A CpG island-encoded mechanism protects genes from premature transcription termination.

Transcription must be tightly controlled to regulate gene expression and development. However, our understanding of the molecular mechanisms that influence transcription and how these are coordinated in cells to ensure normal gene expression remains rudimentary. Here, by dissecting the function of the SET1 chromatin-modifying complexes that bind to CpG island-associated gene promoters, we discover that they play a specific and essential role in enabling the expression of low to moderately transcribed genes. Counterintuitively, this effect can occur independently of SET1 complex histone-modifying activity and instead relies on an interaction with the RNA Polymerase II-binding protein WDR82. Unexpectedly, we discover that SET1 complexes enable gene expression by antagonising premature transcription termination by the ZC3H4/WDR82 complex at CpG island-associated genes. In contrast, at extragenic sites of transcription, which typically lack CpG islands and SET1 complex occupancy, we show that the activity of ZC3H4/WDR82 is unopposed. Therefore, we reveal a gene regulatory mechanism whereby CpG islands are bound by a protein complex that specifically protects genic transcripts from premature termination, effectively distinguishing genic from extragenic transcription and enabling normal gene expression.

Changes in PRC1 activity during interphase modulate lineage transition in pluripotent cells.

The potential of pluripotent cells to respond to developmental cues and trigger cell differentiation is enhanced during the G1 phase of the cell cycle, but the molecular mechanisms involved are poorly understood. Variations in polycomb activity during interphase progression have been hypothesized to regulate the cell-cycle-phase-dependent transcriptional activation of differentiation genes during lineage transition in pluripotent cells. Here, we show that recruitment of Polycomb Repressive Complex 1 (PRC1) and associated molecular functions, ubiquitination of H2AK119 and three-dimensional chromatin interactions, are enhanced during S and G2 phases compared to the G1 phase. In agreement with the accumulation of PRC1 at target promoters upon G1 phase exit, cells in S and G2 phases show firmer transcriptional repression of developmental regulator genes that is drastically perturbed upon genetic ablation of the PRC1 catalytic subunit RING1B. Importantly, depletion of RING1B during retinoic acid stimulation interferes with the preference of mouse embryonic stem cells (mESCs) to induce the transcriptional activation of differentiation genes in G1 phase. We propose that incremental enrolment of polycomb repressive activity during interphase progression reduces the tendency of cells to respond to developmental cues during S and G2 phases, facilitating activation of cell differentiation in the G1 phase of the pluripotent cell cycle.

Distinct roles for CKM-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction.

Precise control of gene expression underpins normal development. This relies on mechanisms that enable communication between gene promoters and other regulatory elements. In embryonic stem cells (ESCs), the cyclin-dependent kinase module Mediator complex (CKM-Mediator) has been reported to physically link gene regulatory elements to enable gene expression and also prime genes for induction during differentiation. Here, we show that CKM-Mediator contributes little to three-dimensional genome organization in ESCs, but it has a specific and essential role in controlling interactions between inactive gene regulatory elements bound by Polycomb repressive complexes (PRCs). These interactions are established by the canonical PRC1 (cPRC1) complex but rely on CKM-Mediator, which facilitates binding of cPRC1 to its target sites. Importantly, through separation-of-function experiments, we reveal that this collaboration between CKM-Mediator and cPRC1 in creating long-range interactions does not function to prime genes for induction during differentiation. Instead, we discover that priming relies on an interaction-independent mechanism whereby the CKM supports core Mediator engagement with gene promoters during differentiation to enable gene activation.

Corrigendum: Socio-Demographic and Clinical Characteristics of Psychiatric Patients Who Have Committed Suicide: Analysis of Bulgarian Regional Suicidal Registry for 10 Years.

[This corrects the article DOI: 10.3389/fpsyt.2021.665154.].

Socio-Demographic and Clinical Characteristics of Psychiatric Patients Who Have Committed Suicide: Analysis of Bulgarian Regional Suicidal Registry for 10 Years.

Introduction: Suicide is a major public health problem but factors determining suicide risk are still unclear. Studies in this field in Bulgaria are limited, especially on a regional level. Methods: By a cross-sectional design, we accessed the medical records of all psychiatric patients committed suicide over a 10-year period (2009-2018) in one major administrative region of Bulgaria. A statistical analysis was performed of the association between age of suicide as an indirect yet measurable expression of the underlying suicidal diathesis and a number of socio-demographic and clinical characteristics. Results: Seventy-seven of 281 suicides (28%) had psychiatric records. Most common diagnoses were mood disorders (44%), followed by schizophrenia (27%), anxiety disorders (10%), substance use disorders (9%) and organic conditions (8%). Male gender, single/divorced marital status, early illness onset, co-occurring substance misuse and lower educational attainment (for patients aged below 70) were significantly associated with earlier age of suicide whereas past suicide attempts and psychiatric hospitalizations, comorbid somatic conditions and unemployment showed insignificant association. Substantial proportion of patients (60%) had contacted psychiatric service in the year preceding suicide, with nearly half of these encounters being within 30 days of the accident. Conclusion: Severe mental disorders are major suicide risk factor with additional contribution of certain socio-demographic and illness-related characteristics. Monitoring for suicidality must be constant in chronic psychiatric patients. Registration of suicide cases in Bulgaria needs improvement in terms of information concerning mental health. More studies with larger samples and longitudinal design are needed to further elucidate distal and proximal suicide risk factors.

CDK-Mediator and FBXL19 prime developmental genes for activation by promoting atypical regulatory interactions.

Appropriate developmental gene regulation relies on the capacity of gene promoters to integrate inputs from distal regulatory elements, yet how this is achieved remains poorly understood. In embryonic stem cells (ESCs), a subset of silent developmental gene promoters are primed for activation by FBXL19, a CpG island binding protein, through its capacity to recruit CDK-Mediator. How mechanistically these proteins function together to prime genes for activation during differentiation is unknown. Here we discover that in mouse ESCs FBXL19 and CDK-Mediator support long-range interactions between silent gene promoters that rely on FBXL19 for their induction during differentiation and gene regulatory elements. During gene induction, these distal regulatory elements behave in an atypical manner, in that the majority do not acquire histone H3 lysine 27 acetylation and no longer interact with their target gene promoter following gene activation. Despite these atypical features, we demonstrate by targeted deletions that these distal elements are required for appropriate gene induction during differentiation. Together these discoveries demonstrate that CpG-island associated gene promoters can prime genes for activation by communicating with atypical distal gene regulatory elements to achieve appropriate gene expression.

FBXL19 recruits CDK-Mediator to CpG islands of developmental genes priming them for activation during lineage commitment.

CpG islands are gene regulatory elements associated with the majority of mammalian promoters, yet how they regulate gene expression remains poorly understood. Here, we identify FBXL19 as a CpG island-binding protein in mouse embryonic stem (ES) cells and show that it associates with the CDK-Mediator complex. We discover that FBXL19 recruits CDK-Mediator to CpG island-associated promoters of non-transcribed developmental genes to prime these genes for activation during cell lineage commitment. We further show that recognition of CpG islands by FBXL19 is essential for mouse development. Together this reveals a new CpG island-centric mechanism for CDK-Mediator recruitment to developmental gene promoters in ES cells and a requirement for CDK-Mediator in priming these developmental genes for activation during cell lineage commitment.

Carcinogenic response and other histopathological alterations in mice exposed to cigarette smoke for varying time periods after birth.

In spite of the outstanding role of tobacco smoking in human carcinogenesis, it is difficult to reproduce its effects in experimental animals. Based on the knowledge that a variety of mechanisms account for a higher susceptibility to carcinogens early in life, we have developed a murine model in which mainstream cigarette smoke becomes convincingly carcinogenic. The standard model involves exposure to smoke for 4 months, starting after birth, followed by an additional 3-4 months in filtered air. We evaluated herein the time- and dose-dependent response, at 7.5 months of life, of Swiss H mice that had been exposed to smoke for either 1, 2 or 4 months after birth. A one-month exposure, corresponding to a period of intense alveolarization, was sufficient to induce most inflammatory, degenerative and preneoplastic pulmonary lesions, including emphysema and alveolar epithelial hyperplasia, blood vessel proliferation and hemangiomas, reflecting an early proangiogenic role of smoking, and microadenomas bearing ki-67-positive proliferating cells as well as urinary bladder epithelial hyperplasia. Two months of exposure were needed to induce pulmonary adenomas and urinary bladder papillomas in males only, which highlights a protective role of estrogens in urinary bladder carcinogenesis. Four months, which in humans would correspond to the postnatal period, puberty, adolescence and early adulthood, were needed to induce other lesions, including tubular epithelial hyperplasia of kidney, bronchial epithelial hyperplasia and especially pulmonary malignant tumors. These findings highlight the concept that preneoplastic and neoplastic lesions occurring in adulthood can be induced by exposure to smoke early in life.

The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin.

Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.

Histone demethylases in chromatin biology and beyond.

Histone methylation plays fundamental roles in regulating chromatin-based processes. With the discovery of histone demethylases over a decade ago, it is now clear that histone methylation is dynamically regulated to shape the epigenome and regulate important nuclear processes including transcription, cell cycle control and DNA repair. In addition, recent observations suggest that these enzymes could also have functions beyond their originally proposed role as histone demethylases. In this review, we focus on recent advances in our understanding of the molecular mechanisms that underpin the role of histone demethylases in a wide variety of normal cellular processes.

Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome.

The Polycomb repressive complexes PRC1 and PRC2 maintain embryonic stem cell (ESC) pluripotency by silencing lineage-specifying developmental regulator genes. Emerging evidence suggests that Polycomb complexes act through controlling spatial genome organization. We show that PRC1 functions as a master regulator of mouse ESC genome architecture by organizing genes in three-dimensional interaction networks. The strongest spatial network is composed of the four Hox gene clusters and early developmental transcription factor genes, the majority of which contact poised enhancers. Removal of Polycomb repression leads to disruption of promoter-promoter contacts in the Hox gene network. In contrast, promoter-enhancer contacts are maintained in the absence of Polycomb repression, with accompanying widespread acquisition of active chromatin signatures at network enhancers and pronounced transcriptional upregulation of network genes. Thus, PRC1 physically constrains developmental transcription factor genes and their enhancers in a silenced but poised spatial network. We propose that the selective release of genes from this spatial network underlies cell fate specification during early embryonic development.

The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.

The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression.

RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.

Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.

The ATRX-ADD domain binds to H3 tail peptides and reads the combined methylation state of K4 and K9.

Mutations in the ATRX protein are associated with the alpha-thalassemia and mental retardation X-linked syndrome (ATR-X). Almost half of the disease-causing mutations occur in its ATRX-Dnmt3-Dnmt3L (ADD) domain. By employing peptide arrays, chromatin pull-down and peptide binding assays, we show specific binding of the ADD domain to H3 histone tail peptides containing H3K9me3. Peptide binding was disrupted by the presence of the H3K4me3 and H3K4me2 modification marks indicating that the ATRX-ADD domain has a combined readout of these two important marks (absence of H3K4me2 and H3K4me3 and presence of H3K9me3). Disease-causing mutations reduced ATRX-ADD binding to H3 tail peptides. ATRX variants, which fail in the H3K9me3 interaction, show a loss of heterochromatic localization in cells, which indicates the chromatin targeting function of the ADD domain of ATRX. Disruption of H3K9me3 binding may be a general pathogenicity pathway of ATRX mutations in the ADD domain which may explain the clustering of disease mutations in this part of the ATRX protein.

A continuous protein methyltransferase (G9a) assay for enzyme activity measurement and inhibitor screening.

The authors describe a continuous protein methylation assay using the G9a protein lysine methyltransferase and its substrate protein WIZ (widely interspaced zinc finger motifs). The assay is based on the coupling of the biotinylated substrate protein to streptavidin-coated FlashPlates and the transfer of radioactive methyl groups from the S-adenosyl-L-methionine to the substrate. The reaction progress is monitored continuously by proximity scintillation counting. The assay is very accurate, convenient, well suited for automation, and highly reproducible with standard errors in the range of 5%. Because of few pipetting steps and continuous data readout, it is ideal for high-throughput applications such as screening of inhibitors, testing many enzyme variants, or analyzing differences in methylation rates of different substrates under various conditions. By using this new assay, the IC(50) of AdoHcy and the G9a inhibitor BIX-01294 were determined for methylation of the G9a nonhistone substrate WIZ.