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1.
Nat Struct Mol Biol ; 25(8): 743, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29995840

ABSTRACT

In this article, the Ponceau staining presented in Fig. 1b (right, bottom) does not follow best practices for figure preparation since itinadvertently included duplications from the Ponceau staining presented in Supplementary Fig. 1b (for which the same preparation ofnucleosomes from HeLa cells had been used). A new Fig. 1b is provided in the Author Correction.

2.
Mol Biosyst ; 6(9): 1719-29, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20577673

ABSTRACT

Histones are highly conserved proteins that organize cellular DNA. These proteins, especially their N-terminal domains, are adorned with many post-translational modifications (PTMs) such as lysine methylation, which are associated with active or repressed transcriptional states. The lysine methyltransferase G9a and its interaction partner Glp1 can mono- or dimethylate histone H3 on lysine (H3K9me1 or me2); possible cross-talk between these modifications and other PTMs on the same or other histone molecules is currently uncharacterized. In this study, we comprehensively analyze the effects of G9a/Glp1 knockdown on the most abundant histone modifications through both Bottom Up and Middle Down mass spectrometry-based proteomics. In addition to the expected decrease in H3K9me1/me2 we find that other degrees of methylation on K9 are affected by the reduction of G9a/Glp1 activity, particularly when K9 methylation occurs in combination with K14 acetylation. In line with this, an increase in K14 acetylation upon G9a knockdown was observed across all H3 variants (H3.1, H3.2 and H3.3), hinting at the potential existence of a binary switch between K9 methylation and K14 acetylation. Interestingly, we also detect changes in the abundance of other modifications (such as H3K79me2) in response to lowered levels of G9a/Glp1 suggesting histone PTM cross-talk amongst the H3 variants. In contrast, we find that G9a/Glp1 knockdown produces little effect on the levels of histone H4 PTMs, indicating low to no trans-histone PTM crosstalk. Lastly, we determined gene expression profiles of control and G9a/Glp1 knockdown cells, and we find that the G9a/Glp1 knockdown influences several genes, including DNA binding proteins and key factors in chromatin. Our results provide new insights into the intra- and inter- histone cross-regulation of histone K9 methylation and its potential downstream gene targets.


Subject(s)
Histone Code/genetics , Methyltransferases/genetics , Proteomics/methods , Cell Line , Chromatography, High Pressure Liquid , Humans , Mass Spectrometry , Oligonucleotide Array Sequence Analysis
3.
Epigenetics Chromatin ; 3(1): 7, 2010 Mar 24.
Article in English | MEDLINE | ID: mdl-20334638

ABSTRACT

BACKGROUND: The linker histone H1 has a key role in establishing and maintaining higher order chromatin structure and in regulating gene expression. Mammals express up to 11 different H1 variants, with H1.2 and H1.4 being the predominant ones in most somatic cells. Like core histones, H1 has high levels of covalent modifications; however, the full set of modifications and their biological role are largely unknown. RESULTS: In this study, we used a candidate screen to identify enzymes that methylate H1 and to map their corresponding methylation sites. We found that the histone lysine methyltransferases G9a/KMT1C and Glp1/KMT1D methylate H1.2 in vitro and in vivo, and we mapped this novel site to lysine 187 (H1.2K187) in the C-terminus of H1. This H1.2K187 methylation is variant-specific. The main target for methylation by G9a in H1.2, H1.3, H1.5 and H1.0 is in the C-terminus, whereas H1.4 is preferentially methylated at K26 (H1.4K26me) in the N-terminus. We found that the readout of these marks is different; H1.4K26me can recruit HP1, but H1.2K187me cannot. Likewise, JMJD2D/KDM4 only reverses H1.4K26 methylation, clearly distinguishing these two methylation sites. Further, in contrast to C-terminal H1 phosphorylation, H1.2K187 methylation level is steady throughout the cell cycle. CONCLUSIONS: We have characterised a novel methylation site in the C-terminus of H1 that is the target of G9a/Glp1 both in vitro and in vivo. To our knowledge, this is the first demonstration of variant-specific histone methylation by the same methyltransferases, but with differing downstream readers, thereby supporting the hypothesis of H1 variants having specific functions.

4.
Nat Struct Mol Biol ; 16(7): 777-81, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19561610

ABSTRACT

Histone modifications are central to the regulation of all DNA-dependent processes. Lys64 of histone H3 (H3K64) lies within the globular domain at a structurally important position. We identify trimethylation of H3K64 (H3K64me3) as a modification that is enriched at pericentric heterochromatin and associated with repeat sequences and transcriptionally inactive genomic regions. We show that this new mark is dynamic during the two main epigenetic reprogramming events in mammals. In primordial germ cells, H3K64me3 is present at the time of specification, but it disappears transiently during reprogramming. In early mouse embryos, it is inherited exclusively maternally; subsequently, the modification is rapidly removed, suggesting an important role for H3K64me3 turnover in development. Taken together, our findings establish H3K64me3 as a previously uncharacterized histone modification that is preferentially localized to repressive chromatin. We hypothesize that H3K64me3 helps to 'secure' nucleosomes, and perhaps the surrounding chromatin, in an appropriately repressed state during development.


Subject(s)
Epigenesis, Genetic , Heterochromatin/metabolism , Histones/metabolism , Lysine/metabolism , Animals , Cell Line , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Methylation , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/metabolism , Heterochromatin/chemistry , Heterochromatin/genetics , Histones/genetics , Humans , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Nucleosomes/chemistry , Nucleosomes/metabolism , Protein Conformation , Repressor Proteins/genetics , Repressor Proteins/metabolism , Xenopus laevis
5.
J Biol Chem ; 280(45): 38090-5, 2005 Nov 11.
Article in English | MEDLINE | ID: mdl-16127177

ABSTRACT

Histone lysine methylation can have positive or negative effects on transcription, depending on the precise methylation site. According to the "histone code" hypothesis these methylation marks can be read by proteins that bind them specifically and then regulate downstream events. Hetero-chromatin protein 1 (HP1), an essential component of heterochromatin, binds specifically to methylated Lys(9) of histone H3 (K9/H3). The linker histone H1.4 is methylated on Lys(26) (K26/H1.4), but the role of this methylation in downstream events remains unknown. Here we identify HP1 as a protein specifically recognizing and binding to methylated K26/H1.4. We demonstrate that the Chromo domain of HP1 is mediating this binding and that phosphorylation of Ser(27) on H1.4 (S27/H1.4) prevents HP1 from binding. We suggest that methylation of K26/H1.4 could have a role in tethering HP1 to chromatin and that this could also explain how HP1 is targeted to those regions of chromatin where it does not colocalize with methylated K9/H3. Our results provide the first experimental evidence for a "phospho switch" model in which neighboring phosphorylation reverts the effect of histone lysine methylation.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Histones/metabolism , Lysine/metabolism , Phosphoserine/metabolism , Animals , Cell Nucleus , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/genetics , HeLa Cells , Histones/chemistry , Humans , Methylation , Mice , Phosphorylation , Protein Binding , Substrate Specificity
6.
Hum Mutat ; 20(1): 77-8, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12112666

ABSTRACT

Mutations in the GJB2 gene encoding the gap-junction protein connexin 26 have been identified in many patients with childhood hearing impairment (HI). One single mutation, 35delG (30delG), accounts for up to 70% of all analyzed European patients with autosomal recessive inherited HI and 10% of patients with HI of unknown origin, respectively. We screened 188 control individuals and 342 German patients with non-syndromic sporadic HI for the 35delG, compound heterozygosity and other GJB2 mutations by PCR, restriction enzyme based screening, SSCP and sequencing. In all patients, non-progressive hearing impairment varied from moderate to profound involving all frequencies. This study revealed one novel silent mutation (438C/T), three novel gene variants resulting in amino acid substitutions (K112E, T123S, K223R) and two novel HI-related mutations (I82M, 313del14).


Subject(s)
Connexins/genetics , Hearing Loss, Sensorineural/genetics , Adolescent , Adult , Base Sequence , Child , Child, Preschool , Connexin 26 , DNA/chemistry , DNA/genetics , DNA Mutational Analysis , Gene Frequency , Germany , Hearing Loss, Sensorineural/physiopathology , Hearing Tests , Humans , Infant , Middle Aged , Mutation , Polymorphism, Single-Stranded Conformational , Sequence Deletion
7.
Hum Mutat ; 19(3): 308-9, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11857751

ABSTRACT

The A1555G mutation in the 12SrRNA gene has been associated with aminoglycoside induced and nonsyndromic sensorineural hearing impairment. In this study we analyzed Hungarian, Polish and German patients with nonsyndromic severe to profound hearing impairment of unknown origin for this mutation. The frequency of the A1555G mutation in the Hungarian hearing impaired population was below 1.8 %. Three out of 125 Polish patients carrying the A1555G mutation were identified. Among German patients one carrier was found (0.7 %) revealing a homoplastic A1555G mutation, whereas no mutation was detected in control individuals with normal hearing (frequency < 0.6%). In summary the frequencies of the A1555G mutation are low in the hearing impaired as well as in the normal population in Hungary, Poland and Germany. Since the importance of this mutation and its relationship with aminoglycoside exposure is not well understood yet, patients with nonsyndromic hearing impairment should be routinely screened for this mutation to avoid aminoglycoside induced hearing impairment due to increased sensitivity of maternal relatives.


Subject(s)
Alanine/genetics , Genetic Predisposition to Disease/epidemiology , Glycine/genetics , Hearing Loss, Sensorineural/epidemiology , Hearing Loss, Sensorineural/genetics , Mutation/genetics , RNA, Ribosomal/genetics , Adult , Aged , Amino Acid Substitution/genetics , Child , Child, Preschool , Female , Genetic Testing , Germany/epidemiology , Humans , Hungary/epidemiology , Infant , Male , Middle Aged , Pedigree , Poland/epidemiology
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