Co-staining of TBP (right) with Pol II P-Ser5 or with H2A mRNA FISH (left). (C) Upper panel, co-staining of TBP (green) with TFIIAL (red). middle panel, co-staining of TFIIB (green) with Lsm11 (red). Lower panel, co-staining of TAF4 (green) with Lsm11 (red). (D) Left and middle panel, chromatin immunoprecipitation of Pol II and TBP on Actin5C and H2A promoter, error bar is Standard Deviation of three independent ChIP. Right panel, ratio of TBP ChIP to Pol II ChIP on Actin5c and H2A promoter, normalized to 1 for Actin5C.
Figure 5
Figure 5
TBP depends on TFIIA but not HLB factors, TFIIB or TFIID for binding to the histone locus. (A) Representative co-staining of TBP (green) with Lsm11 (red) in S2 cells after dsRNA treatment. From first to last row, cells after dsRNA treatment against control, Lsm11, Mxc, TFIIA-L, TFIIB, TAF4. (B) Fraction of TBP dots at His-C showing co-localization with a Lsm11 signal in cells depleted of Lsm11 or Mxc. Average of 2-3 RNAi experiments with standard deviations (N=96-169). (C) Fraction of Lsm11 dots at His-C showing co-localization with a TBP signal in cells depleted of TFIIA-L. Average of 2-3 RNAi experiments with standard deviations.
Figure 6
Figure 6
TBP and TFIIA are required for Pol II transcription of the His genes. (A) Unsynchronized S2 cells were stained for Pol II P-Ser5 (green) and MPM2 (red) after treatment with dsRNA for knock down of TBP, TFIIA-L, TFIIB, and TAF4. Upper panel shows representative pictures. (B) Fraction of MPM-2 dot that co-localized with Pol II signal dots after depletion of different PIC components. Average of 2-3 RNAi experiments (N=72-103, 26 for TFIIB).
Figure 7
Figure 7
FRAP analysis of TBP and TRF2 binding kinetics in the nucleoplasm and at the histone gene locus. (A) FRAP recovery over 20 minutes of GFP-TRF2 (left and right panel) or GFP-TBP at the histone locus, average of 10 cells. (B) Representative FRAP recovery of GFP-TBP at the histone locus. GFP-TBP at His-C fully recovers in 120 minutes. (C) FRAP recovery of GFP-TBP in random locations of the nucleoplasm +/− RNAi of Mot1. Average of 20 FRAP, standard errors are <10%. GFP-TBP in the nucleoplasm fully recovers in one minute. (D) Overexpression of Drosophila Mot1 in S2 cells. Mot1 appears in red TBP is stained in green, and MPM-2 in Yellow. Arrow point to the TBP foci location. Images are maximum projections of Z-stacks.
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