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Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

ABSTRACT: Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of ?-structure in the phosphorylated samples, concomitant to a decrease in ?-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation.

SUBMITTER: Lopez R

PROVIDER: S-EPMC4482070 | biostudies-other | 2015 May

REPOSITORIES: biostudies-other

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Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

Lopez Rita R Sarg Bettina B Lindner Herbert H Bartolomé Salvador S Ponte Inma I Suau Pedro P Roque Alicia A

Nucleic acids research 20150413 9

Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a d ...[more]

PMID: 25870416

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Project description:The linker histone (LH), an auxiliary protein that can bind to chromatin and interact with the linker DNA to form stem motifs, is a key element of chromatin compaction. By affecting the chromatin condensation level, it also plays an active role in gene expression. However, the presence and variable concentration of LH in chromatin fibers with different DNA linker lengths indicate that its folding and condensation are highly adaptable and dependent on the immediate nucleosome environment. Recent experimental studies revealed that the behavior of LH in mononucleosomes markedly differs from that in small nucleosome arrays, but the associated mechanism is unknown. Here we report a structural analysis of the behavior of LH in mononucleosomes and oligonucleosomes (2-6 nucleosomes) using mesoscale chromatin simulations. We show that the adapted stem configuration heavily depends on the strength of electrostatic interactions between LH and its parental DNA linkers, and that those interactions tend to be asymmetric in small oligonucleosome systems. Namely, LH in oligonucleosomes dominantly interacts with one DNA linker only, as opposed to mononucleosomes where LH has similar interactions with both linkers and forms a highly stable nucleosome stem. Although we show that the LH condensation depends sensitively on the electrostatic interactions with entering and exiting DNA linkers, other interactions, especially by nonparental cores and nonparental linkers, modulate the structural condensation by softening LH and thus making oligonucleosomes more flexible, in comparison to to mono- and dinucleosomes. We also find that the overall LH/chromatin interactions sensitively depend on the linker length because the linker length determines the maximal nucleosome stem length. For mononucleosomes with DNA linkers shorter than LH, LH condenses fully, while for DNA linkers comparable or longer than LH, the LH extension in mononucleosomes strongly follows the length of DNA linkers, unhampered by neighboring linker histones. Thus, LH is more condensed for mononucleosomes with short linkers, compared to oligonucleosomes, and its orientation is variable and highly environment-dependent. More generally, the work underscores the agility of LH whose folding dynamics critically controls genomic packaging and gene expression.

Project description:BackgroundHistone H1 is the most mobile histone in the cell nucleus. Defining the positions of H1 on chromatin in situ, therefore, represents a challenge. Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq), is traditionally the method for mapping histones onto DNA elements. But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higher-order structure. Here, we present a new method, xxChIP-seq, employing antibody binding to fixed intact in situ chromatin, followed by extensive washing, a second fixation, sonication and immunoprecipitation. The second fixation is intended to prevent the loss of specifically bound antibody during washing and subsequent sonication and to prevent antibody shifting to epitopes revealed by the sonication process. In many respects, xxChIP-seq is comparable to immunostaining microscopy, which also involves interaction of the primary antibody with fixed and permeabilized intact cells. The only epitopes displayed after immunostaining are the "exposed" epitopes, not "hidden" by the fixation of chromatin higher-order structure. Comparison of immunoprecipitated fragments between xChIP-seq versus xxChIP-seq should indicate which epitopes become inaccessible with fixation and identify their associated DNA elements.ResultsWe determined the genomic distribution of histone variants H1.2 and H1.5 in human myeloid leukemia cells HL-60/S4 and compared their epitope exposure by both xChIP-seq and xxChIP-seq, as well as high-resolution microscopy, illustrating the influences of preserved chromatin higher-order structure in situ. We found that xChIP and xxChIP H1 signals are in general negatively correlated, with differences being more pronounced near active regulatory regions. Among the intriguing observations, we find that transcription-related regions and histone PTMs (i.e., enhancers, promoters, CpG islands, H3K4me1, H3K4me3, H3K9ac, H3K27ac and H3K36me3) exhibit significant deficiencies (depletions) in H1.2 and H1.5 xxChIP-seq reads, compared to xChIP-seq. These observations suggest the existence of in situ transcription-related chromatin higher-order structures stabilized by formaldehyde.ConclusionComparison of H1 xxChIP-seq to H1 xChIP-seq allows the development of hypotheses on the chromosomal localization of (stabilized) higher-order structure, indicated by the generation of "hidden" H1 epitopes following formaldehyde crosslinking. Changes in H1 epitope exposure surrounding averaged chromosomal binding sites or epigenetic modifications can also indicate whether these sites have chromatin higher-order structure. For example, comparison between averaged active or inactive promoter regions suggests that both regions can acquire stabilized higher-order structure with hidden H1 epitopes. However, the H1 xChIP-seq comparison cannot define their differences. Application of the xxChIP-seq versus H1 xChIP-seq method is particularly relevant to chromatin-associated proteins, such as linker histones, that play dynamic roles in establishing chromatin higher-order structure.

Dataset Information

Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

Publications

Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

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