A DNA condensation code for linker histones.

Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.

Hmo1: A versatile member of the high mobility group box family of chromosomal architecture proteins.

Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures, which is facilitated by linker histone H1. Formation of chromatin compacts and protects the genome, but also hinders DNA transactions. Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions. The high mobility group box (HMGB) proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion. They play a major role in chromatin dynamics. The Saccharomyces cerevisiae (yeast hereafter) HMGB protein Hmo1 contains two HMGB motifs. However, unlike a canonical HMGB protein that has an acidic C-terminus, Hmo1 ends with a lysine rich, basic, C-terminus, resembling linker histone H1. Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions. For instance, Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones. Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome. This minireview reviews the functions of Hmo1 and the underlying mechanisms, highlighting recent discoveries.

Anticancer Plant Secondary Metabolites Induce Linker Histone Depletion from Chromatin.

BACKGROUND: Many plant secondary metabolites (PSMs) were shown to intercalate into DNA helix or interact with DNA grooves. This may influence histone-DNA interactions changeing chromatin structure and genome functioning. METHODS: Nucleosome stability and linker histone H1.2, H1.4 and H1.5 localizations were studied in HeLa cells after the treatment with 15 PSMs, which are DNA-binders and possess anticancer activity according to published data. Chromatin remodeler CBL0137 was used as a control. Effects of PSMs were studied using fluorescent microscopy, flowcytometry, quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), western-blotting. RESULTS: We showed that 1-hour treatment with CBL0137 strongly inhibited DNA synthesis and caused intensive linker histone depletion consistent with nucleosome destabilization. None of PSMs caused nucleosome destabilization, while most of them demonstrated significant influence on linker histone localizations. In particular, cell treatment with 11 PSMs at non-toxic concentrations induced significant translocation of the histone H1.5 to nucleoli and most of PSMs caused depletion of the histones H1.2 and H1.4 from chromatin fraction. Curcumin, resveratrol, berberine, naringenin, and quercetin caused significant redistribution of all three variants of the studied linker histones showing some overlap of PSM effects on linker histone DNA-binding. We demonstrated that PSMs, which induced the most significant redistribution of the histone H1.5 (berberine, curcumin and naringenin), influence the proportion of cells synthesizing DNA, expressing or non-expressing cyclin B and influence cell cycle distribution. Berberine induction of H1.5 translocations to nucleoli was shown to occur independently on the phases of cell cycle (metaphase was not analyzed). CONCLUSIONS: For the first time we revealed PSM influence on linker histone location in cell nuclei that opens a new direction of PSM research as anticancer agents.

Multiscale chromatin dynamics and high entropy in plant iPSC ancestors.

Plant protoplasts provide starting material for of inducing pluripotent cell masses that are competent for tissue regeneration in vitro, analogous to animal induced pluripotent stem cells (iPSCs). Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterised by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what factors influence chromatin transitions during culturing are largely unknown. Here, we used high-throughput imaging and a custom supervised image analysis protocol extracting over 100 chromatin features of cultured protoplasts. The analysis revealed rapid, multiscale dynamics of chromatin patterns with a trajectory that strongly depended on nutrient availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating intrinsic entropy as a hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro.

Linker Histone H1.4 Inhibits the Growth, Migration and EMT Process of Non-Small Cell Lung Cancer by Regulating ERK1/2 Expression.

H1.4 is one of the 11 variants of linker histone H1, and is associated with tumorigenesis and development of various cancers. However, it is unclear for the role of histone H1.4 in non-small cell lung cancer (NSCLC). In this study, we found that overexpression of H1.4 significantly inhibited the cell viability, migration, invasion and epithelial-mesenchymal transition (EMT) processes, whereas silencing H1.4 by shRNA knockdown promoted these processes in NSCLC cell lines A549 and H1299. We further showed that H1.4 overexpression reduced ERK1/2 expression or its phosphorylation levels, while H1.4 knockdown increased ERK1/2 expression or phosphorylation levels in NSCLC. Furthermore, we demonstrated that H1.4 bound to the promoter of ERK1/2, and acted as a transcriptional suppressor to inhibit ERK1/2 expression in A549 or H1299 cells. Importantly, we found that ERK ecto-expression can largely recovered the inhibitory effects of H1.4 on cell viability, migration, invasion and EMT processes. In summary, our study reveals that the H1.4-ERK pathway is crucial for cell viability, migration, invasion and EMT of NSCLC and could be a therapeutic target for NSCLC.

H2AK119ub1 differentially fine-tunes gene expression by modulating canonical PRC1- and H1-dependent chromatin compaction.

Polycomb repressive complex 1 (PRC1) is a key transcriptional regulator in development via modulating chromatin structure and catalyzing histone H2A ubiquitination at Lys119 (H2AK119ub1). H2AK119ub1 is one of the most abundant histone modifications in mammalian cells. However, the function of H2AK119ub1 in polycomb-mediated gene silencing remains debated. In this study, we reveal that H2AK119ub1 has two distinct roles in gene expression, through differentially modulating chromatin compaction mediated by canonical PRC1 and the linker histone H1. Interestingly, we find that H2AK119ub1 plays a positive role in transcription through interfering with the binding of canonical PRC1 to nucleosomes and therefore counteracting chromatin condensation. Conversely, we demonstrate that H2AK119ub1 facilitates H1-dependent chromatin condensation and enhances the silencing of developmental genes in mouse embryonic stem cells, suggesting that H1 may be one of several possible pathways for H2AK119ub1 in repressing transcription. These results provide insights and molecular mechanisms by which H2AK119ub1 differentially fine-tunes developmental gene expression.

An antioxidant feedforward cycle coordinated by linker histone variant H1.2 and NRF2 that drives nonsmall cell lung cancer progression.

Nonsmall cell lung cancer (NSCLC) is highly malignant with limited treatment options, platinum-based chemotherapy is a standard treatment for NSCLC with resistance commonly seen. NSCLC cells exploit enhanced antioxidant defense system to counteract excessive reactive oxygen species (ROS), which contributes largely to tumor progression and resistance to chemotherapy, yet the mechanisms are not fully understood. Recent studies have suggested the involvement of histones in tumor progression and cellular antioxidant response; however, whether a major histone variant H1.2 (H1C) plays roles in the development of NSCLC remains unclear. Herein, we demonstrated that H1.2 was increasingly expressed in NSCLC tumors, and its expression was correlated with worse survival. When crossing the H1c knockout allele with a mouse NSCLC model (KrasLSL-G12D/+), H1.2 deletion suppressed NSCLC progression and enhanced oxidative stress and significantly decreased the levels of key antioxidant glutathione (GSH) and GCLC, the catalytic subunit of rate-limiting enzyme for GSH synthesis. Moreover, high H1.2 was correlated with the IC50 of multiple chemotherapeutic drugs and with worse prognosis in NSCLC patients receiving chemotherapy; H1.2-deficient NSCLC cells presented reduced survival and increased ROS levels upon cisplatin treatment, while ROS scavenger eliminated the survival inhibition. Mechanistically, H1.2 interacted with NRF2, a master regulator of antioxidative response; H1.2 enhanced the nuclear level and stability of NRF2 and, thus, promoted NRF2 binding to GCLC promoter and the consequent transcription; while NRF2 also transcriptionally up-regulated H1.2. Collectively, these results uncovered a tumor-driving role of H1.2 in NSCLC and indicate an "H1.2-NRF2" antioxidant feedforward cycle that promotes tumor progression and chemoresistance.

Cryo-ET study from in vitro to in vivo revealed a general folding mode of chromatin with two-start helical architecture.

The organization and dynamics of chromatin fiber play crucial roles in regulating DNA accessibility for gene expression. Here we combine cryoelectron tomography (cryo-ET), sub-volume averaging, and 3D segmentation to visualize the in vitro and in vivo chromatin fibers folding by linker histone. We discover that an increased nucleosome repeat length and prolonged fiber length do not change the two-start helical architecture in reconstituted chromatin of homogeneous composition. Additionally, an isolated chromatin fiber with heterogeneous composition was observed, which includes short-range regions compatible with two-start helix. In vivo, sub-volume averaging reveals similar subunits of two-start helical architecture in transcriptionally inactive chromatin in frog erythrocyte nuclei. Strikingly, unambiguous DNA trajectories that displayed a zigzag pattern universally between alternate N/N+2 nucleosomes were further determined by cryo-ET with voltage phase plate. Therefore, these structural similarities suggest a general folding mode of chromatin induced by linker histone, and heterogeneous compositions mainly affect local conformation rather than changing the overall architecture.

Histone H3 Tail Modifications Alter Structure and Dynamics of the H1 C-Terminal Domain Within Nucleosomes.

The highly positively charged and intrinsically disordered H1 C-terminal domain (CTD) undergoes extensive condensation upon binding to nucleosomes, and stabilizes nucleosomes and higher-order chromatin structures but its interactions in chromatin are not well defined. Using single-molecule FRET we found that about half of the H1 CTDs in H1-nucleosome complexes exhibit well-defined FRET values indicative of distinct, static conformations, while the remainder of the population exhibits exchange between multiple defined FRET structures. Moreover, crosslinking studies indicate that the first 30 residues of the H1 CTD participate in relatively localized contacts with the first ∼25 bp of linker DNA, and that two separate regions in the CTD contribute to H1-dependent organization of linker DNA. Finally, we show that acetylation mimetics within the histone H3 tail markedly reduce the overall extent of H1 CTD condensation and significantly increase the fraction of H1 CTDs undergoing dynamic exchange between FRET states. Our results indicate the nucleosome-bound H1 CTD adopts loosely defined structures that exhibit significantly enhanced dynamics and decondensation upon epigenetic acetylation within the H3 tail.

Histone H1 protects telomeric repeats from H3K27me3 invasion in Arabidopsis.

While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we show that, in Arabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2 cis recruitment. This study provides a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.

Linker H1 is an 'epi'centre of plant defence.

The role of linker H1 histones in plant defence has recently been investigated. Sheikh et al. found that Arabidopsis thaliana plants that were lacking all three H1 proteins showed increased disease resistance, but when primed, failed to induce enhanced resistance. Differences in epigenetic patterns could be the cause of defective priming.

Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast.

Linker histone H1 plays a crucial role in various biological processes, including nucleosome stabilization, high-order chromatin structure organization, gene expression, and epigenetic regulation in eukaryotic cells. Unlike higher eukaryotes, little about the linker histone in Saccharomyces cerevisiae is known. Hho1 and Hmo1 are two long-standing controversial histone H1 candidates in budding yeast. In this study, we directly observed at the single-molecule level that Hmo1, but not Hho1, is involved in chromatin assembly in the yeast nucleoplasmic extracts (YNPE), which can replicate the physiological condition of the yeast nucleus. The presence of Hmo1 facilitates the assembly of nucleosomes on DNA in YNPE, as revealed by single-molecule force spectroscopy. Further single-molecule analysis showed that the lysine-rich C-terminal domain (CTD) of Hmo1 is essential for the function of chromatin compaction, while the second globular domain at the C-terminus of Hho1 impairs its ability. In addition, Hmo1, but not Hho1, forms condensates with double-stranded DNA via reversible phase separation. The phosphorylation fluctuation of Hmo1 coincides with metazoan H1 during the cell cycle. Our data suggest that Hmo1, but not Hho1, possesses some functionality similar to that of linker histone in Saccharomyces cerevisiae, even though some properties of Hmo1 differ from those of a canonical linker histone H1. Our study provides clues for the linker histone H1 in budding yeast and provides insights into the evolution and diversity of histone H1 across eukaryotes. IMPORTANCE There has been a long-standing debate regarding the identity of linker histone H1 in budding yeast. To address this issue, we utilized YNPE, which accurately replicate the physiological conditions in yeast nuclei, in combination with total internal reflection fluorescence microscopy and magnetic tweezers. Our findings demonstrated that Hmo1, rather than Hho1, is responsible for chromatin assembly in budding yeast. Additionally, we found that Hmo1 shares certain characteristics with histone H1, including phase separation and phosphorylation fluctuations throughout the cell cycle. Furthermore, we discovered that the lysine-rich domain of Hho1 is buried by its second globular domain at the C-terminus, resulting in the loss of function that is similar to histone H1. Our study provides compelling evidence to suggest that Hmo1 shares linker histone H1 function in budding yeast and contributes to our understanding of the evolution of linker histone H1 across eukaryotes.

Structural mechanism of LIN28B nucleosome targeting by OCT4.

Pioneer transcription factors are essential for cell fate changes by targeting closed chromatin. OCT4 is a crucial pioneer factor that can induce cell reprogramming. However, the structural basis of how pioneer factors recognize the in vivo nucleosomal DNA targets is unknown. Here, we determine the high-resolution structures of the nucleosome containing human LIN28B DNA and its complexes with the OCT4 DNA binding region. Three OCT4s bind the pre-positioned nucleosome by recognizing non-canonical DNA sequences. Two use their POUS domains while the other uses the POUS-loop-POUHD region; POUHD serves as a wedge to unwrap ∼25 base pair DNA. Our analysis of previous genomic data and determination of the ESRRB-nucleosome-OCT4 structure confirmed the generality of these structural features. Moreover, biochemical studies suggest that multiple OCT4s cooperatively open the H1-condensed nucleosome array containing the LIN28B nucleosome. Thus, our study suggests a mechanism of how OCT4 can target the nucleosome and open closed chromatin.

Involvement of linker histone variant H1a in the regulation of early preimplantation development in mice.

Linker histone variants regulate higher-order chromatin structure and various cellular processes. It has been suggested that linker histone variant H1a loosens chromatin structure and activates transcription. However, its role in early mouse development remains to be elucidated. We investigated the functions of H1a during preimplantation development using H1a gene-deleted mice. Although H1a homozygous knockout (KO) mice were born without any abnormalities, the number of offspring were reduced when the mothers but not fathers were homozygous KO animals. Maternal H1a KO compromised development during the morula and blastocyst stages, but not differentiation of the inner cell mass or trophectoderm. Thus, maternal linker histone H1a is important in early development.

Post-Translation Modifications and Mutations of Human Linker Histone Subtypes: Their Manifestation in Disease.

Linker histones (LH) are a critical component of chromatin in addition to the canonical histones (H2A, H2B, H3, and H4). In humans, 11 subtypes (7 somatic and 4 germinal) of linker histones have been identified, and their diverse cellular functions in chromatin structure, DNA replication, DNA repair, transcription, and apoptosis have been explored, especially for the somatic subtypes. Delineating the unique role of human linker histone (hLH) and their subtypes is highly tedious given their high homology and overlapping expression patterns. However, recent advancements in mass spectrometry combined with HPLC have helped in identifying the post-translational modifications (PTMs) found on the different LH subtypes. However, while a number of PTMs have been identified and their potential nuclear and non-nuclear functions explored in cellular processes, there are very few studies delineating the direct relevance of these PTMs in diseases. In addition, recent whole-genome sequencing of clinical samples from cancer patients and individuals afflicted with Rahman syndrome have identified high-frequency mutations and therefore broadened the perspective of the linker histone mutations in diseases. In this review, we compile the identified PTMs of hLH subtypes, current knowledge of the relevance of hLH PTMs in human diseases, and the correlation of PTMs coinciding with mutations mapped in diseases.

Nucleosome dyad determines the H1 C-terminus collapse on distinct DNA arms.

Nucleosomes are symmetric structures. However, binding of linker histones generates an inherently asymmetric H1-nucleosome complex, and whether this asymmetry is transmitted to the overall nucleosome structure, and therefore also to chromatin, is unclear. Efforts to investigate potential asymmetry due to H1s have been hampered by the DNA sequence, which naturally differs in each gyre. To overcome this issue, we designed and analyzed by cryo-EM a nucleosome reconstituted with a palindromic (601L) 197-bp DNA. As in the non-palindromic 601 sequence, H1 restricts linker DNA flexibility but reveals partial asymmetrical unwrapping. However, in contrast to the non-palindromic nucleosome, in the palindromic nucleosome H1 CTD collapses to the proximal linker. Molecular dynamics simulations show that this could be dictated by a slightly tilted orientation of the globular domain (GD) of H1, which could be linked to the DNA sequence of the nucleosome dyad.

Conformational Change of Nucleosome Arrays prior to Phase Separation.

Chromatin phase transition serves as a regulatory mechanism for eukaryotic transcription. Understanding this process requires the characterization of the nucleosome array structure in response to external stimuli prior to phase separation. However, the intrinsic flexibility and heterogeneity hinders the arrays' structure determination. Here we exploit advances in cryogenic electron tomography (cryo-ET) to determine the three-dimensional (3D) structure of each individual particle of mono-, di-, tri-, and tetranucleosome arrays. Statistical analysis reveals the ionic strength changes the angle between the DNA linker and nucleosome core particle (NCP), which regulate the overall morphology of nucleosome arrays. The finding that one-third of the arrays in the presence of H1 contain an NCP invaded by foreign DNA suggests an alternative function of H1 in constructing nucleosomal networks. The new insights into the nucleosome conformational changes prior to the intermolecular interaction stage extends our understanding of chromatin phase separation regulation.

H1 Linker Histone Gene Regulates Lifespan via Dietary Restriction Pathways in Caenorhabditis elegans / 实验动物与比较医学

Objective To reveal the physiological function of H1 linker histone gene (hil-1) and its molecular mechanism for regulating the lifespan in Caenorhabditis elegans (C. elegans).MethodsC. elegans was used as a model organism and hil-1 gene was knock-down, knock-out and over-expressed via RNA interference technology, hil-1(gk229) mutants backcross purification and microinjection technology. Then the survival and oviposition of C. elegans were observed. Physiological tests including heat shock test, paraquat stress test and heavy metal Cr6+ stress test were conducted to evaluate the stress resistance of hil-1 mutants. After constructing a dual mutant nematode, real-time fluorescence quantitative PCR (RT-qPCR) was used to further identify the signaling pathways and target sites associated with hil-1 gene regulatory lifespan.ResultsCompared with wild-type N2 worms, the lifespan of C. elegans of RNA interference and hil-1(gk229) mutants were significantly shortened (P<0.001), while overexpression of hil-1 in the whole body increased lifespan (P<0.05). The tolerance of hil-1(gk229) mutants to heat stress and oxidative stress was significantly decreased (P<0.001, P<0.05), but the tolerance to heavy metals was not different compared to wild-type N2 worms (P>0.05). In addition, the developmental cycle of hil-1(gk229) mutants was shortened and the time of oviposition was advanced (P<0.001), but there was no significant change in total number of oviposition (P>0.05). After feeding hil-1 RNA interference bacteria to eat-2(ad465) mutants, the down-regulation of hil-1 expression did not affect the lifespan of eat-2(ad465) mutants (P>0.05). Compared with wild-type N2 worms, the expression level of daf-16 in hil-1(gk229) mutants was significantly down-regulated (P<0.001), and the expressions of downstream genes, mtl-1 and ctl-1, were also down-regulated (P<0.05, P<0.001). Compared with daf-2(e1370) mutants, the lifespan of daf-2 (e1370); hil-1(gk229) mutants did not shortened (P>0.05). Compared with daf-16(mu86) mutants, the lifespan of daf-16(mu86); hil-1(gk229) mutants was significantly shortened (P<0.001). The knockdown of hil-1 via RNA interference technology, specifically in epidermis and intestine, was sufficient for lifespan reduction (P<0.001).Conclusion The deletion of hil-1 gene significantly shortened the lifespan of C. elegans and decreased the tolerance to heat and oxidative stress. The hil-1 gene regulates the lifespan of C. elegans via dietary restriction pathway and acts mostly in epidermis and intestine.

The Oocyte-Specific Linker Histone H1FOO Is Not Essential for Mouse Oogenesis and Fertility.

Meiosis is a highly conserved specialized cell division process that generates haploid gametes. Many of its events are associated with dynamically regulated chromosomal structures and chromatin remodeling, which are mainly modulated by histone modifications. Histone H1 is a linker histone essential for packing the nucleosome into higher-order structures, and H1FOO (H1 histone family, member O, oocyte-specific) is a H1 variant whose expression pattern is restricted to growing oocytes and zygotes. To further explore the function of H1FOO, we generated mice lacking the H1foo gene by the CRISPR/Cas9 technique. Herein, we combine mouse genetics and cellular studies to show that H1foo-null mutants have no overt phenotype, with both males and females being fertile and presenting no gross defects in meiosis progression nor in synapsis dynamics. Accordingly, the histological sections show a normal development of gametes in both male and female mice. Considering the important role of oocyte constituents in enhancing mammalian somatic cell reprogramming, we analyzed iPSCs generation in H1foo mutant MEFs and observed no differences in the absence of H1FOO. Taken all together, in this work we present the first in vivo evidence of H1FOO dispensability for mouse fertility, clarifying the debate in the field surrounding its essentiality in meiosis.

Genome-wide characterization and evolutionary analysis of linker histones in castor bean (Ricinus communis).

H1s, or linker histones, are ubiquitous proteins in eukaryotic cells, consisting of a globular GH1 domain flanked by two unstructured tails. Whilst it is known that numerous non-allelic variants exist within the same species, the degree of interspecific and intraspecific variation and divergence of linker histones remain unknown. The conserved basic binding sites in GH1 and evenly distributed strong positive charges on the C-terminal domain (CTD) are key structural characters for linker histones to bind chromatin. Based on these features, we identified five linker histones from 13 GH1-containing proteins in castor bean (Ricinus communis), which were named as RcH1.1, RcH1.2a, RcH1.2b, RcH1.3, and RcH1.4 based on their phylogenetic relationships with the H1s from five other economically important Euphorbiaceae species (Hevea brasiliensis Jatropha curcas, Manihot esculenta Mercurialis annua, and Vernicia fordii) and Arabidopsis thaliana. The expression profiles of RcH1 genes in a variety of tissues and stresses were determined from RNA-seq data. We found three RcH1 genes (RcH1.1, RcH1.2a, and RcH1.3) were broadly expressed in all tissues, suggesting a conserved role in stabilizing and organizing the nuclear DNA. RcH1.2a and RcH1.4 was preferentially expressed in floral tissues, indicating potential involvement in floral development in castor bean. Lack of non-coding region and no expression detected in any tissue tested suggest that RcH1.2b is a pseudogene. RcH1.3 was salt stress inducible, but not induced by cold, heat and drought in our investigation. Structural comparison confirmed that GH1 domain was highly evolutionarily conserved and revealed that N- and C-terminal domains of linker histones are divergent between variants, but highly conserved between species for a given variant. Although the number of H1 genes varies between species, the number of H1 variants is relatively conserved in more closely related species (such as within the same family). Through comparison of nucleotide diversity of linker histone genes and oil-related genes, we found similar mutation rate of these two groups of genes. Using Tajima's D and ML-HKA tests, we found RcH1.1 and RcH1.3 may be under balancing selection.