Histone chaperone-mediated co-expression assembly of tetrasomes and nucleosomes.

The nucleosome, a basic unit of chromatin found in all eukaryotes, is thought to be assembled through the orchestrated activity of several histone chaperones and chromatin assembly factors in a stepwise manner, proceeding from tetrasome assembly, to H2A/H2B deposition, and finally to formation of the mature nucleosome. In this study, we demonstrate chaperone-mediated assembly of both tetrasomes and nucleosomes on the well-defined Widom 601 positioning sequence using a co-expression/reconstitution wheat germ cell-free system. The purified tetrasomes and nucleosomes were positioned around the center of a given sequence. The heights and diameters were measured by atomic force microscopy. Together with the reported unmodified native histones produced by the wheat germ cell-free platform, our method is expected to be useful for downstream applications in the field of chromatin research.

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila.

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

Human TAF-Iα promotes oncogenic transformation via enhancement of cell proliferation and suppression of apoptosis.

Template activating factor-I (TAF-I) is a multifunctional protein involved in various biological processes including the inhibition of histone acetylation, DNA replication, cell cycle regulation, and oncogenesis. Two main TAF-I isoforms with different N-termini, TAF-Iα and TAF-Iß (SET), are expressed in cells. There are numerous data about functional properties of TAF-Iß, whereas the effects of TAF-Iα remain largely unexplored. Here, we employed focus formation and cell proliferation assays, TUNEL staining, cytological analysis, and RT-qPCR to compare the effects of human TAF-Iα and TAF-Iß genes, transiently expressed in Rat2 cells and in Misgurnus fossilis loaches. We found that both TAF-I isoforms possessed equal oncogenic potential in these systems. Furthermore, an overexpression of human TAF-Iα and TAF-Iß in Rat2 cells promoted their proliferation. Accordingly, the mitotic index was increased in the transgenic loaches expressing human TAF-Iα or TAF-Iß. TUNEL assay as well as downregulation of p53 gene and upregulation of bcl-2 gene in these transgenic loaches demonstrated that both isoforms suppressed apoptosis. Thus, TAF-Iα isoform exerts the same oncogenic potential as TAF-Iß, likely by suppressing the apoptosis and promoting cell proliferation.

SET levels contribute to cohesion fatigue.

Chromosome instability (CIN) is a major hallmark of cancer cells and believed to drive tumor progression. Several cellular defects including weak centromeric cohesion are proposed to promote CIN, but the molecular mechanisms underlying these defects are poorly understood. In a screening for SET protein levels in various cancer cell lines, we found that most of the cancer cells exhibit higher SET protein levels than nontransformed cells, including RPE-1. Cancer cells with elevated SET often show weak centromeric cohesion, revealed by MG132-induced cohesion fatigue. Partial SET knockdown largely strengthens centromeric cohesion in cancer cells without increasing overall phosphatase 2A (PP2A) activity. Pharmacologically increased PP2A activity in these cancer cells barely ameliorates centromeric cohesion. These results suggest that compromised PP2A activity, a common phenomenon in cancer cells, may not be responsible for weak centromeric cohesion. Furthermore, centromeric cohesion in cancer cells can be strengthened by ectopic Sgo1 overexpression and weakened by SET WT, not by Sgo1-binding-deficient mutants. Altogether, these findings demonstrate that SET overexpression contributes to impaired centromeric cohesion in cancer cells and illustrate misregulated SET-Sgo1 pathway as an underlying mechanism.

DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network.

From biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Whether mechanisms exist to safeguard the histone fold during histone chaperone handover events or to release trapped intermediates is unclear. Using structure-guided and functional proteomics, we identify and characterize a histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. We elucidate the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates. We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication- and transcription-coupled nucleosome assembly, and to clean up spurious interactions. With its dual functionality, DNAJC9 integrates ATP-resourced protein folding into the histone supply pathway to resolve aberrant intermediates throughout the dynamic lives of histones.

Regulation of chromatin structure and function: insights into the histone chaperone FACT.

In eukaryotic cells, changes in chromatin accessibility are necessary for chromatin to maintain its highly dynamic nature at different times during the cell cycle. Histone chaperones interact with histones and regulate chromatin dynamics. Facilitates chromatin transcription (FACT) is an important histone chaperone that plays crucial roles during various cellular processes. Here, we analyze the structural characteristics of FACT, discuss how FACT regulates nucleosome/chromatin reorganization and summarize possible functions of FACT in transcription, replication, and DNA repair. The possible involvement of FACT in cell fate determination is also discussed.Abbreviations: FACT: facilitates chromatin transcription, Spt16: suppressor of Ty16, SSRP1: structure-specific recognition protein-1, NTD: N-terminal domain, DD: dimerization domain, MD: middle domain, CTD: C-terminus domain, IDD: internal intrinsically disordered domain, HMG: high mobility group, CID: C-terminal intrinsically disordered domain, Nhp6: non-histone chromosomal protein 6, RNAPII: RNA polymerase II, CK2: casein kinase 2, AID: acidic inner disorder, PIC: pre-initiation complex, IR: ionizing radiation, DDSB: DNA double-strand break, PARlation: poly ADP-ribosylation, BER: base-excision repair, UVSSA: UV-stimulated scaffold protein A, HR: homologous recombination, CAF-1: chromatin assembly factor 1, Asf1: anti-silencing factor 1, Rtt106: regulator of Ty1 transposition protein 106, H3K56ac: H3K56 acetylation, KD: knock down, SETD2: SET domain containing 2, H3K36me3: trimethylation of lysine36 in histone H3, H2Bub: H2B ubiquitination, iPSCs: induced pluripotent stem cells, ESC: embryonic stem cell, H3K4me3: trimethylation of lysine 4 on histone H3 protein subunit, CHD1: chromodomain protein.

Histone chaperone FACT represses retrotransposon MERVL and MERVL-derived cryptic promoters.

Endogenous retroviruses (ERVs) were usually silenced by various histone modifications on histone H3 variants and respective histone chaperones in embryonic stem cells (ESCs). However, it is still unknown whether chaperones of other histones could repress ERVs. Here, we show that H2A/H2B histone chaperone FACT plays a critical role in silencing ERVs and ERV-derived cryptic promoters in ESCs. Loss of FACT component Ssrp1 activated MERVL whereas the re-introduction of Ssrp1 rescued the phenotype. Additionally, Ssrp1 interacted with MERVL and suppressed cryptic transcription of MERVL-fused genes. Remarkably, Ssrp1 interacted with and recruited H2B deubiquitinase Usp7 to Ssrp1 target genes. Suppression of Usp7 caused similar phenotypes as loss of Ssrp1. Furthermore, Usp7 acted by deubiquitinating H2Bub and thereby repressed the expression of MERVL-fused genes. Taken together, our study uncovers a unique mechanism by which FACT complex silences ERVs and ERV-derived cryptic promoters in ESCs.

The histone methylation readers MRG1/MRG2 and the histone chaperones NRP1/NRP2 associate in fine-tuning Arabidopsis flowering time.

Histones are highly basic proteins involved in packaging DNA into chromatin, and histone modifications are fundamental in epigenetic regulation in eukaryotes. Among the numerous chromatin modifiers identified in Arabidopsis (Arabidopsis thaliana), MORF-RELATED GENE (MRG)1 and MRG2 have redundant functions in reading histone H3 lysine 36 trimethylation (H3K36me3). Here, we show that MRG2 binds histone chaperones belonging to the NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) family, including NAP1-RELATED PROTEIN (NRP)1 and NRP2. Characterization of the loss-of-function mutants mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 revealed that MRG1/MRG2 and NRP1/NRP2 regulate flowering time through fine-tuning transcription of floral genes by distinct molecular mechanisms. In particular, the physical interaction between NRP1/NRP2 and MRG1/MRG2 inhibited the binding of MRG1/MRG2 to the transcription factor CONSTANS (CO), leading to a transcriptional repression of FLOWERING LOCUS T (FT) through impeded H4K5 acetylation (H4K5ac) within the FT chromatin. By contrast, NRP1/NRP2 and MRG1/MRG2 act together, likely in a multiprotein complex manner, in promoting the transcription of FLOWERING LOCUS C (FLC) via an increase of both H4K5ac and H3K9ac in the FLC chromatin. Because the expression pattern of FLC represents the major category of differentially expressed genes identified by genome-wide RNA-sequencing analysis in the mrg1 mrg2, nrp1 nrp2 and mrg1 mrg2 nrp1 nrp2 mutants, it is reasonable to speculate that the NRP1/NRP2-MRG1/MRG2 complex may be involved in transcriptional activation of genes beyond FLC and flowering time control.

SET protein modulates H4 histone methylation status and regulates miR-137 level in oral squamous cell carcinoma.

Aim: Histone acetylation and methylation control gene expression. We investigated the impact of SET knockdown on histone methylation status and the consequences for the miRNAs levels in oral squamous cell carcinoma (OSCC). Methods: OSCC cells with and without SET knockdown were analyzed by quantitative real-time PCR to determine miRNA levels, and by immunoreactions to histone modifications. Results: The knockdown of SET increased the levels of histone H4K20me2 and miR-137. Still, SET protein binds to the miR-137 promoter region. The transfection of miR-137 mimic reduced the KI67 and Rb proteins and proliferation of OSCC cells. Conclusion: Our results show for the first time a relationship between SET and histone methylation associated with the control of miRNA expression and KI67 and Rb as targets of miR-137 in OSCC.

Histone chaperone FACT complex mediates oxidative stress response to promote liver cancer progression.

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.

The intrinsic stability of H2B-ubiquitylated nucleosomes and their in vitro assembly/disassembly by histone chaperone NAP1.

BACKGROUND: Apart the gene-regulatory functions as docking sites for histone 'readers', some histone modifications could directly affect nucleosome structure. The H2BK34-ubiquitylation deposited by MOF-MSL complex, increases nucleosome dynamics in vitro and promotes donation of one H2A/H2B dimer to histone acceptors. METHODS: We evaluated temperature-depended stability of H2BK34-ubiquitylated nucleosomes under 'physiological' ionic conditions in the presence or absence of histone acceptor, and examined assembly and disassembly of ubiquitylated nucleosomes in vitro by recombinant mouse NAP1. RESULTS: H2BK34ub modification is sufficient to promote selective eviction of only one H2A/H2B dimer independently of histone-binding agents. Despite the robust H2A/H2B dimer-displacement effect of mNAP1 with the H2BK34ub (but not unmodified) nucleosomes, NAP1 could assemble symmetrically- or asymmetrically ubiquitylated nucleosomes under 'physiological' conditions in vitro. CONCLUSIONS AND GENERAL SIGNIFICANCE: The increased mobility of one nucleosomal H2A/H2B dimer is an intrinsic nucleosome destabilizing property of H2BK34 ubiquitylation that has the intranucleosome bases. The ability of NAP to reasonably efficiently assemble H2BK34-ubiquitylated nucleosomes supposes a potential mechanism for deposition/distribution of H2BK34ub mark in the MOF-MSL independent manner (for example, during histone dimer exchange upon transcription elongation).

The SSRP1 subunit of the histone chaperone FACT is required for seed dormancy in Arabidopsis.

SSRP1 is a subunit of the histone chaperone FACT that associates with elongating RNA polymerase II (RNAPII) along the transcribed region of genes. FACT facilitates transcriptional elongation by destabilising nucleosomes in the path of RNAPII, assisting efficient transcription of chromatin templates. In contrast to wild type seeds, freshly harvested seeds of the Arabidopsis ssrp1 mutant germinate efficiently, exhibiting reduced seed dormancy. In line with this phenotype, the ssrp1 seeds have decreased transcript levels of the DOG1 gene, which is a known quantitative trait locus (QTL) for seed dormancy. Analysis of ssrp1 plants harbouring an additional copy of DOG1 show increased levels of DOG1 transcript and consistently more robust seed dormancy. Therefore, our findings indicate that SSRP1 is a novel factor required for the efficient expression of DOG1 and hence a modulator of seed dormancy in Arabidopsis.

Spt6 Is Required for the Fidelity of Promoter Selection.

Spt6 is a conserved factor that controls transcription and chromatin structure across the genome. Although Spt6 is viewed as an elongation factor, spt6 mutations in Saccharomyces cerevisiae allow elevated levels of transcripts from within coding regions, suggesting that Spt6 also controls initiation. To address the requirements for Spt6 in transcription and chromatin structure, we have combined four genome-wide approaches. Our results demonstrate that Spt6 represses transcription initiation at thousands of intragenic promoters. We characterize these intragenic promoters and find sequence features conserved with genic promoters. Finally, we show that Spt6 also regulates transcription initiation at most genic promoters and propose a model of initiation site competition to account for this. Together, our results demonstrate that Spt6 controls the fidelity of transcription initiation throughout the genome.

Analysis of Potential Alterations Affecting SETBP1 as a Novel Contributing Mechanism to Inhibit PP2A in Colorectal Cancer Patients.

BACKGROUND: The functional loss of the tumor suppressor protein phosphatase 2A (PP2A) occurs in a wide variety of human cancers including colorectal cancer (CRC), and SET overexpression has been reported as a key contributing mechanism to inhibit PP2A. Although SET binding protein 1 (SETBP1) overexpression and gain of function mutations have been described in several hematological malignancies as common events that increase the expression levels of the PP2A inhibitor SET, thereby leading to PP2A inactivation, the potential existence of SETBP1 alterations in CRC still remains unexplored. METHODS: We studied the expression profile of SETBP1 by Western blot in a set of CRC cell lines and patient samples. Moreover, we performed co-immunoprecipitation assays to analyze the formation of the previously reported SETBP1-SET-PP2A inhibitory complex. Furthermore, we evaluated the mutational status of SETBP1 by pyrosequencing assays in a cohort of 55 CRC patients with metastatic disease after the immunohistochemical characterization of SET and p-PP2A expression in this cohort. RESULTS: We found high SETBP1 expression in several CRC lines but only in two of the patients analyzed. In addition, we demonstrated the formation of the SETBP1-SET-PP2A heterotrimeric complex in CRC cells. However, we failed to detect SETBP1 mutations in any of the CRC patient samples included in the study. CONCLUSIONS: Our results suggest that SETBP1 expression is mainly similar o lower in colorectal cancer tissue compared to normal colonic mucosa. However, its overexpression is a low prevalent alteration which could contribute to inhibit PP2A in CRC through the formation of a SETBP1-SET-PP2A complex in some CRC patients. Moreover, SETBP1 mutations are, if exist, rare events in CRC patients.

Histone chaperones play crucial roles in maintenance of stem cell niche during plant root development.

Stem cells in both plant and animal kingdoms reside in a specialized cellular context called the stem cell niche (SCN). SCN integrity is crucial for organism development. Here we show that the H3/H4 histone chaperone CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) and the H2A/H2B histone chaperone NAP1-RELATED PROTEIN1/2 (NRP1/2) play synergistic roles in Arabidopsis root SCN maintenance. Compared with either the m56-1 double mutant deprived of NRP1 and NRP2 or the fas2-4 mutant deprived of CAF-1, the combined m56-1fas2-4 triple mutant displayed a much more severe short-root phenotype. The m56-1fas2-4 mutant root lost the normal organizing center Quiescent Center (QC), and some initial stem cells differentiated precociously. Microarray analysis unraveled the deregulation of 2735 genes within the Arabidopsis genome (representing >8% of all genes) in the m56-1fas2-4 mutant roots. Expression of some SCN key regulatory genes (e.g. WOX5, PLT1, SHR) was not limiting, rather the plant hormone auxin gradient maximum at QC was impaired. The mutant roots showed programmed cell death and high levels of the DNA damage marked histone H2A.X phosphorylation (γ-H2A.X). Knockout of either ATAXIA-TELANGIECTASIA MUTATED (ATM) or ATR, encoding a DNA damage response kinase, rescued in part the cell death and the short-root phenotype of the m56-1fas2-4 mutant. Collectively, our study indicated that NRP1/2 and CAF-1 act cooperatively in regulating proper genome transcription, in sustaining chromatin replication and in maintaining genome integrity, which are crucial for proper SCN function during continuous post-embryonic root development.

Characterization of H3.3 and HIRA expression and function in bovine early embryos.

Histone variant H3.3 is encoded by two distinct genes, H3F3A and H3F3B, that are closely associated with actively transcribed genes. H3.3 replacement is continuous and essential for maintaining correct chromatin structure during mouse oogenesis. Upon fertilization, H3.3 is incorporated to parental chromatin, and is required for blastocyst formation in mice. The H3.3 exchange process is facilitated by the chaperone HIRA, particularly during zygote development. We previously demonstrated that H3.3 is required for bovine early embryonic development; here, we explored the mechanisms of its functional requirement. H3F3A mRNA abundance is stable whereas H3F3B and HIRA mRNA are relatively dynamic during early embryonic development. H3F3B mRNA quantity is also considerably higher than H3F3A. Immunofluorescence analysis revealed an even distribution of H3.3 between paternal and maternal pronuclei in zygotes, and subsequent stage-specific localization of H3.3 in early bovine embryos. Knockdown of H3.3 by targeting both H3F3A and H3F3B dramatically decreased the expression of NANOG (a pluripotency marker) and CTGF (Connective tissue growth factor; a trophectoderm marker) in bovine blastocysts. Additionally, we noted that Histone H3 lysine 36 dimethylation and linker Histone H1 abundance is reduced in H3.3-deficient embryos, which was similar to effects following knockdown of CHD1 (Chromodomain helicase DNA-binding protein 1). By contrast, no difference was observed in the abundance of Histone H3 lysine 4 trimethylation, Histone H3 lysine 9 dimethylation, or Splicing factor 3 B1. Collectively, these results established that H3.3 is required for correct epigenetic modifications and H1 deposition, dysregulation of which likely mediate the poor development in H3.3-deficient embryos.

DNA looping mediates nucleosome transfer.

Proper cell function requires preservation of the spatial organization of chromatin modifications. Maintenance of this epigenetic landscape necessitates the transfer of parental nucleosomes to newly replicated DNA, a process that is stringently regulated and intrinsically linked to replication fork dynamics. This creates a formidable setting from which to isolate the central mechanism of transfer. Here we utilized a minimal experimental system to track the fate of a single nucleosome following its displacement, and examined whether DNA mechanics itself, in the absence of any chaperones or assembly factors, may serve as a platform for the transfer process. We found that the nucleosome is passively transferred to available dsDNA as predicted by a simple physical model of DNA loop formation. These results demonstrate a fundamental role for DNA mechanics in mediating nucleosome transfer and preserving epigenetic integrity during replication.

HIRA Is Required for Heart Development and Directly Regulates Tnni2 and Tnnt3.

Chromatin remodelling is essential for cardiac development. Interestingly, the role of histone chaperones has not been investigated in this regard. HIRA is a member of the HUCA (HIRA/UBN1/CABIN1/ASF1a) complex that deposits the variant histone H3.3 on chromatin independently of replication. Lack of HIRA has general effects on chromatin and gene expression dynamics in embryonic stem cells and mouse oocytes. Here we describe the conditional ablation of Hira in the cardiogenic mesoderm of mice. We observed surface oedema, ventricular and atrial septal defects and embryonic lethality. We identified dysregulation of a subset of cardiac genes, notably upregulation of troponins Tnni2 and Tnnt3, involved in cardiac contractility and decreased expression of Epha3, a gene necessary for the fusion of the muscular ventricular septum and the atrioventricular cushions. We found that HIRA binds GAGA rich DNA loci in the embryonic heart, and in particular a previously described enhancer of Tnni2/Tnnt3 (TTe) bound by the transcription factor NKX2.5. HIRA-dependent H3.3 enrichment was observed at the TTe in embryonic stem cells (ESC) differentiated toward cardiomyocytes in vitro. Thus, we show here that HIRA has locus-specific effects on gene expression and that histone chaperone activity is vital for normal heart development, impinging on pathways regulated by an established cardiac transcription factor.

Maintenance of Epigenetic Information.

The genome is subject to a diverse array of epigenetic modifications from DNA methylation to histone posttranslational changes. Many of these marks are somatically stable through cell division. This article focuses on our knowledge of the mechanisms governing the inheritance of epigenetic marks, particularly, repressive ones, when the DNA and chromatin template are duplicated in S phase. This involves the action of histone chaperones, nucleosome-remodeling enzymes, histone and DNA methylation binding proteins, and chromatin-modifying enzymes. Last, the timing of DNA replication is discussed, including the question of whether this constitutes an epigenetic mark that facilitates the propagation of epigenetic marks.

Molecular dynamics of histone H1.

The H1 or linker histones bind dynamically to chromatin in living cells via a process that involves transient association with the nucleosome near the DNA entry/exit site followed by dissociation, translocation to a new location, and rebinding. The mean residency time of H1 on any given nucleosome is about a minute, which is much shorter than that of most core histones but considerably longer than that of most other chromatin-binding proteins, including transcription factors. Here we review recent advances in understanding the kinetic pathway of H1 binding and how it relates to linker histone structure and function. We also describe potential mechanisms by which the dynamic binding of H1 might contribute directly to the regulation of gene expression and discuss several situations for which there is experimental evidence to support these mechanisms. Finally, we review the evidence for the participation of linker histone chaperones in mediating H1 exchange.