Micrococcal nuclease sequencing of porcine sperm suggests enriched co-location between retained histones and genomic regions related to semen quality and early embryo development.

The mammalian spermatozoon has a unique chromatin structure in which the majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The sperm's chromatin structure remains unresolved in most animal species, including the pig. However, mapping the genomic locations of retained nucleosomes in sperm could help understanding the molecular basis of both sperm development and function as well as embryo development. This information could then be useful to identify molecular markers for sperm quality and fertility traits. Here, micrococcal nuclease digestion coupled with high throughput sequencing was performed on pig sperm to map the genomic location of mono- and sub-nucleosomal chromatin fractions in relation to a set of diverse functional elements of the genome, some of which were related to semen quality and early embryogenesis. In particular, the investigated elements were promoters, the different sections of the gene body, coding and non-coding RNAs present in the pig sperm, potential transcription factor binding sites, genomic regions associated to semen quality traits and repeat elements. The analysis yielded 25,293 and 4,239 peaks in the mono- and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison revealed positional conservation of the nucleosome retention in sperm between the pig data and a human dataset that found nucleosome enrichment in genomic regions of importance in development. Both gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and the identification of putative transcription factor binding motifs within the mono- and the sub- nucleosomal peaks showed enrichment for processes related to sperm function and embryo development. There was significant motif enrichment for Znf263, which in humans was suggested to be a key regulator of genes with paternal preferential expression during early embryogenesis. Moreover, enriched positional intersection was found in the genome between the mono-nucleosomal peaks and both the RNAs present in pig sperm and the RNAs related to sperm quality. There was no co-location between GWAS hits for semen quality in swine and the nucleosomal sites. Finally, the data evidenced depletion of mono-nucleosomes in long interspersed nuclear elements and enrichment of sub-nucleosomes in short interspersed repeat elements.These results suggest that retained nucleosomes in sperm could both mark regulatory elements or genes expressed during spermatogenesis linked to semen quality and fertility and act as transcriptional guides during early embryogenesis. The results of this study support the undertaking of ambitious research using a larger number of samples to robustly assess the positional relationship between histone retention in sperm and the reproductive ability of boars.

Towards understanding the Regulation of Histone H1 Somatic Subtypes with OMICs.

Histone H1 is involved in the regulation of chromatin higher-order structure and compaction. In humans, histone H1 is a multigene family with seven subtypes differentially expressed in somatic cells. Which are the regulatory mechanisms that determine the variability of the H1 complement is a long-standing biological question regarding histone H1. We have used a new approach based on the integration of OMICs data to address this issue. We have examined the 3D-chromatin structure, the binding of transcription factors (TFs), and the expression of somatic H1 genes in human cell lines, using data from public repositories, such as ENCODE. Analysis of Hi-C, ChIP-seq, and RNA-seq data, have revealed that transcriptional control has a greater impact on H1 regulation than previously thought. Somatic H1 genes located in topologically associated domains (TADs) show higher expression than in boundary regions. H1 genes are targeted by a variable number of transcription factors including cell cycle-related TFs, and tissue-specific TFs, suggesting a fine-tuned, subtype-specific transcriptional control. We describe, for the first time, that all H1 somatic subtypes are under transcriptional co-regulation. The replication-independent subtypes, which are encoded in different chromosomes isolated from other histone genes, are also co-regulated with the rest of the somatic H1 genes, indicating that transcriptional co-regulation extends beyond the histone cluster. Transcriptional control and transcriptional co-regulation explain, at least in part, the variability of H1 complement, the fluctuations of H1 subtypes during development, and also the compensatory effects observed, in model systems, after perturbation of one or more H1 subtypes.

Histone H1 Post-Translational Modifications: Update and Future Perspectives.

Histone H1 is the most variable histone and its role at the epigenetic level is less characterized than that of core histones. In vertebrates, H1 is a multigene family, which can encode up to 11 subtypes. The H1 subtype composition is different among cell types during the cell cycle and differentiation. Mass spectrometry-based proteomics has added a new layer of complexity with the identification of a large number of post-translational modifications (PTMs) in H1. In this review, we summarize histone H1 PTMs from lower eukaryotes to humans, with a particular focus on mammalian PTMs. Special emphasis is made on PTMs, whose molecular function has been described. Post-translational modifications in H1 have been associated with the regulation of chromatin structure during the cell cycle as well as transcriptional activation, DNA damage response, and cellular differentiation. Additionally, PTMs in histone H1 that have been linked to diseases such as cancer, autoimmune disorders, and viral infection are examined. Future perspectives and challenges in the profiling of histone H1 PTMs are also discussed.

Complex Evolutionary History of the Mammalian Histone H1.1-H1.5 Gene Family.

H1 is involved in chromatin higher-order structure and gene regulation. H1 has a tripartite structure. The central domain is stably folded in solution, while the N- and C-terminal domains are intrinsically disordered. The terminal domains are encoded by DNA of low sequence complexity, and are thus prone to short insertions/deletions (indels). We have examined the evolution of the H1.1-H1.5 gene family from 27 mammalian species. Multiple sequence alignment has revealed a strong preferential conservation of the number and position of basic residues among paralogs, suggesting that overall H1 basicity is under a strong purifying selection. The presence of a conserved pattern of indels, ancestral to the splitting of mammalian orders, in the N- and C-terminal domains of the paralogs, suggests that slippage may have favored the rapid divergence of the subtypes and that purifying selection has maintained this pattern because it is associated with function. Evolutionary analyses have found evidences of positive selection events in H1.1, both before and after the radiation of mammalian orders. Positive selection ancestral to mammalian radiation involved changes at specific sites that may have contributed to the low relative affinity of H1.1 for chromatin. More recent episodes of positive selection were detected at codon positions encoding amino acids of the C-terminal domain of H1.1, which may modulate the folding of the CTD. The detection of putative recombination points in H1.1-H1.5 subtypes suggests that this process may has been involved in the acquisition of the tripartite H1 structure.

Interplay between histone H1 structure and function.

H1 linker histones are involved both in the maintenance of higher-order chromatin structure and in gene regulation. Histone H1 exists in multiple isoforms, is evolutionarily variable and undergoes a large variety of post-translational modifications. We review recent progress in the understanding of the folding and structure of histone H1 domains with an emphasis on the interactions with DNA. The importance of intrinsic disorder and hydrophobic interactions in the folding and function of the carboxy-terminal domain (CTD) is discussed. The induction of a molten globule-state in the CTD by macromolecular crowding is also considered. The effects of phosphorylation by cyclin-dependent kinases on the structure of the CTD, as well as on chromatin condensation and oligomerization, are described. We also address the extranuclear functions of histone H1, including the interaction with the ß-amyloid peptide.

CARACTERIZACIÓN DE LA ESTRUCTURA SECUNDARIA DE SUBTIPOS DE LA HISTONA H1 POR DICROÍSMO CIRCULAR / CHARACTERIZATION OF THE SECONDARY STRUCTURE OF HISTONE H1 SUBTYPES BY CIRCULAR DICHROISM (CD)

Introducción: Las histonas H1 modulan la estructura y la función de la cromatina. Las células somáticas de mamífero contienen los subtipos H1º, H1a, H1b, H1c, H1d y H1e; en células germinales de testículo y en ovocito, se encuentran respectivamente H1t y H1oo. Su estructura está conformada por un dominio central globular flanqueado por los dominios N-Terminal (DNT) y C-Terminal (DCT). Objetivo: Caracterizar la estructura secundaria de subtipos de la histona H1 mediante dicroísmo circular (DC). Materiales y Métodos: La histona H1 total se extrajo de núcleos de cerebro de rata por cromatografía de intercambio catiónico; la H1º se purificó por filtración en gel y las H1a, H1b, H1c y H1e por cromatografía líquida de alta resolución de fase reversa (RF-HPLC). Los espectros de DC se realizaron en tampón fosfato 10 mM; tampón fosfato 10 mM, 20% TFE (trifluoroetanol); tampón fosfato 10 mM, 40% TFE; tampón fosfato 10 mM, 60% TFE; tampón fosfato 10 mM, 150 mM NaCl y tampón fosfato 10 mM, 1 M NaCl. El análisis de los espectros se realizó con el programa Standard Analysis. Resultados: El porcentaje de hélice-alfa se calculó por diferentes métodos matemáticos teniendo en cuenta elipticidad molar a 193 nm y a 222 nm; con programa de deconvolución K2D y con relaciones cualitativas R1 y R2. El TFE induce la estructura en hélice-alfa en cada uno de los subtipos, mientras que NaCl no induce ningún cambio importante. Conclusión: Los subtipos con mayor contenido de hélice-alfa son H1a y H1c. Las diferencias observadas en el porcentaje de hélice-alfa entre los diferentes subtipos puede ser importante para su diferenciación funcional.

H1 histones modulate the structure and function of chromatin. Mammalian somatic cells contain H1º, H1a, H1b, H1c, H1d and H1e subtypes; H1t and H1oo are found in testicular germ cells and oocyte, respectively. Its structure consists of a globular core domain flanked by N-terminal (DNT) and C-terminal (DCT) domains. Objective: To characterize the secondary structure of histone H1 subtypes through circular dichroism (CD). Materials and Methods: Total histone H1 was extracted for rat brain nuclei by cation exchange chromatography; histone H1º was purified by gel filtration and the histones H1a, H1b, H1c and H1e were purified by reversed phase high performance liquid chromatography (RP-HPLC). CD spectra were performed in 10 mM phosphate buffer; 10 mM, 20% TFE phosphate buffer (trifluoroethanol); 10 mM, 40% TFE; phosphate buffer 10 mM, 60% TFE; phosphate buffer 10 mM, 150 mM NaCl and phosphate buffer 10 mm, 1 M NaCl. The analysis of the spectra was performed with JASCO Standard Analysis. Results: The percentage of alpha-helix was calculated using different mathematical methods, taking into account the molar ellipticity at 193 nm, and 222 nm, with K2D deconvolution program and the R1 and R2 qualitative relationships. The results indicate that TFE induced the alpha-helix structure in each of the subtypes, whereas NaCl did not induce any significant change. Conclusion: H1a and H1c are subtypes with highest content of alpha-helix. The observed differences in the percentage of alpha-helix between different subtypes may be important for their functional differentiation.

Histone H1 Favors Folding and Parallel Fibrillar Aggregation of the 1-42 Amyloid-ß Peptide.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases of the central nervous system. The aggregation of the amyloid-ß peptide, Aß(1-42), is believed to play an important role in the pathogenesis of AD. Histone H1 is found in the cytoplasm of neurons in AD, and it has been shown to interact with aggregated amyloid-ß peptides and with amyloid fibrils. We have used Thioflavin T (ThT) fluorescence enhancement, circular dichroism spectroscopy (CD), coprecipitation, and transmission electron microscopy (TEM) to study the interaction of histone H1 with Aß(1-42). Both freshly prepared (monomeric) Aß(1-42) and histone H1 solutions showed negative CD bands typical of the random coil. Mixing Aß(1-42) and histone H1 led to the loss of the random coil, which was replaced mostly by ß-structure. Therefore, both Aß(1-42) and histone H1 behave as intrinsically disordered proteins with coupled binding and folding. Mutual structure induction demonstrates the interaction of Aß(1-42) and histone H1. The interaction was confirmed by coprecipitation followed by SDS-PAGE. Mutual structure induction was also observed with the H1 terminal domains. Incubation of Aß(1-42) for 1 week in the presence of histone H1 led to the formation of laminar aggregates and thick bundles, characterized by the parallel association of large numbers of fibrils. The aggregates were particularly large and ordered with the H1 subtype H1.2. Further aging of the complexes led to tight compaction of fibril bundles and to fiber growth. Stabilization of fibril-fibril interactions appeared to be determined by the C-terminal domain of histone H1. In summary, these observations indicate that histone H1 has at least two effects: it helps the folding of Aß monomers and stabilizes the parallel association of fibrils.

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

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 ß-structure in the phosphorylated samples, concomitant to a decrease in α-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.

Identification of novel post-translational modifications in linker histones from chicken erythrocytes.

Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC-ESI-MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including N(α)-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics. BIOLOGICAL SIGNIFICANCE: Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to establish the interplay between PTMs of linker and core histones in order to fully understand chromatin regulation. A protein sequence alignment summarizing the PTMs found to date in chicken, mouse, rat and humans showed that, while many of the modified positions were conserved between these species, the type of modification often varied depending on the species or the cellular type. This finding suggests an important role for the PTMs in the regulation of linker histone functions.

Sequence conservation of linker histones between chicken and mammalian species.

The percent identity matrices of two sequence multiple alignments between linker histones from chicken and mammalian species are described. Linker histone protein sequences for chicken, mouse, rat and humans, available on public databases were used. This information is related to the research article entitled "Identification of novel post-translational modifications in linker histones from chicken erythrocytes"published in the Journal of Proteomics [1].

Contribution of hydrophobic interactions to the folding and fibrillation of histone H1 and its carboxy-terminal domain.

Histone H1 is involved in chromatin structure and gene regulation. H1 also performs functions outside cell nuclei, which may depend on its properties as a lipid-binding protein. The H1 CTD behaves as an intrinsically disordered protein (IDP) with coupled binding and folding. Here, we used neutral detergents and anionic SDS to study the contribution of hydrophobic interactions to the folding of the CTD. In the presence of neutral detergents, the CTD folded with proportions of secondary structure motifs similar to those observed in the DNA complexes. These results identify a folding pathway for the CTD based on hydrophobic interactions, and independent of charge compensation. The CTD is phosphorylated to different extents by cyclin-dependent kinases. The general effect of phosphorylation in the presence of detergents was a decrease in the α-helix content and an increase in that of the ß-structure. The greatest effect was observed in the fully phosphorylated CTD (three phosphate groups) in the presence of anionic SDS (7:1, detergent/CTD molar ratio); in these conditions, the CTD became an all-ß protein, with 83% ß-structure and no α-helix. The CTD in all-ß conformation readily formed ribbon-like fibers. The entire H1 also formed fibers when fully phosphorylated in the CTD. Fibers were of the amyloid type, as judged by strong birefringence in the presence of Congo red and thioflavin fluorescence enhancement. Amyloid fiber formation was only observed in SDS, suggesting that it requires the joint effects of partial charge neutralization and hydrophobic interactions, together with the all-ß potential provided by full phosphorylation.

Secondary structure of protamine in sperm nuclei: an infrared spectroscopy study.

BACKGROUND: Protamines are small basic proteins that condense the DNA in mature spermatozoa. Typical protamines are of simple composition and very arginine-rich, usually in the range of 60-80%. Arginine residues are distributed in a number of stretches separated by neutral amino acids. We have used Fourier transform infrared spectroscopy (FTIR) to gain access for the first time to the secondary structure of protamines in sperm nuclei. This technique is particularly well suited to the study of DNA-bound protamine in whole nuclei since it is not affected by turbidity. RESULTS: We show that DNA -bound salmon (salmine) and squid protamines contain α-helix, ß-turns and a proportion of other structures not stabilized by intramolecular hydrogen bonding. No ß-sheet was observed. In salmine, the α-helix amounted to ~20%, while in squid protamine it reached ~40%. In contrast, the structure not stabilized by intermolecular hydrogen bonding was more abundant in salmine (~40%) than in squid protamine (~20%). Both protamines contained ~40% ß-turns. The different helical potential of salmine and squid protamine was confirmed by structure predictions and CD in the presence of trifluoroethanol. CONCLUSION: DNA-bound protamine in sperm nuclei contains large amounts of defined secondary structure stabilized by intramolecular hydrogen bonding. Both salmine and squid protamine contain similar amounts of ß-turns, but differ in the proportions of α-helix and non-hydrogen bonded conformations. In spite of the large differences in the proportions of secondary structure motifs between salmon and squid protamines, they appear to be equally efficient in promoting tight hexagonal packing of the DNA molecules in sperm nuclei.

Role of charge neutralization in the folding of the carboxy-terminal domain of histone H1.

H1 linker histones are involved in chromatin structure and gene regulation. The carboxy-terminal domain (CTD) of histone H1 is very basic with approximately 40% Lys residues, approximately 75% of which are present as doublets. The CTD has little structure in diluted solution but becomes cooperatively folded upon interaction with DNA. The DNA-bound CTD contains alpha-helix, beta-structure, turns, and flexible regions. We studied the effects of charge neutralization on the secondary structure of the CTD independently of DNA interaction through deprotonation of the epsilon-amino groups of the Lys side chains at alkaline pH. Alkaline pH induces extensive folding of the CTD with proportions of secondary structure similar to those observed in the complexes with DNA. The CTD is phosphorylated by cyclin-dependent kinases. In the fully phosphorylated CTD, alkaline pH induces a higher amount of beta-sheet and a lower amount of alpha-helix, as observed in the complexes with DNA. These results, together with structure predictions, suggest that the increased hydrophobicity of Lys side chains accompanying charge neutralization is responsible for the folding of the CTD upon interaction with DNA.

Phosphorylation of the carboxy-terminal domain of histone H1: effects on secondary structure and DNA condensation.

Linker histone H1 plays an important role in chromatin folding. Phosphorylation by cyclin-dependent kinases is the main post-translational modification of histone H1. We studied the effects of phosphorylation on the secondary structure of the DNA-bound H1 carboxy-terminal domain (CTD), which contains most of the phosphorylation sites of the molecule. The effects of phosphorylation on the secondary structure of the DNA-bound CTD were site-specific and depended on the number of phosphate groups. Full phosphorylation significantly increased the proportion of beta-structure and decreased that of alpha-helix. Partial phosphorylation increased the amount of undefined structure and decreased that of alpha-helix without a significant increase in beta-structure. Phosphorylation had a moderate effect on the affinity of the CTD for the DNA, which was proportional to the number of phosphate groups. Partial phosphorylation drastically reduced the aggregation of DNA fragments by the CTD, but full phosphorylation restored to a large extent the aggregation capacity of the unphosphorylated domain. These results support the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of H1 partial phosphorylation in interphase chromatin relaxation. More generally, our results suggest that the effects of phosphorylation are mediated by specific structural changes and are not simply a consequence of the net charge.

Macromolecular crowding induces a molten globule state in the C-terminal domain of histone H1.

We studied the secondary structure of the C-terminal domains of the histone H1 subtypes H1 degrees (C-H1 degrees ) and H1t (C-H1t) in the presence of macromolecular crowding agents (Ficoll 70 and PEG 6000) by IR spectroscopy. The carboxyl-terminal domain has little structure in aqueous solution but became extensively folded in the presence of crowding agents. In 30% PEG, C-H1 degrees contained 19% alpha-helix, 28% beta-sheet, 16% turns, and 31% open loops. Similar proportions were observed in 30% Ficoll 70 and for C-H1t in both crowding agents. The proportions of secondary structure motifs were comparable to those of the DNA-bound domain. Kratky plots of the small-angle x-ray scattering showed that in crowding agents the C-terminus had the compaction of a globular state. Progressive dissipation of the secondary structure and a linear increase in partial heat capacity with temperature together with increased binding of ANS indicated that the C-terminus is not cooperatively folded in crowded conditions. Native-like secondary structure and compactness in absence of folding cooperativity indicate that the C-terminus in crowding agents is in a molten globule state. Folding of the C-terminus in crowded conditions may increase the rate of the transition toward the DNA-bound state and facilitate H1 diffusion inside cell nuclei.