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Results for “chromatin recording”

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At least 19 recordsLinked to original sources
PubMed2026

Genome-wide chromatin recording resolves dynamic cell state changes.

Understanding how the chromatin state of a cell influences its future behavior is a major challenge throughout biology. However, most chromatin profiling methods are limited to endpoint assays. Here, we present LagTag, a method for recovery of earlier and endpoint chromatin states in the same mammalian cells. In this approach, transient activity of bacterial adenine methyltransferase fusions records the DNA-binding profiles of chromatin-associated proteins of interest at earlier time points. Subsequent tagmentation and sequencing recover the earlier chromatin profile from adenine methylation profiles, alongside endpoint profiles of endogenous chromatin-associated proteins. We verified that LagTag profiles aligned with those from established methods in mouse and human cells. We then applied LagTag to record and recover dynamic chromatin state transitions during mouse embryonic stem cell differentiation, capturing transcriptional signatures from pre- and post-differentiation time points within the same cell population. LagTag thus provides a foundation for temporally resolved chromatin profiling. A record of this paper's transparent peer review process is included in the supplemental information.

chromatin organization↗
PubMed2026

Genome-wide chromatin recording resolves dynamic cell state changes.

Understanding how the chromatin state of a cell influences its future behavior is a major challenge throughout biology. However, most chromatin profiling methods are limited to endpoint assays. Here, we present LagTag, a method for recovery of earlier and endpoint chromatin states in the same mammalian cells. In this approach, transient expression of bacterial adenine methyltransferase fusions records the DNA binding profiles of chromatin-associated proteins of interest at earlier timepoints. Subsequent tagmentation and sequencing recovers the earlier chromatin profile from adenine methylation profiles, alongside endpoint profiles of endogenous chromatin-associated proteins. We verified that LagTag profiles aligned with those from established methods in mouse and human cells. We then applied LagTag to record and recover dynamic chromatin state transitions during mouse embryonic stem cell differentiation, capturing transcriptional signatures from pre- and post-differentiation timepoints within the same cell population. LagTag thus provides a foundation for temporally resolved chromatin profiling.

Journal Article↗
PubMed2025

CUT&TIME captures the history of open chromatin in developing neurons.

Chromatin structure plays a central role in defining cell identity by regulating gene expression. During development, shifts in chromatin structure facilitate changes in gene expression needed to specify distinct cell types. To understand how changes in chromatin structure influence the developmental trajectory of neural progenitor cells, we developed CUT&TIME, a technique that uses a hyperactive 6-methyl adenosine (6mA) methyltransferase pulsed in living cells to map historical chromatin accessibility genome-wide in single cells. We show that CUT&TIME produces a record of the chromatin landscape during neurogenesis in the developing retina, specifically as neural progenitors produce the major projection neuron type, retinal ganglion cells (RGCs). We further show that this method is compatible with single cell profiling technologies, which allows us to visualize and capture the diversity of chromatin states that produce RGCs. Additionally, we identify changes in promoter accessibility associated with the transition from progenitor to RGC. Together, these data demonstrate that CUT&TIME captures a historical record of chromatin structure, which can be used to identify early changes in accessibility associated with cell-fate commitment.

Journal Article↗
PubMed2006

EM measurements define the dimensions of the "30-nm" chromatin fiber: evidence for a compact, interdigitated structure.

Chromatin structure plays a fundamental role in the regulation of nuclear processes such as DNA transcription, replication, recombination, and repair. Despite considerable efforts during three decades, the structure of the 30-nm chromatin fiber remains controversial. To define fiber dimensions accurately, we have produced very long and regularly folded 30-nm fibers from in vitro reconstituted nucleosome arrays containing the linker histone and with increasing nucleosome repeat lengths (10 to 70 bp of linker DNA). EM measurements show that the dimensions of these fully folded fibers do not increase linearly with increasing linker length, a finding that is inconsistent with two-start helix models. Instead, we find that there are two distinct classes of fiber structure, both with unexpectedly high nucleosome density: arrays with 10 to 40 bp of linker DNA all produce fibers with a diameter of 33 nm and 11 nucleosomes per 11 nm, whereas arrays with 50 to 70 bp of linker DNA all produce 44-nm-wide fibers with 15 nucleosomes per 11 nm. Using the physical constraints imposed by these measurements, we have built a model in which tight nucleosome packing is achieved through the interdigitation of nucleosomes from adjacent helical gyres. Importantly, the model closely matches raw image projections of folded chromatin arrays recorded in the solution state by using electron cryo-microscopy.

Animals↗
PubMed1991

Accumulation of DNA within chromocentres of terminally differentiating chick embryo chondrocytes.

Automated television (TV) densitometric technique which allows the recognition and recording of chromatin compartments was applied to the study of chromatin rearrangement during chondrogenesis. Genetically active chondroblasts and inactive definite chondrocytes of E7 chick cartilage model, stained on the imprints for DNA, were a subject for the comparative study. Large chromatin granules with constant morphometric parameters, displaying positive staining for C-heterochromatin and identified as chromocentres, were found to accumulate 30% of cellular DNA, doubling its concentration during chondrogenic maturation. A test for the DNA content ethalonization proved this to be due to redistribution of DNA from the euchromatinic compartment.

Animals↗
PubMed2002

Sex-chromosome heterochromatin variation in the wood mouse, Apodemus sylvaticus.

Variation in heterochromatin content, as revealed by G- and C-banding, was studied in the sex chromosomes of the wood mouse, Apodemus sylvaticus. The sex-chromosome heterochromatin was also characterized by DAPI staining. Variation in sex chromatin was recorded in extremely large (giant) sex chromosomes in certain individuals and populations. In some individuals, the Y chromosome was the largest element of the complement. Different variants of both the X and Y chromosomes were found within a single population. The variation is therefore a type of population polymorphism and should not be used for taxonomic discrimination.

Animals↗
PubMed1994

Selective capacity of glass-wool filtration for the separation of human spermatozoa with condensed chromatin: a possible therapeutic modality for male-factor cases?

PURPOSE: The aim of this study was to evaluate chromatin condensation of human spermatozoa following swim-up compared to glass-wool separation. Semen aliquots from men attending an andrological outpatient clinic were processed by means of a swim-up procedure and glass-wool filtration. Chromatin condensation was recorded using aniline blue staining and results were reported according to color intensity of stained sperm heads. Morphometric measurements of sperm heads were performed on stained sperm samples. RESULTS: Glass-wool filtration resulted (i) in a significantly higher total motile sperm count (P < 0.0005) compared to swim-up and (ii) in a significantly higher percentage of normal chromatin-condensed spermatozoa compared to the ejaculate (P < 0.01). CONCLUSION: In contrast, comparing swim-up to the ejaculate, the percentage of matured nuclei (unstained spermatozoa) retrieved following swim-up was significantly lower (P < 0.005). Glass-wool filtration separates human spermatozoa according to motility and size of the sperm head. The size of the sperm head closely correlated with the chromatin condensation quality.

Aniline Compounds↗
PubMed1999

Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining.

The successful implementation of ICSI has provided a unique means of allowing couples suffering from severe male infertility to achieve their reproductive goals. However, despite the great therapeutic advantages of the technique, ICSI often provides solutions to clinicians in the absence of an aetiological or pathophysiological diagnosis. The development of a sequential diagnostic schedule for patients consulting for fertility disturbances would be an ideal method of approach. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology and chromatin staining among fertile and subfertile men. Both chromomycin A3 (CMA3) and acidic aniline blue (AAB) were employed to record chromatin packaging quality among 58 men visiting the andrology laboratory. Intra- and interassay variations were initially recorded for fertile sperm donors. The coefficients of variation (CV) for all intra- and inter-assay assessments were < 12%. Chromatin packaging was significantly and negatively correlated with normal sperm morphology, namely r = 0.40 (P = 0.001) and r = 0.33 (P = 0.001) for CMA3 and AAB, respectively. Receiver operator characteristics illustrated sensitivity and specificity values of 75% and 82% for CMA3 and 60% and 91% for AAB, respectively. Significantly different CMA3 and AAB staining was recorded among men with severe teratozoospermia (< 4% normal forms) when compared with normozoospermic men (> 14% normal forms), namely 49% vs. 29% for CMA3 and 51% vs. 26% for AAB staining, respectively. Chromatin packaging assessments should be a valuable addition to the sequential diagnostic programme in an assisted reproduction arena.

Aniline Compounds↗
PubMed1987

The effect of FSH, LH, oestradiol-17 beta, and progesterone on cytoplasmic maturation of bovine follicular oocytes in vitro.

Bovine oocytes removed from follicles 2-5 mm in diameter were cultured for 24 h in the medium without hormone (control) and in the medium supplemented with 1 microgram FSH/ml, 1 microgram LH/ml, 250 ng oestradiol-17 beta/ml and 250 ng progesterone/ml. At the end of culture the zonae pellucidae were dissolved by pronase and the eggs were transferred to droplets of preincubated bovine epididymal spermatozoa. After incubation for 14-16 h, the number of penetrated oocytes, the average number of spermatozoa per one oocyte, and the stage of sperm nucleus chromatin decondensation were recorded. The highest number of penetrated oocytes (99.2%) was found in cultures with progesterone, but 83.7% oocytes were polyspermic and in 77.0% oocytes a normal male and a female pronucleus was formed, it was the highest incidence among all hormones tested. Oocytes cultured with oestradiol-17 beta were penetrated in 76.5% of cases, but in this group the largest number of monospermic oocytes with a female and a male pronucleus was seen. Also, the lowest number of spermatozoa per polyspermic oocyte was recorded. In cultures with FSH and LH, the percentage of fertilization was almost equal (62.2 and 61.1%, respectively). With FSH the lowest number of polyspermic oocytes was observed. From the results it follows that progesterone has a positive effect on the synthesis of MPGF in the cytoplasm of oocytes and on the number of penetrated oocytes. Oestradiol-17 beta and FSH probably participate in the formation of a polyspermic block via cortical granules.

Animals↗
PubMed1984

Packing of a specific gene into higher order structures following repression of RNA synthesis.

Transcription of the Balbiani ring (BR) genes of the dipteran Chironomus tentans was inhibited by teh nucleoside analogue DRB (5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole). The BR genes were emptied of RNA polymerases and the subsequent packing of the genes was monitored by transmission electron microscopy. The thin chromatin axis of the transcriptionally active genes condensed into a thick (20-25 nm) chromatin fiber, which was recorded as a linear structure, an open loop or a supercoiled loop. The compacted genes were finally packed into dense clumps of chromatin. It was proposed that upon repression of RNA synthesis the BR gene template attains the following consecutive stages with increasing compaction: transcription loop----linear thick fiber----open thick fiber loop----supercoiled thick fiber loop----dense chromatin. Within the chromatin blocks structures that resembled the supercoiled loops were discerned, suggesting that the final packing of the template might be accomplished by a close alignment of supercoiled loops.

Animals↗
PubMed1998

Calorimetric investigation of ethidium and netropsin binding to chicken erythrocyte chromatin.

We have investigated the thermodynamic aspects of the ligand binding to chromatin, using isothermal titration calorimetry. Two classical DNA ligands were used: an intercalator, ethidium bromide, and a minor groove binder, netropsin. Stoichiometry, affinity constant, and thermodynamic parameters were determined at various salt concentrations and different temperatures. The effect of ionic strength was analyzed according to the Record theory applied to chromatin. We also compared the binding parameters on naked DNA, H1/H5-depleted chromatin, and chromatin. We demonstrated that the presence of histones on DNA still allows the ligand binding that takes place according to a simple one single-site model. For both ligand types, the thermodynamic driving force is enthalpic and the association is characterized by a somewhat weaker affinity and more scattered ligand distribution than on naked DNA. The ligand affinity is weakly altered by the salt-induced compaction of the chromatin and the binding is accompanied by a release of one counterion per ligand molecule. The temperature-dependent studies revealed the existence of a small heat capacity change associated with ligand binding to chromatin, together with an enthalpy-entropy compensation that maintains the free energy constant over the investigated temperature range.

Animals↗
PubMed1988

Circular dichroism microscopy of compact forms of DNA and chromatin in vivo and in vitro: cholesteric liquid-crystalline phases of DNA and single dinoflagellate nuclei.

Two highly condensed structures of DNA have been analyzed in the circular dichroism (CD) microscope: the cholesteric liquid-crystalline phase of DNA and the nucleus of a dinoflagellate (Prorocentrum micans). In both cases, the DNA shows a helical cholesteric organization, but the helical pitch equals about 2500 nm in the first case and 250 nm in the second one. Since the absorption band of DNA is located at 260 nm, the reflection and absorption bands are well separated in the cholesteric phase of DNA and are overlapping in the dinoflagellate nucleus. However, both structures give a very strong negative CD signal at 265 nm. We show that this very strong signal cannot correspond to a Borrmann effect, i.e., to a superposition of the absorption and reflection bands, but is a differential absorption of left versus right circularly polarized light. This anomalous differential absorption is probably due to a significant scattering of light, inside of the structure, which produces a resonance phenomenon in the absorption band of the chromophore. Therefore, for any helical structure containing a chromophore, the apparent CD can be expressed as CD = [(epsilon L - epsilon R)cl] + (psi L - psi R) + (SL - SR) The first term is true absorption and is located in the absorption band of the chromophore, and the last term is true scattering and is observed at the wavelength corresponding to the helical pitch of the structure. The second term (psi L - psi R) corresponds to the anomalous differential absorption observed in dense superhelical structures of DNA. It superimposes to the first term in the absorption band of the chromophore. psi L - psi R is a measure of the perfection of the helical structure and of the density of chromophores in the material. Intercalative dyes [ethidium bromide and meso-tetrakis(4-N-methylpyridyl)porphine (H2TMpyP-4) and its nickel(II) derivative (NiIITMpyP-4)] were inserted in the dinoflagellate chromatin. The CD signal recorded in their absorption band mimics the signal observed in the absorption band of DNA. In both structures, the negative sign of the CD at 265 nm indicates that the twist occurring between DNA. In both structures, the negative sign of the CD at 265 nm indicates that the twist occurring between DNA molecules is left-handed, and we show that this situation is the most frequently encountered in vivo and vitro.

Animals↗
PubMed1994

Chromatin fibers observed in situ in frozen hydrated sections. Native fiber diameter is not correlated with nucleosome repeat length.

Chromatin fibers have been observed and measured in frozen hydrated sections of three types of cell (chicken erythrocytes and sperm of Patiria miniata and Thyone briareus) representing an approximately 20-bp range of nucleosomal repeat lengths. For sperm of the starfish P. miniata, it was possible to obtain images of chromatin fibers from cells that were swimming in seawater up to the moment of cryo-immobilization, thus providing a record of the native morphology of the chromatin of these cells. Glutaraldehyde fixation produced no significant changes in the ultrastructure or diameter of chromatin fibers, and fiber diameters observed in cryosections were similar to those recorded after low temperature embedding in Lowicryl K11M. Chromatin fiber diameters measured from cryosections of the three types of nuclei were similar, a striking contrast to the situation for chromatin isolated from these cell types, where a strong positive correlation between diameter and nucleosomal repeat length has been established. The demonstration of chromatin fibers in unfixed whole cells establishes an unequivocal baseline for the study of native chromatin and chromosome architecture. The significant differences between chromatin fibers in nucleo and after isolation supports a previous observation (P. J. Giannasca, R. A. Horowitz, and C. L. Woodcock. 1993. J. Cell Sci. 105:551-561), and suggests that structural studies on isolated material should be interpreted with caution until the changes that accompany chromatin isolation are understood.

Animals↗