H1 Binding Research Articles

Post-translational modifications of the intrinsically disordered terminal domains of histone H1: effects on secondary structure and chromatin dynamics.

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.

Change in linker DNA conformation upon histone H1.5 binding to nucleosome: Fluorescent microscopy of single complexes

A method for fluorescently labeled DNA synthesis, which makes it possible to assemble mononucleosomes with 40 bp linkers, was developed. Cy3 and Cy5 labels were introduced into the linkers at distances of 10 bp before the first nucleotide and 15 bp after the last nucleotide of the nucleosome positioning DNA sequence, respectively. Without histone H1.5, f luorescence microscopy of single complexes revealed two equally probable states of nucleosomes. The states were different in the linker conformation: the open one with the energy transfer efficiency (E) between the labels of 0.06 and the closed one with E = 0.37, when the linkers are brought together. Binding of histone H1.5 with nucleosomes occurs in a range of nanomolar concentrations, and the complex formation rate is significantly higher as compared with its dissociation rate. The significant convergence of the DNA linkers (E = 0.73) is observed in these complexes together with the higher conformation uniformity in the region where the labels are localized. The developed nucleosomal constructs represent highly sensitive f luorescent sensors that can be used for the analysis of structural linker rearrangements. Also, in combination with microscopy of single complexes, they are suitable for studying the structure of complexes of nucleosomes with different chromatin architectural proteins.

Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome.

Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.

A JUMONJI Protein with E3 Ligase and Histone H3 Binding Activities Affects Transposon Silencing in Arabidopsis.

Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity.

Atrasentan increased the expression of klotho by mediating miR-199b-5p and prevented renal tubular injury in diabetic nephropathy

Atrasentan is a promising therapy for treating diabetic nephropathy (DN). Here we evaluated whether atrasentan down-regulated the miR-199b-5p expression, thereby increasing klotho and preventing renal tubular injury in DN. One-hundred patients with type 2 diabetes mellitus (T2DM) and 40 healthy subjects were included. A DN mice model was established by an injection of streptozotocin (STZ). Human renal proximal tubular epithelial HK-2 cells were exposed to high glucose (20 mmol/L). Treated the mice and HK-2 cells with atrasentan, and we then investigated whether and how miR-199b-5p and Klotho were involved in preventing renal tubular injury in DN. In patients, the serum miR-199b-5p level increased and the klotho concentration decreased in accordance with elevated albuminuria. Atrasentan down-regulated miR-199b-5p and up-regulated klotho of the DN mice and HK-2 cells exposed to high glucose. High glucose promoted the binding of histone H3 to the miR-199b-5p promoter, and atrasentan canceled this effect. MiR-199b-5p targeted the 3′ UTR of klotho. Overexpression of miR-199b-5p canceled the effects of atrasentan on klotho expression and apoptosis of renal tubular cells in both in vivo and in vitro. The increased serum klotho, mediated by miR-199b-5p, is a possible mechanism by which atrasentan prevents renal tubular injury in DN.

Design and biological testing of peptidic dimerization inhibitors of human Hsp90 that target the C-terminal domain

BackgroundSmall molecules targeting the dimerization interface of the C-terminal domain of Hsp90, a validated target for cancer treatment, have yet to be identified. MethodsThree peptides were designed with the aim to inhibit the dimerization of Hsp90. Computational and biophysical methods examined the α-helical structure for the three peptides. Based on the Autodisplay technology, a novel flow cytometer dimerization assay was developed to test inhibition of Hsp90 dimerization. Microscale thermophoresis was used to determine the KD of the peptides towards the C-terminal domain of Hsp90. ResultsMD simulations and CD spectroscopy indicated an α-helical structure for two of the three peptides. By flow cytometer analysis, IC50 values of 2.08μM for peptide H2 and 8.96μM for peptide H3 were determined. Dimer formation of the C-terminal dimerization domain was analyzed by microscale thermophoresis, and a KD of 1.29nM was determined. Furthermore, microscale thermophoresis studies demonstrated a high affinity binding of H2 and H3 to the C-terminal domain, with a KD of 1.02μM and 1.46μM, respectively. ConclusionsThese results revealed the first peptidic inhibitors of Hsp90 dimerization targeting the C-terminal domain. Furthermore, it has been shown that these peptides bind to the C-terminal domain with a low micromolar affinity. General significanceThese results can be used to design and screen for small molecules that inhibit the dimerization of the C-terminal domain of Hsp90, which could open a new route for cancer therapy.

The replication foci targeting sequence (RFTS) of DNMT1 functions as a potent histone H3 binding domain regulated by autoinhibition

DNA methyltransferase 1 (DNMT1) plays an essential role in propagation of the DNA methylation pattern to daughter cells. The replication foci targeting sequence (RFTS) of DNMT1 is required for the recruitment of DNMT1 to DNA methylation sites through direct binding to ubiquitylated histone H3 mediated by UHRF1 (Ubiquitin-like containing PHD and RING finger domains 1). Recently, it has been reported that the RFTS plugs the catalytic pocket of DNMT1 in an intermediated manner and inhibits its DNA methyltransferase activity. However, it is unclear whether this binding affects RFTS function in terms of recruitment to DNA methylation sites. Using Xenopus egg extracts, we demonstrate here that abrogation of the interaction between the RFTS and the catalytic center of DNMT1, by deletion of the C-terminal portion or disruption of the hydrogen bond, results in non-ubiquitylated histone H3 binding and abnormal accumulation of DNMT1 on the chromatin. Interestingly, DNMT1 mutants identified in patients with a neurodegenerative disease, ADCA-DN, bound to non-ubiquitylated histone H3 and accumulated on chromatin during S phase in Xenopus egg extracts. These results suggest that the interaction between the RFTS and the catalytic center of DNMT1 serves as an autoinhibitory mechanism for suppressing the histone H3 binding of DNMT1 and ensuring the accurate recruitment of DNMT1 to sites of DNA methylation. The autoinhibitory mechanism may play an important role in the regulation of gene expression in neurogenesis.

High-throughput analyses of hnRNP H1 dissects its multi-functional aspect

ABSTRACThnRNPs are polyvalent RNA binding proteins that have been implicated in a range of regulatory roles including splicing, mRNA decay, translation, and miRNA metabolism. A variety of genome wide studies have taken advantage of methods like CLIP and RIP to identify the targets and binding sites of RNA binding proteins. However, due to the complex nature of RNA-binding proteins, these studies are incomplete without assays that characterize the impact of RBP binding on mRNA target expression. Here we used a suite of high-throughput approaches (RIP-Seq, iCLIP, RNA-Seq and shotgun proteomics) to provide a comprehensive view of hnRNP H1s ensemble of targets and its role in splicing, mRNA decay, and translation. The combination of RIP-Seq and iCLIP allowed us to identify a set of 1,086 high confidence target transcripts. Binding site motif analysis of these targets suggests the TGGG tetramer as a prevalent component of hnRNP H1 binding motif, with particular enrichment around intronic hnRNP H1 sites. Our analysis of the target transcripts and binding sites indicates that hnRNP H1s involvement in splicing is 2-fold: it directly affects a substantial number of splicing events, but also regulates the expression of major components of the splicing machinery and other RBPs with known roles in splicing regulation. The identified mRNA targets displayed function enrichment in MAPK signaling and ubiquitin mediated proteolysis, which might be main routes by which hnRNP H1 promotes tumorigenesis.

HMGNs: The enhancer charmers.

The DNase I hypersensitive sites (DHSs) of chromatin constitute one of the best landmarks of eukaryotic genes that are poised and/or activated for transcription. For over 35 years, the high-mobility group nucleosome-binding chromosomal proteins HMGN1 and HMGN2 have been shown to play a role in the establishment of these chromatin-accessible domains at transcriptional regulatory elements, namely promoters and enhancers. The critical presence of HMGNs at enhancers, as highlighted by a recent publication, suggests a role for them in the structural and functional fine-tuning of the DHSs in vertebrates. As we review here, while preferentially out-competing histone H1 binding and invading neighbor nucleosomes, HMGNs may also modulate histone H3 at serine 10 (H3S10ph), which plays an important role in enhancer function and transcriptional initiation.

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. This article is part of a Special Issue entitled: Histone H1, edited by Dr. Albert Jordan.

The PZP Domain of AF10 Senses Unmodified H3K27 to Regulate DOT1L-Mediated Methylation of H3K79

AF10, a DOT1L cofactor, is required for H3K79 methylation and cooperates with DOT1L in leukemogenesis. However, the molecular mechanism bywhich AF10 regulates DOT1L-mediated H3K79 methylation is not clear. Here we report that AF10 contains a "reader" domain that couples unmodified H3K27 recognition to H3K79 methylation. An AF10 region consisting of a PHD finger-Zn knuckle-PHD finger (PZP) folds into a single module that recognizes amino acids 22-27 of H3, and this interaction is abrogated by H3K27 modification. Structural studies reveal that H3 binding triggers rearrangement of the PZP module to form an H3(22-27)-accommodating channel and that the unmodified H3K27 side chain is encased in a compact hydrogen-bond acceptor-lined cage. In cells, PZP recognition of H3 is required for H3K79 dimethylation, expression of DOT1L-target genes, and proliferation of DOT1L-addicted leukemic cells. Together, our results uncover a pivotal role for H3K27-via readout by the AF10 PZP domain-in regulating the cancer-associated enzyme DOT1L.

Novel nucleosomal particles containing core histones and linker DNA but no histone H1.

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.

SRSF3 and hnRNP H1 regulate a splicing hotspot of HER2 in breast cancer cells

Overexpression of the oncogene HER2 occurs in 20–30% of invasive breast cancer and is associated with poor prognosis. A number of different splice variants of HER2 have been identified which produce functionally different proteins. Previously these splice variants have been investigated separately, but in the present study we collectively look at the expression and regulation of a group of HER2 splice variants produced by a splicing hotspot. Initial investigation in a cohort of tumor samples showed large variations in HER2 variant expression between patient samples. RNA interference studies identified 2 splicing factors involved in the regulation of splicing within this region, hnRNP H1 and SRSF3. siRNA targeting hnRNP H1 increases levels of X5 and the oncogenic variant Δ16HER2. Furthermore RNA chromatography assays demonstrated binding of hnRNP H1 to RNA in this region. Additionally the proto-oncogene SRSF3 was also identified as an important regulator of splicing with SRSF3 knockdown resulting in changes in all the splice variants located at the hotspot. Most notably knockdown of SRSF3 resulted in a switch from the oncogenic Δ16HER2 to p100 which inhibits cell proliferation. Binding of SRSF3 to RNA within this region was also demonstrated by RNA chromatography and more specifically 2 SRSF3 binding sites were identified within exon 15. SRSF3 and hnRNP H1 are the first splicing factors identified which regulate the production of these functionally distinct HER2 splice variants and therefore maybe important for the regulation of HER2 signaling.

Zinc finger transcription factor CASZ1 interacts with histones, DNA repair proteins and recruits NuRD complex to regulate gene transcription.

The zinc finger transcription factor CASZ1 has been found to control neural fate-determination in flies, regulate murine and frog cardiac development, control murine retinal cell progenitor expansion and function as a tumor suppressor gene in humans. However, the molecular mechanism by which CASZ1 regulates gene transcription to exert these diverse biological functions has not been described. Here we identify co-factors that are recruited by CASZ1b to regulate gene transcription using co-immunoprecipitation (co-IP) and mass spectrometry assays. We find that CASZ1b binds to the nucleosome remodeling and histone deacetylase (NuRD) complex, histones and DNA repair proteins. Mutagenesis of the CASZ1b protein assay demonstrates that the N-terminus of CASZ1b is required for NuRD binding, and a poly(ADP-ribose) binding motif in the CASZ1b protein is required for histone H3 and DNA repair proteins binding. The N-terminus of CASZ1b fused to an artificial DNA-binding domain (GAL4DBD) causes a significant repression of transcription (5xUAS-luciferase assay), which could be blocked by treatment with an HDAC inhibitor. Realtime PCR results show that the transcriptional activity of CASZ1b mutants that abrogate NuRD or histone H3/DNA binding is significantly decreased. This indicates a model in which CASZ1b binds to chromatin and recruits NuRD complexes to orchestrate epigenetic-mediated transcriptional programs.

Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25.

The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38–68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM–MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator–transactivator interactions.

Interaction of chromatin with a histone H1 containing swapped N- and C-terminal domains.

Although the details of the structural involvement of histone H1in the organization of the nucleosome are quite well understood, the sequential events involved in the recognition of its binding site are not as well known. We have used a recombinant human histone H1 (H1.1) in which the N- and C-terminal domains (NTD/CTD) have been swapped and we have reconstituted it on to a 208-bp nucleosome. We have shown that the swapped version of the protein is still able to bind to nucleosomes through its structurally folded wing helix domain (WHD); however, analytical ultracentrifuge analysis demonstrates its ability to properly fold the chromatin fibre is impaired. Furthermore, FRAP analysis shows that the highly dynamic binding association of histone H1 with the chromatin fibre is altered, with a severely decreased half time of residence. All of this suggests that proper binding of histone H1 to chromatin is determined by the simultaneous and synergistic binding of its WHD-CTD to the nucleosome.

Deregulation of Retinaldehyde Dehydrogenase 2 Leads to Defective Angiogenic Function of Endothelial Colony-Forming Cells in Pediatric Moyamoya Disease.

Moyamoya disease (MMD) is a common cause of childhood stroke, in which the abnormal function of the endothelial colony-forming cell (ECFC) plays a key role in the pathogenesis of the disease. This study was designed to identify genes involved in MMD pathogenesis using gene expression profiling and to understand the defective function of MMD ECFCs. We compared gene expression profiles of ECFCs isolated from patients with MMD and normal controls. Among the differentially expressed genes, we selected a gene with the most downregulated expression, retinaldehyde dehydrogenase 2 (RALDH2). The activity of RALDH2 in MMD ECFCs was assessed by in vitro tube formation assay and in vivo Matrigel plug assay in the presence of all-trans retinoic acid. The transcriptional control of RALDH2 was tested using ChIP assays on acetyl-histone H3. In the results, MMD ECFCs inefficiently formed capillary tubes in vitro and capillaries in vivo, a defect restored by all-trans retinoic acid treatment. Knockdown of RALDH2 mRNA in normal ECFCs also induced decreased activity of capillary formation in vitro. The decreased level of RALDH2 mRNA in MMD ECFCs was attributed to defective acetyl-histone H3 binding to the promoter region. From these results, we conclude that the expression of RALDH2 was epigenetically suppressed in ECFCs from patients with MMD, which may play a key role in their functional impairment.

Helicobacter pylori vesicles carrying CagA localize in the vicinity of cell-cell contacts and induce histone H1 binding to ATP in epithelial cells.

Helicobacter pylori produces outer membrane vesicles (OMV), delivering bacterial substances including the oncogenic cytotoxin-associated CagA protein to their surroundings. We investigated the effects of H. pylori OMV carrying CagA (OMV-CagA) on cell junctions and ATP-binding proteome of epithelial monolayers, using proteomics, mass spectrometry and imaging. OMV-CagA localized in close vicinity of ZO-1 tight junction protein and induced histone H1 binding to ATP. We suggest the expression of novel events in the interactions between H. pylori OMV and epithelia, which may have an influence on host gene transcription and lead to different outcomes of an infection and development of cancer.

The Dnmt3L ADD Domain Controls Cytosine Methylation Establishment during Spermatogenesis.

A critical aspect of mammalian gametogenesis is the reprogramming of genomic DNA methylation. The catalytically inactive adaptor Dnmt3L is essential to ensuring this occurs correctly, but the mechanism by which it functions is unclear. Using gene targeting to engineer a single-amino-acid mutation, we show that the Dnmt3L histone H3 binding domain (ADD) is necessary for spermatogenesis. Genome-wide single-base-resolution DNA methylome analysis of mutant germ cells revealed overall reductions in CG methylation at repetitive sequences and non-promoter CpG islands. Strikingly, we also observe an even more severe loss of non-CG methylation, suggesting an unexpected role for the ADD in this process. These epigenetic deficiencies were coupled with defects in spermatogonia, with mutant cells displaying marked changes in gene expression and reactivation of retrotransposons. Our results demonstrate that the Dnmt3L ADD is necessary for Dnmt3L function and full reproductive fitness.

WD Repeat-containing Protein 5 (WDR5) Localizes to the Midbody and Regulates Abscission

Cytokinesis partitions the cytoplasm of a parent cell into two daughter cells and is essential for the completion of cell division. The final step of cytokinesis in animal cells is abscission, which is a process leading to the physical separation of two daughter cells. Abscission requires membrane traffic and microtubule disassembly at a specific midbody region called the secondary ingression. Here, we report that WD repeat-containing protein 5 (WDR5), a core subunit of COMPASS/MLL family histone H3 lysine 4 methyltransferase (H3K4MT) complexes, resides at the midbody and associates with a subset of midbody regulatory proteins, including PRC1 and CYK4/MKLP1. Knockdown of WDR5 impairs abscission and increases the incidence of multinucleated cells. Further investigation revealed that the abscission delay is primarily due to slower formation of secondary ingressions in WDR5 knockdown cells. Consistent with these defects, midbody microtubules in WDR5 knockdown cells also display enhanced resistance to depolymerization by nocodazole. Recruitment of WDR5 to the midbody dark zone appears to require integrity of the WDR5 central arginine-binding cavity, as mutations that disrupt histone H3 and MLL1 binding to this pocket also abolish the midbody localization of WDR5. Taken together, these data suggest that WDR5 is specifically targeted to the midbody in the absence of chromatin and that it promotes abscission, perhaps by facilitating midbody microtubule disassembly.