Spleen Nuclei Research Articles

Cell atlas of the Atlantic salmon spleen reveals immune cell heterogeneity and cell-specific responses to bacterial infection

The spleen is a conserved secondary lymphoid organ that emerged in parallel to adaptive immunity in early jawed vertebrates. Recent studies have applied single cell transcriptomics to reveal the cellular composition of spleen in several species, cataloguing diverse immune cell types and subpopulations. In this study, 51,119 spleen nuclei transcriptomes were comprehensively investigated in the commercially important teleost Atlantic salmon (Salmo salar L.), contrasting control animals with those challenged with the bacterial pathogen Aeromonas salmonicida. We identified clusters of nuclei representing the expected major cell types, namely T cells, B cells, natural killer-like cells, granulocytes, mononuclear phagocytes, endothelial cells, mesenchymal cells, erythrocytes and thrombocytes. We discovered heterogeneity within several immune lineages, providing evidence for resident macrophages and melanomacrophages, infiltrating monocytes, several candidate dendritic cell subpopulations, and B cells at distinct stages of differentiation, including plasma cells and an igt + subset. We provide evidence for twelve candidate T cell subsets, including cd4+ T helper and regulatory T cells, one cd8+ subset, three γδT subsets, and populations double negative for cd4 and cd8. The number of genes showing differential expression during the early stages of Aeromonas infection was highly variable across immune cell types, with the largest changes observed in macrophages and infiltrating monocytes, followed by resting mature B cells. Our analysis provides evidence for a local inflammatory response to infection alongside B cell maturation in the spleen, and upregulation of ccr9 genes in igt + B cells, T helper and cd8+ cells, and monocytes, consistent with the recruitment of immune cell populations to the gut to deal with Aeromonas infection. Overall, this study provides a new cell-resolved perspective of the immune actions of Atlantic salmon spleen, highlighting extensive heterogeneity hidden to bulk transcriptomics. We further provide a large catalogue of cell-specific marker genes that can be leveraged to further explore the function and structural organization of the salmonid immune system.

Clonal Evolution and Sub-Clonal Architecture In Paediatric T-Cell Acute Lymphoblastic Leukaemia (T-ALL)

IntroductionThe issue of clonal heterogeneity in leukaemia has diverse implications for disease monitoring, targeted therapy design and understanding the biology of disease relapse. In this study, we aimed to perform single cell analysis to improve understanding of clonal evolution in T-ALL. We used fluorescence in situ hybridisation (FISH) and phylogenetic analysis of diagnostic T-ALL samples to infer the order of acquisition of genetic events and define the early and likely founder events in this subtype of leukaemia. Screening of paired diagnostic and xenograft material from two STIL-TAL1 positive cases allowed analysis of clonal heterogeneity and evolution in leukaemia initiating cells. MethodsDNA from seven diagnostic T-ALL patients aged 1 – 25 years (five STIL-TAL1 fusion positive, one TLX1 translocation positive, one TLX3 translocation positive) was screened using the Affymetrix® SNP 6.0 platform (analysed using CNAG version 3.3.0 software) and standard Sanger Sequencing to detect genome wide copy number alterations (CNA), mutations in NOTCH1 (exons 26, 27, 34) FBXW7 (exons 9,10), PTEN (exon 7) and IL7R (exon 6) and to define the genomic DNA sequence of the STIL-TAL1 fusion in relevant cases. Genomic localisation of CNA was used to design patient-specific FISH probes for the STIL-TAL1 fusion and losses of CDKN2A, PTEN, LEF1, SYNCRIP (marker of 6q deletion), MYC (marker of chromosome 8 gain) and ETV6 (marker of 12p loss). TLX1 status was assessed using a commercial Cytocell FISH probe and TLX3 status using an in-house probe set. Interphase FISH scoring of one to two hundred methanol-acetic acid fixed nuclei per patient with four - five differentially labelled FISH probes allowed phylogenetic analysis and reconstruction of the evolution of the malignant clone. In two of the STIL-TAL1 cases, we completed xenograft transplants using NOD/ShiSCID/IL2Rgamma null (NOG) mice. Mutation screening, copy number analysis and FISH were performed on xenograft spleen DNA and methanol-acetic acid fixed spleen nuclei. ResultsConstruction of phylogenetic trees for each patient based on single cell four - five colour FISH analysis demonstrated the presence of two - four abnormal sub-clones per patient. Both linear clonal progression and branching sub-clonal patterns were observed. STIL-TAL1 fusion, TLX1 and TLX3 translocations were placed at the root of each phylogenetic tree as they were present in all cells with an abnormal FISH pattern signal; the majority (but not all) cells also demonstrated loss of CDKN2A. Copy number losses of PTEN and LEF1 as well as losses of 12p and 6q and gain of chromosome 8 occurred at a lower level than CDKN2A loss. Sequencing of the STIL-TAL1 fusion sequence in the two paired diagnosis/xenograft cases (A and B) demonstrated that the fusion sequence was conserved in the xenografts. Case A harboured a NOTCH1 PEST domain truncating mutation detected in the xenograft. Sequencing of the diagnostic sample was suggestive of the presence of the same mutation at low level. At diagnosis, case B was found to have a PTEN exon 7 truncating mutation which was not detectable by Sanger Sequencing in the xenograft. In case B, the dominant diagnostic clone assessed by FISH had the STIL-TAL1 fusion plus losses of CDKN2A, 4p and 6q, compared to the dominant clone in the paired xenograft, which harboured the STIL-TAL1 fusion and loss of CDKN2A but retained both copies of 4p and 6q. This dominant xenograft clone was present at low level in the diagnostic sample. ConclusionsThese results support the hypothesis that, in accordance with other types of leukaemia, intra or inter-chromosomal rearrangements (STIL-TAL1 fusion, TLX1 and TLX3 translocation) are early events. As the STIL-TAL1 fusion was present in all cells with an abnormal FISH signal pattern in all five cases and the fusion sequence was conserved in the xenograft transplants, this provides evidence to support the hypothesis that it is a founder event in the development of T-ALL. CDKN2A loss also occurred early in the evolution of the leukaemogenic clone, in line with its key role in T-ALL. Assessment of the order of acquisition of genetic events in T-ALL and their stability as evolution of the malignant clone unfolds are key factors to determine rational treatment targets for molecularly targeted therapies. Future work will incorporate mutations and gene fusions in to the single cell phylogenetic analysis using a multiplex Q-PCR approach. Disclosures:Ferrando:Pfizer: Research Funding; BMS: Research Funding.

Tissue microchimerism is increased during pregnancy: a human autopsy study.

Microchimerism is the occurrence of small populations of cells with a different genetic background within an individual. Tissue microchimerism is considered to be primarily pregnancy-derived and is often studied relative to female-dominant autoimmune diseases, pregnancy complications, malignancies, response to injury, and transplantation outcomes. A particular distribution pattern of chimeric cells across various organs was recently described in a model of murine pregnancies. Our aim was to determine the frequency and distribution of tissue microchimerism across organs during and after pregnancy in humans. We performed in situ hybridization of the Y chromosome on paraffin-embedded autopsy samples of kidneys, livers, spleens, lungs, hearts and brains that were collected from 26 women who died while pregnant or within 1 month after delivery of a son. Frequencies of chimeric cells in various tissues were compared with those of a control group of non-pregnant women who had delivered sons. Tissue microchimerism occurred significantly more frequently in the lungs, spleens, livers, kidneys and hearts of pregnant women compared with non-pregnant women (all P < 0.01). We showed that some of the chimeric cells were CD3+ or CD34+. After correction for cell density, the lung was most chimeric (470 Y chromosome-positive nuclei per million nuclei scored), followed by the spleen (208 Y+/10(6) nuclei), liver (192 Y+/10(6) nuclei), kidney (135 Y+/10(6) nuclei), brain (85 Y+/10(6) nuclei) and heart (40 Y+/10(6) nuclei). Data from this unique study group of women who died while pregnant or shortly after delivery provide information about the number and physiologic distribution of chimeric cells in organs of pregnant women. We demonstrate that during pregnancy, a boost of chimeric cells is observed in women, with a distribution across organs, that parallels findings in mouse models.

Editorial: Functional intracellular renin-angiotensin systems: potential for pathophysiology of disease

the renin-angiotensin system (RAS) is a coordinated hormonal cascade playing a major role in cardiovascular, renal, and adrenal homeostasis ([4][1]). Derangements in the RAS have been implicated in the pathogenesis of many disease states, including hypertension and of target organ damage related to

Application of cellular ELISA (CELISA) to the detection of human monoclonal autoantibodies

This work describes the application of cellular enzyme-linked immunosorbent assay (CELISA) for the detection of both polyreactive and monospecific human monoclonal antibodies against autoantigens. The CELISA is ideally suited for the screening of a large number of hybridoma culture supernatants, being, in this way, superior to other methods commonly used for the detection of autoantibody activity, such as indirect immunofluorescence on tissue sections and slide cell preparations, in terms of speed and sensitivity. This assay demonstrated higher sensitivity than ELISA using autoantigenic extracts from rabbit thymus, human spleen, nucleoprotamine, and salmon sperm nuclei, and enzyme immunoassays using ssDNA, dsDNA, and affinity purified autoantigens as substrate. The CELISA has been also successfully applied to the detection of autolymphocytotoxic antibody activity in heterohybridoma supernatants.

Tissue-specific interactions between nuclear proteins and the aminopeptidase N promoter.

Aminopeptidase N/CD13 is a metallopeptidase found in many tissues. Aminopeptidase N activity is high in the small intestinal mucosa, moderate in the liver, and low in the spleen. Using DNase I footprinting and electrophoretic mobility shift assays with nuclear extracts from these tissues, three cis elements (DF, LF-B1, UF) were identified in the aminopeptidase N promoter. The DF region (-53 to -30) interacts with the ubiquitously expressed transcription factor Sp1. The LF-B1 region (-85 to -58) interacts with the liver transcription factor LF-B1 (HNF-1) which was detected as well in nuclei from small intestinal mucosa. The UF region (-112 to -90) interacts with nuclear factors which seem to be expressed differentially in the liver and the small intestine. Transfection of promoter deletions into HepG2 cells showed that the LF-B1 region is necessary for high expression of the aminopeptidase N gene in liver cells. LF-B1 could not be detected in spleen nuclei. In accordance with this, RNA analysis demonstrated that the aminopeptidase N promoter operating in the small intestine and in the liver is inactive in the spleen. In this tissue initiation of transcription from the aminopeptidase N gene occurs from an upstream promoter.

A liver-specific nuclear protein that binds to the distal promoter element of the rat tyrosine aminotransferase gene

Using a gel retardation assay and exonuclease III footprinting, we have analyzed sequence-specific DNA-binding nuclear factors which interact with the distal promoter element of the rat tyrosine aminotransferase gene. A factor called LspA1, binding to a sequence that resembles the consensus binding site for the transcription factor Ap-1, was shown to be present in adult rat-liver nuclear protein extracts but not in the extracts from embryonic liver or spleen nuclei.

The interplay of DNA-binding proteins on the promoter of the mouse albumin gene.

The promoter of the mouse albumin gene contains at least six binding sites for specific DNA-binding proteins (A to F). Four of these sites (A, D, E, and F) can be occupied by transcription factors that are considerably enriched in liver nuclei, as compared to spleen or brain nuclei. These factors consist of a heat-stable protein that fills sites A, D, and F, and a member of a family of nuclear factor I (NF-I) related proteins that occupies site E. Site C binds a protein that is equally abundant in liver, brain, and spleen nuclei. Occupancy of this site and the binding of the heat-stable factor to the immediately adjacent site D appear to be mutually exclusive. However, both of these competing binding sites are required for maximal in vitro transcription.

Two species of chromatin-RNA polymerase II complex are commonly present in nuclei of various tissues of rats.

When rat liver nuclei were digested with nuclease, we found that the chromatin-bound RNA polymerase II was liberated as two distinct complexes, peak 1 and peak 2, which seemed to reflect different functional states in cell nuclei. We further examined their occurrence in nuclear digests of various tissues of rats and the following results were obtained. Upon digestion with micrococcal nuclease of nuclei from brain, spleen, testis and kidney, chromatin-bound RNA polymerase II was liberated as two distinct forms which sedimented differently in a sucrose density gradient. The sedimentation rate of peak 1 varied depending on the tissue nuclei examined. After high salt or RNase treatment of the nuclear digests, peak 1 from liver, brain, spleen and testis nuclei showed the same sedimentation rate as did kidney peak 1, the rate for which remained unchanged by these treatments. The results suggested that peak 1 complexes from various tissue nuclei had basically the same structural organization, and we confirmed this by electrophoretic studies on RNase-treated liver and kidney nuclear digests. Peak 2 from various tissue nuclei exhibited identical sedimentation rates. Thus, the chromatin-bound RNA polymerase II seems to exist commonly in two distinct states in cell nuclei of rats.

Inhibition of DNA methyltransferases in vitro by benzo[a]pyrene diol epoxide-modified substrates.

Covalent adducts formed from the ultimate carcinogen 7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene inhibit the enzyme-catalyzed transfer of methyl groups from S-adenosylmethionine to cytosine residues in DNA. Two DNA methyltransferase enzymes, isolated from the bacterium Haemophilus and mouse spleen nuclei, were tested for their ability to methylate carcinogen-modified substrates in vitro. These model enzymes possess the known methylation activities found in mammalian cells, de novo, and maintenance methylation of CpG-containing nucleotide sequences. The in vitro alkylation of DNA substrates by the carcinogen effectively decreases the methyltransferase reaction of both enzymes in a manner that is directly dependent upon the level of covalent modification of the DNA. Inhibition of de novo methylation activity can be detected at very low levels of carcinogen modification, 1 hydrocarbon residue per 20,000-40,000 nucleotides. Adduct levels in this range are capable of initiating transformation. Both enzymes are inactivated by direct reaction with the carcinogen in the absence of DNA. We also find that carcinogen adducts are capable of inhibiting DNA methylation at CpG sites removed from the primary lesion. These results support the proposal that carcinogen-induced DNA damage can cause alterations in methylation patterns that may eventually lead to heritable changes in gene expression.

Evidence for attachment of interphase chromatin to the nuclear matrix via matrix-bound nucleosomes

Chromatin structure has been studied in the sites of attachment to the nuclear matrix in interphase mouse liver and spleen nuclei. The patterns of fragmentation of the DNA belonging to these sites (0.3–2% of total DNA in spleen and liver, respectively) with staphylococcal nuclease and DNAase I were very close to those of usual nucleosomal chains. Moreover, the nuclear matrix preparations contained all five major histones, including H1, in almost stoichiometric amounts. The histone/DNA ratios for the matrix were also similar to those found in nuclei. These findings and the size of the matrix-protected DNA indicated that interphase chromatin was attached to the nuclear matrix via matrix-bound nucleosomes and, to a much lesser extent, oligonucleosomes up to 5–6 units long. Two-dimensional electrophoretic separation of the matrix-bound histones revealed that modifications of histone H1 and, probably, of other histones were distinguished from those in bulk chromatin. Study of binding of exogenously added labeled histone octamers or mononucleosomal size DNA to nuclear matrix excluded the possibility of their artifactual trapping during the isolation procedure.

Comparison of thyroid hormone binding to heptic nuclei of the rat and a teleost (Oncorhynchus kisutch).

A modified T3 binding assay was used with coho salmon liver nuclei. Purified nuclei were incubated with [125I]T3 (or [125I]T4), nonradioactive thyroid hormone, or thyroid hormone analog. Bound hormone was separated from hormone by use of polyethylene glycol. Salmon liver nuclei specifically bound T3 with a high affinity [Ka = 1.031 +/- 0.174 x 10(9) M-1 (mean +/- SE); n = 12] which was 50% of the affinity measured with rat liver nuclei using the same assay. Salmon nuclear T3-binding capacity was 0.127 +/- 0.035 pmol T3/mg DNA (n = 7). Rat liver nuclear capacity for T3 binding was 3.4-fold greater than in salmon liver nuclei. T3 binding to salmon liver nuclei was greater than to nuclei from salmon brain; T3 binding to salmon spleen nuclei was low. Binding competition studies with hormone analogs indicated that binding to the salmon nuclei was dependent on molecular structure. These data indicate that the T3 binding to salmon nuclei may be due to T3 receptor activity. Analysis of Scatchard plots showed that the affinity of salmon hepatic nuclei for T3 is 10-fold greater than the affinity for T4. Reciprocal binding competition experiments with T3 and T4 indicate that both hormones bind to the same site. This suggests that in salmon, as in mammals, the liver nuclei predominantly bind thyroid hormones at T3-binding sites. Hepatic nuclei of human, rat, chick embryo, amphibian larva, and salmon have similar affinities for thyroid hormones and similar relative affinities for several thyroid hormone analogs, suggesting that the receptor molecule has been highly conserved during evolution.

Specific nuclear binding of angiotensin II by rat liver and spleen nuclei.

1. Nuclei were isolated from rat liver and spleen by a standard technique which preserved the nuclear membrane. Electron microscopy, DNA/protein ratios and 5′-nucleotidase determinations confirmed the purity of the preparation. 2. Both liver and spleen nuclei specifically bound 125I-labelled angiotensin II (ANG II) with high affinity (Kd ≅ 1.0 nmol/l). Saralasin ANG III and ANG II competed with tracer for ligand whereas ANG I did so less effectively and neurotensin not at all. 3. These results suggest the presence of specific nuclear intracellular ANG II receptors and raise important questions regarding the mechanism of action of this peptide.

Glutathione reductase activity of chromatin isolated from mouse spleen nuclei

1. 1. Chromatin isolated from either lymphoid or erythroblastic mouse spleen cell nuclei exhibit distinct glutathione reductase activity but only insignificant activities of disulphide reductases independent of GSSG. 2. 2. Glutathione reductase activity of the isolated chromatin has been found to be affected neither by repeated washing of chromatin nor supplementation of the nucleus lysate with cytosol containing high amount of GSSG reductase. 3. 3. Disulphide reductase activities of the same order were found in nuclear material from normal and erythroblastic spleen nuclei.

Role of Alkaline Endonucleases in the Release of Soluble Chromatin from Thymus, Spleen and Liver Nuclei of Normal and Irradiated Mice

Thymus, spleen and liver nuclei released a large fraction of soluble chromatin in vitro when incubation was carried out in sucrose media containing low concentrations of CaCl2 and/or MgCl2. A significant fraction of deoxyribopolynucleotides (DPN) was also extracted from nuclei. After 30 min of incubation at 37 degrees C, the maximum release of soluble chromatin was observed near a pH of 8, which corresponds to the optimum pH of the alkaline endonuclease activity from thymus, spleen and liver. The soluble chromatin and DPN were precipitated by increasing the bivalent ion concentration of the medium. The protein/DNA ratio and the molecular weight of DNA suggest that the soluble chromatin and DPN represent nucleosome-like particles. The release of soluble chromatin in the first 4 hours of incubation was significantly increased if the nuclear fraction was isolated from the thymus and spleen of whole-body irradiated mice (1000 rad). This effect was absent in the liver nuclei.

Hemoglobin switching in sheep: Only the γ gene is in the active conformation in fetal liver but all the β and γ genes are in the active conformation in bone marrow

Differential expression of the closely linked gamma, beta(A) (or beta(B)), and beta(C) globin genes in sheep results in the production of fetal hemoglobin (Hb F, alpha(2)gamma(2)) during gestation and the adult hemoglobins (Hb A, alpha(2)beta(2) (A), and Hb B, alpha(2)beta(2) (B)) after birth. Erythropoietic stress in certain animals leads to production of Hb C (alpha(2)beta(2) (C)). The molecular mechanism of differential expression of these genes in nuclei of fetal and adult erythroid cells has been investigated by analysis of their susceptibility to digestion by DNase I (genes that are in the conformation associated with active transcription are sensitive to this nuclease). The concentration of globin gene sequences in DNA from control and DNase I-digested nuclei was determined by annealing to synthetic DNAs and analogous cDNA probes derived from recombinant plasmids containing one of the sheep globin genes. In nuclei from sheep fetal liver erythroid cells, the gamma genes but not the beta genes were digested by DNase I; the gamma locus was open but the beta(A) or beta(C) loci was closed, consistent with synthesis of only Hb F by these cells. DNase I digestion of nuclei from bone marrow of anemic sheep making only Hb C or Hb B resulted in equivalent digestion of the beta and gamma gene sequences, although gamma mRNA was not detected in these cells. Digestion by DNase I did not decrease the globin gene sequence concentration in residual DNA of spleen nuclei. As a further control, DNA from digested bone marrow and spleen nuclei were shown to anneal equally well to a cDNA prepared from liver polysomal mRNA. Differential expression of the gamma and beta globin genes in sheep fetal erythroid cell appears to be based on differences in chromatin structure. The gamma globin gene remains in the active conformation in adult erythroid cells; failure of gamma mRNA to accumulate in these cells probably reflects transcriptional or post-transcriptional regulation.

Effects of disulphides and α-oxoglutarate on nuclear thiol formation and thiol content of chromatin in lysed rat spleen nuclei

1. 1. During incubation of lysed rat spleen nuclei, there was a noted increase in total SH, accompanied by a slight decrease of chromatin SH and a more pronounced increase in SH of the non-sedimentable fraction. 2. 2. During incubation, chromatin nucleolytic cleavage was completely inhibited by 1 mM cystamine and 1 mM oxidized glutathione (GSSG). 3. 3. In nucleus lysates, glutathione reductase activity dependent on NADPH, and disulphide reductase NADH-dependent reducing disulphides of the non-sedimentable fraction, but not affecting GSSG, were demonstrated. 4. 4. In the presence of 1 mM GSSG there was a transfer of SH from the non-sedimentable fraction to the rat spleen chromatin, while 1 mM 6,8-thioctic acid exerted an opposite effect. Cystamine (1 mM) was found to decrease SH content of either chromatin and the non-sedimentable fraction. 5. 5. Reduced glutathione (GSH) and cysteamine (MEA) and CoA did not change chromatin SH. 6. 6. In the presence of α-oxoglutarate, as in the presence of GSSG, an increase in chromatin SH was found, indicating the role of dihydrolipoate in the control of chromatin SH level.

Sensitivity to α-amanitin of multiple DNA-dependent RNA polymerases from experimental tumors

Summary The distribution and properties of DNA-dependent RNA polymerases separated by DEAE-Sephadex chromatography from extracts obtained after lysis of transplanted tumor nuclei were compared with those from rat spleen and kidney nuclei. The effect of α-amanitin has been investigated on the multiple forms of the enzyme isolated from normal and neoplastic extracts. The results show that while from rat spleen and kidney only two peaks containing polymerase activity were obtained, from the nuclei of four experimental tumors investigated, three distinct peaks were separated. On the basis of the degree of inhibition by α-amanitin, it was possible to classify the first two activities from tumor tissue as belonging to Class A and the third to Class B.

Binding of cytoplasmic 5alpha-dihydrotestosterone-receptor complex by nuclei from the ventral prostate, liver, and spleen of rats.

Cytoplasmic 3H-5alpha-dihydrotestosterone-receptor complex of the rat ventral prostate was efficiently taken up by nuclei isolated from the liver, the ventral prostate and the spleen. The amount of the complex bound to liver nuclei was approximately 3 times that to prostate nuclei, and a relatively small amount of the complex was taken up by spleen nuclei under the incubation conditions: 0.16 nM complex (2.0 mg as protein) was used. Binding of the complex to liver nuclei was low-affinity and non-saturable, while in nuclei from the ventral prostate and the spleen, high-affinity and saturable binding was observed. Kd of nuclear binding in the latter two tissues was roughly of the order of 10(-10) M. Castration caused a reduction in the binding of the complex to prostate nuclei and replacement therapy with testosterone propionate completely blocked the effect of castration on the nuclear binding. The affinity of high-affinity binding was not changed significantly after castration. No significant effects of castration and testosterone treatment were observed in the binding of nuclei from the other two tissues examined. The physiological significance of the nuclear binding of hormone-receptor complexes in the action of steroid hormones is discussed.

Incorporation of 35S-cystamine into rat spleen nuclear proteins and its influence on the SH level and on chromatin nucleolytic degradation

1. 1. Incorporation of 345S-cystamine in vitro to rat spleen nuclei, chromatin, histones and non-histone proteins, was investigated. Whole nuclei incorporate labelled disulphide quicker than chromatin. Among chromatin proteins, the highest cystamine. level was found in non-histone proteins. 2. 2. The process of incorporation of cystamine into nuclei was accompanied by a decrease in SH groups of chromatin was well as in the whole bulk of nuclear SH. It is supposed that cystamine suppresses enzymatic formation of SH groups in the nucleus. 3. 3. Cystamine completely inhibits the process of chromatin degradation by endogenic nucleases, which may be the result of either inhibition of the respective enzyme or disulphide modification of chromatin.