Mice without macroH2A histone variants.

MacroH2A core histone variants have a unique structure that includes a C-terminal nonhistone domain. They are highly conserved in vertebrates and are thought to regulate gene expression. However, the nature of genes regulated by macroH2As and their biological significance remain unclear. Here, we examine macroH2A function in vivo by knocking out both macroH2A1 and macroH2A2 in the mouse. While macroH2As are not required for early development, the absence of macroH2As impairs prenatal and postnatal growth and can significantly reduce reproductive efficiency. The distributions of macroH2A.1- and macroH2A.2-containing nucleosomes show substantial overlap, as do their effects on gene expression. Our studies in fetal and adult liver indicate that macroH2As can exert large positive or negative effects on gene expression, with macroH2A.1 and macroH2A.2 acting synergistically on the expression of some genes and apparently having opposing effects on others. These effects are very specific and in the adult liver preferentially involve genes related to lipid metabolism, including the leptin receptor. MacroH2A-dependent gene regulation changes substantially in postnatal development and can be strongly affected by fasting. We propose that macroH2As produce adaptive changes to gene expression, which in the liver focus on metabolism.

Genome-wide distribution of macroH2A1 histone variants in mouse liver chromatin.

Studies of macroH2A histone variants indicate that they have a role in regulating gene expression. To identify direct targets of the macroH2A1 variants, we produced a genome-wide map of the distribution of macroH2A1 nucleosomes in mouse liver chromatin using high-throughput DNA sequencing. Although macroH2A1 nucleosomes are widely distributed across the genome, their local concentration varies over a range of 100-fold or more. The transcribed regions of most active genes are depleted of macroH2A1, often in sharply localized domains that show depletion of 4-fold or more relative to bulk mouse liver chromatin. We used macroH2A1 enrichment to help identify genes that appear to be directly regulated by macroH2A1 in mouse liver. These genes functionally cluster in the area of lipid metabolism. All but one of these genes has increased expression in macroH2A1 knockout mice, indicating that macroH2A1 functions primarily as a repressor in adult liver. This repressor activity is further supported by the substantial and relatively uniform macroH2A1 enrichment along the inactive X chromosome, which averages 4-fold. Genes that escape X inactivation stand out as domains of macroH2A1 depletion. The rarity of such genes indicates that few genes escape X inactivation in mouse liver, in contrast to what has been observed in human cells.

The Sgp3 locus derived from the 129 strain is responsible for enhanced endogenous retroviral expression in macroH2A1-deficient mice.

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine lupus nephritis is secreted by hepatocytes, and its expression is largely regulated by the Sgp3 (serum gp70 production 3) locus derived from lupus-prone mice. Because of the localization of the macroH2A1 gene encoding macroH2A histone variants within the Sgp3 interval and of an up-regulated transcription of endogenous retroviral sequences in macroH2A1-deficient C57BL/6 (B6) mice, we investigated whether macroH2A1 is a candidate gene for Sgp3. macroH2A1-deficient B6 mice carrying the 129-derived Sgp3 locus, which was co-transferred with the 129 macroH2A1 mutant gene, displayed increased levels of serum gp70 and hepatic retroviral gp70 RNAs comparable to those of B6.NZB-Sgp3 congenic mice bearing the Sgp3 locus of lupus-prone NZB mice. In contrast, the abundance of retroviral gp70 RNAs in macroH2A1-deficient 129 mice was not elevated at all as compared with wild-type 129 mice. Furthermore, Sgp3 subcongenic B6 mice devoid of the NZB-derived macroH2A1 gene displayed an Sgp3 phenotype identical to that of B6.NZB-Sgp3 congenic mice carrying the NZB-derived macroH2A1 gene, thus excluding macroH2A1 as a candidate Sgp3 gene. Collectively, our data indicate that enhanced transcription of endogenous retroviral sequences observed in macroH2A1-deficient B6 mice is not a result of the macroH2A1 mutation, but due to the presence of the 129-derived Sgp3 locus. In contrast, the effect of a macroH2A1 knockout mutation on the expression of several non-retroviral cellular genes was very similar on the B6 and 129 backgrounds, indicating that these effects were due to the macroH2A1 knockout.

macroH2A1-dependent silencing of endogenous murine leukemia viruses.

We show that macroH2A1 histone variants are important for repressing the expression of endogenous murine leukemia viruses (MLVs) in mouse liver. Intact MLV proviruses and proviruses with deletions in env were nearly silent in normal mouse liver and showed substantial derepression in macroH2A1 knockout liver. In contrast, MLV proviruses with a deletion in the 5' end of pro-pol were expressed in normal liver and showed relatively low levels of derepression in knockout liver. macroH2A1 nucleosomes were enriched on endogenous MLVs, with the highest enrichment occurring on the 5' end of pro-pol. The absence of macroH2A1 also led to a localized loss of DNA methylation on the 5' ends of MLV proviruses. These results demonstrate that macroH2A1 histones have a significant role in silencing endogenous MLVs in vivo and suggest that specific internal MLV sequences are targeted by a macroH2A1-dependent silencing mechanism.

A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis.

Histone variants play an important role in numerous biological processes through changes in nucleosome structure and stability and possibly through mechanisms influenced by posttranslational modifications unique to a histone variant. The family of histone H2A variants includes members such as H2A.Z, the DNA damage-associated H2A.X, macroH2A (mH2A), and H2ABbd (Barr body-deficient). Here, we have undertaken the challenge to decipher the posttranslational modification-mediated "histone code" of mH2A, a variant generally associated with certain forms of condensed chromatin such as the inactive X chromosome in female mammals. By using female human cells as a source of mH2A, endogenous mH2A was purified and analyzed by mass spectrometry. Although mH2A is in low abundance compared with conventional histones, we identified a phosphorylation site, S137ph, which resides within the "hinge" region of mH2A. This lysine-rich hinge is an approximately 30-aa stretch between the H2A and macro domains, proposed to bind nucleic acids. A specific antibody to S137ph was raised; by using this reagent, S137 phosphorylation was found to be present in both male and female cells and on both splice variants of the mH2A1 gene. Although mH2A is generally enriched on the inactive X chromosome in female cells, mH2AS137ph is excluded from this heterochromatic structure. Thus, a phosphorylated subpopulation of mH2A appears to play a unique role in chromatin regulation beyond X inactivation. We provide evidence that S137ph is enriched in mitosis, suggestive of a role in the regulation of mH2A posttranslational modifications throughout the cell cycle.

Developmental changes in histone macroH2A1-mediated gene regulation.

macroH2A histone variants have been implicated to function in gene silencing by several studies, including ones showing a preferential association of macroH2A on the inactive X chromosome. To examine macroH2A function in vivo, we knocked out macroH2A1. macroH2A1 knockout mice are viable and fertile. A broad screen of liver gene expression showed no evidence of defects in X inactivation but did identify genes that have increased expression levels in macroH2A1 knockouts. macroH2A1-containing nucleosomes are enriched on the coding and/or upstream regions of these genes, suggesting that their increased expression levels are a direct effect of the absence of macroH2A1. The concentrations of macroH2A1 nucleosomes on these genes are low in the livers of newborn mice, and the macroH2A1 knockout had little effect on the expression levels of these genes in newborn liver. Our results indicate that an increase in liver macroH2A1 during the transition from newborn to young-adult status contributes to a decrease in the expression levels of these genes. These genes cluster in the area of lipid metabolism, and we observed metabolic effects in macroH2A1 knockouts. Our results indicate that the function of macroH2A1 histones is not restricted to gene silencing but also involves fine tuning the expression of specific genes.

macroH2A1 histone variants are depleted on active genes but concentrated on the inactive X chromosome.

Using a novel thiol affinity chromatography approach to purify macroH2A1-containing chromatin fragments, we examined the distribution of macroH2A1 histone variants in mouse liver chromatin. We found that macroH2A1 was depleted on the transcribed regions of active genes. This depletion was observed on all of the 20 active genes that we probed, with only one site showing a small amount of enrichment. In contrast, macroH2A1 was concentrated on the inactive X chromosome, consistent with our previous immunofluorescence studies. This preferential localization was seen on genes that are active in liver, genes that are inactive in liver, and intergenic regions but was absent from four regions that escape X inactivation. These results support the hypothesis that macroH2As function as transcriptional repressors. Also consistent with this hypothesis is our finding that the heterochromatin protein HP1beta copurifies with the macroH2A1-containing chromatin fragments. This study presents the first detailed examination of the distribution of macroH2A1 variants on specific sequences. Our results indicate that macroH2As have complex distribution patterns that are influenced by both local factors and long-range mechanisms.

Reconstitution of nucleosomes with histone macroH2A1.2.

MacroH2A histones have an unusual hybrid structure, consisting of an N-terminal domain that is approximately 65% identical to a full-length histone H2A and a large C-terminal nonhistone domain. To develop an in vitro approach for investigating the effects of macroH2A proteins on chromatin structure and function, we reconstituted nucleosomes with recombinant macroH2A1.2, substituting for conventional H2A. Recombinant macroH2A1.2 was able to efficiently replace both of the conventional H2As in reconstituted nucleosomes. The substitution of macroH2A1.2 for H2A did not appear to grossly perturb the basic structure of the nucleosome core, as assessed by sedimentation and by digestion with micrococcal nuclease or DNase I. However, two differences were observed. First, the region around the midpoint of the nucleosomal core DNA was more resistant to digestion by DNase I in nucleosome core particles reconstituted with macroH2A1.2. Second, preparations of core particles reconstituted with macroH2A1.2 had a greater amount of material that sedimented more rapidly than mononucleosomes, suggesting that macroH2A1.2 may promote interactions between nucleosomes. Recombinant macroH2A proteins should be valuable tools for examining the effects of macroH2A on nucleosome and chromatin structure.