Gal4-VP16 directs ATP-independent chromatin reorganization in a yeast chromatin assembly system.
Major insights into the regulation of chromatin organization have stemmed from biochemical studies using Gal4-VP16, a chimeric transcriptional activator in which the DNA binding domain of Gal4p is fused to the activation domain of viral protein VP16. Unexpectedly, given previous intensive efforts to understand how Gal4-VP16 functions in the context of chromatin, we have uncovered a new mode of chromatin reorganization that is dependent on Gal4-VP16. This reorganization is performed by an activity in a crude DEAE (CD) fraction from budding yeast which also supports ATP-dependent assembly of physiologically spaced nucleosome arrays. Biochemical analysis reveals that the activity tightly associates with chromatin and reorganizes nucleosome arrays by a mechanism which is insensitive to ATP depletion after nucleosome assembly. It generates a chromatin organization in which a nucleosome is stably positioned immediately adjacent to Gal4p binding sites in the template DNA. Individual deletion of genes previously implicated in chromatin assembly and remodeling, namely, the histone chaperones NAP1, ASF1, and CAC1 and the SNF2-like DEAD/H ATPases SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20, does not significantly perturb reorganization. Therefore, Gal4-VP16-directed chromatin reorganization in yeast can occur by an ATP-independent mechanism that does not require SAGA, SWI/SNF, Isw1, or Isw2 chromatin remodeling complexes.
Robinson
Karen M
KM
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Schultz
Michael C
MC
D000255:Adenosine Triphosphate / Q000502:physiology*; D002843:Chromatin / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004983:Ethanolamines / Q000737:chemistry; D017353:Gene Deletion; D018390:Gene Targeting; D017405:Herpes Simplex Virus Protein Vmw65 / Q000737:chemistry* / Q000502:physiology; D009707:Nucleosomes / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology; D015534:Trans-Activators / Q000737:chemistry* / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry* / Q000502:physiology
D002843:Chromatin; D004268:DNA-Binding Proteins; D004983:Ethanolamines; C052130:GAL4 protein, S cerevisiae; C070633:Gal-VP16; D017405:Herpes Simplex Virus Protein Vmw65; D009707:Nucleosomes; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific; C007369:2-diethylaminoethanol