Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis

Recent studies indicate that, similar to other covalent modifications, histone lysine methylation is subject to enzyme-catalysed reversion. So far, LSD1 (also known as AOF2) and the jumonji C (JmjC)-domain-containing proteins have been shown to possess histone demethylase activity. LSD1 catalyses removal of H3K4me2/H3K4me1 through a flavin-adenine-dinucleotide-dependent oxidation reaction. In contrast, JmjC-domain-containing proteins remove methyl groups from histones through a hydroxylation reaction that requires alpha-ketoglutarate and Fe(II) as cofactors. Although an increasing number of histone demethylases have been identified and biochemically characterized, their biological functions, particularly in the context of an animal model, are poorly characterized. Here we use a loss-of-function approach to demonstrate that the mouse H3K9me2/1-specific demethylase JHDM2A (JmjC-domain-containing histone demethylase 2A, also known as JMJD1A) is essential for spermatogenesis. We show that Jhdm2a-deficient mice exhibit post-meiotic chromatin condensation defects, and that JHDM2A directly binds to and controls the expression of transition nuclear protein 1 (Tnp1) and protamine 1 (Prm1) genes, the products of which are required for packaging and condensation of sperm chromatin. Thus, our work uncovers a role for JHDM2A in spermatogenesis and reveals transition nuclear protein and protamine genes as direct targets of JHDM2A.     

The RCAF complex mediates chromatin assembly during DNA replication and repair

Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome. In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin. Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro. RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4. The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones. Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.     

Active genes are tri-methylated at K4 of histone H3

Lysine methylation of histones in vivo occurs in three states: mono-, di- and tri-methyl. Histone H3 has been found to be di-methylated at lysine 4 (K4) in active euchromatic regions but not in silent heterochromatic sites. Here we show that the Saccharomyces cerevisiae Set1 protein can catalyse di- and tri-methylation of K4 and stimulate the activity of many genes. Using antibodies that discriminate between the di- and tri-methylated state of K4 we show that di-methylation occurs at both inactive and active euchromatic genes, whereas tri-methylation is present exclusively at active genes. It is therefore the presence of a tri-methylated K4 that defines an active state of gene expression. These findings establish the concept of methyl status as a determinant for gene activity and thus extend considerably the complexity of histone modifications.     

Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation

Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the 'histone code' hypothesis, in which histone modifications are integral to choreographing the expression of the genome. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5-8), by K79-specific methyltransferase Dot1 (KMT4). However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.