Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.

Enhancers are activated by p300/CBP activity-dependent PIC assembly, RNAPII recruitment, and pause release.

The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a "recruit-and-release" mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.

Synergy between Variant PRC1 Complexes Defines Polycomb-Mediated Gene Repression.

The Polycomb system modifies chromatin and plays an essential role in repressing gene expression to control normal mammalian development. However, the components and mechanisms that define how Polycomb protein complexes achieve this remain enigmatic. Here, we use combinatorial genetic perturbation coupled with quantitative genomics to discover the central determinants of Polycomb-mediated gene repression in mouse embryonic stem cells. We demonstrate that canonical Polycomb repressive complex 1 (PRC1), which mediates higher-order chromatin structures, contributes little to gene repression. Instead, we uncover an unexpectedly high degree of synergy between variant PRC1 complexes, which is fundamental to gene repression. We further demonstrate that variant PRC1 complexes are responsible for distinct pools of H2A monoubiquitylation that are associated with repression of Polycomb target genes and silencing during X chromosome inactivation. Together, these discoveries reveal a new variant PRC1-dependent logic for Polycomb-mediated gene repression.

A Family of Vertebrate-Specific Polycombs Encoded by the LCOR/LCORL Genes Balance PRC2 Subtype Activities.

The polycomb repressive complex 2 (PRC2) consists of core subunits SUZ12, EED, RBBP4/7, and EZH1/2 and is responsible for mono-, di-, and tri-methylation of lysine 27 on histone H3. Whereas two distinct forms exist, PRC2.1 (containing one polycomb-like protein) and PRC2.2 (containing AEBP2 and JARID2), little is known about their differential functions. Here, we report the discovery of a family of vertebrate-specific PRC2.1 proteins, "PRC2 associated LCOR isoform 1" (PALI1) and PALI2, encoded by the LCOR and LCORL gene loci, respectively. PALI1 promotes PRC2 methyltransferase activity in vitro and in vivo and is essential for mouse development. Pali1 and Aebp2 define mutually exclusive, antagonistic PRC2 subtypes that exhibit divergent H3K27-tri-methylation activities. The balance of these PRC2.1/PRC2.2 activities is required for the appropriate regulation of polycomb target genes during differentiation. PALI1/2 potentially link polycombs with transcriptional co-repressors in the regulation of cellular identity during development and in cancer.

Polycomb-mediated histone modifications and gene regulation.

Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are transcriptional repressor complexes that play a fundamental role in epigenomic regulation and the cell-fate decision; these complexes are widely conserved in multicellular organisms. PRC1 is an E3 ubiquitin (ub) ligase that generates histone H2A ubiquitinated at lysine (K) 119 (H2AK119ub1), whereas PRC2 is a histone methyltransferase that specifically catalyzes tri-methylation of histone H3K27 (H3K27me3). Genome-wide analyses have confirmed that these two key epigenetic marks highly overlap across the genome and contribute to gene repression. We are now beginning to understand the molecular mechanisms that enable PRC1 and PRC2 to identify their target sites in the genome and communicate through feedback mechanisms to create Polycomb chromatin domains. Recently, it has become apparent that PRC1-induced H2AK119ub1 not only serves as a docking site for PRC2 but also affects the dynamics of the H3 tail, both of which enhance PRC2 activity, suggesting that trans-tail communication between H2A and H3 facilitates the formation of the Polycomb chromatin domain. In this review, we discuss the emerging principles that define how PRC1 and PRC2 establish the Polycomb chromatin domain and regulate gene expression in mammals.

Defining super-enhancers by highly ranked histone H4 multi-acetylation levels identifies transcription factors associated with glioblastoma stem-like properties.

BACKGROUND: Super-enhancers (SEs), which activate genes involved in cell-type specificity, have mainly been defined as genomic regions with top-ranked enrichment(s) of histone H3 with acetylated K27 (H3K27ac) and/or transcription coactivator(s) including a bromodomain and extra-terminal domain (BET) family protein, BRD4. However, BRD4 preferentially binds to multi-acetylated histone H4, typically with acetylated K5 and K8 (H4K5acK8ac), leading us to hypothesize that SEs should be defined by high H4K5acK8ac enrichment at least as well as by that of H3K27ac. RESULTS: Here, we conducted genome-wide profiling of H4K5acK8ac and H3K27ac, BRD4 binding, and the transcriptome by using a BET inhibitor, JQ1, in three human glial cell lines. When SEs were defined as having the top ranks for H4K5acK8ac or H3K27ac signal, 43% of H4K5acK8ac-ranked SEs were distinct from H3K27ac-ranked SEs in a glioblastoma stem-like cell (GSC) line. CRISPR-Cas9-mediated deletion of the H4K5acK8ac-preferred SEs associated with MYCN and NFIC decreased the stem-like properties in GSCs. CONCLUSIONS: Collectively, our data highlights H4K5acK8ac's utility for identifying genes regulating cell-type specificity.

Impact of early initiation of renin-angiotensin blockade on renal function and clinical outcomes in patients with hypertensive emergency: a retrospective cohort study.

BACKGROUND: Hypertensive emergency is a critical disease that causes multifaceted sequelae, including end-stage kidney disease and cardiovascular disease. Although the renin-angiotensin-aldosterone (RAA) system is enormously activated in this disease, there are few reports that attempt to characterize the effect of early use of RAA inhibitors (RASi) on the temporal course of kidney function. METHODS: This retrospective cohort study was conducted to clarify whether the early use of RASi during hospitalization offered more favorable benefits on short-term renal function and long-term renal outcomes in patients with hypertensive emergencies. We enrolled a total of 49 patients who visited our medical center with acute severe hypertension and multiple organ dysfunction between April 2012 and August 2020. Upon admission, the patients were treated with intravenous followed by oral antihypertensive drugs, including RASi and Ca channel blockers (CCB). Kidney function as well as other laboratory and clinical parameters were compared between RASi-treated and CCB- treated group over 2 years. RESULTS: Antihypertensive treatment effectively reduced blood pressure from 222 ± 28/142 ± 21 to 141 ± 18/87 ± 14 mmHg at 2 weeks and eGFR was gradually restored from 33.2 ± 23.3 to 40.4 ± 22.5 mL/min/1.73m2 at 1 year. The renal effect of antihypertensive drugs was particularly conspicuous when RASi was started in combination with other conventional antihypertensive drugs at the early period of hospitalization (2nd day [IQR: 1-5.5]) and even in patients with moderately to severely diminished eGFR (< 30 mL/min/1.73 m2) on admission. In contrast, CCB modestly restored eGFR during the observation period. Furthermore, renal survival probabilities were progressively deteriorated in patients who had manifested reduced eGFR (< 15 mL/min/1.73 m2) or massive proteinuria (urine protein/creatinine ≥ 3.5 g/gCr) on admission. Early use of RASi was associated with a favorable 2-year renal survival probability (0.90 [95%CI: 0.77-1.0] vs. 0.63 [95%CI: 0.34-0.92] for RASi ( +) and RASi (-), respectively, p = 0.036) whereas no apparent difference in renal survival was noted for CCB. CONCLUSIONS: Early use of RASi contributes to the renal functional recovery from acute reduction in eGFR among patients with hypertensive emergencies. Furthermore, RASi offers more favorable effect on 2-year renal survival, compared with CCB.

Polycomb in Transcriptional Phase Transition of Developmental Genes.

Combinatorial associations between distinct chromatin domains, namely promoters and cis-regulatory elements, determine transcriptional status. Developmental regulatory gene expression, mostly regulated by the Polycomb/Trithorax group of chromatin regulators, is often temporally and spatially specific, and sometimes changes repeatedly within the same cell lineage. Dysregulated expression of these genes causes morphological and/or functional disorganization of tissues and organs. Therefore, maintenance of both the active and negative states of transcription is equally important. In this review, we summarize the mechanisms of transition between transcriptional status of developmental regulators, including complex processes for enhancer activation and promoter-enhancer association. In particular, we propose testable models in which Polycomb group factors contribute to promoter-enhancer associations and thus proper gene expression.

Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells.

Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.

MMXD, a TFIIH-independent XPD-MMS19 protein complex involved in chromosome segregation.

Xeroderma pigmentosum group D (XPD) protein is one of the subunits of TFIIH that is required for nucleotide excision repair and transcription. We found a XPD protein complex containing MMS19 that was assumed to be a regulator of TFIIH. However, the MMS19-XPD complex did not contain any other subunits of TFIIH. Instead, it included FAM96B (now designated MIP18), Ciao1, and ANT2. MMS19, MIP18, and XPD localized to the mitotic spindle during mitosis. The siRNA-mediated knockdown of MMS19, MIP18, or XPD led to improper chromosome segregation and the accumulation of nuclei with abnormal shapes. In addition, the frequency of abnormal mitosis and nuclei was increased in XP-D and XP-D/CS patients' cells. These results indicate that the MMS19-XPD protein complex, now designated MMXD (MMS19-MIP18-XPD), is required for proper chromosome segregation, an abnormality of which could contribute to the pathogenesis in some cases of XP-D and XP-D/CS.

Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells.

Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. The recent demonstration that members of the Ten-eleven translocation (Tet) family of proteins can convert 5-methylcytosine to 5-hydroxymethylcytosine raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, here we show in mouse ES cells that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite an increase in levels of DNA methylation at many Tet1-binding sites, Tet1 depletion does not lead to downregulation of all the Tet1 targets. Interestingly, although Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also involved in repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators.

Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification.

DNA methylation is one of the best-characterized epigenetic modifications. Although the enzymes that catalyse DNA methylation have been characterized, enzymes responsible for demethylation have been elusive. A recent study indicates that the human TET1 protein could catalyse the conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC), raising the possibility that DNA demethylation may be a Tet1-mediated process. Here we extend this study by demonstrating that all three mouse Tet proteins (Tet1, Tet2 and Tet3) can also catalyse a similar reaction. Tet1 has an important role in mouse embryonic stem (ES) cell maintenance through maintaining the expression of Nanog in ES cells. Downregulation of Nanog via Tet1 knockdown correlates with methylation of the Nanog promoter, supporting a role for Tet1 in regulating DNA methylation status. Furthermore, knockdown of Tet1 in pre-implantation embryos results in a bias towards trophectoderm differentiation. Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but also demonstrate a role for Tet1 in ES cell maintenance and inner cell mass cell specification.

The impact of hearing aids on cognitive function and quality of life in patients with hearing impairment: A cross-sectional study.

OBJECTIVE: Age-related cognitive decline involves a complex set of factors. Among these factors, hearing loss is considered to have a significant impact, but the effect of hearing aid use remains unresolved. The purpose of this study was to evaluate the effects of hearing aid use by simultaneously assessing various factors not only cognitive function but also frailty, anxiety, depression, and quality of life (QOL) in patients with hearing loss. METHODS: The cross-sectional study at the Hearing Aid (HA) Center was conducted between 2020 and 2021. Initially, associations with cognitive function, QOL, frailty, and mental state among patients with hearing loss were examined, irrespective of whether they wore a hearing aid or not. Next, these patients were divided into HA users (using HA for more than 1 year) and non-users (no prior use of HA) with 42 patients in each group. The average age and 6-frequency pure tone audiometry (PTA) was 74.5 ± 6.5 years and 50.6 ± 12.1 dB, respectively. All participants filled out the questionnaire about their life style, medical condition. Mini-Mental State Examination (MMSE) for cognitive function, Hospital Anxiety and Depression Scale for mental state, Short Form 36 version 2 (SF-36v2) for QOL, and Kihon Checklist for frailty were compared between HA users and non-users and correlated with the auditory data (PTA and speech discrimination). RESULTS: Among 84 patients, 40 had an MMSE score ≦26. All eight scores and three components of SF-36v2 were lower than those of the control group. The patients with hypertension were significantly more in HA user than in non-HA user, whereas there was no difference in diabetes, heart attack, stroke and education. Although HA users were older and showed hypertension more their PTA was worse than that of non-users, MMSE scores were not different between the groups. MMSE scores correlated with both PTA and speech discrimination in non-users but not in HA users. However, a multivariate analysis of the effect of HA use on MMSE scores adjusting for age, hypertension, and hearing loss, could not be revealed. The vitality and mental component summary of the SF-36v2 was better in HA users than in non-users. CONCLUSION: Elderly patients with hearing loss were cognitively impaired and had low QOL. HA users showed better QOL score than non-HA user, especially about the mental condition. The absence of a correlation between MMSE scores and hearing loss in HA users suggests the potential use of HA in preventing cognitive decline.

H2A Ubiquitination Alters H3-tail Dynamics on Linker-DNA to Enhance H3K27 Methylation.

Polycomb repressive complex 1 (PRC1) and PRC2 are responsible for epigenetic gene regulation. PRC1 ubiquitinates histone H2A (H2Aub), which subsequently promotes PRC2 to introduce the H3 lysine 27 tri-methyl (H3K27me3) repressive chromatin mark. Although this mechanism provides a link between the two key transcriptional repressors, PRC1 and PRC2, it is unknown how histone-tail dynamics contribute to this process. Here, we have examined the effect of H2A ubiquitination and linker-DNA on H3-tail dynamics and H3K27 methylation by PRC2. In naïve nucleosomes, the H3-tail dynamically contacts linker DNA in addition to core DNA, and the linker-DNA is as important for H3K27 methylation as H2A ubiquitination. H2A ubiquitination alters contacts between the H3-tail and DNA to improve the methyltransferase activity of the PRC2-AEBP2-JARID2 complex. Collectively, our data support a model in which H2A ubiquitination by PRC1 synergizes with linker-DNA to hold H3 histone tails poised for their methylation by PRC2-AEBP2-JARID2.

Sustained effect of leukocytapheresis/granulocytapheresis versus anti-human TNF-α monoclonal antibody on ulcerative colitis: A 2-year retrospective study.

Although anti-tumor necrosis factor-α monoclonal antibody biological preparations (BP) agents are widely used as an established treatment tool for refractory ulcerative colitis (UC), whether leukocytapheresis/granulocytapheresis (L/G-CAP) has similar beneficial impact on the disease activity remains undetermined. Furthermore, the costs defrayed for the treatment with these 2 modalities have not been compared. We retrospectively evaluated whether L/G-CAP offered sustained beneficial effects over 2-year period. The patients who had moderately to severely active UC (Rachmilewitz clinical activity index (CAI) ≧ 5) and were treated with a series (10 sessions) of L/G-CAP (n = 19) or BP (n = 7) as an add-on therapy to conventional medications were followed. Furthermore, the cost-effectiveness pertaining to the treatment with L/G-CAP and BP was assessed over 12 months. At baseline, L/G-CAP and BP groups manifested similar disease activity (CAI, L/G-CAP; 7.0 [6.0-10.0], BP; 10.0 [6.0-10.0], P = .207). The L/G-CAP and BP treatment suppressed the activity, with CAI 1 or less attained on day 180. When the L/G-CAP group was dichotomized into L/G-CAP-high and L/G-CAP-low group based on CAI values (≥3 or < 3) on day 365, CAI was gradually elevated in L/G-CAP-high group but remained suppressed in L/G-CAP-low group without additional apheresis for 2 years. Anemia was corrected more rapidly and hemoglobin levels were higher in BP group. The cost of the treatment with L/G-CAP over 12 months was curtailed to 76% of that with BP (1.79 [1.73-1.92] vs 2.35 [2.29-3.19] million yen, P = .028). L/G-CAP is as effective as BP in a substantial number of patients over 2 years. The cost for the treatment of UC favors L/G-CAP although the correction of anemia may prefer BP. Thus, L/G-CAP can effectively manage the disease activity with no additional implementation for 2 years although further therapeutic modalities might be required in a certain population with high CAI observed on day 365.

Factors and Mechanisms That Influence Chromatin-Mediated Enhancer-Promoter Interactions and Transcriptional Regulation.

Eukaryotic gene expression is regulated through chromatin conformation, in which enhancers and promoters physically interact (E-P interactions). How such chromatin-mediated E-P interactions affect gene expression is not yet fully understood, but the roles of histone acetylation and methylation, pioneer transcription factors, and architectural proteins such as CCCTC binding factor (CTCF) and cohesin have recently attracted attention. Moreover, accumulated data suggest that E-P interactions are mechanistically involved in biophysical events, including liquid-liquid phase separation, and in biological events, including cancers. In this review, we discuss various mechanisms that regulate eukaryotic gene expression, focusing on emerging views regarding chromatin conformations that are involved in E-P interactions and factors that establish and maintain them.

A three-dimensional cephalometric analysis of Japanese adults and its usefulness in orthognathic surgery: A retrospective study.

This study aimed to establish a three-dimensional (3D) cephalometric analysis of craniofacial morphology and discuss its theoretical usefulness in orthognathic patients. Cone-beam computed tomography (CBCT) images of Japanese subjects with skeletal Class I malocclusion before treatment were selected from among 1000 patients so that samples matched a historic 2D cephalometric cohort with normal occlusion using propensity score matching. In each CBCT image, 67 3D measurements were calculated based on manually identified landmarks. The mean and standard deviation of the measurements were calculated and used as the normative range for each sex. To confirm the usefulness of the 3D measurements, pre- and post-treatment CT data of nine jaw deformity patients who underwent orthognathic surgery with two-dimensional planning (2DP) in the past were used. Pre- and post-treatment CT values were evaluated with a paired t-test as well as a Z-score, which was calculated using the aforementioned normative range, and then categorized into five groups ("deteriorated", "no improvement", "over-treatment", "no change", "improvement") with -1 < Z-score < 1 considered normal. Fifty-six patients were matched to normal skeletal 1 subjects. The normative range of 67 items indicating 3D craniofacial morphology of the Japanese was calculated. Postoperatively, the horizontal position of the pogonion to the mid-sagittal plane significantly decreased (p = 0.043) and "improved"; however, the ramus axis on the right side significantly increased (p = 0.005) and "deteriorated". Maxillary yaw and the horizontal position of the gonion also tended to "deteriorated". The normative range for the 3D cephalometric analysis in Japanese has been established. Given findings of deteriorated maxillomandibular yawing after surgery when using conventional 2DP, 3D cephalometric measurements should be used when planning jaw positions after surgery for orthognathic patients.

Estimated number and prevalence of patients with delayed endolymphatic hydrops in Japan: a nationwide survey.

BACKGROUND: Delayed endolymphatic hydrops (DEH) is a rare disease, and the actual number of patients in Japan remains unknown. OBJECTIVE: To investigate the number and prevalence of patients with DEH in Japan. METHODS: In total, 781 departments of otolaryngology in Japan were selected for survey by stratified random sampling according to the total number of hospital beds. We sent questionnaires to the target departments and collected data regarding the number of patients with DEH who visited those departments in 2019. RESULTS: The overall response rate was 68.0% (531 departments). The estimate number of patients with DEH in Japan was 962, and the prevalence was calculated to be 0.8 per 100,000 population. CONCLUSION: Patients with DEH were extremely rare in Japan. SIGNIFICANCE: This may be the first nationwide epidemiological study on the number and prevalence of patients with DEH in Japan or in the world.

Flap suturing endonasal dacryocystorhinostomy assisted by ultrasonic bone aspirator.

BACKGROUND: In the external dacryocystorhinostomy (DCR), a sutured anastomosis technique performed between the nasal mucosal and lacrimal sac flaps reported by Dupuy-Dutemps and Bourguet was the gold standard and was believed to lead to the success of the surgery. However, because of the small working space, a flap suturing technique has not been completely established in endonasal DCR (END-DCR). OBJECTIVES: The effect of the modified flap suture anastomosis technique using a Sonopet ultrasonic bone aspirator was retrospectively compared to that using a diamond burr in patients with nasolacrimal duct obstruction. MATERIALS AND METHODS: One hundred ten patients underwent the modified flap suturing technique using the Sonopet, and 30 patients were operated on using a diamond burr. RESULTS: Successful patency of the lacrimal ostium (LO) was obtained in all patients in both groups. The rates of successful suturing during the operation and of a large diameter of the LO 3 months after the operation were significantly higher in patients in whom the Sonopet rather than the burr was used. CONCLUSIONS AND SIGNIFICANCE: The Sonopet might offer similar surgical outcome to the traditional microdrill DCR and is a safer means of bone removal in END-DCR in the small working space.