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BackgroundThe mammalian mature spermatozoon has a unique chromatin structure in which the vast majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The chromatin structure of sperm remains unresolved in most livestock species, including the pig. However, its resolution could provide further light into the identification of the genomic regions related to sperm biology and embryo development and it could also help identifying molecular markers for sperm quality and fertility traits. Here, for the first time in swine, we performed Micrococcal Nuclease coupled with high throughput sequencing on pig sperm and characterized the mono-nucleosomal (MN) and sub-nucleosomal (SN) chromatin fractions.ResultsWe identified 25,293 and 4,239 peaks in the mono-nucleosomal and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison of nucleosome-associated DNAs in sperm revealed positional conservation of the nucleosome retention between human and pig. Gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and identification of putative transcription factor binding motifs within the mono-nucleosomal peaks showed enrichment for sperm function and embryo development related processes. We found motif enrichment for the transcription factor Znf263, which in humans was suggested to be a key regulator of the genes with paternal preferential expression during early embryo development. Moreover, we found enriched co-occupancy between the RNAs present in pig sperm and the RNA related to sperm quality, and the mono-nucleosomal peaks. We also found preferential co-location between GWAS hits for semen quality in swine and the mono-nucleosomal sites identified in this study.ConclusionsThese results suggest a clear relationship between nucleosome positioning in sperm and sperm and embryo development.

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, β-turns and a proportion of other structures not stabilized by intramolecular hydrogen bonding. No β-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% β-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 β-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.

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., This work was supported by the Grants from Ministerio de Economía y Competitividad (MINECO) BFU2017-82805-C2-2-P and BFU2017-82805-C2-1-P, awarded to Alicia Roque and Albert Jordan, respectively.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases of the central nervous system. The aggregation of the amyloid-β peptide, Aβ(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-β 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 Aβ(1-42). Both freshly prepared (monomeric) Aβ(1-42) and histone H1 solutions showed negative CD bands typical of the random coil. Mixing Aβ(1-42) and histone H1 led to the loss of the random coil, which was replaced mostly by β-structure. Therefore, both Aβ(1-42) and histone H1 behave as intrinsically disordered proteins with coupled binding and folding. Mutual structure induction demonstrates the interaction of Aβ(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 Aβ(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 Aβ monomers and stabilizes the parallel association of fibrils.

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 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.

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 Nα-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.

H1 linker histones are involved both in the maintenance of chromatin higher-order structure and in gene regulation. H1 binds to linker DNA regions on the surface of the nucleosome. In higher eukaryotes, H1 contains three distinct domains: a short N-terminal domain (NTD), a central globular domain, and a long C-terminal domain (CTD). Terminal domains determine subtype specificity and to a large extent the linker DNA binding and chromatin condensing properties of histone H1. This review is focused on the recent numerous studies that have provided insights in the role of H1 terminal domains in chromatin dynamics. The N- and C-terminal domains behave as intrinsically disordered proteins with coupled binding and folding. We examine the potential kinetic advantages of intrinsic disorder in the recognition of the specific H1 binding sites in chromatin. As typical intrinsically disordered regions, H1 terminal domains are post-translationally modified. Post-translational modifications in the NTD determine the interaction of histone H1 with other proteins involved in heterochromatin formation and transcriptional regulation, while phosphorylation by cyclin-dependent kinases modulates the secondary structure of the CTD and chromatin condensation. We review the arguments in favor of the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of partial phosphorylation in interphase chromatin relaxation. In addition, the interplay of histone H1 and other chromatin architectural proteins, such as proteins of the high-mobility group, protamines, and MeCP2, is associated with changes in chromatin structure.

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 β-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-β protein, with 83% β-structure and no α-helix. The CTD in all-β 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-β potential provided by full phosphorylation.

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.

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 β-amyloid peptide.

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].

Histone H1 subtypes are involved in chromatin higher-order structure. The representation of the subtypes varies greatly depending on the cellular and developmental context. We have estimated the rates of nucleotide substitution for several H1 subtypes, including mammalian and amphibian H1 degree, avian H5, and mammalian H1a-e and H1t, with the aim of finding evidence for their functional differentiation. The rates of nonsynonymous substitution differ among the subtypes by almost one order of magnitude. Such a wide variation in the degree of tolerance of amino acid substitutions is consistent with the functional differentiation of the subtypes. H1 has a characteristic three-domain structure. The rate ratios among the domains of the molecule are not systematically maintained in the different subtypes. This suggests the assumption of differentiated functions by the individual domains in chromatin structure. We have estimated the average time of divergence of H1a-e and H1t paralogs as 406 +/- 80 Myr. The lack of evidence for concerted evolution of H1a-e and H1t since long before the mammalian radiation further supports the functional differentiation of the subtypes.

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.