Anat Cell Biol.  2010 Mar;43(1):78-85. 10.5115/acb.2010.43.1.78.

Chromatin organization and transcriptional activation of Hox genes

• Lee JY
• Min H
• Wang X
• Khan AA
• Kim MH

• KMID: 2168900
• DOI: http://doi.org/10.5115/acb.2010.43.1.78

Abstract

Spatially and temporally programmed expression of the Hox genes along the antero-posterior (A-P) axis is essential for correct pattern formation during embryonic development. An accumulating body of evidence indicates the pivotal role of spatial chromatin organization for the coordination of gene regulation. Recently, chromosome conformation capture (3C) technique has been developed and opened a new way to study chromosomal interactions in the nucleus. In this study, we describe 3C method we applied in F9 embryonic teratocarcinoma cells and demonstrate that the chromosomal interactions at Hox loci are successfully detected. Interestingly, at Hoxc loci, the abundance of intrachromosomal interactions with neighboring fragments was drastically decreased when the genes are expressed. These results indicate the possibility of the dynamic pattern of chromosomal interaction in association with the transcriptional regulation of Hox genes.

Keyword

Hox genes; chromosome conformation capture; spatial organization; chromosomal interaction; gene expression

MeSH Terms

Axis, Cervical Vertebra
Chromatin
Embryonic Development
Female
Gene Expression
Genes, Homeobox
Pregnancy
Teratocarcinoma
Transcriptional Activation
Chromatin

Figure

• 

  Fig. 1 Expression pattern of Hoxa and Hoxc cluster genes following RA treatment. (A) Hoxa and Hoxc gene organization in each cluster. (B) F9 cells were cultured with RA for 4 days. Total RNAs were isolated each day from Day 1 to Day 4 (D0: control cells with no RA treatment), and then RT-PCR was performed with each Hox primers. β-actin was used as a positive control for amplifiable cDNA. Data are representative of one of three replicate experiments. (C) The relative expression level of each Hox gene was determined by using Multi Gauge software. The amount of each RT-PCR product was normalized with that of β-actin to obtain relative intensities. Each point represents the average of three separate experiments. Error bars represent SEM.

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  Fig. 2 PCR analysis of restriction digestion and ligation efficiency used for the evaluation of 3C template (A) To determine digestion efficiency, primer pairs D1/D2 and D3/D4 were used. When the HindIII restriction digestion was completed, D1/D2 primer pair gives a faint PCR band whereas D3/D4 primers generate a band whether DNA templates are undigested (-) and digested (+) with HindIII. (B) To determine the efficiency of self ligation, the primer L1/L2 was used. DNA was prepared before (-) and after (+) the ligation step. The 3C templates from Day 3 of RA treatment were also tested with same ways and obtained similar results (Data not shown).

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  Fig. 3 Schematic presentation of the Hoxc (A) and Hoxa (B) loci showing the primer as well as HindIII regcognition sites. The UCSC Genes track includes both protein-coding and putative non-coding transcripts. The black bar depicts the HindIII recognition sites. The position of each primer we used for 3C analysis is indicated as arrowhead along with a serial number (cP1-cP22 and aP1-aP24 in Hoxc and Hoxa clusters, respectively). Each Hoxc and Hoxa region we analyzed covers 130 kb in mouse chromosome 15 and 6, respectively.

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  Fig. 4 Identification of intrachromosomal interaction of RA-untreated (D0) samples at Hoxc loci. 3C-PCR was performed with the primers indicated. The primer cP12 was used as an anchor. 3C DNA templates of 250 ng were used per each PCR. This experiment was performed three times (D0-#1, D0-#2, D0-#3) for validation of this method.

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  Fig. 5 Identification of intrachromosomal interaction of RA-treated (D3) samples at Hoxc loci. 3C-PCR was performed with the primers indicated. The primer cP12 was used as an anchor. 3C DNA templates of 250 ng were used per each PCR. This experiment was performed three times (D3-#1, D3-#2, D3-#3) for validation. Compare to those of Fig. 4, many PCR bands were disappeared indicating the disappearance of intrachromosomal interaction.

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  Fig. 6 Identification of intrachromosomal interaction of RA-treated (D3) samples at Hoxa loci. 3C-PCR was performed with the primers indicated. The primer aP16 was used as an anchor. 3C DNA templates of 250 ng were used per each PCR. This experiment was performed three times (D3-#1, D3-#2, D3-#3) for validation.

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