Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster

Abstract

The Spt4, Spt5, and Spt6 proteins are conserved throughout eukaryotes and are believed to play critical and related roles in transcription. They have a positive role in transcription elongation in Saccharomyces cerevisiae and in the activation of transcription by the HIV Tat protein in human cells. In contrast, a complex of Spt4 and Spt5 is required in vitro for the inhibition of RNA polymerase II (Pol II) elongation by the drug DRB, suggesting also a negative role in vivo. To learn more about the function of the Spt4/Spt5 complex and Spt6 in vivo, we have identified Drosophila homologs of Spt5 and Spt6 and characterized their localization on Drosophila polytene chromosomes. We find that Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock. Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II. These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo.

Figure 1

Conservation and domain structure of the Drosophila melanogaster Spt5 and Spt6 proteins. Overall regions of homology indicated by dashed lines. (A) Drosophila Spt5 domain structure illustrated with murine Spt5 as a comparison. Spt5 proteins have acidic amino termini (region B), sequence homology to Escherichia coli NusG (region C) (Hartzog et al. 1998; Wada et al. 1998a; Wu-Baer et al. 1998), and serine-, threonine-, and proline-rich carboxy-terminal repeat regions noted in Yamaguchi et al. (1999b) and defined as CTR1 and CTR2 in Stachora et al. (1997) (regions D and E). Region E of Drosophila Spt5 has characteristics of CTR1 and CTR2 but the repeats appear degenerate. Homology of D. melanogaster Spt5 determined by BLAST (Altschul et al. 1990) with murine Spt5 is 50% amino-acid identity (E value = 0.0) and with Saccharomyces cerevisiae is 26% amino-acid identity (E value = 1e-58). The amino-terminal RS domain of D. melanogaster (region A) is novel for the Spt5 proteins. (B) D. melanogaster Spt6 domain structure illustrated with murine Spt6 as a comparison. Like Spt5 proteins, Spt6 proteins have acidic amino-termini (region A). Spt6 proteins also have sequence homology with a prokaryotic family of proteins implicated in transcription regulation (region B, named after the Bordetella pertussis Tex protein (Fuchs et al. 1996) and this region contains a conserved helix–hairpin–helix fold (HhH, region C, [Doherty et al. 1996]). Most Spt6 proteins are also predicted to contain RNA-binding S1 domains (region D, [Bycroft et al. 1997]). All Spt6 proteins except S. cerevisiae Spt6 have extended, divergent carboxyl termini rich in certain amino acids such as serine, threonine, and glycine (Drosophila) or glutamine (mouse) (region E and data not shown). Homology (by BLAST) of D. melanogaster Spt6 with murine Spt6 is 48% amino-acid identity (E value = 0.0) and S. cerevisiae Spt6 is 22% amino-acid identity (E value = 3e-79).

Figure 2

Detection of Spt5 and Spt6 mRNA and protein in Drosophila embryos. (A) Northern analysis of Spt5 and Spt6 transcripts in Drosophila embryos using Poly(A)⁺ RNA isolated from Drosophila embryos at specific times (indicated in hours) after egg deposition (1.5 μg RNA/lane). The lower panel shows the control probe RP49. Numbers to the left indicate sizes (kb) of molecular weight markers. (B) Western analysis of Spt5 and Spt6. Each lane contains 200 μg of Drosophila 2–12 h embryonic extract. Lanes were then separated and probed with antibodies directed against Spt5, Spt6, or Cyclin T. Antibodies used: lane 1, HMGP11 (directed against amino terminus Spt5); lane 2, HMGP14 (carboxyl terminus Spt5); lane 3, HMGP15 (directed against amino terminus Spt6); lane 4, HMGP17 (carboxyl terminus Spt6). Numbers to the left indicate sizes (kD) of molecular weight markers. (C) Whole mount immunofluorescence detection of Spt5 in Drosophila embryos. A precellularization embryo probed with HMGP11 (left panel); or with an antibody directed against the Pol II CTD as a nuclear marker (middle panel); (right panel) overlay of left and middle panels showing colocalization of red and green signals (appears yellow). All Spt5 and Spt6 antibodies give the same ubiquitous, nuclear staining pattern in both early- and later-staged embryos (data not shown).

Figure 3

Colocalization of Spt5 and Spt6 with Pol IIo^ser2 on Drosophila polytene chromosomes: (A) Immunofluorescence detection of Spt5; (B) Pol IIo^ser2; (C) overlay of A and B; (D) Spt6; (E) Pol IIo^ser2; (F) Overlay of D and E. Colocalization of red and green signals in C and F appear as yellow.

Figure 4

Partial colocalization of Spt5 and Spt6 with Pol IIa. Immunofluorescence detection of Spt5 (red) and Pol IIa (green) (A) or Spt6 (red) and Pol IIa (green) (B) in PS1–2 third instar salivary glands. Detection of Spt6 (red) and Pol IIa (green) in PS3–4 (C) and PS7–8 (D) third instar salivary glands. Loci labeled in magenta correspond to Sgs gene containing intermolt puffs; loci in white correspond to intermolt puffs that do not contain Sgs genes (A) or early or late puffs (B–D). An intermolt puff is located at 71CD (Ashburner 1972). Sgs-6 has been mapped by recombination to 71C-F (Velissariou and Ashburner 1981). Because 71E stains in a similar fashion to other Sgs loci and contains a gene homologous to other Sgs genes (data not shown) we tentatively assign Sgs-6 to 71E and distinguish it from 71CD.

Figure 5

Heat-shock induced localization of Spt5, Spt6, and cyclin T to heat-shock loci. Immunofluorescence detection of Spt proteins at uninduced hsp70 loci: (A) Pol IIa; (B) Spt5; (C) overlay of A and B; (D) Pol IIo^ser2; (E) Spt6, (F) overlay of D and E. Polytene bands containing hsp70 loci labeled in orange. Panels G–I show recruitment of Spt proteins to heat-shock loci upon 5 min heat-shock: (G) Spt5 (red) and Pol IIa (green); (H) Spt5 (red) and cyclin T (green); (I) Spt6 (red) and Pol IIo^ser2 (green).

Figure 6

Deficiency of Pol IIo^ser2 at loci containing Sgs genes and presence of Pol IIo^ser5. Immunofluorescence detection of Spt proteins and different forms of phosphorylated Pol II on PS1–2 polytenes. (A) Spt6 (red) and Pol IIo^ser2 (green). Enlargement of chromosome 2L from A showing: (B) Spt6 staining (red); (C) Pol IIo^ser2 (green); (D) overlay of B and C. Staining of region shown in B–D for Spt6 and Pol IIo^ser5: (E) Spt6 (red); (F) Pol IIo^ser5; (G) overlay of E and F. Intermolt puffs containing Sgs genes labeled in magenta. Other intermolt puffs in A labeled in white.

Figure 7

Colocalization of Spt6 and cyclin T and enrichment at loci containing Sgs genes. Immunofluorescence detection of Spt6 (red) and cyclin T (green) on PS1–2 polytenes. Polytene bands containing Sgs genes are labeled.

Figure 8

Localization of Spt5, Spt6, cyclin T, and Pol IIa to Sgs-4 transgenes and recruitment dependent on the Sgs-4 enhancer. (A) Schematic representation of Sgs-4 transgenes. P(WT) contains the wild-type Sgs-4 gene. The Pig-1 gene is divergently transcribed from Sgs-4 under control of a shared set of enhancer elements. Transcription switches from Pig-1 to Sgs-4 during third larval instar development (Mougneau et al. 1993). The SAX transgene contains the Sgs-4/Pig-1 enhancer region fused to ADH (A. Hofmann, pers. comm.). (B) Localization of Spt5, Spt6, Pol IIa, and cyclin T to Sgs-4 transgenes. Top two rows: Spt5 and Pol IIa staining of P(WT) (top) and SAX (bottom). Bottom two rows: Spt6 and cyclin T staining of P(WT) (top) and SAX (bottom). Positions of transgenes indicated by arrows. In the nontransgenic wild-type strain, there is little or no staining for Spt5, Spt6, Pol IIa, or cyclin T at the sites at which transgenes are inserted in the transformed lines (data not shown).