Identification of lamin B–regulated chromatin regions based on chromatin landscapes

Lamins, the major structural components of the nuclear lamina (NL) found beneath the nuclear envelope, are known to interact with most of the nuclear peripheral chromatin in metazoan cells. Although NL–chromatin associations correlate with a repressive chromatin state, the role of lamins in tethering chromatin to NL and how such tether influences gene expression have remained challenging to decipher. Studies suggest that NL proteins regulate chromatin in a context-dependent manner. Therefore understanding the context of chromatin states based on genomic features, including chromatin–NL interactions, is important to the study of lamins and other NL proteins. By modeling genome organization based on combinatorial patterns of chromatin association with lamin B1, core histone modification, and core and linker histone occupancy, we report six distinct large chromatin landscapes, referred to as histone lamin landscapes (HiLands)-red (R), -orange (O), -yellow (Y), -green (G), -blue (B), and -purple (P), in mouse embryonic stem cells (mESCs). This HiLands model demarcates the previously mapped lamin-associated chromatin domains (LADs) into two HiLands, HiLands-B and HiLands-P, which are similar to facultative and constitutive heterochromatins, respectively. Deletion of B-type lamins in mESCs caused a reduced interaction between regions of HiLands-B and NL as measured by emerin–chromatin interaction. Our findings reveal the importance of analyzing specific chromatin types when studying the function of NL proteins in chromatin tether and regulation.     

Translocation of dna polymerase-α during the mitotic cycle of Physarum polycephalum

The location of DNA polymerase-α within nuclei of a macroplasmodium of Physarum polycephalum was identified at successive stages of the highly synchronous mitotic cycle by indirect immunofluorescence. The distribution of α-tubulin, the nucleolar protein fibrillarin and DNA were also identified.In G2-phase the polymerase-α was found mainly in the region of the nucleolus. In prophase the polymerase became dispersed into the nucleus with the breakdown of the nucleolus. In metaphase, the polymerase-α was located in two zones separated by the metaphase plate. In S-phase, which follows immediately after mitosis, the distribution of the polymerase was initially punctate but later segments were noted during nucleolar reconstruction. We infer that DNA polymerase-α is stored mainly in the region of the nucleolus (or the reconstructing nucleolus) when it is not needed for DNA replication.     

Aging by epigenetics--a consequence of chromatin damage?

Chromatin structure is not fixed. Instead, chromatin is dynamic and is subject to extensive developmental and age-associated remodeling. In some cases, this remodeling appears to counter the aging and age-associated diseases, such as cancer, and extend organismal lifespan. However, stochastic non-deterministic changes in chromatin structure might, over time, also contribute to the break down of nuclear, cell and tissue function, and consequently aging and age-associated diseases.     

CTCF shapes chromatin by multiple mechanisms: the impact of 20 years of CTCF research on understanding the workings of chromatin

More than 10⁹ base pairs of the genome in higher eucaryotes are positioned in the interphase nucleus such that gene activation, gene repression, remote gene regulation by enhancer elements, and reading as well as adjusting epigenetic marks are possible. One important structural and functional component of chromatin organization is the zinc finger factor CTCF. Two decades of research has advanced the understanding of the fundamental role that CTCF plays in regulating such a vast expanse of DNA.     

The effect of heparin on the porcine lymphocyte chromatin—I. The comparative study of DNA in different chromatin fractions

• 1.1. Porcine lymphocyte chromatin in the solution of 0.15 M NaCl + 0.01 M Tris. pH 7 treated with heparin liberated 30% protein and 7.5% DNA to the supernatant.
• 2.2. DNA from the supernatant and the pellet fractions as well as from control chromatin were isolated in identical conditions.
• 3.3. No significant changes were observed in spectral properties and melting points in SSC of comparable DNA specimens.
• 4.4. It was noted, however, that DNA of the supernatant is subject to denaturation in the process of isolation, which, apart from the difference in protein composition of the supernatant and the pellet fractions, suggests different chromatin structure of these fractions.

     

Characterization of dna polymerase α from untransformed and pSV3.neo-transformed human fibroblasts

• 1.1. The specific activity of DNA-polymerase α isolated from pSV3.neo-transformed cells was more than 9-fold higher than that of polymerase a from untransformed cells.
• 2.2. Western blot analysis, using anti-SV40 large T antigen, of both a crude cellular extract and of partially purified polymerase a from pSV3.neo-transformed cells revealed a single 76 kDa immunoreactive band not found in either crude extracts or partially purified enzyme from untransformed cells.
• 3.3. The α polymerases from untransformed and transformed cells differed in molecular size, sensitivity to various inhibitors, specificity of template-primer utilization, and binding affinity for DNA cellulose, but showed essentially no differences in K_m or V_max.
• 4.4. These data suggest that polymerase α isolated from pSV3.neo-transformed cells exhibits altered physical and catalytic characteristics compared with its untransformed cell counterpart, and that those alterations may be associated with increased replication of the genome in plasmid-transformed cells.

     

Neutrophil extracellular traps: Is immunity the second function of chromatin?

Neutrophil extracellular traps (NETs) are made of processed chromatin bound to granular and selected cytoplasmic proteins. NETs are released by white blood cells called neutrophils, maybe as a last resort, to control microbial infections. This release of chromatin is the result of a unique form of cell death, dubbed "NETosis." Here we review our understanding of how NETs are made, their function in infections and as danger signals, and their emerging importance in autoimmunity and coagulation.     

Quantitative analysis of chromatin compaction in living cells using FLIM–FRET

We present a quantitative Förster resonance energy transfer (FRET)–based assay using multiphoton fluorescence lifetime imaging microscopy (FLIM) to measure chromatin compaction at the scale of nucleosomal arrays in live cells. The assay uses a human cell line coexpressing histone H2B tagged to either enhanced green fluorescent protein (FP) or mCherry FPs (HeLa^H2B-2FP). FRET occurs between FP-tagged histones on separate nucleosomes and is increased when chromatin compacts. Interphase cells consistently show three populations of chromatin with low, medium, or high FRET efficiency, reflecting spatially distinct regions with different levels of chromatin compaction. Treatment with inhibitors that either increase chromatin compaction (i.e., depletion of adenosine triphosphate) or decrease chromosome compaction (trichostatin A) results in a parallel increase or decrease in the FLIM–FRET signal. In mitosis, the assay showed variation in compaction level, as reflected by different FRET efficiency populations, throughout the length of all chromosomes, increasing to a maximum in late anaphase. These data are consistent with extensive higher order folding of chromatin fibers taking place during anaphase.     

Is adult stem cell aging driven by conflicting modes of chromatin remodeling?

Epigenetic control of gene expression by chromatin remodeling is critical for adult stem cell function. A decline in stem cell function is observed during aging, which is accompanied by changes in the chromatin structure that are currently unexplained. Here, we hypothesize that these epigenetic changes originate from the limited cellular capability to inherit epigenetic information. We suggest that spontaneous loss of histone modification, due to fluctuations over short time scales, gives rise to long-term changes in DNA methylation and, accordingly, in gene expression. These changes are assumed to impair stem cell function and, thus, to contribute to aging. We discuss cell replication as a major source of fluctuations in histone modification patterns. Gene silencing by our proposed mechanism can be interpreted as a manifestation of the conflict between the stem cell plasticity required for tissue regeneration and the permanent silencing of potentially deleterious genomic sequences.     

Non-histone chromatin protein fractions associated with ‘active’ chromatin in embryonic chicken liver

Non-histone chromatin proteins synthesized during chicken embryonic liver development were labeled with [3H]tryptophan and [3H]methionine and characterized by electrophoresis. During embryonic development protein/DNA ratio in chromatin was low (1.30-1.62) but synthesis of non-histone protein was high. Especially one characteristic fraction K (MW 18 000), tightly bound with DNA was preferentially associated with DNAase II sensitive, active transcribed sequences. In 7-day old and adult chicken synthesis of all non-histone proteins was low, fraction K was absent or synthesized only in small amounts in association with non-active sequences, however protein/DNA ratio in chromatin was high (2.30-2.33).     

The NuRD chromatin–remodeling complex regulates signaling and repair of DNA damage

Cells respond to ionizing radiation (IR)–induced DNA double-strand breaks (DSBs) by orchestrating events that coordinate cell cycle progression and DNA repair. How cells signal and repair DSBs is not yet fully understood. A genome-wide RNA interference screen in Caenorhabditis elegans identified egr-1 as a factor that protects worm cells against IR. The human homologue of egr-1, MTA2 (metastasis-associated protein 2), is a subunit of the nucleosome-remodeling and histone deacetylation (NuRD) chromatin-remodeling complex. We show that knockdown of MTA2 and CHD4 (chromodomain helicase DNA-binding protein 4), the catalytic subunit (adenosine triphosphatase [ATPase]) of NuRD, leads to accumulation of spontaneous DNA damage and increased IR sensitivity. MTA2 and CHD4 accumulate in DSB-containing chromatin tracks generated by laser microirradiation. Directly at DSBs, CHD4 stimulates RNF8/RNF168-dependent formation of ubiquitin conjugates to facilitate the accrual of RNF168 and BRCA1. Finally, we show that CHD4 promotes DSB repair and checkpoint activation in response to IR. Thus, the NuRD chromatin–remodeling complex is a novel regulator of DNA damage responses that orchestrates proper signaling and repair of DSBs.