Acidic Tail Research Articles

The Anti-Inflammatory Activity of HMGB1 A Box Is Enhanced When Fused with C-Terminal Acidic Tail

HMGB1, composed of the A box, B box, and C tail domains, is a critical proinflammatory cytokine involved in diverse inflammatory diseases. The B box mediates proinflammatory activity, while the A box alone acts as a specific antagonist of HMGB1. The C tail contributes to the spatial structure of A box and regulates HMGB1 DNA binding specificity. It is unknown whether the C tail can enhance the anti-inflammatory effect of A box. In this study, we generated fusion proteins consisting of the A box and C tail, in which the B box was deleted and the A box and C tail were linked either directly or by the flexible linker sequence (Gly4Ser)3. In vitro and in vivo experiments showed that the two fusion proteins had a higher anti-inflammatory activity compared to the A box alone. This suggests that the fused C tail enhances the anti-inflammatory effect of the A box.

Molecular Dynamics Simulations of Alpha-Synuclein at Various Temperatures

Alpha-synuclein (αS) is a natively unfolded protein with a C-terminus that is enriched with acidic residues. Three independent mutants (i.e., A30P, A53T and E46K) were identified in the genetic study of familial Parkinson's disease. Wild-type αS has been shown to possess a consistent secondary structure composition (Thomas D. Kim et al, 2000) at various temperatures. Other studies indicate that the acidic tail of αS plays an important role in preventing the protein's aggregation (Sang Myun Park et al, 2002). Here we show that both the wild-type and mutated proteins have a similar response to heat in our MD simulations. The mutant models were obtained by mutating the NMR minimized average structure of αS (PDB ID = 1XQ8). Atomistic simulations for both wild-type and mutated αS in explicit water were conducted for at least 30 ns at four different temperatures (i.e., 310 K, 330 K, 350 K and 372 K) using the CHARMM22/CMAP force field. For wild-type αS, the percentage of α-helix changed by less than 5% from that of the NMR structure regardless of simulation temperature. For each mutant αS, the percentage of α-helix differed by 10% or less regardless of simulation temperature. In all simulations, the acidic tail of αS remained predominantly random coil. Moreover, at 372 K, beta sheet was temporarily observed within a small region (approximately 3%) for the wild-type and A30P mutant.

The Acidic Tail of the Cdc34 Ubiquitin-conjugating Enzyme Functions in Both Binding to and Catalysis with Ubiquitin Ligase SCFCdc4

Ubiquitin ligases, together with their cognate ubiquitin-conjugating enzymes, are responsible for the ubiquitylation of proteins, a process that regulates a myriad of eukaryotic cellular functions. The first cullin-RING ligase discovered, yeast SCFCdc4, functions with the conjugating enzyme Cdc34 to regulate the cell cycle. Cdc34 orthologs are notable for their highly acidic C-terminal extension. Here we confirm that the Cdc34 acidic C-terminal tail has a role in Cdc34 binding to SCFCdc4 and makes a major contribution to the submicromolar Km of Cdc34 for SCFCdc4. Moreover, we demonstrate that a key functional property of the tail is its acidity. Our analysis also uncovers an unexpected new function for the acidic tail in promoting catalysis. We demonstrate that SCF is functional when Cdc34 is fused to the C terminus of Cul1 and that this fusion retains partial function even when the acidic tail has been deleted. The Cdc34-SCF fusion proteins that lack the acidic tail must interact in a fundamentally different manner than unfused SCF and wild type Cdc34, demonstrating that distinct mechanisms of E2 recruitment to E3, as is seen in nature, can sustain substrate ubiquitylation. Finally, a search of the yeast proteome uncovered scores of proteins containing highly acidic stretches of amino acids, hinting that electrostatic interactions may be a common mechanism for facilitating protein assembly.

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170–MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly ( cytoskeleton- associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.

Rapid E2-E3 Assembly and Disassembly Enable Processive Ubiquitylation of Cullin-RING Ubiquitin Ligase Substrates

Degradation by the ubiquitin-proteasome system requires assembly of a polyubiquitin chain upon substrate. However, the structural and mechanistic features that enable template-independent processive chain synthesis are unknown. We show that chain assembly by ubiquitin ligase SCF and ubiquitin-conjugating enzyme Cdc34 is facilitated by the unusual nature of Cdc34-SCF transactions: Cdc34 binds SCF with nanomolar affinity, nevertheless the complex is extremely dynamic. These properties are enabled by rapid association driven by electrostatic interactions between the acidic tail of Cdc34 and a basic 'canyon' in the Cul1 subunit of SCF. Ab initio docking between Cdc34 and Cul1 predicts intimate contact between the tail and the basic canyon, an arrangement confirmed by crosslinking and kinetic analysis of mutants. Basic canyon residues are conserved in both Cul1 paralogs and orthologs, suggesting that the same mechanism underlies processivity for all cullin-RING ubiquitin ligases. We discuss different strategies by which processive ubiquitin chain synthesis may be achieved.

Interactions between EB1 and Microtubules

Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.

The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1ΔC) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1ΔC were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

Comparative analysis and evolutionary significance of the HMG1 gene in crucian carp, blunt snout bream, and their polyploid progeny

The full-length mRNA of the high mobility group protein 1 coding gene ( HMG1) was obtained by RACE-PCR from red crucian carp ( Carassius auratus red var.), blunt snout bream ( Megalobrama amblycephala), and their triploid and tetraploid progeny. The sequence contained an open reading frame of 579 nucleotides coding for 193 amino acids. The nucleotide identity of HMG1 was higher between the tetraploid hybrid and the maternal red crucian carp (99%) than between the tetraploid hybrid and the paternal blunt snout bream (97%). The nucleotide identity between the triploid hybrids and each parent (95%) was lower than that between the parents (98%). The protein identity between the tetraploid hybrid and each parent (100%) was higher than that between the triploid hybrid and each parent (97%). Our results suggest that interspecific hybridization generates a shock to the HMG1 gene in triploid hybrids, causing divergence of nucleotides. The HMG1 protein of the tetraploid hybrids was consistent with that of its parents, which reduced the barrier of cross incompatibility between alleles, providing the basis for the bisexual fertile tetraploid hybrids forming a new polyploid species in nature. The secondary and tertiary structures of the HMG1 protein contain eight helices, three switches, two DNA-binding domains in the N-terminus, and a long acidic tail in the C-terminus. Together, these data suggest that the HMG1 protein plays a role of protein–DNA interactions, facilitating various DNA-dependent activities in the nucleus. We also investigated the phylogeny of fish, amphibian, reptilian, bird, and mammalian HMG1 proteins. Our results suggest that HMG1 is an ancestral protein that has been highly conserved. These data provide clues as to how interspecific hybridization may form polyploid hybrids.

Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1

BackgroundAntibacterial activity is a novel function of high-mobility group box 1 (HMGB1). However, the functional site for this new effect is presently unknown.Methods and ResultsIn this study, recombinant human HMGB1 A box and B box (rHMGB1 A box, rHMGB1 B box), recombinant human HMGB1 (rHMGB1) and the truncated C-terminal acidic tail mutant (tHMGB1) were prepared by the prokaryotic expression system. The C-terminal acidic tail (C peptide) was synthesized, which was composed of 30 amino acid residues. Antibacterial assays showed that both the full length rHMGB1 and the synthetic C peptide alone could efficiently inhibit bacteria proliferation, but rHMGB1 A box and B box, and tHMGB1 lacking the C-terminal acidic tail had no antibacterial function. These results suggest that C-terminal acidic tail is the key region for the antibacterial activity of HMGB1. Furthermore, we prepared eleven different deleted mutants lacking several amino acid residues in C-terminal acidic tail of HMGB1. Antibacterial assays of these mutants demonstrate that the amino acid residues 201-205 in C-terminal acidic tail region is the core functional site for the antibacterial activity of the molecule.ConclusionIn sum, these results define the key region and the crucial site in HMGB1 for its antibacterial function, which is helpful to illustrating the antibacterial mechanisms of HMGB1.

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.

Direct Bre1-Paf1 Complex Interactions and RING Finger-independent Bre1-Rad6 Interactions Mediate Histone H2B Ubiquitylation in Yeast

Recent yeast genetic studies have implicated the ubiquitin-conjugating enzyme and ubiquitin ligase functions of yRad6 and yBre1, respectively, in H2B ubiquitylation. However, there have been no corresponding biochemical analyses demonstrating intrinsic enzyme activities of yRad6 and yBre1 or related mechanistic details. Here, we describe a robust in vitro chromatin ubiquitylation assay that involves purified H2B ubiquitylation factors and natural nucleosomes. Our results indicate that yRad6 has an in vitro ability to nonspecifically ubiquitylate all core histones in the absence of an ubiquitin ligase but that yBre1 functions, through direct interactions with yRad6, to direct the ubiquitin conjugating activity of yRad6 toward the physiological H2B ubiquitylation site. Moreover, a yRad6 domain mapping analysis shows that an intact UBC domain is required for binding to yBre1, whereas the C-terminal acidic tail domain that is not required for a stable yBre1-yRad6 interaction is necessary for full enzyme activity of yRad6. We also find that, analogous to heteromeric complex formation by BRE1 paralogues in other organisms, yBre1 forms a homo-multimeric complex. Of special significance, our detailed biochemical analyses further show that the yBre1 RING finger domain is essential for H2B ubiquitylation but, surprisingly, dispensable for interaction of yBre1 with yRad6. In further support of the genetically identified requirement of the RNA polymerase II-associated yPaf1 complex for H2B ubiquitylation, protein interaction studies reveal that a purified yPaf1 complex directly and selectively interacts with yBre1 and thus serves to link the H2B ubiquitylation and general transcription machineries. These studies provide a more detailed mechanistic basis for H2B ubiquitylation in yeast.

Macroscopic Differences in HMGA Oncoproteins Post-Translational Modifications: C-Terminal Phosphorylation of HMGA2 Affects Its DNA Binding Properties

HMGA is a family of nuclear proteins involved in a huge number of functions at the chromatin level. It consists of three members, HMGA1a, HMGA1b, and HMGA2, having high sequence homology and sharing the same structural organization (three highly conserved DNA-binding domains, an acidic C-terminal tail, and a protein-protein interaction domain). They are considered important nodes in the chromatin context, establishing a complex network of interactions with both promoter/enhancer sequences and nuclear factors. They are involved in a plethora of biological processes and their activities are finely tuned by several different post-translational modifications. We have performed an LC/MS screening on several different cell lines to investigate HMGA proteins expression and their post-translational modifications in order to detect distinctive modification patterns for each. Our analyses evidenced relevant macroscopic differences in the phosphorylation and methylation patterns of these proteins. These differences occur both within the HMGA family members and in the different cell types. Focusing on HMGA2, we have mapped its in vivo phosphorylation sites demonstrating that, similarly to the HMGA1 proteins, it is highly phosphorylated on the acidic C-terminal tail and that these modifications affect its DNA binding properties.

Identification of distinct SET/TAF-Iβ domains required for core histone binding and quantitative characterisation of the interaction

BackgroundThe assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Iβ belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Iβ, we designed several SET/TAF-Iβ truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays.ResultsWild type SET/TAF-Iβ binds to histones H2B and H3 with Kd values of 2.87 and 0.15 μM, respectively. The preferential binding of SET/TAF-Iβ to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Iβ, as well as the H3 amino-terminal tail, are dispensable for this interaction.ConclusionThis type of analysis allowed us to assess the relative affinities of SET/TAF-Iβ for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Iβ and can be valuable to understand the role of SET/TAF-Iβ in chromatin function.

E46K Parkinson’s-Linked Mutation Enhances C-Terminal-to-N-Terminal Contacts in α-Synuclein

Parkinson's disease (PD) is associated with the deposition of fibrillar aggregates of the protein α-synuclein (αS) in neurons. Intramolecular contacts between the acidic C-terminal tail of αS and its N-terminal region have been proposed to regulate αS aggregation, and two originally described PD mutations, A30P and A53T, reportedly reduce such contacts. We find that the most recently discovered PD-linked αS mutation E46K, which also accelerates the aggregation of the protein, does not interfere with C-terminal-to-N-terminal contacts and instead enhances such contacts. Furthermore, we do not observe a substantial reduction in such contacts in the two previously characterized mutants. Our results suggest that C-terminal-to-N-terminal contacts in αS are not strongly protective against aggregation, and that the dominant mechanism by which PD-linked mutations facilitate αS aggregation may be altering the physicochemical properties of the protein such as net charge (E46K) and secondary structure propensity (A30P and A53T).

Native HMGB1 protein inhibits repair of cisplatin-damaged nucleosomes in vitro

The high mobility group box (HMGB) 1 protein, one of the most abundant nuclear non-histone proteins has been known for its inhibitory effect on repair of DNA damaged by the antitumor drug cisplatin. Here, we report the first results that link HMGB1 to repair of cisplatin-treated DNA at nucleosome level. Experiments were carried out with three types of reconstituted nucleosomes strongly positioned on the damaged DNA: linker DNA containing nucleosomes (centrally and end-positioned) and core particles. The highest repair synthesis was registered with end-positioned nucleosomes, two and three times more efficient than that with centrally positioned nucleosomes and core particles, respectively. HMGB1 inhibited repair of linker DNA containing nucleosomes more efficiently than that of core particles. Just the opposite was the effect of the in vivo acetylated HMGB1: stronger repair inhibition was obtained with core particles. No inhibition was observed with HMGB1 lacking the acidic tail. Binding of HMGB1 proteins to different nucleosomes was also analysed. HMGB1 bound preferentially to damage nucleosomes containing linker DNA, while the binding of the acetylated protein was linker independent. We show that both the repair of cisplatin-damaged nucleosomes and its inhibition by HMGB1 are nucleosome position-dependent events which are accomplished via the acidic tail and modulated by acetylation.

High mobility group B2 is secreted by myeloid cells and has mitogenic and chemoattractant activities similar to high mobility group B1

High mobility group B box (HMGB) proteins are a family of chromatin proteins made up of two basic DNA binding domains, HMG box A and B, and a C-terminal acidic tail. HMGB have a highly conserved sequence, but different expression pattern: HMGB1 is almost ubiquitous, whereas the others are highly expressed in only a few tissues in adults. We previously demonstrated that HMGB1 is released by necrotic cells and has chemoattractant activity for inflammatory and stem cells, via binding to receptor for advanced glycation endproducts (RAGE). HMGB1 can be actively secreted by inflammatory cells. Here, we report that also HMGB2 can be secreted by THP-1 cells, and promotes proliferation and migration of endothelial cells. These functions of HMGB2 are exerted via engagement of RAGE, whose blockade completely abrogates cell responses. Since extracellular HMGB2 has been detected in the blood and other biological fluids, it might be necessary to target HMGB2 at the same time as HMGB1 for therapeutical efficacy.

Enhanced DNA dynamics due to cationic reagents, topological states of dsDNA and high mobility group box 1 as probed by PicoGreen

We report a study of dynamics with a dsDNA-specific dye called PicoGreen bound to plasmid DNA (3.4 kb), and show that at low dye/DNA phosphate ratios (1 : 100 and below), PicoGreen dynamics reflect the motional dynamics of dsDNA. We further evaluated the usefulness of this probe by measuring the time-resolved fluorescence dynamics of PicoGreen bound to dsDNA in the presence of cationic reagents that affect DNA dynamics [MgCl2 and polyethyleneimine (PEI)] and also with plasmid DNA in different topological states. Among these conditions, MgCl2, PEI and the supercoiled form of plasmid resulted in increases in the very short component (0.2-0.4 ns) of the rotational correlation time. Separately, HMGB1 protein enhanced DNA dynamics, as observed from the rotational correlation times of very short (0.2-0.4 ns) and short (2-4 ns) rotational correlation timescale components. By studying the effect of specific deletion mutants HMGB1-DeltaA (deletion of 98 N-terminal amino acids) and HMGB1-DeltaC (deletion of 30 C-terminal amino acids), we show that the acidic C-terminal tail is required for enhancement of DNA dynamics. We then discuss the possible mechanisms and implications of HMGB1-mediated flexibility of DNA in the context of formation of large nucleoprotein complexes.

Distinct Domains in HMGB1 Are Involved in Specific Intramolecular and Nucleosomal Interactions

HMGB1 is composed of two DNA-binding domains and a long acidic tail at the C-terminus. The acidic tail interacts with the DNA-binding domains of HMGB1 and with core histone H3 in the nucleosome. These interactions are important for modulation of the DNA and chromatin binding activities of HMGB1, as well as biological functions of HMGB1. However, the interactions are not fully characterized, because the tertiary structure of full-length HMGB1 is still unknown. Here we use chemical cross-linking, mass spectrometry, and epitope masking analysis to perform a detailed characterization of the inter- and intramolecular protein interactions of the acidic tail of HMGB1. We show that specific regions of the acidic tail participate in intramolecular interactions with Lys2 of HMGB1 and in intermolecular interactions with Lys36 and Lys37 of histone H3. The acidic tail is oriented by its location adjacent to the C-terminus of helix III of DNA-binding HMG box A in the HMGB1 molecule. These results suggest that the acidic tail modulates the biological functions of HMGB1 through these specific interactions.

HMGB4, a Novel Member of the HMGB Family, Is Preferentially Expressed in the Mouse Testis and Localizes to the Basal Pole of Elongating Spermatids1

We identified HMGB4, a novel member of the HMGB family lacking the acidic tail typically found in this family. HMGB4 is strongly and preferentially expressed in the adult mouse testis and weakly in the brain, but not in many other tissues. HMGB4 associates with chromatin, and in transfection assays, in contrast to HMGB1, it acts as a potent transcriptional repressor. During spermatogenesis, HMGB4 is present in the euchromatin of late pachytene spermatocytes and haploid round spermatids, whereas stronger expression is observed during the elongation phase, where it localizes to the basal pole of the nucleus in a manner mutually exclusive with H1FNT (H1T2) localized at the apical pole. HMGB4 basal localization is lost in H1FNT-mutant spermatids, showing that H1FNT provides a positional cue for organizing chromatin domains within the nucleus. These results show that HMGB4 and H1FNT specify distinct chromatin domains at the apical and basal poles of the elongating spermatid nucleus.