Postnatal development- and age-related changes in DNA-methylation patterns in the human genome.

Alterations in DNA methylation have been reported to occur during development and aging; however, much remains to be learned regarding post-natal and age-associated epigenome dynamics, and few if any investigations have compared human methylome patterns on a whole genome basis in cells from newborns and adults. The aim of this study was to reveal genomic regions with distinct structure and sequence characteristics that render them subject to dynamic post-natal developmental remodeling or age-related dysregulation of epigenome structure. DNA samples derived from peripheral blood monocytes and in vitro differentiated dendritic cells were analyzed by methylated DNA Immunoprecipitation (MeDIP) or, for selected loci, bisulfite modification, followed by next generation sequencing. Regions of interest that emerged from the analysis included tandem or interspersed-tandem gene sequence repeats (PCDHG, FAM90A, HRNR, ECEL1P2), and genes with strong homology to other family members elsewhere in the genome (FZD1, FZD7 and FGF17). Our results raise the possibility that selected gene sequences with highly homologous copies may serve to facilitate, perhaps even provide a clock-like function for, developmental and age-related epigenome remodeling. If so, this would represent a fundamental feature of genome architecture in higher eukaryotic organisms.

The circadian expression of c-MYC is modulated by the histone deacetylase inhibitor trichostatin A in synchronized murine neuroblastoma cells.

Circadian clocks govern the mammalian physiology in a day/night-dependent manner. The circadian oscillator of peripheral organs is composed of the same elements as the central pacemaker at the suprachiasmatic nucleus (SCN). The interaction between the circadian clock and several cell cycle components has been established in recent years, since many key regulators of cell cycle and growth control were proved to be rhythmically expressed. In particular, the proto-oncogene c-Myc has been documented to be under circadian regulation. Given that it is overexpressed in many malignancies, the study of c-Myc mRNA and c-MYC protein regulation by the circadian clock is of great interest. Thus, the aim of this work was to: (a) analyze in detail the circadian oscillations of c-Myc steady-state mRNA levels and to investigate whether c-MYC protein levels display any oscillating pattern, and (b) ascertain whether circadian time is important for reducing c-MYC levels after drug application. For this purpose, we selected trichostatin A (TSA), since it is known that long (>or=12 h) treatment durations negatively influence the expression levels of c-Myc and short 2 h treatments up regulate the expression of the central oscillator gene Per1 resulting in the resetting of its rhythm. TSA is a specific inhibitor of histone deacetylases (HDACs), and its application results in increased acetylation levels of histone and non-histone proteins. Our results, using the murine neuroblastoma cell line N2A, show that Per1 and c-Myc steady-state mRNA levels oscillate with the same phase. Moreover, a short 2 h TSA treatment causes a phase-dependent decrease of oscillating c-Myc transcript levels only when applied at the trough of its mRNA rhythm, where a general decrease of c-MYC protein levels is also observed. At the peak of its rhythm, no apparent changes can be observed. These experiments demonstrate for the first time that a significant decrease in c-Myc transcript and protein levels can be achieved after a short TSA treatment applied only at specific circadian times. This is also followed by a reduction in the proliferation rate of the cell population.

Histone H1 subtype preferences of DFF40 and possible nuclear localization of DFF40/45 in normal and trichostatin A-treated NB4 leukemic cells.

A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.

Differential sensitivity of human leukemic cell lines to the histone deacetylase inhibitor, trichostatin A.

Histone deacetylase inhibitors (HDACIs) inhibit deacetylases and the accumulation of high levels of acetylation results in chromatin remodeling events which may lead to cell cycle arrest and apoptosis. This work investigates the sensitivity of four leukemic cell lines to the HDACI, trichostatin A (TSA) as compared to normal lymphocytes with respect to acetylation and apoptotic levels. Specifically, this study analyzes the time kinetics of histone H4 and alpha-tubulin acetylation and associates these findings to the time course of TSA-induced PARP cleavage and DFF45 proteolysis. The results of this study show (1) that a non-responsive leukemic cell line to the apoptotic effects of TSA does not have increased acetylation levels in contrast to the responsive leukemic cell lines that show a hyperacetylated profile. This indicates that acetylation levels may be of special importance in accessing the potential sensitivities of leukemic cells to HDACIs, (2) TSA induced apoptosis in lymphocytes but at lower levels and (3) the lack of PARP cleavage and DFF45 proteolysis found in lymphocytes clearly differentiates the final stages apoptosis of human peripheral blood lymphocytes from those of the TSA-sensitive leukemic cell lines. Of value is that the results of this study show that the evaluation of the acetylation levels of target proteins may possibly have the potential of being used as additional indicators of the responsiveness or sensitivity of different cancer cell types to this continuously growing class of anticancer agents.

H1 histone subtype constitution and phosphorylation state of the ageing cell system of human peripheral blood lymphocytes.

Until a few years ago, the H1 histones were exclusively considered to be the architectural proteins of chromatin involved in chromatin condensation. However there is now increasing data to support the hypothesis that the H1 subtypes are involved in genomic integrity and that they may have unexpected functional roles in various biological processes such as in differentiation and DNA repair, apoptosis and lifespan. Moreover, the H1 histones are phosphorylated to a great extent. Recent work has implicated phosphorylation of H1 in the regulation of chromatin remodeling. In light of the fact that chromatin reorganization and heterochromatin formation has been shown to take place during ageing and senescence, in the present investigation, we have analyzed the changes that take place in the somatic H1 linker histone subtype profile and their phosphorylation states in human peripheral blood lymphocytes as a function of donor age. Results from this work show that there is a significant age-related dephosphorylation of H1.4 and H1.5 and an increase in the heterochromatin protein HP1alpha as a function of donor age. These results indicate that dephosphorylation of H1 histones may be related to an increase in senescence-associated heterochromatin formation during the in vivo ageing of human peripheral blood lymphocytes.

The differentiation-associated linker histone, H1.0, during the in vitro aging and senescence of human diploid fibroblasts.

There are numerous similarities between aging/senescence and differentiation. One key similarity is that in both biological processes chromatin remodeling events occur. It is now known that during both processes there is a reorganization of eu- and heterochromatic domains and an increase in heterochromatin, known as heterochromatinization. Previous work of more than two decades has shown that the replacement H1 linker histone subtype, H1.0, accumulates during terminal differentiation in numerous cell/tissue systems. However, work with this differentiation-associated H1 subtype in aging cell systems has only recently been accomplished. In this article, we outline the cumulative results from our investigations of H1.0 protein and mRNA levels in the in vitro aging cell system of human diploid fibroblasts (HDFs) and discuss the potential rationale of why this particular subtype was found to accumulate during both these processes.

Age-dependent response of lymphocytes in the induction of the linker histone variant, H1 degrees and histone H4 acetylation after treatment with the histone deacetylase inhibitor, trichostatin A.

In the present study we investigated the age-related response of Phytohemaglutinin (PHA)-activated S phase human lymphocytes isolated from peripheral blood from donors of four different age groups, namely young (25-30 years), mid-aged (40-45 years), senior (60-65 years) and elderly (80-95 years) on the induction of the linker histone variant, H1 degrees and histone H4 acetylation after treatment with the very specific histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). The cell system of peripheral blood lymphocytes is ideal for the study of H1 degrees induction since they do not synthesize this particular linker histone variant. Lymphocytes isolated from peripheral blood were activated with PHA (5 microg/10(6) cells/ml medium) and placed in culture for a duration of 72 h at which time cells are in the S phase. Forty-eight hours after inoculation, TSA (250 ng/10(6) cells/ml medium) was added to the cell cultures for a period of 24 h. Assays were performed 72 h after initiation of cultures. The results showed that the induction of H1 degrees after TSA treatment increased to a statistically significant degree in the elderly age group with respect to both the young and the mid-aged age groups. Moreover histone H4 acetylation was found to increase as a function of increasing donor age. A hyperacetylation pattern was observed even in the youngest age group analyzed. Specifically, the tetra-acetylated (H4.4) H4 form increased to a statistically significant degree with the concomitant decrease in the non-acetylated H4 for (H4.0) as a function of donor age. The other acetylated H4 forms (H4.1, H4.2, and H4.3) remained more or less constant, irrespective of donor age. These results show that the sensitivity of lymphocytes to TSA is enhanced with increasing donor age. Since to date, 11 class I and II HDACs have been isolated that have been found by other investigators to have differential responses to HDAC inhibitors, these findings may indicate that there is also a differential age-related response of certain HDACs or perhaps a senescent-specific HDAC. This line of research warrants further study.

Lymphocytes from bipolar and schizophrenic patients share common biochemical markers related to histone synthesis and histone cell membrane localization characteristic of an activated state.

In a previous communication, based on the total histone and histone variants' synthesis rates, biochemical parameters used for the characterization of the activation state of lymphocytes, we showed that a portion of the lymphocyte population obtained from peripheral blood of patients with bipolar disorder in the manic and/or depressed phases of the illness were in an activated state as opposed to normothymic patients and control subjects whose lymphocytes are in a resting, Go, state. In light of these previous findings, in the present investigation, we have analyzed total histone synthesis rates and the H2A and H3 histone variants' synthesis pattern of acid-extracted histones from the lymphocytes' nuclear fraction obtained from control subjects, patients with bipolar disorder in all phases of the illness, and patients with schizophrenia. Additional biochemical parameters, such as total cellular protein and DNA synthesis rates, were also studied. Moreover, recent findings from other investigators showed the association of histones on the plasma membrane fraction of PHA-activated, but not Go resting lymphocytes. Based on these results, acid-extracted proteins from the plasma membrane fraction obtained from control, bipolar patients in all phases of the illness, and schizophrenic patients were analyzed by immunoblotting using a polyclonal histone antibody, anti-H2B. All biochemical parameters tested show that a portion of the lymphocyte population from bipolar, i.e. manic and depressive, as well as schizophrenic patients are in an activated state and clearly indicate that the unusual for lymphocytes cell cycle-related histone biochemical properties are common to both disorders.

The effect of the histone deacetylase inhibitor, trichostatin A, on total histone synthesis, H1(0) synthesis and histone H4 acetylation in peripheral blood lymphocytes increases as a function of increasing age: a model study.

A pilot study was initiated in order to ascertain whether the age of the donor might affect either the induction of the expression of H1(0) or histone H4 acetylation by the very specific histone deacetylase inhibitor, trichostatin A. This was investigated in a cell system which normally does not express this linker histone variant, i.e. peripheral blood lymphocytes (PBL), which were obtained from donors of different ages (25-95 years). Forty-eight hours after activation by the mitogen phytohemaglutinin (PHA), 250 ng of trichostatin A per 10(6) cells per ml culture medium was added and cultured for an additional 24h. Assays were performed 72 h after initiation of cultures, i.e. during the S phase. It was found that in PBL, trichostatin A induced the expression of the linker histone variant, H1(0) as well as histone H4 acetylation, and, more importantly, that these effects were enhanced with increasing age of the donor. More specifically, under the influence of trichostatin A, PBL showed increasing H1(0) synthesis rates and increasing levels of histone H4 acetylation as a function of increasing age of the donor. Moreover, although trichostatin A induced an increasing expression of H1(0) with increasing age, it also concomitantly partially inhibited S phase total histone synthesis. This inhibition also increased as a function of increasing age of the donor.