Beta-globin intergenic transcription and histone acetylation dependent on an enhancer - PubMed (original) (raw)

Beta-globin intergenic transcription and histone acetylation dependent on an enhancer

Aeri Kim et al. Mol Cell Biol. 2007 Apr.

Abstract

Histone acetyltransferases are associated with the elongating RNA polymerase II (Pol II) complex, supporting the idea that histone acetylation and transcription are intertwined mechanistically in gene coding sequences. Here, we studied the establishment and function of histone acetylation and transcription in noncoding sequences by using a model locus linking the beta-globin HS2 enhancer and the embryonic epsilon-globin gene in chromatin. An intact HS2 enhancer that recruits RNA Pol II is required for intergenic transcription and histone H3 acetylation and K4 methylation between the enhancer and target gene. RNA Pol II recruitment to the target gene TATA box is not required for the intergenic transcription or intergenic histone modifications, strongly implying that they are properties conferred by the enhancer. However, Pol II recruitment at HS2, intergenic transcription, and intergenic histone modification are not sufficient for transcription or modification of the target gene: these changes require initiation at the TATA box of the gene. The results suggest that intergenic and genic transcription complexes are independent and possibly differ from one another.

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Figures

FIG. 1.

FIG. 1.

Structure of the human β-globin locus and minichromosomal locus containing the ɛ-globin gene and LCR HS2. The human β-globin locus is diagrammed across the top of the figure. A domain of histone modification mapped over the LCR and ɛ- and γ-globin genes in K562 cells is indicated by the colored rectangle. LCR HS2 (1.46 kb) was fused to the ɛ-globin gene (3.7 kb) in the minichromosome locus (see Materials and Methods). Two mutant loci were created in which either the HS2 NF-E2 site or the ɛ-globin promoter TATA box was destroyed by clustered point mutations. The positions of TaqMan probes used for real-time PCR are indicted at the bottom.

FIG. 2.

FIG. 2.

RNA FISH indicates active transcription on the multiple copies of minichromosomes in K562 cells. RNA FISH was carried out as described in Materials and Methods with K562 cells and a K562 clone carrying HS2ɛ minichromosomes. The ɛ-globin transcripts were labeled with FITC (green). The slides were then fixed, and DNA FISH was carried out with a chromosome 11-specific centromeric probe labeled with Spectrum Orange. One hundred random nuclei from each cell line on each of three slides were counted to determine the number of transcribing endogenous and minichromosomal templates. (A) DAPI-stained K562 nucleus containing three copies of chromosome 11 (red). Two ɛ-globin transcriptional foci are observed at some distance from each centromere, as expected (green). (B) A nucleus from a cell clone with HS2ɛ minichromosomes has many additional foci (8.5 ± 0.9) of ɛ-globin transcription, in good agreement with the Southern blot assessment of the number of minichromosomal templates (not shown).

FIG. 3.

FIG. 3.

Intergenic transcription and Pol II association are dependent on NF-E2 binding in HS2. (A) cDNA was prepared by reverse transcription using 1 μg of RNA isolated from K562 cells containing minichromosomes and then amplified by real-time PCR using the indicated probes. The cDNA signal was compared with that of genomic DNA purified from K562 cells containing minichromosomes and was corrected by the amount of actin cDNA compared with genomic DNA. No signal was produced without RT. The results of three independent experiments ± standard errors of the means are graphed. (B) Chromatin was prepared from 0.4% formaldehyde-cross-linked K562 cells carrying wild-type minichromosomes. Chromatin was sonicated to 100- to 500-bp fragments, and immunoprecipitation was performed with antibodies to Pol II. ChIP DNA was amplified by real-time PCR using the indicated probes. Control samples were incubated without antibody (ab). The relative intensity was determined as described in Materials and Methods. The results of three independent experiments ± standard errors of the means are graphed. (C) Chromatin was prepared from K562 cells carrying NF-E2 mutant minichromosomes, and ChIP was carried out as described for panel B.

FIG. 4.

FIG. 4.

TATA box mutation does not alter intergenic transcription or Pol II recruitment at HS2. (A) RNase protection analysis was performed with RNA isolated from K562 cells containing minichromosomes with a mutated ɛ-globin TATA box. Two RNA preparations for each of three ΔTATA clones are shown. The bands protected by endogenous (end) and minichromosomal ɛ-globin (mini) RNA are indicated by arrows. Controls include K562 cell RNA and RNA from K562 cells carrying wild-type minichromosomes. Actin served as the loading control, and the results were corrected for minichromosome copy numbers. The results for multiple clones ± standard errors of the means are depicted graphically. (B) ChIP was carried out as described in the legend to Fig. 3B. The relative intensity of detection at each position was determined as described in Materials and Methods. The results of three independent experiments ± standard errors of the means are presented. (C) cDNA was prepared by reverse transcription using 1 μg of RNA isolated from K562 cells containing minichromosomes and then amplified by real-time PCR using the indicated probes. No signal was produced without RT. The results of three independent experiments ± standard errors of the means are graphed.

FIG. 5.

FIG. 5.

The TATA box mutation affects coding sequence histone modifications. Mono- and dinucleosomes were prepared from non-cross-linked chromatin by MNase digestion (see Materials and Methods) and reacted with antibodies to diacetylated H3 K9 and K14. Input and immunoprecipitated DNAs were analyzed by real-time PCR using the indicated probes. The enrichment was determined by comparing the amount of target sequence in immunoprecipitated DNA to the amount of target sequence in input DNA. Three independent experiments were carried out, and the average results ± standard errors of the means are shown. (B) ChIP was carried out using 1% formaldehyde cross-linking (see Materials and Methods) before immunoprecipitation with antibodies to dimethylated H3 K4. The relative intensity of detection at each position was determined as described in Materials and Methods. The results of three independent experiments ± standard errors of the means are presented.

FIG. 6.

FIG. 6.

NF-E2 mutation inhibits recruitment of CBP and p300 to HS2. Chromatin was prepared from 1% formaldehyde-cross-linked K562 cells carrying minichromosomes and fragmented by MNase digestion and sonication (see Materials and Methods). Chromatin was subjected to immunoprecipitation using antibodies to CBP or p300 followed by amplification using real-time PCR with the indicated probes. The difference for each primer pair was determined by comparing antibody-precipitated DNA with input DNA. The results for three chromatin preparations ± standard errors of the means are presented.

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