QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 23 February 2005
doi: 10.1242/dev.01713

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Srinivasan, S. Articles by Tamkun, J. W. Search for Related Content PubMed PubMed Citation Articles by Srinivasan, S. Articles by Tamkun, J. W.

The Drosophila trithorax group protein Kismet facilitates an early step in transcriptional elongation by RNA Polymerase II

Shrividhya Srinivasan¹, Jennifer A. Armstrong^1,*, Renate Deuring¹, Ina K. Dahlsveen^2,, Helen McNeill² and John W. Tamkun^1,

¹ Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA
² Cancer Research UK, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

View larger version (11K): [in a new window]   Fig. 1. KIS-L is related to chromatin-remodeling factors. The two major KIS isoforms (KIS-S and KIS-L) are compared with other Drosophila ATPases involved in chromatin remodeling, including BRM, Mi-2, CHD1 and ISWI. The ATPase domain of KIS-L is most closely related to that of BRM (44% identical) and CHD family members (50% identical). The C-terminal segment common to KIS-S and KIS-L (shaded light gray) contains a BRK domain, but lacks the bromodomain (BD) found in BRM and other SWI2/SNF2 family members. The N-terminal extension unique to KIS-L contains an ATPase domain and two chromodomains (CD), but lacks the PHD fingers (PHD) and putative DNA-binding domains found in other CHD proteins. KIS-L also lacks a SANT domain, a nucleosome-recognition module found in ISWI and other proteins. A and B indicate regions of KIS proteins against which antibodies were raised (residues 100-300 and 3902-4200, respectively).

View larger version (51K): [in a new window]   Fig. 2. kis encodes multiple large nuclear proteins that are subunits of distinct complexes. (A) Embryo and salivary gland extracts were resolved on a 5% SDS-polyacrylamide gel and analyzed by western blotting. Molecular masses were determined relative to prestained markers and cytoplasmic dynein (detected by western blotting). Antibodies against the common C-terminal segment detect both KIS-L and KIS-S in embryo extracts (lane 1) but only KIS-L in salivary gland extracts (lane 2). Antibodies against the N-terminal segment unique to KIS-L recognize only this protein in embryo extracts (lane 3). The asterisk marks minor bands that are occasionally observed and probably represent degradation products or minor isoforms of KIS. (B) Drosophila embryos were stained with antibodies that specifically recognize KIS-L. DNA was visualized using propidium iodide. There is uniform nuclear distribution of KIS-L. (C) Native embryo extracts were fractionated on a Superose 6 gel filtration column and assayed for chromatin-remodeling factors by western blotting. Fraction numbers are indicated at the top. Void and elution volumes of native molecular mass standards are shown by vertical arrows. Denatured molecular masses of the proteins are indicated on the right.

View larger version (47K): [in a new window]   Fig. 3. KIS-L binds a large number of sites on polytene chromosomes. (A) KIS-L (green) binds to 300 sites on wild-type polytene chromosomes and is not associated with the heterochromatic chromocenter (white arrowhead). (B) Higher magnification of the distal region of chromosome arm 2R. KIS-L (green) is compared with DNA (blue); the last two panels show the merged and split images of the first two panels. KIS-L is primarily associated with interband regions.

View larger version (77K): [in a new window]   Fig. 4. KIS-L is associated with regions of active transcription. (A-C) KIS-L (red) and Pol II (green) were detected on wild-type polytene chromosomes using antibodies against KIS-L and the 140 kDa subunit of Pol II (Pol IIc); the merged image is shown in C. (D-G) Magnifications of the distal regions of the X chromosome and chromosome arm 3L (white rectangle in C) are shown. The distributions of KIS-L and Pol II are highly overlapping, although the levels vary from site to site, as evident in the split image (G).

View larger version (63K): [in a new window]   Fig. 5. KIS-L co-localizes with both the paused and elongating forms of Pol II. The distributions of CHD1 and Pol IIoser2 (A); KIS-L and Pol IIoser2 (B); KIS-L and Pol IIa (C); and KIS-L and Pol IIoser5 (D) are shown. Magnifications of the distal chromosome arm 2R are shown in A-C. A magnification of the distal chromosome arm 2L is shown in D. The distribution of KIS-L is highly coincident with all forms of Pol II, whereas CHD1 is associated primarily with the elongating form of Pol II (Pol IIoser2).

View larger version (49K): [in a new window]   Fig. 6. KIS-L colocalizes with BRM and other chromatin-remodeling factors. Merged images of the distributions of KIS-L (red) and BRM (green; A), CHD1 (green; B) and Mi-2 (green; C) are shown. Extensive overlap between KIS-L and the other proteins are evident in the enlarged merged and split images of the regions bound by white rectangles. The distribution of KIS-L is identical to that of BRM and very similar to that of Mi-2. Although not as striking, there is also considerable overlap between the chromosomal distributions of KIS-L and CHD1.

View larger version (29K): [in a new window]   Fig. 7. KIS-L is not physically associated with other chromatin-remodeling factors or Pol II. Proteins were immunoprecipitated from embryo extracts using rabbit IgG (control) or antibodies against the N-terminal segment unique to KIS-L (-KIS-L). One-fifth of the total input extract (I) and supernatant (S), and four-fifths of the total pellet (P) were separated by SDS-PAGE and analyzed by western blotting.

View larger version (41K): [in a new window]   Fig. 8. The levels of KIS-L are dramatically reduced on polytene chromosomes of kis mutant larvae. KIS-L (red; C and D) was detected on chromosomes of wild type (A,C) and kisk13416 (B,D) larvae. DNA (blue; A,B) was visualized by DAPI staining. The level of KIS-L associated with chromatin is drastically reduced in kis mutants, but the overall banding pattern and morphology of polytene chromosomes is normal.

View larger version (33K): [in a new window]   Fig. 9. Loss of KIS-L does not affect the association of PC with chromatin. The distribution of KIS-L (red; A) is predominantly non-overlapping with PC (green; B) on wild-type chromosomes as seen in merged (C) and magnified (D,E) images corresponding to the region bounded by the white rectangle in C. The number and intensity of PC bands are similar on the chromosomes of wild-type (B) and kisk13416 (G) larvae.

View larger version (41K): [in a new window]   Fig. 10. KIS-L is required for the association of Pol IIoser2 with polytene chromosomes. The distribution of KIS-L (red; A,D,G,J), Pol IIa (green; B,E,H,K), Pol IIoser5 (blue; C,F) and Pol IIoser2 (blue; I.L) on chromosomes isolated from wild type (A-C,G-I) and kisk13416 mutant larvae (D-F,J-L) is shown. The levels of both Pol IIa and Pol IIoser5 are unaffected by the loss of KIS-L. By contrast, loss of KIS-L results in a dramatic reduction in the level of Pol IIoser2 associated with chromosomes.

View larger version (31K): [in a new window]   Fig. 11. KIS-L is required for the recruitment of the elongation factors SPT6 and CHD1, but not BRM and Mi-2, to chromatin. The distributions of SPT6 (A,E), BRM (B,F), Mi-2 (C,G) and CHD1 (D,H) on wild type (A-D) and kisk13416 (E-H) chromosomes are compared. Loss of KIS-L function does not affect binding of BRM and Mi-2 to chromosomes but the levels of both SPT6 and CHD1 are greatly reduced. Insets (E,H) show DAPI staining of chromosomes.

View larger version (22K): [in a new window]   Fig. 12. Chromatin-remodeling factors facilitate distinct stages of transcription. The stage at which each chromatin-remodeling factor is hypothesized to function is marked by an unbroken line. BRM physically interacts with transcriptional activators and facilitates a step prior to the recruitment of Pol II to promoters. CHD1 has been implicated in the later stages of transcriptional elongation. Our findings suggest that KIS-L is required for the transition from the early to late stages of elongation. The activities of KIS-L and CHD1 may be influenced by the methylation of histone H3 lysine 4 and lysine 36 near the 5' end and in the body of transcribed genes, respectively.