Dissecting molecular steps in chromatin domain activation during hematopoietic differentiation.
GATA factors orchestrate hematopoiesis via multistep transcriptional mechanisms, but the interrelationships and importance of individual steps are poorly understood. Using complementation analysis with GATA-1-null cells and mice containing a hypomorphic allele of the chromatin remodeler BRG1, we dissected the pathway from GATA-1 binding to cofactor recruitment, chromatin loop formation, and transcriptional activation. Analysis of GATA-1-mediated activation of the beta-globin locus, in which GATA-1 assembles dispersed complexes at the promoters and the distal locus control region (LCR), revealed molecular intermediates, including GATA-1-independent and GATA-1-containing LCR subcomplexes, both defective in promoting loop formation. An additional intermediate consisted of an apparently normal LCR complex and a promoter complex with reduced levels of total RNA polymerase II (Pol II) and Pol II phosphorylated at serine 5 of the carboxy-terminal domain. Reduced BRG1 activity solely compromised Pol II and serine 5-phosphorylated Pol II occupancy at the promoter, phenocopying the LCR-deleted mouse. These studies defined a hierarchical order of GATA-1-triggered events at a complex locus and establish a novel mechanism of long-range gene regulation.
Kim
Shin-Il
SI
University of Wisconsin School of Medicine, Department of Pharmacology, 383 Medical Sciences Center, Madison, WI 53706, USA.
Bultman
Scott J
SJ
Jing
Huie
H
Blobel
Gerd A
GA
Bresnick
Emery H
EH
D000483:Alleles; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D041905:Erythroid Cells / Q000166:cytology / Q000378:metabolism; D050982:GATA1 Transcription Factor / Q000235:genetics / Q000502:physiology*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics; D006413:Hematopoietic System / Q000502:physiology*; D019901:Locus Control Region; D051379:Mice; D018345:Mice, Knockout; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D002843:Chromatin; D050982:GATA1 Transcription Factor; C494689:Gata1 protein, mouse; D009687:Nuclear Proteins; D014157:Transcription Factors; D005914:Globins; D012319:RNA Polymerase II; C513194:Smarca4 protein, mouse; D004265:DNA Helicases