Histone H3 lysine 27 crotonylation mediates gene transcriptional repression in chromatin.

Histone lysine acylation, including acetylation and crotonylation, plays a pivotal role in gene transcription in health and diseases. However, our understanding of histone lysine acylation has been limited to gene transcriptional activation. Here, we report that histone H3 lysine 27 crotonylation (H3K27cr) directs gene transcriptional repression rather than activation. Specifically, H3K27cr in chromatin is selectively recognized by the YEATS domain of GAS41 in complex with SIN3A-HDAC1 co-repressors. Proto-oncogenic transcription factor MYC recruits GAS41/SIN3A-HDAC1 complex to repress genes in chromatin, including cell-cycle inhibitor p21. GAS41 knockout or H3K27cr-binding depletion results in p21 de-repression, cell-cycle arrest, and tumor growth inhibition in mice, explaining a causal relationship between GAS41 and MYC gene amplification and p21 downregulation in colorectal cancer. Our study suggests that H3K27 crotonylation signifies a previously unrecognized, distinct chromatin state for gene transcriptional repression in contrast to H3K27 trimethylation for transcriptional silencing and H3K27 acetylation for transcriptional activation.

GAS41 promotes H2A.Z deposition through recognition of the N terminus of histone H3 by the YEATS domain.

Glioma amplified sequence 41 (GAS41), which has the Yaf9, ENL, AF9, Taf14, and Sas5 (YEATS) domain that recognizes lysine acetylation (Kac), regulates gene expression as a subunit of the SRCAP (SNF2-related CREBBP activator protein) complex that deposits histone H2A.Z at promoters in eukaryotes. The YEATS domains of the proteins AF9 and ENL recognize Kac by hydrogen bonding the aromatic cage to arginine situated just before K9ac or K27ac in the N-terminal tail of histone H3. Curiously, the YEATS domain of GAS41 binds most preferentially to the sequence that contains K14ac of H3 (H3K14ac) but lacks the corresponding arginine. Here, we biochemically and structurally elucidated the molecular mechanism by which GAS41 recognizes H3K14ac. First, stable binding of the GAS41 YEATS domain to H3K14ac required the N terminus of H3 (H3NT). Second, we revealed a pocket in the GAS41 YEATS domain responsible for the H3NT binding by crystallographic and NMR analyses. This pocket is away from the aromatic cage that recognizes Kac and is unique to GAS41 among the YEATS family. Finally, we showed that E109 of GAS41, a residue essential for the formation of the H3NT-binding pocket, was crucial for chromatin occupancy of H2A.Z and GAS41 at H2A.Z-enriched promoter regions. These data suggest that binding of GAS41 to H3NT via its YEATS domain is essential for its intracellular function.

Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation.

The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.

Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3' end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3' splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger.

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

The Impact of FGFR3 Alterations on the Tumor Microenvironment and the Efficacy of Immune Checkpoint Inhibitors in Bladder Cancer.

BACKGROUND: Currently, only limited knowledge is available regarding the phenotypic association between fibroblast growth factor receptor 3 (FGFR3) alterations and the tumor microenvironment (TME) in bladder cancer (BLCA). METHODS: A multi-omics analysis on 389 BLCA and 35 adjacent normal tissues from a cohort of OMPU-NCC Consortium Japan was retrospectively performed by integrating the whole-exome and RNA-sequence dataset and clinicopathological record. A median follow-up duration of all BLCA cohort was 31 months. RESULTS: FGFR3 alterations (aFGFR3), including recurrent mutations and fusions, accounted for 44% of non-muscle invasive bladder cancer (NMIBC) and 15% of muscle-invasive bladder cancer (MIBC). Within MIBC, the consensus subtypes LumP was significantly more prevalent in aFGFR3, whereas the Ba/Sq subtype exhibited similarity between intact FGFR3 (iFGFR3) and aFGFR3 cases. We revealed that basal markers were significantly increased in MIBC/aFGFR3 compared to MIBC/iFGFR3. Transcriptome analysis highlighted TIM3 as the most upregulated immune-related gene in iFGFR3, with differential immune cell compositions observed between iFGFR3 and aFGFR3. Using EcoTyper, TME heterogeneity was discerned even within aFGFR cases, suggesting potential variations in the response to checkpoint inhibitors (CPIs). Among 72 patients treated with CPIs, the objective response rate (ORR) was comparable between iFGFR3 and aFGFR3 (20% vs 31%; p = 0.467). Strikingly, a significantly higher ORR was noted in LumP/aFGFR3 compared to LumP/iFGFR3 (50% vs 5%; p = 0.022). This trend was validated using data from the IMvigor210 trial. Additionally, several immune-related genes, including IDO1, CCL24, IL1RL1, LGALS4, and NCAM (CD56) were upregulated in LumP/iFGFR3 compared to LumP/aFGFR3 cases. CONCLUSIONS: Differential pathways influenced by aFGFR3 were observed between NMIBC and MIBC, highlighting the upregulation of both luminal and basal markers in MIBC/aFGFR3. Heterogeneous TME was identified within MIBC/aFGFR3, leading to differential outcomes for CPIs. Specifically, a favorable ORR in LumP/aFGFR3 and a poor ORR in LumP/iFGFR3 were observed. We propose TIM3 as a potential target for iFGFR3 (ORR: 20%) and several immune checkpoint genes, including IDO1 and CCL24, for LumP/iFGFR3 (ORR: 5%), indicating promising avenues for precision immunotherapy for BLCA.

Functional insight into a neurodevelopmental disorder caused by missense variants in an RNA-binding protein, RBM10.

The RNA-binding motif protein 10, RBM10, is an RNA splicing regulator essential for development. Loss-of-function RBM10 variants are associated with TARP syndrome, a severe X-linked recessive condition in males. We report a 3-year-old male with a mild phenotype, consisting of cleft palate, hypotonia, developmental delay, and minor dysmorphisms, associated with a missense RBM10 variant, c.943T>C, p.Ser315Pro, affecting the RRM2 RNA-binding domain. His clinical features were similar to a previously reported case associated with a missense variant. The p.Ser315Pro mutant protein was expressed normally in the nucleus, but its expression level and protein stability were slightly reduced. Nuclear magnetic resonance spectroscopy showed that the structure and the RNA-binding ability of the RRM2 domain with the p.Ser315Pro were unaffected. However, it affects the alternative splicing regulations of downstream genes, NUMB and TNRC6A, and its splicing alteration patterns were variable depending on target transcripts. In summary, a novel germline missense RBM10 p.Ser315Pro variant that causes functional changes in the expression of its downstream genes results in a non-lethal phenotype associated with developmental delays. The functional alteration effects depend on the residues affected by missense variants. Our findings are expected to bring broader insights into the RBM10-associated genotype-phenotype relationships by delineating the molecular mechanism of RBM10 functions.

Crystal structure of higher plant heme oxygenase-1 and its mechanism of interaction with ferredoxin.

Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (∼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light.

Single-Cell Native Mass Spectrometry of Human Erythrocytes.

Native mass spectrometry (MS) enables the determination of the molecular mass of protein complexes. Generally, samples for native MS are isolated, purified, and prepared in volatile solutions. However, to understand the function of proteins in living cells, it is essential to characterize the protein complex as is, without isolation/purification of the protein, using the smallest possible amount of the sample. In the present study, we modified the "live single-cell MS" method, which has mainly been used in metabolomics, and applied it to observe hemoglobin directly sampled from human erythrocytes. By optimizing the experimental methods and conditions, we obtained native mass spectra of hemoglobin using only a single erythrocyte, which was directly sampled into a nanoelectrospray ionization emitter using a micromanipulator and microinjector system. That is, our method enables the analysis of ∼0.45 fmol of hemoglobin directly sampled from an erythrocyte. To our knowledge, this is the first report of native MS for endogenous proteins using a single intact human cell.

A novel missense variant in RBM10 can cause a mild form of TARP syndrome with developmental delay and dysmorphic features.

RBM10, is an RNA binding protein that is important for development by regulating the expression of multiple genes. RBM10 is on the X chromosome, and nonsense and frameshift RBM10 variants cause TARP syndrome in males. In a 4-year-old male, we identified a novel maternally inherited missense RBM10 variant in the RRM2 RNA binding domain, c.965C>T, p.Pro322Leu. His clinical features included intellectual disability, developmental delay, growth restriction, hypotonia, and craniofacial malformations. These features were much milder than those described in previously reported cases of TARP syndrome. By in vitro assays, we found that the mutant p.Pro322Leu RBM10 protein retained its specific RNA binding capacity, while gaining a low-affinity nonspecific RNA binding. It was normally localized to the nucleus, but its expression level was significantly reduced with a significantly short half-life. These results indicated that the p.Pro322Leu missense variant causes a developmental disorder in humans through a unique loss-of-function mechanism.

Screening of protein-ligand interactions under crude conditions by native mass spectrometry.

A convenient analytical system for protein-ligand interactions under crude conditions was developed using native mass spectrometry (MS). As a model protein, Escherichia coli (E. coli) dihydrofolate reductase (DHFR) with and without a histidine tag was used for the study. First, overexpressed DHFR with a His-tag was roughly purified with a Ni-sepharose resin and subjected to native mass spectrometry with or without incubation with an inhibitor, Methotrexate (MTX). Even only with the minimum cleanup by the Ni-sepharose resin, intact ions of DHFR-nicotinamide adenine dinucleotide phosphate (NADPH) and DHFR-NADPH-ligand complexes were successfully observed. By optimizing the preparation procedures of the crude sample for native MS, e.g., avoiding sonication for cell lysis, we successfully observed intact ions of the specific DHFR-NADPH-MTX ternary complex starting with cultivation of E. coli in ≤ 25 mL medium. When the crude DHFR sample was mixed with two, four, or eight candidate compounds, only ions of the specific protein-ligand complex were observed. This indicates that the present system can be used as a rapid and convenient method for the rough determination of binding of specific ligands to the target protein without the time-consuming purification of protein samples. Moreover, it is important to rapidly determine specific interactions with target proteins under conditions similar to those in "real" biological systems. Graphical abstract.

A New Quinoline BRD4 Inhibitor Targets a Distinct Latent HIV-1 Reservoir for Reactivation from Other "Shock" Drugs.

Upon HIV-1 infection, a reservoir of latently infected resting T cells prevents the eradication of the virus from patients. To achieve complete depletion, the existing virus-suppressing antiretroviral therapy must be combined with drugs that reactivate the dormant viruses. We previously described a novel chemical scaffold compound, MMQO (8-methoxy-6-methylquinolin-4-ol), that is able to reactivate viral transcription in several models of HIV latency, including J-Lat cells, through an unknown mechanism. MMQO potentiates the activity of known latency-reversing agents (LRAs) or "shock" drugs, such as protein kinase C (PKC) agonists or histone deacetylase (HDAC) inhibitors. Here, we demonstrate that MMQO activates HIV-1 independently of the Tat transactivator. Gene expression microarrays in Jurkat cells indicated that MMQO treatment results in robust immunosuppression, diminishes expression of c-Myc, and causes the dysregulation of acetylation-sensitive genes. These hallmarks indicated that MMQO mimics acetylated lysines of core histones and might function as a bromodomain and extraterminal domain protein family inhibitor (BETi). MMQO functionally mimics the effects of JQ1, a well-known BETi. We confirmed that MMQO interacts with the BET family protein BRD4. Utilizing MMQO and JQ1, we demonstrate how the inhibition of BRD4 targets a subset of latently integrated barcoded proviruses distinct from those targeted by HDAC inhibitors or PKC pathway agonists. Thus, the quinoline-based compound MMQO represents a new class of BET bromodomain inhibitors that, due to its minimalistic structure, holds promise for further optimization for increased affinity and specificity for distinct bromodomain family members and could potentially be of use against a variety of diseases, including HIV infection.IMPORTANCE The suggested "shock and kill" therapy aims to eradicate the latent functional proportion of HIV-1 proviruses in a patient. However, to this day, clinical studies investigating the "shocking" element of this strategy have proven it to be considerably more difficult than anticipated. While the proportion of intracellular viral RNA production and general plasma viral load have been shown to increase upon a shock regimen, the global viral reservoir remains unaffected, highlighting both the inefficiency of the treatments used and the gap in our understanding of viral reactivation in vivo Utilizing a new BRD4 inhibitor and barcoded HIV-1 minigenomes, we demonstrate that PKC pathway activators and HDAC and bromodomain inhibitors all target different subsets of proviral integration. Considering the fundamental differences of these compounds and the synergies displayed between them, we propose that the field should concentrate on investigating the development of combinatory shock cocktail therapies for improved reservoir reactivation.

Non-Native α-Helices in the Initial Folding Intermediate Facilitate the Ordered Assembly of the ß-Barrel in ß-Lactoglobulin.

The roles of non-native α-helices frequently observed in the initial folding stage of ß-sheet proteins have been examined for many years. We herein investigated the residue-level structures of several mutants of bovine ß-lactoglobulin (ßLG) in quenched-flow pH-pulse labeling experiments. ßLG assumes a collapsed intermediate with a non-native α-helical structure (I0) in the early stage of folding, although its native form is predominantly composed of ß-structures. The protection profile in I0 of pseudo-wild type (WT*) ßLG was found to deviate from the pattern of the "average area buried upon folding" (AABUF). In particular, the level of protection at the region of strand A, at which non-native α-helices form in the I0 state, was significantly low compared to AABUF. G17E, the mutant with an increased helical propensity, showed a similar protection pattern. In contrast, the protection pattern for I0 of E44L, the mutant with an increased ß-sheet propensity, was distinct from that of WT* and resembled the AABUF pattern. Transverse relaxation measurements demonstrated that the positions of the residual structures in the unfolded states of these mutants were consistent with those of the protected residues in the respective I0 states. On the basis of the slower conversion of I0 to the native state for E44L to that for WT*, non-native α-helices facilitate the ordered assembly of the ß-barrel by preventing interactions that trap folding.

Dynamics of the Extended String-Like Interaction of TFIIE with the p62 Subunit of TFIIH.

General transcription factor II E (TFIIE) contains an acid-rich region (residues 378-393) in its α-subunit, comprising 13 acidic and two hydrophobic (Phe387 and Val390) residues. Upon binding to the p62 subunit of TFIIH, the acidic region adopts an extended string-like structure on the basic groove of the pleckstrin homology domain (PHD) of p62, and inserts Phe387 and Val390 into two shallow pockets in the groove. Here, we have examined the dynamics of this interaction by NMR and molecular dynamics (MD) simulations. Although alanine substitution of Phe387 and/or Val390 greatly reduced binding to PHD, the binding mode of the mutants was similar to that of the wild-type, as judged by the chemical-shift changes of the PHD. NMR relaxation dispersion profiles of the interaction exhibited large amplitudes for residues in the C-terminal half-string in the acidic region (Phe387, Glu388, Val390, Ala391, and Asp392), indicating a two-site binding mode: one corresponding to the final complex structure, and one to an off-pathway minor complex. To probe the off-pathway complex structure, an atomically detailed free-energy landscape of the binding mode was computed by all-atom multicanonical MD. The most thermodynamically stable cluster corresponded to the final complex structure. One of the next stable clusters was the off-pathway structure cluster, showing the reversed orientation of the C-terminal half-string on the PHD groove, as compared with the final structure. MD calculations elucidated that the C-terminal half-acidic-string forms encounter complexes mainly around the positive groove region with nearly two different orientations of the string, parallel and antiparallel to the final structure. Interestingly, the most encountered complexes exhibit a parallel-like orientation, suggesting that the string has a tendency to bind around the groove in the proper orientation with the aid of Phe387 and/or Val390 to proceed smoothly to the final complex structure.

Use of glass capillaries to suppress thermal convection in NMR tubes in diffusion measurements.

Diffusion ordered spectroscopy (DOSY) is used to determine the translational diffusion coefficients of molecules in solution. However, DOSY is highly susceptible to spurious spectral peaks resulting from thermal convection occurring in the NMR tube. Thermal convection therefore must be suppressed for accurate estimation of translational diffusion coefficients. In this study, we developed a new method to effectively suppress thermal convection using glass capillaries. A total of 6 to 18 capillaries (0.8-mm outer diameter) were inserted into a regular 5-mm NMR tube. The capillaries had minimal effect on magnetic field homogeneity and enabled us to obtain clean DOSY spectra of a mixture of small organic compounds. Moreover, the capillaries did not affect chemical shifts or signal intensities in two-dimensional heteronuclear single quantum coherence spectra. Capillaries are a simple and inexpensive means of suppressing thermal convection and thus can be used in a wide variety of DOSY experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Solution structure of the ubiquitin-associated (UBA) domain of human autophagy receptor NBR1 and its interaction with ubiquitin and polyubiquitin.

NBR1 (neighbor of BRCA1 gene 1) is a protein commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Due to its high architectural similarity to the well studied autophagy receptor protein p62/SQSTM1, NBR1 has been thought to analogously bind to ubiquitin-marked autophagic substrates via its C-terminal ubiquitin-associated (UBA) domain and deliver them to autophagosomes for degradation. Unexpectedly, we find that NBR1 differs from p62 in its UBA structure and accordingly in its interaction with ubiquitin. Structural differences are observed on helix α-3, which is tilted farther from helix α-2 and extended by approximately one turn in NBR1. This results not only in inhibition of a p62-type self-dimerization of NBR1 UBA but also in a significantly higher affinity for monoubiquitin as compared with p62 UBA. Importantly, the NBR1 UBA-ubiquitin complex structure shows that the negative charge of the side chain in front of the conserved MGF motif in the UBA plays an integral role in the recognition of ubiquitin. In addition, NMR and isothermal titration calorimetry experiments show that NBR1 UBA binds to each monomeric unit of polyubiquitin with similar affinity and by the same surface used for binding to monoubiquitin. This indicates that NBR1 lacks polyubiquitin linkage-type specificity, in good agreement with the nonspecific linkages observed in intracellular ubiquitin-positive inclusions. Consequently, our results demonstrate that the structural differences between NBR1 UBA and p62 UBA result in a much higher affinity of NBR1 for ubiquitin, which in turn suggests that NBR1 may form intracellular inclusions with ubiquitylated autophagic substrates more efficiently than p62.

Extracting protein dynamics information from overlapped NMR signals using relaxation dispersion difference NMR spectroscopy.

Protein dynamics plays important roles in many biological events, such as ligand binding and enzyme reactions. NMR is mostly used for investigating such protein dynamics in a site-specific manner. Recently, NMR has been actively applied to large proteins and intrinsically disordered proteins, which are attractive research targets. However, signal overlap, which is often observed for such proteins, hampers accurate analysis of NMR data. In this study, we have developed a new methodology called relaxation dispersion difference that can extract conformational exchange parameters from overlapped NMR signals measured using relaxation dispersion spectroscopy. In relaxation dispersion measurements, the signal intensities of fluctuating residues vary according to the Carr-Purcell-Meiboon-Gill pulsing interval, whereas those of non-fluctuating residues are constant. Therefore, subtraction of each relaxation dispersion spectrum from that with the highest signal intensities, measured at the shortest pulsing interval, leaves only the signals of the fluctuating residues. This is the principle of the relaxation dispersion difference method. This new method enabled us to extract exchange parameters from overlapped signals of heme oxygenase-1, which is a relatively large protein. The results indicate that the structural flexibility of a kink in the heme-binding site is important for efficient heme binding. Relaxation dispersion difference requires neither selectively labeled samples nor modification of pulse programs; thus it will have wide applications in protein dynamics analysis.

Distinct Histone H3 Lysine 27 Modifications Dictate Different Outcomes of Gene Transcription.

Site-specific histone modifications have long been recognized to play an important role in directing gene transcription in chromatin in biology of health and disease. However, concrete illustration of how different histone modifications in a site-specific manner dictate gene transcription outcomes, as postulated in the influential "Histone code hypothesis", introduced by Allis and colleagues in 2000, has been lacking. In this review, we summarize our latest understanding of the dynamic regulation of gene transcriptional activation, silence, and repression in chromatin that is directed distinctively by histone H3 lysine 27 acetylation, methylation, and crotonylation, respectively. This represents a special example of a long-anticipated verification of the "Histone code hypothesis."

Biophysical insights into the dimer formation of human Sirtuin 2.

Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild-type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 (hSIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT hSIRT2 HDAC (residues 52-357) forms a homodimer in a concentration-dependent manner with a dimer-monomer dissociation constant of 8.3 ± 0.5 µM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of hSIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of hSIRT2 HDAC revealed that some non-dimerizing mutants exhibited deacetylation activity for the N-terminal peptide of histone H3, similar to the wild type. The hSIRT2 HDAC mutant Δ292-306, which lacks a SIRT2-specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X-ray structure of the mutant Δ292-306 was almost identical to that of the WT hSIRT2 HDAC bound to an inhibitor. These results indicate that hSIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of hSIRT2 based on our biophysical experimental results.

1H, 13C and 15N backbone resonance assignments of hepatocyte nuclear factor-1-beta (HNF1ß) POUS and POUHD.

Hepatocyte nuclear factor 1ß (HNF1ß) is a transcription factor that plays a key role in the development and function of the liver, pancreas, and kidney. HNF1ß plays a key role in early vertebrate development and the morphogenesis of these organs. In humans, heterozygous mutations in the HNF1B gene can result in organ dysplasia, making it the most common cause of developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. Pathogenic variants in the HNF1B gene are known to cause various diseases, including maturity-onset diabetes of the young and developmental renal diseases. This study presents the backbone resonance assignments of HNF1ß POUS and POUHD domains, which are highly conserved domains required for the recognition of double-stranded DNA. Our data will be useful for NMR studies to verify the altered structures and functions of mutant HNF1B proteins that can induce developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. This study will provide the structural basis for future studies to elucidate the molecular mechanisms underlying how mutations in HNF1ß cause diseases.