Crosstalk between chromatin, chromosomes, and epigenetics.

The International Asilomar Chromatin, Chromosomes, and Epigenetics Conference was held online from 8 to 10 December 2022. Topics of this year's conference included chromosome dysregulation, genome integrity, nuclear organization, regulation of chromatin, epigenetics, transcription, and gene regulation in cell differentiation and disease. The meeting featured four keynote speakers, including Yamini Dalal (National Cancer Institute, USA), Meaghan Jones (University of Manitoba, Canada), Pedro Rocha (National Institute of Child Health and Human Development, USA), and Vincent Pasque (University of Leuven, Belgium). The meeting brought together scientists at all career stages to present and discuss their work in the fields of chromatin and epigenetics.

The ChIP-Exo Method to Identify Genomic Locations of DNA-Binding Proteins at Near Single Base-Pair Resolution.

Chromatin immunoprecipitation (ChIP) is a technique to determine whether a protein interacts with a specific DNA sequence. ChIP-sequencing (ChIP-seq) is one of the most widely used methods to identify genome-wide DNA-binding sites of nuclear proteins. Here, we describe the ChIP-exo method, which is a refined version of ChIP-seq combined with lambda exonuclease digestion. ChIP-exo can identify genomic locations of DNA-binding proteins at a near single base-pair (bp) resolution. It removes most of the background DNA signals. ChIP-exo has emerged as a powerful technique to study the genome-wide organization of DNA-binding proteins.

Extended intergenic DNA contributes to neuron-specific expression of neighboring genes in the mammalian nervous system.

Mammalian genomes comprise largely intergenic noncoding DNA with numerous cis-regulatory elements. Whether and how the size of intergenic DNA affects gene expression in a tissue-specific manner remain unknown. Here we show that genes with extended intergenic regions are preferentially expressed in neural tissues but repressed in other tissues in mice and humans. Extended intergenic regions contain twice as many active enhancers in neural tissues compared to other tissues. Neural genes with extended intergenic regions are globally co-expressed with neighboring neural genes controlled by distinct enhancers in the shared intergenic regions. Moreover, generic neural genes expressed in multiple tissues have significantly longer intergenic regions than neural genes expressed in fewer tissues. The intergenic regions of the generic neural genes have many tissue-specific active enhancers containing distinct transcription factor binding sites specific to each neural tissue. We also show that genes with extended intergenic regions are enriched for neural genes only in vertebrates. The expansion of intergenic regions may reflect the regulatory complexity of tissue-type-specific gene expression in the nervous system.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo).

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5' to 3' exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

The Efficacy of a Novel Tissue Grasper-Clips Technique for Large Perforations of the Sigmoid Colon in an Experimental Animal Model (Video).

BACKGROUND: The incidence of iatrogenic colonic perforation has been gradually increasing. In particular, sigmoid colon perforations are difficult to handle because of excess mobility. AIM: The aim of this study was to evaluate the efficacy of the twin grasper-clips technique for large perforations of the sigmoid colon. METHODS: This study was designed as a prospective, randomized, experimental study using ex vivo porcine colorectal specimens. Thirty standardized and variable artificial perforations were closed in the hemoclip group (hemoclips) and twin grasper group (hemoclips with a novel tissue grasper). We counted the number of hemoclips used per case to assess the cost and efficacy of the procedure. RESULTS: In the hemoclip group (n = 15), among the 20-, 25-, and 30-mm defects, the mean number of clips (4.8 ± 0.8, 6.0 ± 1.6, and 8.4 ± 2.1, respectively, p = 0.011) and closure time (7.6 ± 0.5, 9.9 ± 3.3, and 13.9 ± 4.1 min, respectively, p = 0.020) differed significantly. In the twin grasper group (n = 15), among the 20-, 25-, and 30-mm defects, the mean number of clips (4.0 ± 1.0, 5.0 ± 0.7, and 5.4 ± 1.1, respectively, p = 0.101) and closure time (7.7 ± 0.6, 8.3 ± 1.9, and 9.1 ± 2.7 min, respectively, p = 0.506) did not differ significantly. In 30-mm defects, the mean number of hemoclips used per case and total closure time were significantly lower in the twin grasper group than the hemoclip group. CONCLUSIONS: The twin grasper-clips technique seems to reduce the use of hemoclips and to result in more effective and rapid closure than does the conventional technique in large perforations of the ex vivo porcine sigmoid colon.

Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers.

Generic spinal motor neuron identity is established by cooperative binding of programming transcription factors (TFs), Isl1 and Lhx3, to motor-neuron-specific enhancers. How expression of effector genes is maintained following downregulation of programming TFs in maturing neurons remains unknown. High-resolution exonuclease (ChIP-exo) mapping revealed that the majority of enhancers established by programming TFs are rapidly deactivated following Lhx3 downregulation in stem-cell-derived hypaxial motor neurons. Isl1 is released from nascent motor neuron enhancers and recruited to new enhancers bound by clusters of Onecut1 in maturing neurons. Synthetic enhancer reporter assays revealed that Isl1 operates as an integrator factor, translating the density of Lhx3 or Onecut1 binding sites into transient enhancer activity. Importantly, independent Isl1/Lhx3- and Isl1/Onecut1-bound enhancers contribute to sustained expression of motor neuron effector genes, demonstrating that outwardly stable expression of terminal effector genes in postmitotic neurons is controlled by a dynamic relay of stage-specific enhancers.

Usefulness of the Forrest Classification to Predict Artificial Ulcer Rebleeding during Second-Look Endoscopy after Endoscopic Submucosal Dissection.

BACKGROUND/AIMS: Delayed post-endoscopic submucosal dissection (ESD) bleeding (DPEB) is difficult to predict and there is controversy regarding the usefulness of prophylactic hemostasis during second-look endoscopy. This study evaluated the risk factors related to DPEB, the relationship between clinical outcomes and the Forrest classification, and the results of prophylactic hemostasis during second-look endoscopy. METHODS: Second-look endoscopy was performed on the day after ESD to check for recent hemorrhage or potential bleeding and the presence of artificial ulcers in all patients. RESULTS: DPEB occurred in 42 of 581 patients (7.2%). Multivariate analysis determined that a specimen size ≥40 mm (odds ratio [OR], 3.03; p=0.003), and a high-risk Forrest classification (Forrest Ib+IIa+IIb; OR, 6.88; p<0.001) were risk factors for DPEB. DPEB was significantly more likely in patients classified with Forrest Ib (OR, 24.35; p<0.001), IIa (OR, 12.91; p<0.001), or IIb (OR, 8.31; p<0.001) ulcers compared with Forrest III ulcers. There was no statistically significant difference between the prophylactic hemostasis and non-hemostasis groups (Forrest Ib, p=0.938; IIa, p=0.438; IIb, p=0.397; IIc, p=0.773) during second-look endoscopy. CONCLUSIONS: The Forrest classification of artificial gastric ulcers during second-look endoscopy seems to be a useful tool for predicting delayed bleeding. However, routine prophylactic hemostasis during second-look endoscopy seemed to not be useful for preventing DPEB.

Subnucleosomal structures and nucleosome asymmetry across a genome.

Genes are packaged into nucleosomal arrays, each nucleosome typically having two copies of histones H2A, H2B, H3, and H4. Histones have distinct posttranslational modifications, variant isoforms, and dynamics. Whether each histone copy within a nucleosome has distinct properties, particularly in relation to the direction of transcription, is unknown. Here we use chromatin immunoprecipitation-exonuclease (ChIP-exo) to resolve the organization of individual histones on a genomic scale. We detect widespread subnucleosomal structures in dynamic chromatin, including what appear to be half-nucleosomes consisting of one copy of each histone. We also detect interactions of H3 tails with linker DNA between nucleosomes, which may be negatively regulated by methylation of H3K36. Histone variant H2A.Z is enriched on the promoter-distal half of the +1 nucleosome, whereas H2BK123 ubiquitylation and H3K9 acetylation are enriched on the promoter-proximal half in a transcription-linked manner. Subnucleosome asymmetries might serve as molecular beacons that guide transcription.

A comprehensive and high-resolution genome-wide response of p53 to stress.

Tumor suppressor p53 regulates transcription of stress-response genes. Many p53 targets remain undiscovered because of uncertainty as to where p53 binds in the genome and the fact that few genes reside near p53-bound recognition elements (REs). Using chromatin immunoprecipitation followed by exonuclease treatment (ChIP-exo), we associated p53 with 2,183 unsplit REs. REs were positionally constrained with other REs and other regulatory elements, which may reflect structurally organized p53 interactions. Surprisingly, stress resulted in increased occupancy of transcription factor IIB (TFIIB) and RNA polymerase (Pol) II near REs, which was reduced when p53 was present. A subset associated with antisense RNA near stress-response genes. The combination of high-confidence locations for p53/REs, TFIIB/Pol II, and their changes in response to stress allowed us to identify 151 high-confidence p53-regulated genes, substantially increasing the number of p53 targets. These genes composed a large portion of a predefined DNA-damage stress-response network. Thus, p53 plays a comprehensive role in regulating the stress-response network, including regulating noncoding transcription.

Takotsubo cardiomyopathy associated with severe hypocalcemia secondary to idiopathic hypoparathyroidism.

The etiology and pathophysiology of takotsubo cardiomyopathy have not yet been fully clarified. We report a case of takotsubo cardiomyopathy associated with severe hypocalcemia secondary to hypoparathyroidism. A 69-year-old woman presented with acute pulmonary edema caused by severe left ventricular dysfunction with apical ballooning compatible with takotsubo cardiomyopathy. Laboratory tests revealed severe hypocalcemia secondary to idiopathic hypoparathyroidism. Coronary angiography showed normal coronary artery function. Her symptoms and signs of heart failure improved dramatically with the correction of hypocalcemia through calcium and calcitriol replacement.

Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing.

Pausing of RNA polymerase II (Pol II) 20-60 bp downstream of transcription start sites is a major checkpoint during transcription in animal cells. Mechanisms that control pausing are largely unknown. We developed permanganate-ChIP-seq to evaluate the state of Pol II at promoters throughout the Drosophila genome, and a biochemical system that reconstitutes promoter-proximal pausing to define pausing mechanisms. Stable open complexes of Pol II are largely absent from the transcription start sites of most mRNA genes but are present at snRNA genes and the highly transcribed heat shock genes following their induction. The location of the pause is influenced by the timing between when NELF loads onto Pol II and how fast Pol II escapes the promoter region. Our biochemical analysis reveals that the sequence-specific transcription factor, GAF, orchestrates efficient pausing by recruiting NELF to promoters before transcription initiation and by assisting in loading NELF onto Pol II after initiation.

ChIP-exo method for identifying genomic location of DNA-binding proteins with near-single-nucleotide accuracy.

This unit describes the ChIP-exo methodology, which combines chromatin immunoprecipitation (ChIP) with lambda exonuclease digestion followed by high-throughput sequencing. ChIP-exo allows identification of a nearly complete set of the binding locations of DNA-binding proteins at near-single-nucleotide resolution with almost no background. The process is initiated by cross-linking DNA and associated proteins. Chromatin is then isolated from nuclei and subjected to sonication. Subsequently, an antibody against the desired protein is used to immunoprecipitate specific DNA-protein complexes. ChIP DNA is purified, sequencing adaptors are ligated, and the adaptor-ligated DNA is then digested by lambda exonuclease, generating 25- to 50-nucleotide fragments for high-throughput sequencing. The sequences of the fragments are mapped back to the reference genome to determine the binding locations. The 5' ends of DNA fragments on the forward and reverse strands indicate the left and right boundaries of the DNA-protein binding regions, respectively.

Genome-wide structure and organization of eukaryotic pre-initiation complexes.

Transcription and regulation of genes originate from transcription pre-initiation complexes (PICs). Their structural and positional organization across eukaryotic genomes is unknown. Here we applied lambda exonuclease to chromatin immunoprecipitates (termed ChIP-exo) to examine the precise location of 6,045 PICs in Saccharomyces. PICs, including RNA polymerase II and protein complexes TFIIA, TFIIB, TFIID (or TBP), TFIIE, TFIIF, TFIIH and TFIIK were positioned within promoters and excluded from coding regions. Exonuclease patterns were in agreement with crystallographic models of the PIC, and were sufficiently precise to identify TATA-like elements at so-called TATA-less promoters. These PICs and their transcription start sites were positionally constrained at TFIID-engaged downstream +1 nucleosomes. At TATA-box-containing promoters, which are depleted of TFIID, a +1 nucleosome was positioned to be in competition with the PIC, which may allow greater latitude in start-site selection. Our genomic localization of messenger RNA and non-coding RNA PICs reveals that two PICs, in inverted orientation, may occupy the flanking borders of nucleosome-free regions. Their unambiguous detection may help distinguish bona fide genes from transcriptional noise.

Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution.

Chromatin immunoprecipitation (ChIP-chip and ChIP-seq) assays identify where proteins bind throughout a genome. However, DNA contamination and DNA fragmentation heterogeneity produce false positives (erroneous calls) and imprecision in mapping. Consequently, stringent data filtering produces false negatives (missed calls). Here we describe ChIP-exo, where an exonuclease trims ChIP DNA to a precise distance from the crosslinking site. Bound locations are detectable as peak pairs by deep sequencing. Contaminating DNA is degraded or fails to form complementary peak pairs. With the single bp accuracy provided by ChIP-exo, we show an unprecedented view into genome-wide binding of the yeast transcription factors Reb1, Gal4, Phd1, Rap1, and human CTCF. Each of these factors was chosen to address potential limitations of ChIP-exo. We found that binding sites become unambiguous and reveal diverse tendencies governing in vivo DNA-binding specificity that include sequence variants, functionally distinct motifs, motif clustering, secondary interactions, and combinatorial modules within a compound motif.

Interaction of transcriptional regulators with specific nucleosomes across the Saccharomyces genome.

A canonical nucleosome architecture around promoters establishes the context in which proteins regulate gene expression. Whether gene regulatory proteins that interact with nucleosomes are selective for individual nucleosome positions across the genome is not known. Here, we examine on a genomic scale several protein-nucleosome interactions, including those that (1) bind histones (Bdf1/SWR1 and Srm1), (2) bind specific DNA sequences (Rap1 and Reb1), and (3) potentially collide with nucleosomes during transcription (RNA polymerase II). We find that the Bdf1/SWR1 complex forms a dinucleosome complex that is selective for the +1 and +2 nucleosomes of active genes. Rap1 selectively binds to its cognate site on the rotationally exposed first and second helical turn of nucleosomal DNA. We find that a transcribing RNA polymerase creates a delocalized state of resident nucleosomes. These findings suggest that nucleosomes around promoter regions have position-specific functions and that some gene regulators have position-specific nucleosomal interactions.

Momordin I, an inhibitor of AP-1, suppressed osteoclastogenesis through inhibition of NF-kappaB and AP-1 and also reduced osteoclast activity and survival.

Osteoclasts originating from hematopoietic precursor cells differentiate into multinucleated cells through multiple steps. The essential roles of NF-kappaB and AP-1 in osteoclast differentiation have been clearly demonstrated in numerous studies. c-Fos, a component of AP-1 transcription factor, plays a key role in osteoclast differentiation. Recently, we found a strong inhibitor of AP-1 transcriptional activity, named momordin I, based on the structure of oleanolic acid glycosides and originally isolated from Ampelopsis radix. So, we hypothesized that momordin I might be able to regulate osteoclast formation, activity, and survival. Here, we report the ability of momordin I to suppress osteoclastogenesis in a co-cultured system and a RANKL-induced osteoclast precursor system. Momordin I remarkably inhibited the activation of NF-kappaB as well as AP-1 in RANKL-induced RAW264.7 cells, in which momordin I appeared to target IkappaB degradation and c-Fos expression, respectively, but not MAPK signaling pathways. The ability of momordin I to change the ratio of RANKL and OPG in primary osteoblasts was partially responsible for the reduction of osteoclast formation. Furthermore, pit formation on dentin slices was suppressed by momordin I with stimulating actin ring disruption. Our results also showed that momordin I highly shortened osteoclast lifespan and induced osteoclast apoptosis. In conclusion, the present results demonstrate for the first time that momordin I is a potent inhibitor of osteoclast differentiation via the reduction of NF-kappaB and AP-1, and also suppresses osteoclast function and survival.

Negative regulation of beta-catenin/Tcf signaling by naringenin in AGS gastric cancer cell.

Functional activation of beta-catenin/Tcf signaling plays an important role in early events in carcinogenesis. We examined the effect of naringenin against beta-catenin/Tcf signaling in gastric cancer cells. Reporter gene assay showed that naringenin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by naringenin in HEK293 cells transiently transfected with constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism was related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that the beta-catenin distribution and the levels of nuclear beta-catenin and Tcf-4 proteins were unchanged after naringenin treatment. Moreover, the binding activities of Tcf complexes to consensus DNA were not affected by naringenin. Taken together, these data suggest that naringenin inhibits beta-catenin/Tcf signaling in gastric cancer with unknown mechanisms.

Determination of the dissociation constants for recombinant c-Myc, Max, and DNA complexes: the inhibitory effect of linoleic acid on the DNA-binding step.

c-Myc, the protein product of protooncogene c-myc, functions in cell proliferation, differentiation, and neoplastic disease. In this study, recombinant c-Myc and Max proteins, encompassing DNA binding (basic region) and dimerization (helix-loop-helix/leucine zipper) domain of human origin, were expressed in bacteria as Myc87 and Max85. Myc87 was purified under denatured conditions and was renatured again. The dissociation constant for the protein dimers and for dimer/DNA complexes were not detectable by isothermal titration calorimetry because of the low degree of solubility of Myc87 and Max85. Therefore, we set up equations which were used to determine the dissociation constants from the proportion of protein-DNA complexes. The dimer dissociation constants in TBS were 5.90(+/-0.54)x10(-7)M for Max85/Max85 homodimer, 6.85(+/-0.25)x10(-3)M for Myc87/Myc87 homodimer, and 2.55(+/-0.29)x10(-8)M for Myc87/Max85 heterodimer, and the DNA-binding dissociation constants in TBS were 1.33(+/-0.21)x10(-9)M for Max85/Max85/DNA, 2.27(+/-0.08)x10(-12)M for Myc87/Myc87/DNA, and 4.43(+/-0.37)x10(-10)M for Myc87/Max85/DNA. In addition, we revealed that linoleic acid which is known as an inhibitor for the formation of Max/Max/DNA complex reduced the affinity of Max homodimer for DNA. This result indicates that linoleic acid may bind to the DNA-binding region of Max homodimer.

Ionomycin downregulates beta-catenin/Tcf signaling in colon cancer cell line.

Functional activation of beta-catenin/Tcf signaling plays an important role in the early events in colorectal carcinogenesis. We examined the effect of ionomycin against beta-catenin/Tcf signaling in colon cancer cells. Reporter gene assay showed that ionomycin inhibited beta-catenin/Tcf signaling efficiently. In addition, the inhibition of beta-catenin/Tcf signaling by ionomycin in HEK293 cells transiently transfected with a constitutively mutant beta-catenin gene, whose product is not phosphorylated by GSK3beta, indicates that its inhibitory mechanism is related to beta-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunoprecipitation analysis, western blot and electrophoretic mobility shift assay. As a result, our data reveal that the association of beta-catenin and Tcf-4 is disrupted and the amount of beta-catenin product in the nucleus is decreased by ionomycin in a concentration-dependent manner. Moreover, ionomycin strongly suppressed the binding of the Tcf complexes to its specific DNA-binding sites. The significance of the current work is that ionomycin is a negative regulator of beta-catenin/Tcf signaling in colon cancer cells and its inhibitory mechanism is related to the decreased nuclear beta-catenin products and to the suppressed binding of Tcf complexes to consensus DNA.