Fine Tuning of Globin Gene Expression by DNA Methylation

Expression patterns in the globin gene cluster are subject to developmental regulation in vivo. While the γ(A) and γ(G) genes are expressed in fetal liver, both are silenced in adult erythrocytes. In order to decipher the role of DNA methylation in this process, we generated a YAC transgenic mouse system that allowed us to control γ(A) methylation during development. DNA methylation causes a 20-fold repression of γ(A) both in non-erythroid and adult erythroid cells. In erythroid cells this modification works as a dominant mechanism to repress γ gene expression, probably through changes in histone acetylation that prevent the binding of erythroid transcription factors to the promoter. These studies demonstrate that DNA methylation serves as an elegant in vivo fine-tuning device for selecting appropriate genes in the globin locus. In addition, our findings provide a mechanism for understanding the high levels of γ-globin transcription seen in patients with Hereditary Persistence of Fetal Hemoglobin, and help explain why 5azaC and butyrate compounds stimulate γ-globin expression in patients with β-hemoglobinopathies

Coordinated allele-specific histone acetylation at the differentially methylated regions of imprinted genes

Genomic imprinting is an epigenetic inheritance system characterized by parental allele-specific gene expression. Allele-specific DNA methylation and chromatin composition are two epigenetic modification systems that control imprinted gene expression. To get a general assessment of histone lysine acetylation at imprinted genes we measured allele-specific acetylation of a wide range of lysine residues, H3K4, H3K18, H3K27, H3K36, H3K79, H3K64, H4K5, H4K8, H4K12, H2AK5, H2BK12, H2BK16 and H2BK46 at 11 differentially methylated regions (DMRs) in reciprocal mouse crosses using multiplex chromatin immunoprecipitation SNuPE assays. Histone acetylation marks generally distinguished the methylation-free alleles from methylated alleles at DMRs in mouse embryo fibroblasts and embryos. Acetylated lysines that are typically found at transcription start sites exhibited stronger allelic bias than acetylated histone residues in general. Maternally methylated DMRs, that usually overlap with promoters exhibited higher levels of acetylation and a 10% stronger allele-specific bias than paternally methylated DMRs that reside in intergenic regions. Along the H19/Igf2 imprinted domain, allele-specific acetylation at each lysine residue depended on functional CTCF binding sites in the imprinting control region. Our results suggest that many different histone acetyltransferase and histone deacetylase enzymes must act in concert in setting up and maintaining reciprocal parental allelic histone acetylation at DMRs

More than Insulator: Multiple Roles of CTCF at the H19-Igf2 Imprinted Domain

CTCF-mediated insulation at the H19-Igf2 imprinted domain is a classic example for imprinted gene regulation. DNA methylation difference in the imprinting control region (ICR) is inherited from the gametes and subsequently determines parental allele-specific enhancer blocking and imprinted expression in the soma. Recent genetic studies showed that proper monoallelic enhancer blocking at the H19-Igf2 ICR is critical for development. Strict biallelic insulation at this locus causes perinatal lethality, whereas leaky biallelic insulation results in smaller size but no lethality. Apart from enhancer blocking, CTCF is also the master organizer of chromatin composition in the maternal allele along this imprinted domain, affecting not only histone tail covalent modifications but also those in the histone core. Additionally, CTCF binding in the soma protects the maternal allele from de novo DNA methylation. CTCF binding is not involved in the establishment of the gametic marks at the ICR, but it slightly delays de novo methylation in the maternally inherited ICR allele in prospermatogonia. This review focuses on the developmental and epigenetic consequences of CTCF binding at the H19-Igf2 ICR

Effect of DNA methylation on gene expression

<div><p>A. RNA from purified adult or fetal liver erythroblasts and MEFs from Cre (γ^A promoter methylated) or Mx-cre (γ^A promoter unmethylated) crossed transgenic (lines 47, 64 or 113)) mice were subjected to semi-quantitative RT-PCR on 1 or 3 µl samples to detect human β (HBB) or γ(HBG)-globin RNA. </p> <p>We measured Aprt expression to control for the amount of cDNA in the reaction.</p> <p>Genes expressed at high levels were quantitated by diluting the input sample (e.g. 1/10⁵ for Aprt in MEFs).</p> <p>In order to distinguish between the two γ genes, we took advantage of a PstI restriction site that is present in γ^A and not γ^G<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Tanimoto2" target="_blank">[51]</a>.</p> <p>A smaller induction of γ^A was observed in line 66 which is only partially unmethylated at the promoter (data not shown).</p> <p>The degree of IE excision in purified erythroblasts from induced Mx-cre carrying mice was found to be >90% by PCR analysis.</p> <p>The data for fetal erythroblasts was taken from an animal with a “methylated” γ^A gene, but we have demonstrated that it is indeed unmethylated in fetal liver (data not shown), as expected.</p> <p>B. Quantitative analysis of RNA. Levels of globin expression were obtained from dilution analysis and compared to Aprt (set to 1) in the same cells. </p> <p>Each experiment was repeated 2–3 times (coefficient of variance = 14%).</p> <p>γ/γ+β was calculated on the basis of total γ globin (γ^A+γ^G).</p> <p>Results (average of 3 experiments) for real time PCR are included for some samples.</p> <p>The degree of γ globin induction is shown for adult erythroblast cells.</p></div

Programmed methylation of the human γ^A-globin promoter

<div><p> <b>A.</b> Two IEs (yellow) bounded by loxP elements (stippled black) were inserted into the γ^A promoter region of a YAC containing the human β-globin locus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Tanimoto1" target="_blank">[18]</a>, and this was used to generate transgenic mice that were then crossed with two different cre-expressing lines. </p> <p>In the first line (Cre) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Lallemand1" target="_blank">[19]</a>, cre is expressed (red) prior to implantation.</p> <p>In mice carrying this construct the IE is deleted before the wave of de novo methylation, and surrounding CpG sites thus become methylated (red circles).</p> <p>The second cre-expressing line carries interferon-inducible cre <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Kuhn1" target="_blank">[20]</a>.</p> <p>In these mice (Mx-cre), the IE remains present during implantation and protects adjacent regions from methylation.</p> <p>By treating adult animals with polyI-polyC, cre activity could be induced (red) and the IE removed, generating an unmethylated version of the transgene.</p> <p> <b>B.</b> Tail DNA from four transgenic founders (lines 47, 64, 66 and 113) was cut with AvrII with or without the methylation sensitive restriction enzyme BsrFI, and subjected to Southern blotting using a radioactive probe (green arrow). </p> <p>Line 47 was crossed with cre mice and offspring analyzed in the same way.</p> <p> <b>C.</b> The region upstream of the γ^A and γ^G coding sequences (blue triangles) was analyzed for CpG methylation using the bisulfite technique.</p> <p>To this end we generated MEFs from line 64 (IE present at time of implantation) and infected them with an adenovirus that expresses Cre activity.</p> <p>Alternatively, we derived MEFs from line 64 mice crossed with Cre (IE removed prior to implantation).</p> <p>Five CpG residues were analyzed for methylation in the γ^G promoter.</p> <p>The normal γ^A promoter also carries 5 CpGs, but one is replaced by the inserted loxP element.</p> <p>Following bisulfite treatment, PCR products were cloned and subjected to sequencing.</p> <p>Each row represents a single molecule.</p> <p>Black circles indicate a methylated CpG, whereas open circles indicate a lack of methylation.</p> <p>The numbers in the margin indicate how many clones were fully methylated.</p></div

γ promoter methylation pattern in patients with HPFH

<div><p>The γ^A and γ^G promoter regions were analyzed for CpG methylation on individual clones by the bisulfite technique.</p> <p>To this end we used Fibach culture erythroblasts derived from normal individuals, as well as heterozygote HPFH1 or β⁰ Thalassemia (IVS-II-I, G⇑A) patients.</p> <p>Black circles indicate methylated CpGs, whereas open circles indicate a lack of methylation.</p> <p>The numbers in the margin indicate how many clones were fully methylated.</p></div

Effect of DNA methylation on promoter structure

<div><p>Mononucleosomes were prepared from either non-erythroid (<b>A</b>) or erythroid (<b>B</b>) cells taken from Cre (methylated) and Mx-cre (unmethylated) founder (64) transgene crosses and subjected to ChIP analysis using anti-Ac-H4.</p> <p>Input (I) and bound (B) DNA fractions were used for semi-quantitative PCR on 1 or 3 µl samples using primer sets specific for the γ^G or loxP-inserted γ^A promoter regions.</p> <p>For each ChIP preparation, the Acta1 gene (green) was assayed as a negative control and the Actb gene (yellow) as a positive control.</p> <p>The results are summarized in graphic form after normalizing to Acta1 (green) enrichment (set at 1.0) (coefficient of variance = 17%).</p> <p>The results for the Cre (blue) and Mx-cre (red) mice are shown.</p> <p>Results for β globin are presented for comparison.</p> <p>The data shown for non-erythroid cells was obtained using mononucleosomes from MEFs, but the graph summarizes results from 3–4 ChIP experiments on both MEFs and lymphocytes.</p> <p>(<b>C</b>) In vivo footprinting of γ promoter.</p> <p>Erythroblasts (in vivo) or purified erythroblast DNA (in vitro) from mice carrying either a methylated or unmethylated γ^A promoter were treated with DMS. LMPCR gel.</p> <p>Maxam-Gilbert lanes (AG and CT) are sequencing controls.</p> <p>The numbers are according to PubMed accession number NG_000007.</p> <p>Putative protein factor binding regions (rectangles) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Ikuta1" target="_blank">[26]</a>, DMS footprints, as in vivo protected (open circle) or hyper-reactive (closed circle) nucleotides, are indicated.</p> <p>The sizes of the circles represent relative differences in footprint intensities.</p> <p>DMS footprinting on spleen lymphocytes did not show any hyperreactive sites on unmethylated DNA, but did reveal some slight reactivity over the distal CCAAT box on methylated DNA.</p></div