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A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes.

Anosova I, Melnik S, Tripsianes K, Kateb F, Grummt I, Sattler M - Nucleic Acids Res. (2015)

Bottom Line: Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins.Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo.Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg D-85764, Germany Biomolecular NMR and Center for Integrated Protein Science Munich (CIPSM), Department Chemie, Technische Universität München, Garching D-85747, Germany.

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Domain organization of TIP5 and its TAM domain. (A) Top: domain organization of human TIP5. Bottom: the role of pRNA in NoRC-mediated silencing of rRNA genes (1). Intergenic transcripts (red dashed line) originating from the rDNA intergenic spacer are processed into 150–250 nt transcripts, termed pRNA (red waved lines). The interaction of pRNA with TIP5 guides NoRC to the rDNA promoter, an essential step in NoRC-dependent silencing of rRNA genes. (B) Multiple sequence alignment of the TAM domain of hTIP5 (BAZ2A) with phylogenetically related BAZ2A homologs (14) and canonical human MBDs. Sequences were aligned using MUSCLE algorithm (47), color coding according to CLUSTAL-X (48). Secondary structure observed in human TAM (Figure 3) is indicated above the sequence. Structural features of the TAM domain that are not present in MBD folds are indicated by black boxes. Black stars mark residues that are strongly affected in NMR titrations of TIP5/TAM-AT with pRNAmini. Red and black filled circles indicate residues that were mutated to probe the TAM/pRNA interaction interface showing strong or no effect on RNA binding, respectively. Black triangles indicate key residues of the MBD1 that are crucial for its interaction with methylated CpG (52). Many of these residues are not conserved in TAM domains, i.e. TIP5 TAM R538, Q547, Q562. Residue numbering refers to human TIP5.
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Figure 1: Domain organization of TIP5 and its TAM domain. (A) Top: domain organization of human TIP5. Bottom: the role of pRNA in NoRC-mediated silencing of rRNA genes (1). Intergenic transcripts (red dashed line) originating from the rDNA intergenic spacer are processed into 150–250 nt transcripts, termed pRNA (red waved lines). The interaction of pRNA with TIP5 guides NoRC to the rDNA promoter, an essential step in NoRC-dependent silencing of rRNA genes. (B) Multiple sequence alignment of the TAM domain of hTIP5 (BAZ2A) with phylogenetically related BAZ2A homologs (14) and canonical human MBDs. Sequences were aligned using MUSCLE algorithm (47), color coding according to CLUSTAL-X (48). Secondary structure observed in human TAM (Figure 3) is indicated above the sequence. Structural features of the TAM domain that are not present in MBD folds are indicated by black boxes. Black stars mark residues that are strongly affected in NMR titrations of TIP5/TAM-AT with pRNAmini. Red and black filled circles indicate residues that were mutated to probe the TAM/pRNA interaction interface showing strong or no effect on RNA binding, respectively. Black triangles indicate key residues of the MBD1 that are crucial for its interaction with methylated CpG (52). Many of these residues are not conserved in TAM domains, i.e. TIP5 TAM R538, Q547, Q562. Residue numbering refers to human TIP5.

Mentions: Despite the fact that mammalian cells contain several hundreds of rRNA genes (rDNA) and rRNA genes are the most actively transcribed genes in eukaryotes, a large fraction of rDNA is silenced by heterochromatin formation to prevent aberrant homologous recombination, thus safeguarding rDNA stability and nucleolar integrity (1). The transcriptionally silent state of rDNA is established by the chromatin remodeling complex NoRC, which comprises the DNA-dependent adenosine triphosphatase SNF2h and a large subunit, termed TIP5 (also known as BAZ2A) (2) (Figure 1a). The C-terminal part of TIP5 harbors a tandem PHD finger/bromodomain, a cooperative unit that interacts with chromatin modifying enzymes, which set specific heterochromatic histone marks and trigger de novo DNA methylation (3,4). Targeting NoRC to rDNA leads to repositioning of the promoter-bound nucleosome, changes in histone modifications, increased DNA methylation and silencing of rRNA genes (5–8). Consistent with epigenetic regulation representing an intimate and balanced interplay of both RNA and chromatin fields, NoRC function requires the association of TIP5 with specific 150–250 nt RNA, named pRNA (‘promoter-associated RNA’) as its sequence overlaps the rDNA promoter (6,9). pRNA folds into a phylogenetically conserved hairpin structure that is recognized by TIP5 and this specific secondary structure of the pRNA is required for guiding NoRC to nucleoli (10). Mutations that disrupt this specific stem-loop structure impair binding of TIP5 to pRNA and abolish the nucleolar localization of NoRC,whereas compensatory mutations that restore the hairpin structure re-establish binding and targeting NoRC to nucleoli. The interaction with pRNA is mediated by the TAM (TIP5/ARBP/MBD) domain in the N-terminal region of TIP5, and is indispensable for nucleolar localization of NoRC and heterochromatin formation at rDNA. RNase footprinting and protease sensitivity experiments suggest that TIP5 associates with pRNA in an induced fit mechanism, resulting in structural changes that may facilitate the interaction with co-repressors to promote heterochromatin formation and rDNA silencing (6). A recent report demonstrated that the poly (ADP-ribose)-polymerase-1 (PARP1) associates with TIP5, thus acting as a co-repressor that mediates the inheritance of silent histone marks through cell cycle progression (11). Significantly, NoRC function is not restricted to rDNA silencing, but it has been shown to also establish a repressive heterochromatic structure at centromeres and telomeres, thus preserving the structural integrity of these repetitive genomic loci (12). Paradoxically, TIP5 may serve as a useful marker for the metastatic potential in prostate cancer. By interacting with EZH2, TIP5 hypermethylates and silences genes that are repressed in metastasis (13).


A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes.

Anosova I, Melnik S, Tripsianes K, Kateb F, Grummt I, Sattler M - Nucleic Acids Res. (2015)

Domain organization of TIP5 and its TAM domain. (A) Top: domain organization of human TIP5. Bottom: the role of pRNA in NoRC-mediated silencing of rRNA genes (1). Intergenic transcripts (red dashed line) originating from the rDNA intergenic spacer are processed into 150–250 nt transcripts, termed pRNA (red waved lines). The interaction of pRNA with TIP5 guides NoRC to the rDNA promoter, an essential step in NoRC-dependent silencing of rRNA genes. (B) Multiple sequence alignment of the TAM domain of hTIP5 (BAZ2A) with phylogenetically related BAZ2A homologs (14) and canonical human MBDs. Sequences were aligned using MUSCLE algorithm (47), color coding according to CLUSTAL-X (48). Secondary structure observed in human TAM (Figure 3) is indicated above the sequence. Structural features of the TAM domain that are not present in MBD folds are indicated by black boxes. Black stars mark residues that are strongly affected in NMR titrations of TIP5/TAM-AT with pRNAmini. Red and black filled circles indicate residues that were mutated to probe the TAM/pRNA interaction interface showing strong or no effect on RNA binding, respectively. Black triangles indicate key residues of the MBD1 that are crucial for its interaction with methylated CpG (52). Many of these residues are not conserved in TAM domains, i.e. TIP5 TAM R538, Q547, Q562. Residue numbering refers to human TIP5.
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Figure 1: Domain organization of TIP5 and its TAM domain. (A) Top: domain organization of human TIP5. Bottom: the role of pRNA in NoRC-mediated silencing of rRNA genes (1). Intergenic transcripts (red dashed line) originating from the rDNA intergenic spacer are processed into 150–250 nt transcripts, termed pRNA (red waved lines). The interaction of pRNA with TIP5 guides NoRC to the rDNA promoter, an essential step in NoRC-dependent silencing of rRNA genes. (B) Multiple sequence alignment of the TAM domain of hTIP5 (BAZ2A) with phylogenetically related BAZ2A homologs (14) and canonical human MBDs. Sequences were aligned using MUSCLE algorithm (47), color coding according to CLUSTAL-X (48). Secondary structure observed in human TAM (Figure 3) is indicated above the sequence. Structural features of the TAM domain that are not present in MBD folds are indicated by black boxes. Black stars mark residues that are strongly affected in NMR titrations of TIP5/TAM-AT with pRNAmini. Red and black filled circles indicate residues that were mutated to probe the TAM/pRNA interaction interface showing strong or no effect on RNA binding, respectively. Black triangles indicate key residues of the MBD1 that are crucial for its interaction with methylated CpG (52). Many of these residues are not conserved in TAM domains, i.e. TIP5 TAM R538, Q547, Q562. Residue numbering refers to human TIP5.
Mentions: Despite the fact that mammalian cells contain several hundreds of rRNA genes (rDNA) and rRNA genes are the most actively transcribed genes in eukaryotes, a large fraction of rDNA is silenced by heterochromatin formation to prevent aberrant homologous recombination, thus safeguarding rDNA stability and nucleolar integrity (1). The transcriptionally silent state of rDNA is established by the chromatin remodeling complex NoRC, which comprises the DNA-dependent adenosine triphosphatase SNF2h and a large subunit, termed TIP5 (also known as BAZ2A) (2) (Figure 1a). The C-terminal part of TIP5 harbors a tandem PHD finger/bromodomain, a cooperative unit that interacts with chromatin modifying enzymes, which set specific heterochromatic histone marks and trigger de novo DNA methylation (3,4). Targeting NoRC to rDNA leads to repositioning of the promoter-bound nucleosome, changes in histone modifications, increased DNA methylation and silencing of rRNA genes (5–8). Consistent with epigenetic regulation representing an intimate and balanced interplay of both RNA and chromatin fields, NoRC function requires the association of TIP5 with specific 150–250 nt RNA, named pRNA (‘promoter-associated RNA’) as its sequence overlaps the rDNA promoter (6,9). pRNA folds into a phylogenetically conserved hairpin structure that is recognized by TIP5 and this specific secondary structure of the pRNA is required for guiding NoRC to nucleoli (10). Mutations that disrupt this specific stem-loop structure impair binding of TIP5 to pRNA and abolish the nucleolar localization of NoRC,whereas compensatory mutations that restore the hairpin structure re-establish binding and targeting NoRC to nucleoli. The interaction with pRNA is mediated by the TAM (TIP5/ARBP/MBD) domain in the N-terminal region of TIP5, and is indispensable for nucleolar localization of NoRC and heterochromatin formation at rDNA. RNase footprinting and protease sensitivity experiments suggest that TIP5 associates with pRNA in an induced fit mechanism, resulting in structural changes that may facilitate the interaction with co-repressors to promote heterochromatin formation and rDNA silencing (6). A recent report demonstrated that the poly (ADP-ribose)-polymerase-1 (PARP1) associates with TIP5, thus acting as a co-repressor that mediates the inheritance of silent histone marks through cell cycle progression (11). Significantly, NoRC function is not restricted to rDNA silencing, but it has been shown to also establish a repressive heterochromatic structure at centromeres and telomeres, thus preserving the structural integrity of these repetitive genomic loci (12). Paradoxically, TIP5 may serve as a useful marker for the metastatic potential in prostate cancer. By interacting with EZH2, TIP5 hypermethylates and silences genes that are repressed in metastasis (13).

Bottom Line: Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins.Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo.Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg D-85764, Germany Biomolecular NMR and Center for Integrated Protein Science Munich (CIPSM), Department Chemie, Technische Universität München, Garching D-85747, Germany.

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