Poly(ADP-ribose) polymerase 1 is inhibited by a histone H2A variant, MacroH2A, and contributes to silencing of the inactive X chromosome

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in modulating chromatin structure, regulation of gene expression, and sensing DNA damage. Here, we report that PARP-1 enzymatic activity is inhibited by macroH2A, a vertebrate histone H2A variant that is enriched on facultative heterochromatin. MacroH2A family members have a large C-terminal non-histone domain (NHD) and H2A-like histone domain. MacroH2A1.2 and PARP-1 interact in vivo and in vitro via the NHD. The NHD of each macroH2A family member was sufficient to inhibit PARP-1 enzymatic activity in vitro. The NHD of macroH2A1.2 was a mixed inhibitor of PARP-1 catalytic activity, with affects on both catalytic activity and the substrate binding affinity of PARP-1. Depletion of PARP-1 by RNA interference caused reactivation of a reporter gene on the inactive X chromosome, demonstrating that PARP-1 participates in the maintenance of silencing. These results suggest that one function of macroH2A in gene silencing is to inhibit PARP-1 enzymatic activity, and this may affect PARP-1 association with chromatin.     

Developmental and tissue expression patterns of histone macroH2A1 subtypes

MacroH2A is a novel nucleosomal core histone that contains a large nonhistone region and a region that closely resembles a full length histone H2A. We have cloned a cDNA that contains the entire coding region of macroH2A1.2, one of the two identified subtypes of macroH2A1. MacroH2A1.2 was found to differ from the other known subtype, macroH2A1.1, in a single segment of the nonhistone region. MacroH2A1 specific antibodies revealed relatively high levels of both subtypes in adult liver and kidney. MacroH2A1.1 was much lower in fetal liver and kidney in comparison to their adult counterparts, and was not detected in adult thymus and testis, tissues with active cell division and differentiation. Both subtypes were present at very low levels or absent from mouse embryonic stem cells maintained in an undifferentiated state by growth in the presence of leukemia inhibitory factor. MacroH2A1.2 increased when the embryonic stem cells were induced to differentiate in vitro, while macroH2A1.1 remained undetectable. These results support the idea that macroH2A1.1 and macroH2A1.2 are functionally distinct, and suggest that changes in their expression may play a role in developmentally regulated changes in chromatin structure and function. J. Cell. Biochem. 65:107–113. © 1997 Wiley-Liss, Inc.     

Splicing regulates NAD metabolite binding to histone macroH2A

Histone macroH2A is a hallmark of mammalian heterochromatin. Here we show that human macroH2A1.1 binds the SirT1-metabolite O-acetyl-ADP-ribose (OAADPR) through its macro domain. The 1.6-A crystal structure and mutants reveal how the metabolite is recognized. Mutually exclusive exon use in the gene H2AFY produces macroH2A1.2, whose tissue distribution differs. MacroH2A1.2 shows only subtle structural changes but cannot bind nucleotides. Alternative splicing may thus regulate the binding of nicotinamide adenine dinucleotide (NAD) metabolites to chromatin.     

Structural characterization of the histone variant macroH2A

macroH2A is an H2A variant with a highly unusual structural organization. It has a C-terminal domain connected to the N-terminal histone domain by a linker. Crystallographic and biochemical studies show that changes in the L1 loop in the histone fold region of macroH2A impact the structure and potentially the function of nucleosomes. The 1.6-A X-ray structure of the nonhistone region reveals an alpha/beta fold which has previously been found in a functionally diverse group of proteins. This region associates with histone deacetylases and affects the acetylation status of nucleosomes containing macroH2A. Thus, the unusual domain structure of macroH2A integrates independent functions that are instrumental in establishing a structurally and functionally unique chromatin domain.     

The basic linker of macroH2A stabilizes DNA at the entry/exit site of the nucleosome

MacroH2A is a histone H2A variant that is typically found in heterochromatic regions of the genome. A positively charged linker that connects the histone-fold with the macro-domain was suggested to have DNA-binding properties, and has been shown to promote oligomerization of chromatin fibers. Here we examine the influence of this basic linker on DNA of mononucleosomes. We find that the macro-linker reduces accessibility to extranucleosomal DNA, and appears to increase compaction of the nucleosome. These properties arise from interactions between the H1-like basic linker region and DNA around the entry/exit site, which increases protection of nucleosomal DNA from exonuclease III digestion by ∼10 bp. By stabilizing the wrapping of DNA around the histone core, this basic linker of macroH2A may alter the distribution of nucleosome-associated factors, and potentially contribute to the more compacted nature of heterochromatin.     

QKI-mediated alternative splicing of the histone variant MacroH2A1 regulates cancer cell proliferation

The histone variant macroH2A1 contains a carboxyl-terminal ～30-kDa domain called a macro domain. MacroH2A1 is produced as one of two alternatively spliced forms, macroH2A1.1 and macroH2A1.2. While the macro domain of macroH2A1.1 can interact with NAD(+)-derived small molecules, such as poly(ADP-ribose), macroH2A1.2's macro domain cannot. Here, we show that changes in the alternative splicing of macroH2A1 pre-mRNA, which lead to a decrease in macroH2A1.1 expression, occur in a variety of cancers, including testicular, lung, bladder, cervical, breast, colon, ovarian, and endometrial. Furthermore, reintroduction of macroH2A1.1 suppresses the proliferation of lung and cervical cancer cells in a manner that requires the ability of macroH2A1.1 to bind NAD(+)-derived metabolites. MacroH2A1.1-mediated suppression of proliferation occurs, at least in part, through the reduction of poly(ADP-ribose) polymerase 1 (PARP-1) protein levels. By analyzing publically available expression and splicing microarray data, we identified splicing factors that correlate with alterations in macroH2A1 splicing. Using RNA interference, we demonstrate that one of these factors, QKI, regulates the alternative splicing of macroH2A1 pre-mRNA, resulting in increased levels of macroH2A1.1. Finally, we demonstrate that QKI expression is significantly reduced in many of the same cancer types that demonstrate a reduction in macroH2A1.1 splicing.     

MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP‐ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1‐dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform‐specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.     

The histone domain of macroH2A1 contains several dispersed elements that are each sufficient to direct enrichment on the inactive X chromosome

Histone variants replace the core histones in a substantial fraction of nucleosomes, affecting chromatin structure and impacting chromatin-templated processes. In many instances incorporation of histone variants results in formation of specialized regions of chromatin. Proper localization of histone variants to distinct regions of the genome is critical for their function, yet how this specific localization is achieved remains unclear. macroH2A1 is enriched on the inactive X chromosome in female mammalian cells, where it functions to maintain gene silencing. macroH2A1 consists of a histone H2A-like histone domain and a large, globular C-terminal macro domain that is not present in other histone proteins. The histone domain of macroH2A1 is alone sufficient to direct enrichment on the inactive X chromosome when expressed in female cells, indicating that sequences important for correct localization lie in this domain. Here we investigate whether divergent sequences of the H2A variant macroH2A1 contribute to its correct localization. We mapped the regions of the macroH2A1 histone domain that are sufficient for localization to the inactive X chromosome using chimeras between H2A and the histone domain of macroH2A1. Multiple short sequences dispersed along the macroH2A1 histone domain individually supported enrichment on the inactive X chromosome when introduced into H2A. These sequences map to the surface of the macroH2A1/H2B dimer, but are buried in the crystal structure of the macroH2A1 containing nucleosome, suggesting that they may contribute to recognition by macroH2A1/H2B deposition factors.     

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.     

Histone variant macroH2A contains two distinct macrochromatin domains capable of directing macroH2A to the inactive X chromosome

Chromatin on the inactive X chromosome (Xi) of female mammals is enriched for the histone variant macroH2A that can be detected at interphase as a distinct nuclear structure referred to as a macro chromatin body (MCB). Green fluorescent protein-tagged and Myc epitope-tagged macroH2A readily form an MCB in the nuclei of transfected female, but not male, cells. Using targeted disruptions, we have identified two macrochromatin domains within macroH2A that are independently capable of MCB formation and association with the Xi. Complete removal of the non-histone C-terminal tail does not reduce the efficiency of association of the variant histone domain of macroH2A with the Xi, indicating that the histone portion alone can target the Xi. The non-histone domain by itself is incapable of MCB formation. However, when directed to the nucleosome by fusion to core histone H2A or H2B, the non-histone tail forms an MCB that appears identical to that of the endogenous protein. Mutagenesis of the non-histone portion of macroH2A localized the region required for MCB formation and targeting to the Xi to an ~190 amino acid region.     

The crystal structure of AF1521 a protein from Archaeoglobus fulgidus with homology to the non-histone domain of macroH2A

MacroH2A is an unusual histone H2A variant that has an extensive C-terminal tail that comprises approximately two thirds of the protein. The C-terminal non-histone domain of macroH2A is also found in a number of other proteins and has been termed the macro domain. Here we report the crystal structure to 1.7A of AF1521, a protein consisting of a stand-alone macro domain from Archaeoglobus fulgidus. The structure has a mixed alpha/beta fold that closely resembles the N-terminal DNA binding domain of the Escherichia coli leucine aminopeptidase PepA. The structure also shows some similarity to members of the P-loop family of nucleotide hydrolases.     

A maternal store of macroH2A is removed from pronuclei prior to onset of somatic macroH2A expression in preimplantation embryos

MacroH2A histones are variants of canonical histone H2A that are conserved among vertebrates. Previous studies have implicated macroH2As in epigenetic gene-silencing events including X chromosome inactivation. Here we show that macroH2A is present in developing and mature mouse oocytes. MacroH2A is localized to chromatin of germinal vesicles (GV) in both late growth stage (lg-GV) and fully grown (fg-GV) stage oocytes. In addition, macroH2A is associated with the chromosomes of mature oocytes, and abundant macroH2A is present in the first polar body. However, maternal macroH2A is lost from zygotes generated by normal fertilization by the late 2 pronuclei (2PN) stage. Normal embryos at 2-, 4-, and 8-cell stages lack macroH2A except in residual polar bodies. MacroH2A protein expression reappears in embryos after the 8-cell stage and persists in morulae and blastocysts, where nuclear macroH2A is present in both the trophectodermal and inner cell mass cells. We followed the loss of macroH2A from pronuclei in parthenogenetic embryos generated by oocyte activation. Abundant macroH2A is present upon the metaphase II plate and persists through parthenogenetic anaphase, but macroH2A is progressively lost during pronuclear decondensation prior to synkaryogamy. Examination of embryos generated by intracytoplasmic sperm injection (ICSI) revealed that macroH2A is associated exclusively with female pronuclei prior to loss in late pronucleus stage embryos. These results outline a surprising finding that a maternal store of macroH2A is removed from the maternal genome prior to synkaryogamy, resulting in embryos that execute three to four mitotic divisions in the absence of macroH2A prior to the onset of embryonic macroH2A expression.     

A Distinct Switch in Interactions of the Histone H4 Tail Domain upon Salt-dependent Folding of Nucleosome Arrays

The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes (1, 2). Likewise, H4 tail-H2A contacts stabilize array folding (3). However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.     

The core histone N termini function independently of linker histones during chromatin condensation

The relationships between the core histone N termini and linker histones during chromatin assembly and salt-dependent chromatin condensation were investigated using defined chromatin model systems reconstituted from tandemly repeated 5 S rDNA, histone H5, and either native "intact" core histone octamers or "tailless" histone octamers lacking their N-terminal domains. Nuclease digestion and sedimentation studies indicate that H5 binding and the resulting constraint of entering and exiting nucleosomal DNA occur to the same extent in both tailless and intact chromatin arrays. However, despite possessing a normal chromatosomal structure, tailless chromatin arrays can neither condense into extensively folded structures nor cooperatively oligomerize in MgCl(2). Tailless nucleosomal arrays lacking linker histones also are unable to either fold extensively or oligomerize, demonstrating that the core histone N termini perform the same functions during salt-dependent condensation regardless of whether linker histones are components of the array. Our results further indicate that disruption of core histone N termini function in vitro allows a linker histone-containing chromatin fiber to exist in a decondensed state under conditions that normally would promote extensive fiber condensation. These findings have key implications for both the mechanism of chromatin condensation, and the regulation of genomic function by chromatin.     

Identification of specific functional subdomains within the linker histone H10 C-terminal domain

Linker histone binding to nucleosomal arrays in vitro causes linker DNA to form an apposed stem motif, stabilizes extensively folded secondary chromatin structures, and promotes self-association of individual nucleosomal arrays into oligomeric tertiary chromatin structures. To determine the involvement of the linker histone C-terminal domain (CTD) in each of these functions, and to test the hypothesis that the functions of this highly basic domain are mediated by neutralization of linker DNA negative charge, four truncation mutants were created that incrementally removed stretches of 24 amino acids beginning at the extreme C terminus of the mouse H1(0) linker histone. Native and truncated H1(0) proteins were assembled onto biochemically defined nucleosomal arrays and characterized in the absence and presence of salts to probe primary, secondary, and tertiary chromatin structure. Results indicate that the ability of H1(0) to alter linker DNA conformation and stabilize condensed chromatin structures is localized to specific C-terminal subdomains, rather than being equally distributed throughout the entire CTD. We propose that the functions of the linker histone CTD in chromatin are linked to the characteristic intrinsic disorder of this domain.