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Transvection-like interchromosomal interaction is not observed at the transcriptional level when tested in the Rosa26 locus in mouse - PubMed

  • ️Tue Jan 01 2019

Transvection-like interchromosomal interaction is not observed at the transcriptional level when tested in the Rosa26 locus in mouse

Keiji Tanimoto et al. PLoS One. 2019.

Abstract

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a comprehensive view of intra- and interchromosomal contacts between specific genomic sites. The locus control region of the β-globin genes (β-LCR) is a super-enhancer that is capable of activating all of the β-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3'HS1 in the human β-globin locus, in this way thought to contribute to the formation of a "chromatin hub". The β-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared "transcription factory" for co-transcription. Theoretically, enhancers could also activate target gene promoters at the identical loci, yet on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the β-LCR and the β-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because of a lack of suitable regulatory elements that might facilitate such trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and β-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the β-globin gene in trans even in this idealized experimental context.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Generation of enhancer and promoter knock-in alleles in mice.

(A) Structure of the human β-globin gene locus shown in 1D (left) and 3D (right) views. (B) The enhancer targeting vector carrying the human β-globin LCR and β-globin gene that is marked by an ε-globin sequence, wild-type ROSA26 locus, and the correctly targeted enhancer knock-in locus are shown. In the targeting vector, neomycin resistance (Neor) and diphtheria toxin (DT)-A genes are shown as striped and solid boxes, respectively. The solid triangles indicate the loxP sequences. Probes used for Southern blot analyses in (D) are shown as filled rectangles. Expected restriction fragments with their sizes are shown beneath the partial restriction enzyme maps. (C) The promoter targeting vector carrying the human β-globin gene (marked by an γ-globin sequence) and 3’HS1, wild-type ROSA26 locus, and the correctly targeted promoter knock-in locus are shown. Probes used for Southern blot analyses in (E) are shown as filled rectangles. (D and E) Genomic DNA from ES clones was digested with restriction enzymes, separated on agarose gels, and Southern blots were hybridized to the probes. Asterisks denote nonspecific bands.

Fig 2
Fig 2. Derivation of enhancer/promoter-allele variants by in vivo Cre-loxP recombination.

(A) Enhancer knock-in mouse bearing the LCR+β(ε)+Neor locus was mated with Cre-TgM to induce in utero, partial cre-loxP recombination, which resulted in selective excision of either Neor or Neor+β(ε)-globin sequences to generate LCR+β(ε) or LCR alleles, respectively. A, AseI; B, BamHI; 36, Bsu36I. (B) Similarly, β(γ)+3’HS1 or β(γ) alleles were derived from the promoter knock-in mouse bearing the β(γ)+3'HS1+Neor locus by deletion of the Neor or Neor+3’HS1 sequences, respectively. (C) Successful cre-loxP recombination was confirmed by Southern blot analysis. Tail genomic DNA of mutant mice was digested with AseI (enhancer knock-in series) or Bsu36I (promoter knock-in series), separated on agarose gels, and Southern blots were hybridized to the ROSA-3’-383 probe shown in panels A and B. (D) Each allele was discriminated by multiplex PCR analyses of tail genomic DNA from mutant mice. The LCR, ε-βand Neor sequences in the enhancer knock-in alleles were amplified by PCR primers shown by paired open arrowheads in panel A. The γ-β, 3’HS1 and Neor sequences in the promoter knock-in alleles were amplified by PCR primers shown by paired open arrowheads in panel B.

Fig 3
Fig 3. Expression of human β-globin genes in knock-in mice.

(A) Total RNA was prepared from spleens of 1-month-old anemic mice (N = 4 for each genotype). Expression levels of the human β(γ)- or β(ε)-globin genes (analyzed by common primer set targeted at β-globin sequence; BT-4S1 and BT-4A1) and endogenous mouse α (mα)-globin gene were analyzed by qRT-PCR. The ratio of hβ/mα-globin genes was calculated (the expression value of the β(γ)-globin was set at 1). P values (vs β(γ): *<0.05; **<0.01. (B) Total RNA was prepared from spleens, livers and kidneys of 1-month-old anemic mice (N = 4 for each tissues). Expression of mα-globin, β(γ)-globin and endogenous mouse (m)GAPDH genes was analyzed by qRT-PCR. The expression levels of mα- (left panel) or β(γ)-globin (right) genes, both compared to that of mGAPDH gene were calculated (the expression values in spleen samples were set at 100). (C and D) ChIP was conducted for CTCF in the spleen cells of anemic animals bearing both LCR+β(ε) and β(γ)+3’HS1 alleles. The Necdin gene and the H19 ICR sequences were analyzed as negative and positive controls, respectively. Quantitative PCR was repeated at least three times for each sample. Fold enrichment of CTCF relative to IgG control (average values with S.D.) was calculated and graphically depicted (average value of negative controls was set at 1.0). P values (vs Necdin): *<0.05; **<0.01.

Fig 4
Fig 4. Expression of β(γ)-globin genes in knock-in mice.

(A) Schematic representation of four different combinations of enhancer and promoter knock-in alleles to test for enhancer-promoter interaction in trans. (B) Mouse genotypes shown in (A) were confirmed by multiplex PCR analyses of tail genomic DNA of mutant mice. The ε-β, LCR, γ-β and 3’HS1 sequences were amplified by PCR primers shown by paired open arrowheads in panel A. (C-F) Total RNA was prepared from spleens of 1-month-old anemic mice. Numbers analyzed for each genotype are shown in the S1 Fig. Expression of β(γ)-globin and endogenous mα-globin genes was analyzed by qRT-PCR. The ratio of hβ(γ)-globin / mα-globin genes was calculated and average value with S.D. was graphically depicted for each genotype group (Although values are arbitrary, they can be quantitatively compared between the panels).

Fig 5
Fig 5. Expression of endogenous genes in knock-in mice.

(A and B) Schematic representation of combinations of enhancer/promoter knock-in alleles to test for intra- and interchromosomal enhancer-promoter interactions in the adult spleen (A) or fetal liver (B) cells. (C-J) The adult spleen was analyzed by qRT-PCR. In addition to the β(γ) promoter alleles, animals used in the panels C-F and G-J carried the LCR or the LCR+β(ε) enhancer alleles (shown in A), respectively. (K, L) The fetal liver of the animals bearing the LCR+β(ε) enhancer allele (shown in B) was analyzed by qRT-PCR. The ratios of Thumpd3 and Setd5 genes expression to that of the GAPDH gene (C, D, G and H) or those of β-major- and βh1-globin genes expression to that of the α-globin gene (E, F, I, J, K and L) were calculated and average value with S.D. was graphically depicted. Although values are arbitrary, they can be quantitatively compared between the panels. Sample numbers analyzed in the panels C-F, G-J and K-L are 28, 13 and 16, respectively, in each group.

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This work was supported in part by research grants from JSPS (Japan Society for the Promotion of Science, https://www.jsps.go.jp/j-grantsinaid/) KAKENHI Grant Number 26292189 [Grant-in-Aid for Scientific Research (B) to K.T.]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.