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Histone deacetylase activity is necessary for left-right patterning during vertebrate development.

Carneiro K, Donnet C, Rejtar T, Karger BL, Barisone GA, Díaz E, Kortagere S, Lemire JM, Levin M - BMC Dev. Biol. (2011)

Bottom Line: To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery.The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator.The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology Center for Regenerative and Developmental Biology Tufts University, Medford, MA 02155 USA.

ABSTRACT

Background: Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFβ superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known.

Results: To understand how an early physiological gradient is transduced into a late, stable pattern of Nr1 expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked Nr1 expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of Nr1 expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the Nr1 gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of Nr1 transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3's biological activity is dependent on 5HT binding.

Conclusion: HDAC activity is a new LR determinant controlling the epigenetic state of Nr1 from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

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HDAC inhibition leads to increased levels of acetylated histones and H3K4me2 on the XNr-1 gene. (A) Schematic showing the structure of the Xenopus Nr-1 gene. Light gray boxes represent the protein-coding and the dark gray box represents the promoter region (adapted from [53]). Intronic regions 1 and 2 are indicated. Red and blue arrows represent the primer set used for qPCR reaction for the promoter and intronic region, respectively. (A1) The red lines indicate the sequence of the XNr-1 promoter region used to design the primer set for qPCR reaction. (A2) The regions underlined show the sequence used for primer set design. In purple are highlighted the FAST binding domains as in [53] and the black CATTTG indicates two putative Mad binding sites. (B) Chromatin isolated from embryos exposed to NaB from stage 1-7 and allowed to develop until stage 21 in 0.1X MMR was used for ChIP with anti-acetyl H4, anti-acetyl H3, anti-H3K4me2 and rabbit IgG (Control) followed by qPCR analysis against the promoter region (gray bar) and intronic region (black bar) of XNr-1. The levels of acetylated H4 and H3 and H3K4me2 were increased only on the intronic region (black bars).
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Figure 3: HDAC inhibition leads to increased levels of acetylated histones and H3K4me2 on the XNr-1 gene. (A) Schematic showing the structure of the Xenopus Nr-1 gene. Light gray boxes represent the protein-coding and the dark gray box represents the promoter region (adapted from [53]). Intronic regions 1 and 2 are indicated. Red and blue arrows represent the primer set used for qPCR reaction for the promoter and intronic region, respectively. (A1) The red lines indicate the sequence of the XNr-1 promoter region used to design the primer set for qPCR reaction. (A2) The regions underlined show the sequence used for primer set design. In purple are highlighted the FAST binding domains as in [53] and the black CATTTG indicates two putative Mad binding sites. (B) Chromatin isolated from embryos exposed to NaB from stage 1-7 and allowed to develop until stage 21 in 0.1X MMR was used for ChIP with anti-acetyl H4, anti-acetyl H3, anti-H3K4me2 and rabbit IgG (Control) followed by qPCR analysis against the promoter region (gray bar) and intronic region (black bar) of XNr-1. The levels of acetylated H4 and H3 and H3K4me2 were increased only on the intronic region (black bars).

Mentions: Our data indicate that very early epigenetic modifications control expression of Xnr-1 at a much later developmental stage. Thus, we tested the prediction that this epigenetic modification was made directly on the Xnr-1 gene. The Xnr-1 genomic region is composed of 2 main regulatory elements (RE). The RE on the promoter region (PRE) is located 230 bp from the start codon [52]. The second RE is the ASE (asymmetric element) in the intron 1 of the Xnr-1 gene. It is known to drive the asymmetric expression of Xnr-1 in the Lateral Plate Mesoderm (LPM) [53] (Figure 3A).


Histone deacetylase activity is necessary for left-right patterning during vertebrate development.

Carneiro K, Donnet C, Rejtar T, Karger BL, Barisone GA, Díaz E, Kortagere S, Lemire JM, Levin M - BMC Dev. Biol. (2011)

HDAC inhibition leads to increased levels of acetylated histones and H3K4me2 on the XNr-1 gene. (A) Schematic showing the structure of the Xenopus Nr-1 gene. Light gray boxes represent the protein-coding and the dark gray box represents the promoter region (adapted from [53]). Intronic regions 1 and 2 are indicated. Red and blue arrows represent the primer set used for qPCR reaction for the promoter and intronic region, respectively. (A1) The red lines indicate the sequence of the XNr-1 promoter region used to design the primer set for qPCR reaction. (A2) The regions underlined show the sequence used for primer set design. In purple are highlighted the FAST binding domains as in [53] and the black CATTTG indicates two putative Mad binding sites. (B) Chromatin isolated from embryos exposed to NaB from stage 1-7 and allowed to develop until stage 21 in 0.1X MMR was used for ChIP with anti-acetyl H4, anti-acetyl H3, anti-H3K4me2 and rabbit IgG (Control) followed by qPCR analysis against the promoter region (gray bar) and intronic region (black bar) of XNr-1. The levels of acetylated H4 and H3 and H3K4me2 were increased only on the intronic region (black bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113753&req=5

Figure 3: HDAC inhibition leads to increased levels of acetylated histones and H3K4me2 on the XNr-1 gene. (A) Schematic showing the structure of the Xenopus Nr-1 gene. Light gray boxes represent the protein-coding and the dark gray box represents the promoter region (adapted from [53]). Intronic regions 1 and 2 are indicated. Red and blue arrows represent the primer set used for qPCR reaction for the promoter and intronic region, respectively. (A1) The red lines indicate the sequence of the XNr-1 promoter region used to design the primer set for qPCR reaction. (A2) The regions underlined show the sequence used for primer set design. In purple are highlighted the FAST binding domains as in [53] and the black CATTTG indicates two putative Mad binding sites. (B) Chromatin isolated from embryos exposed to NaB from stage 1-7 and allowed to develop until stage 21 in 0.1X MMR was used for ChIP with anti-acetyl H4, anti-acetyl H3, anti-H3K4me2 and rabbit IgG (Control) followed by qPCR analysis against the promoter region (gray bar) and intronic region (black bar) of XNr-1. The levels of acetylated H4 and H3 and H3K4me2 were increased only on the intronic region (black bars).
Mentions: Our data indicate that very early epigenetic modifications control expression of Xnr-1 at a much later developmental stage. Thus, we tested the prediction that this epigenetic modification was made directly on the Xnr-1 gene. The Xnr-1 genomic region is composed of 2 main regulatory elements (RE). The RE on the promoter region (PRE) is located 230 bp from the start codon [52]. The second RE is the ASE (asymmetric element) in the intron 1 of the Xnr-1 gene. It is known to drive the asymmetric expression of Xnr-1 in the Lateral Plate Mesoderm (LPM) [53] (Figure 3A).

Bottom Line: To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery.The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator.The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology Center for Regenerative and Developmental Biology Tufts University, Medford, MA 02155 USA.

ABSTRACT

Background: Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFβ superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known.

Results: To understand how an early physiological gradient is transduced into a late, stable pattern of Nr1 expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked Nr1 expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of Nr1 expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the Nr1 gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of Nr1 transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3's biological activity is dependent on 5HT binding.

Conclusion: HDAC activity is a new LR determinant controlling the epigenetic state of Nr1 from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

Show MeSH
Related in: MedlinePlus