Structural characterization of macroH2A containing chromatin

MacroH2A (mH2A) is one of the most recently identified members of the heteromorphous histone variant family. It is unique among the members of this group because it contains an unusually large non-histone C-terminal end, from where its name derives, and appears to be restricted to subphylum vertebrata. Although a concerted effort has been carried out in order to characterize the physiological relevance of mH2A, little is known in comparison about the structural importance of the molecule. Elucidating the biophysical and conformational proprieties of mH2A in chromatin may provide clues into the links between this histone variant and its unique function(s). In this paper, we look first at the heterogeneous tissue-specific distribution of this protein in different vertebrate classes. This is followed by a structural comparison between mH2A and H2A protein and by the characterization of the nucleosome core particles with which these histone subtypes are associated. We find that the highly alpha-helical C-terminus of mH2A confers an asymmetric conformation to nucleosomes and that this variant is tightly bound to chromatin fragments in a way that does not depend on the overall extent of acetylation of the other core histones.     

X chromosome inactivation and differentiation occur readily in ES cells doubly-deficient for macroH2A1 and macroH2A2

Macrohistones (mH2As) are unusual histone variants found exclusively in vertebrate chromatin. In mice, the H2afy gene encodes two splice variants, mH2A1.1 and mH2A1.2 and a second gene, H2afy2, encodes an additional mH2A2 protein. Both mH2A isoforms have been found enriched on the inactive X chromosome (Xi) in differentiated mammalian female cells, and are incorporated into the chromatin of developmentally-regulated genes. To investigate the functional significance of mH2A isoforms for X chromosome inactivation (XCI), we produced male and female embryonic stem cell (ESC) lines with stably-integrated shRNA constructs that simultaneously target both mH2A1 and mH2A2. Surprisingly, we find that female ESCs deficient for both mH2A1 and mH2A2 readily execute and maintain XCI upon differentiation. Furthermore, male and female mH2A-deficient ESCs proliferate normally under pluripotency culture conditions, and respond to several standard differentiation procedures efficiently. Our results show that XCI can readily proceed with substantially reduced total mH2A content.     

Weak but uniform enrichment of the histone variant macroH2A1 along the inactive X chromosome

We studied the enrichment and distribution of the histone variant mH2A1 in the condensed inactive X (Xi) chromosome. By using highly specific antibodies against mH2A1 and stable HEK 293 cell lines expressing either green fluorescent protein (GFP)-mH2A1 or GFP-H2A, we found that the Xi chromosome contains ~1.5-fold more mH2A1 than the autosomes. To determine the in vivo distribution of mH2A1 along the X chromosome, we used a native chromatin immunoprecipitation-on-chip technique. DNA isolated from mH2A1-immunoprecipitated nucleosomes from either male or female mouse liver were hybridized to tiling microarrays covering 5 kb around most promoters or the entire X chromosome. The data show that mH2A1 is uniformly distributed across the entire Xi chromosome. Interestingly, a stronger mH2A1 enrichment along the pseudoautosomal X chromosome region was observed in both sexes. Our results indicate a potential role for macroH2A in large-scale chromosome structure and genome stability.     

Age-associated increase in heterochromatic marks in murine and primate tissues

Chromatin is highly dynamic and subject to extensive remodeling under many physiologic conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We found increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1β) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver, we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In the skeletal muscle, we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene-induced cellular senescence, and are prominently found in terminally differentiated, postmitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to investigate further the progressive epigenetic changes that occur during aging.     

MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism‐like behaviors

The development of the nervous system requires precise regulation. Any disturbance in the regulation process can lead to neurological developmental diseases, such as autism and schizophrenia. Histone variants are important components of epigenetic regulation. The function and mechanisms of the macroH2A (mH2A) histone variant during brain development are unknown. Here, we show that deletion of the mH2A isoform mH2A1.2 interferes with neural stem cell differentiation in mice. Deletion of mH2A1.2 affects neurodevelopment, enhances neural progenitor cell (NPC) proliferation, and reduces NPC differentiation in the developing mouse brain. mH2A1.2‐deficient mice exhibit autism‐like behaviors, such as deficits in social behavior and exploratory abilities. We identify NKX2.2 as an important downstream effector gene and show that NKX2.2 expression is reduced after mH2A1.2 deletion and that overexpression of NKX2.2 rescues neuronal abnormalities caused by mH2A1.2 loss. Our study reveals that mH2A1.2 reduces the proliferation of neural progenitors and enhances neuronal differentiation during embryonic neurogenesis and that these effects are at least in part mediated by NKX2.2. These findings provide a basis for studying the relationship between mH2A1.2 and neurological disorders.     

A genetic analysis of human minor histocompatibility antigens demonstrates Mendelian segregation independent of HLA

An analysis of the genetic traits of human minor histocompatibility (mH) antigens is, unlike with inbred mice, rather complicated. Moreover, the fact that mH antigens are recognized in the context of MHC molecules creates an additional complication for reliable segregation analysis. To gain insight into the mode of inheritance of the mH antigens, we relied upon a series of HLA-A2-restricted cytotoxic T-cell (CTL) clones specific for four mH antigens. To perform segregation analysis independent of HLA-A2 gene: we transfected HLA-A2-negative cells with the HLA-A2 gene: this results in the cell surface expression of the HLA-A2 gene product and, if present, mH antigen recognition. The mode of inheritance of the HLA-A2-restricted mH antigens HA-1, -2, -4, and -5 was analysed in 25 families whoese members either naturally expressed positive. Analysis of distribution of the mH antigens in the parent population among the mating types, together with their inheritance patterns in the families, demonstrated that the four mH antigens behaved as Mendelian traits, whereby each can be considered a product of a gene with two alleles, one expressing and one not expressing the detected specificity. We also showed that the loci encoding the HA-1 and HA-2 antigens are not closely linked to HLa (lod scores Z (0 = 0.05) <–4.0). Some indication was obtained that the HA-4- and HA-5-encoding loci may be losely linked to HLA. While we are aware of the limited results of this nonetheless comprehensive study, we feel the similarity in immunogenetic traits between human and mouse mH antigens is at least striking.     

Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells

Hybridoma cell lines were prepared from spleen cells of mouse immunized with mitotic HeLa cells. A monoclonal antibody (mH1), which intensively reacted with cleavage furrows of dividing HeLa cells in immunofluorescence, was obtained. In interphase, this antibody diffusely stained whole HeLa cells. Immunoelectron microscopy showed that mH1 antigens were localized at microvillus projections at the surface of dividing HeLa cells, but definite localization of that antigen was not observed in interphasic cells. Immunoblot analysis showed that mH1 is reactive to 42-kDa and 130-kDa components. Further, the 42-kDa component was identified as a gamma-actin homolog by N-terminal amino acid sequence analysis.     

Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms

The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.     

Histone H2A/H2B dimer exchange by ATP-dependent chromatin remodeling activities

ATP-dependent chromatin remodeling activities function to manipulate chromatin structure during gene regulation. One of the ways in which they do this is by altering the positions of nucleosomes along DNA. Here we provide support for the ability of these complexes to move nucleosomes into positions in which DNA is unraveled from one edge. This is expected to result in the loss of histone-DNA contacts that are important for retention of one H2A/H2B dimer within the nucleosome. Consistent with this we find that several chromatin remodeling complexes are capable of catalyzing the exchange of H2A/H2B dimers between chromatin fragments in an ATP-dependent reaction. This provides eukaryotes with additional means by which they may manipulate chromatin structure.     

Immunogenetics of human minor histocompatibility antigens: their polymorphism and immunodominance

Minor Histocompatibility (mH) antigens are polymorphic endogenously synthesized products that can be recognized by alloreactive T cells in the context of major histocompatibility complex molecules. In transplant situations where tissue donor and recipient are matched for HLA, mH antigens may trigger strong cellular immune responses. To gain insight into the polymorphism of mH antigens we studied their frequencies in the healthy population. Five HLA class I restricted mH antigens recognized by distinct cytotoxic T-cell (CTL) clones were used in the population genetic analysis consisting of a panel (N=100) of HLA typed target cells. Three mH antigens showed phenotype frequencies of 69% or higher, this contrasted the frequencies of two other mH antigens with 16 and 7% respectively. To gain insight into the functional polymorphism of the T-cell response to mH antigens, we analyzed the specificity of CTL clones within individuals. Three out of five individuals investigated shared a CTL response to one single HLA-A2 restricted mH antigen. These results indicate limited allelic polymorphism for some mH antigens in the healthy population and are suggestive of the existence of immunodominant human mH antigens. Address correspondence and offprint requests to: E. Goulmy.     

Physicochemical studies of the folding of the 100 A nucleosome filament into the 300 A filament. Cation dependence

The cation-induced refolding of the 100 A nucleosome filament into the 300 A filament has been studied over a wide range of concentrations of Na+, Mg2+, Co(NH3)3+6 and other cations. X-ray diffraction, electron microscopy and analytical ultracentrifugation have been used to determine the conditions under which the 300 A filament is formed. It is shown that cations induce chromatin refolding by acting as general DNA counterions. The concentration of any cation required to induce refolding is greatly dependent on the valence of that cation. Na+ (and, presumably, other monovalent cations) has dual effects: at high concentrations (greater than 45 to 65 mM) it stabilizes the 300 A filament state of chromatin; however, at low concentrations (less than approximately equal to 45 mM), when cations of higher valence are present and stabilizing the 300 A filament state, Na+ has the opposite effect, competing with the higher-valence cation for binding to the chromatin and destabilizing the 300 A filament state. It is shown that further addition of cations to chromatin in the 300 A filament state causes a further folding of the chromatin in which the sedimentation coefficient increases and the X-ray diffraction bands resulting from nucleosomal packing sharpen. This may reflect subtle structural changes within the 300 A filament, or it may reflect a shift in equilibrium constant for chromatin fluctuating between the 100 A and 300 A filament states. It is also shown that, with continued addition of cation, the 300 A filaments precipitate before any "endpoint" is reached in this further folding. The tendency of 300 A filaments to aggregate in vitro appears to be a built-in property, and may reflect the packing of 300 A filaments within metaphase chromosomes in vivo.     

Chemically modified short interfering hybrids (siHYBRIDS): nanoimmunoliposome delivery in vitro and in vivo for RNAi of HER-2

A blunt-ended 19-mer short interfering hybrid (siHybrid) (H) comprised of sense-DNA/antisense-RNA targeting HER-2 mRNA was encapsulated in a liposomal nanoplex with anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the targeting moiety for clinically relevant tumor-specific delivery. In vitro delivery to a human pancreatic cell line (PANC-1) was shown to exhibit sequence-specific inhibition of 48-h cell growth with an IC50 value of 37 nM. The inhibitory potency of this siHybrid was increased (IC50 value of 7.8 nM) using a homologous chemically modified siHybrid (mH) in which the 19-mer sense strand had the following pattern of 2 '-deoxyinosine (dI) and 2 '-O-methylribonucleotide (2 '-OMe) residues: 5'-d(TITIT)-2'OMe(GCGGUGGUU)-d(GICIT). These modifications were intended to favor antisense strand-mediated RNAi while mitigating possible sense strand-mediated off-target effects and RNase H-mediated cleavage of the antisense RNA strand. The presently reported immunoliposomal delivery system was successfully used in vivo to inhibit HER-2 expression, and thus induce apoptosis in human breast carcinoma tumors (MDA-MB-435) in mice upon repeated i.v. treatment at a dose of 3 mg/kg of H or mH. The in vivo potency of modified siHybrid mH appeared to be qualitatively greater than that of H, as was the case in vitro.     

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

We have used gene disruptions and nuclease probes to assess the roles of yeast 2 micron plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) control copy number by regulating the level of the gene A recombinase.     

Differential effects of spermine on phosphatidylinositol 3-kinase and phosphatidylinositol phosphate 5-kinase

The metabolism of phosphoinositides plays an important role in the signal transduction pathways. We report here that naturally occuring polyamines affect the activities of phosphatidylinositol (PI) 3-kinase and PI 4-phosphate (PIP) 5-kinase differently. While polyamines inhibited the PI 3-kinase activity, they stimulated the activity of PIP 5-kinase in the order of spermine > spermidine > putrescine. Spermine inhibited the PI 3-kinase activity in a concentration-dependent manner with an IC₅₀ of 100 μM. On the other hand, spermine (5 mH) stimulated the activity of PIP 5-kinase 2–3 fold. Kinetic studies of spermine-mediated inhibition of PI 3-kinase revealed that it was noncompetitive with respect to ATP. The effect of Mg²⁺ and PIP, concentration on kinase activity was sigmoidal, with spermine inhibiting PI 3-kinase activity at all PIP₂ concentrations. While 1 mH calcium stimulated PI 3-kinase activity at submaximal concentrations of Mg²⁺ (1.25 mH), inhibition was observed at optimal concentration of Mg²⁺(2 mM). We propose that spermine may modulate the cellular signal by virtue of its differential effects on phosphoinositide kinases.