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Summary: Leaf senescence is an orderly process regulated by multiple internal factors and diverse environmental stresses including nutrient deficiency. Histone variants are involved in regulating plant growth and development. However, their functions and underlying regulatory mechanisms in leaf senescence remain largely unclear.Here, we found that H2B histone variant HTB4 functions as a negative regulator of leaf senescence. Loss of function of HTB4 led to early leaf senescence phenotypes that were rescued by functional complementation. RNA‐seq analysis revealed that several Ib subgroup basic helix–loop–helix (bHLH) transcription factors (TFs) involved in iron (Fe) homeostasis, including bHLH038, bHLH039, bHLH100, and bHLH101, were suppressed in the htb4 mutant, thereby compromising the expressions of FERRIC REDUCTION OXIDASE 2 (FRO2) and IRON‐REGULATED TRANSPORTER (IRT1), two important components of the Fe uptake machinery.Chromatin immunoprecipitation‐quantitative polymerase chain reaction analysis revealed that HTB4 could bind to the promoter regions of Ib bHLH TFs and enhance their expression by promoting the enrichment of the active mark H3K4me3 near their transcriptional start sites. Moreover, overexpression of Ib bHLH TFs or IRT1 suppressed the premature senescence phenotype of the htb4 mutant.Our work established a signaling pathway, HTB4‐bHLH TFs‐FRO2/IRT1‐Fe homeostasis, which regulates the onset and progression of leaf senescence. See also the Commentary on this article by Dubos, 240: 461–463. [ABSTRACT FROM AUTHOR]

Eukaryotic organisms adapt to environmental fluctuations by altering their epigenomic landscapes and transcriptional programs. Nucleosomal histones carry vital epigenetic information and regulate gene expression, yet the mechanisms underlying chromatin‐bound histone exchange remain elusive. Here, we found that histone H2Bs are globally degraded in Caenorhabditis elegans during starvation. Our genetic screens identified mutations in ubiquitin and ubiquitin‐related enzymes that block H2B degradation in starved animals, identifying lysine 31 as the crucial residue for chromatin‐bound H2B ubiquitination and elimination. Retention of aberrant nucleosomal H2B increased the association of the FOXO transcription factor DAF‐16 with chromatin, generating an ectopic gene expression profile detrimental to animal viability when insulin/IGF signaling was reduced in well‐fed animals. Furthermore, we show that the ubiquitin‐proteasome system regulates chromosomal histone turnover in human cells. During larval development, C. elegans epidermal cells undergo H2B turnover after fusing with the epithelial syncytium. Thus, histone degradation may be a widespread mechanism governing dynamic changes of the epigenome. Synopsis: The exchange of histones and their variants within nucleosomes plays a crucial role in modulating the epigenome and gene expression. This study shows that ubiquitin‐dependent histone H2B exchange and degradation are crucial in determining C. elegans diapause by regulating the transcriptional activity of DAF‐16/FoxO.Histone H2B undergoes global exchange and degradation during development and starvation in C. elegans.The components of the ubiquitin‐proteasome system (UPS), including ubiquitin/UBQ‐1, E1/UBA‐1, E2/UBC‐20, and E3/HECD‐1, regulate H2B exchange and degradation by ubiquitinating H2B at lysine 31.Disruption of H2B exchange and degradation enhances the binding of DAF‐16/FoxO to chromatin, resulting in aberrant activation or suppression of target genes, and ultimately leading to larval death.Ubiquitin‐dependent histone H2B exchange and degradation are conserved in mammalian cells. [ABSTRACT FROM AUTHOR]

Small‐molecule responsive protein switches are powerful tools for controlling cellular processes. These switches are designed to respond rapidly and specifically to their inducer. They have been used in numerous applications, including the regulation of gene expression, post‐translational protein modification, and signal transduction. Typically, small‐molecule responsive protein switches consist of two proteins that interact with each other in the presence or absence of a small molecule. Recent advances in computational protein design already contributed to the development of protein switches with an expanded range of small‐molecule inducers and increasingly sophisticated switch mechanisms. Further progress in the engineering of small‐molecule responsive switches is fueled by cutting‐edge computational design approaches, which will enable more complex and precise control over cellular processes and advance synthetic biology applications in biotechnology and medicine. Here, we discuss recent milestones and how technological advances are impacting the development of chemical switches. [ABSTRACT FROM AUTHOR]

The telomeric repeat‐containing RNA, TERRA, associates with both telomeric DNA and telomeric proteins, often forming RNA:DNA hybrids (R‐loops). TERRA is most abundant in cancer cells utilizing the alternative lengthening of telomeres (ALT) pathway for telomere maintenance, suggesting that persistent TERRA R‐loops may contribute to activation of the ALT mechanism. Therefore, we sought to identify the enzyme(s) that regulate TERRA metabolism in mammalian cells. Here, we identify that the 5′–3′ exoribonuclease XRN2 regulates the stability of TERRA RNA. Moreover, while stabilization of TERRA alone was insufficient to drive ALT, depletion of XRN2 in ALT‐positive cells led to a significant increase in TERRA R‐loops and exacerbated ALT activity. Together, our findings highlight XRN2 as a key determinant of TERRA metabolism and telomere stability in cancer cells that rely on the ALT pathway. [ABSTRACT FROM AUTHOR]

We report that preexisting (old) and newly synthesized (new) histones H3 and H4 are asymmetrically partitioned during the division of Drosophila intestinal stem cells (ISCs). Furthermore, the inheritance patterns of old and new H3 and H4 in postmitotic cell pairs correlate with distinct expression patterns of Delta, an important cell fate gene. To understand the biological significance of this phenomenon, we expressed a mutant H3T3A to compromise asymmetric histone inheritance. Under this condition, we observe an increase in Delta‐symmetric cell pairs and overpopulated ISC‐like, Delta‐positive cells. Single‐cell RNA‐seq assays further indicate that H3T3A expression compromises ISC differentiation. Together, our results indicate that asymmetric histone inheritance potentially contributes to establishing distinct cell identities in a somatic stem cell lineage, consistent with previous findings in Drosophila male germline stem cells. Synopsis: Old and newly synthesized histones are asymmetrically distributed and inherited in Drosophila intestinal stem cell divisions. Disruption of asymmetric histone inheritance compromises stem cell differentiation, indicating a role in regulating cell fates in adult stem lineages in vivo. Old and newly synthesized histones H3 and H4 are asymmetrically distributed and partitioned in Drosophila intestinal stem cell divisions.Compromising asymmetric histone inheritance leads to intestinal stem cell differentiation defects. [ABSTRACT FROM AUTHOR]

Summary: The histone variant H2A.Z plays key functions in transcription and genome stability in all eukaryotes ranging from yeast to human, but the molecular mechanisms by which H2A.Z is incorporated into chromatin remain largely obscure.Here, we characterized the two homologs of yeast Chaperone for H2A.Z‐H2B (Chz1) in Arabidopsis thaliana, AtChz1A and AtChz1B. AtChz1A/AtChz1B were verified to bind to H2A.Z‐H2B and facilitate nucleosome assembly in vitro. Simultaneous knockdown of AtChz1A and AtChz1B, which exhibit redundant functions, led to a genome‐wide reduction in H2A.Z and phenotypes similar to those of the H2A.Z‐deficient mutant hta9‐1hta11‐2, including early flowering and abnormal flower morphologies.Interestingly, AtChz1A was found to physically interact with ACTIN‐RELATED PROTEIN 6 (ARP6), an evolutionarily conserved subunit of the SWR1 chromatin‐remodeling complex. Genetic interaction analyses showed that atchz1a‐1atchz1b‐1 was hypostatic to arp6‐1. Consistently, genome‐wide profiling analyses revealed partially overlapping genes and fewer misregulated genes and H2A.Z‐reduced chromatin regions in atchz1a‐1atchz1b‐1 compared with arp6‐1.Together, our results demonstrate that AtChz1A and AtChz1B act as histone chaperones to assist the deposition of H2A.Z into chromatin via interacting with SWR1, thereby playing critical roles in the transcription of genes involved in flowering and many other processes. [ABSTRACT FROM AUTHOR]

Summary: Plants can sense temperature changes and adjust their development and morphology accordingly in a process called thermomorphogenesis. This phenotypic plasticity implies complex mechanisms regulating gene expression reprogramming in response to environmental alteration. Histone variants often associate with specific chromatin states; yet, how their deposition/eviction modulates transcriptional changes induced by environmental cues remains elusive.In Arabidopsis thaliana, temperature elevation‐induced transcriptional activation at thermo‐responsive genes entails the chromatin eviction of a histone variant H2A.Z by INO80, which is recruited to these loci via interacting with a key thermomorphogenesis regulator PIF4.Here, we show that both INO80 and the deposition chaperones of another histone variant H3.3 associate with ELF7, a critical component of the transcription elongator PAF1 complex. H3.3 promotes thermomorphogenesis and the high temperature‐enhanced RNA Pol II transcription at PIF4 targets, and it is broadly required for the H2A.Z removal‐induced gene activation. Reciprocally, INO80 and ELF7 regulate H3.3 deposition, and are necessary for the high temperature‐induced H3.3 enrichment at PIF4 targets.Our findings demonstrate close coordination between H2A.Z eviction and H3.3 deposition in gene activation induced by high temperature, and pinpoint the importance of histone variants dynamics in transcriptional regulation. [ABSTRACT FROM AUTHOR]

The centrosome, a non‐membranous organelle, constrains various soluble molecules locally to execute its functions. As the centrosome is surrounded by various dense components, we hypothesized that it may be bordered by a putative diffusion barrier. After quantitatively measuring the trapping kinetics of soluble proteins of varying size at centrosomes by a chemically inducible diffusion trapping assay, we find that centrosomes are highly accessible to soluble molecules with a Stokes radius of less than 5.8 nm, whereas larger molecules rarely reach centrosomes, indicating the existence of a size‐dependent diffusion barrier at centrosomes. The permeability of this barrier is tightly regulated by branched actin filaments outside of centrosomes and it decreases during anaphase when branched actin temporally increases. The actin‐based diffusion barrier gates microtubule nucleation by interfering with γ‐tubulin ring complex recruitment. We propose that actin filaments spatiotemporally constrain protein complexes at centrosomes in a size‐dependent manner. Synopsis: Centrosomes maintain a discrete protein composition without a membrane boundary. This study shows that a size‐dependent diffusion barrier composed of branched actin filaments restricts access to centrosomes and gates microtubule nucleation by interfering with γ‐TuRC recruitment. Large molecules (≥ 6.0 nm in stokes radius) rarely or cannot access centrosome core regions including the centriolar lumen and pericentriolar matrix.The permeability of the centrosome diffusion barrier is tightly regulated by branched actin filaments.The permeability of centrosomal diffusion barriers decreases in anaphase correlating with a temporal increase in branched actin filaments.The centrosomal diffusion barrier gates γ‐TuRC recruitment and interferes with microtubule nucleation. [ABSTRACT FROM AUTHOR]

Epigenome reprogramming after fertilization enables transcriptionally quiescent maternal and paternal chromatin to acquire a permissive state for subsequent zygotic genome activation (ZGA). H3K27 acetylation (H3K27ac) is a well‐established chromatin marker of active enhancers and promoters. However, reprogramming dynamics of H3K27ac during maternal‐to‐zygotic transition (MZT) in mammalian embryos are not well‐studied. By profiling the allelic landscape of H3K27ac during mouse MZT, we show that H3K27ac undergoes three waves of rapid global transitions between oocyte stage and 2‐cell stage. Notably, germinal vesicle oocyte and zygote chromatin are globally hyperacetylated, with noncanonical, broad H3K27ac domains that correlate with broad H3K4 trimethylation (H3K4me3) and open chromatin. H3K27ac marks genomic regions primed for activation including ZGA genes, retrotransposons, and active alleles of imprinted genes. We show that CBP/p300 and HDAC activities play important roles in regulating H3K27ac dynamics and are essential for preimplantation development. Specifically, CBP/p300 acetyltransferase broadly deposits H3K27ac in zygotes to induce the opening of condensed chromatin at putative enhancers and ensure proper ZGA. On the contrary, HDACs revert broad H3K27ac domains to canonical domains and safeguard ZGA by preventing premature expression of developmental genes. In conclusion, coordinated activities of CBP/p300 and HDACs during mouse MZT are essential for ZGA and preimplantation development. Synopsis: Reprogramming of the epigenome after fertilization is required for subsequent zygotic genome activation (ZGA). Here, profiling of H3K27 acetylation (H3K27ac) associated with active enhancers/promotors reveals its dynamic reprogramming during maternal‐to‐zygotic transition in mouse preimplantation embryos. H3K27ac undergoes three waves of rapid global reprogramming during mouse maternal‐to‐zygotic transition.Germinal vesicle oocyte and zygote are globally hyperacetylated with noncanonical broad H3K27ac domains that correlate with broad H3K4me3 domains and open chromatin.CBP/p300 acetyltransferase broadly deposits H3K27ac in zygotes, regulates minor ZGA, and opens condensed chromatin at putative enhancers to ensure proper ZGA.HDAC‐dependent transition from broad‐to‐canonical H3K27ac domains between zygote and 2‐cell stage safeguards ZGA by preventing premature expression of developmental genes. [ABSTRACT FROM AUTHOR]

In some types of cancer, telomere length is maintained by the alternative lengthening of telomeres (ALT) mechanism. In many ALT cancers, the α‐thalassemia/mental retardation syndrome X‐linked (ATRX) gene is mutated leading to the conclusion that the ATRX complex represses ALT. Here, we report that most high‐grade pediatric osteosarcomas maintain their telomeres by ALT, and that the majority of these ALT tumors are ATRX wild‐type (wt) and instead carry an amplified 17p11.2 chromosomal region containing TOP3A. We found that TOP3A was overexpressed in the ALT‐positive ATRX‐wt tumors consistent with its amplification. We demonstrated the functional significance of these results by showing that TOP3A overexpression in ALT cancer cells countered ATRX‐mediated ALT inhibition and that TOP3A knockdown disrupted the ALT phenotype in ATRX‐wt cells. Moreover, we report that TOP3A is required for proper BLM localization and promotes ALT DNA synthesis in ALT cell lines. Collectively, our results identify TOP3A as a major ALT player and potential therapeutic target. Synopsis: There is an urgent need to treat pediatric osteosarcomas that present high frequency of alternative lengthening of telomeres (ALT). This study identifies TOP3A amplification as mutually exclusive with ATRX inactivation, promoting ALT and TOP3A action at telomeres as a novel therapeutic target. 17p11 amplification and ATRX mutation are exclusive genomic events in ALT high‐grade pediatric osteosarcomas.TOP3A overexpression is a feature of ATRX‐wt ALT osteosarcomas.Over‐expression of TOP3A correlates with the presence of ATRX in ATRX‐wt ALT cell lines.TOP3A is required for proper BLM localization in ALT osteosarcoma cell lines and promotes ALT‐mediated telomeric DNA synthesis. [ABSTRACT FROM AUTHOR]

Over the last decade, developments in molecular profiling have radically altered the diagnosis, classification, and management of numerous cancer types, with primary brain tumors being no exception. Although historically brain tumors have been classified based on their morphological characteristics, recent advances have allowed refinement of tumor classification based on molecular alterations. This shift toward molecular classification of primary brain tumors is reflected in the 2021 5th edition of the WHO classification of central nervous system tumors (WHO 2021). In this review, we will discuss the most recent updates to the classification of adult‐type diffuse gliomas, a group of highly infiltrative and largely incurable CNS malignancies. It is our hope continued that refinement of molecular criteria will improve diagnosis, prognostication, and eventually treatment of these devastating tumors. [ABSTRACT FROM AUTHOR]

Quantitative analysis of chromatin protein–nucleosome interactions is essential to understand regulation of genome‐templated processes. However, current methods to measure nucleosome interactions are limited by low throughput, low signal‐to‐noise, and/or the requirement for specialized instrumentation. Here, we report a Lanthanide Chelate Excite Time‐Resolved Fluorescence Resonance Energy Transfer (LANCE TR‐FRET) assay to efficiently quantify chromatin protein–nucleosome interactions. The system makes use of commercially available reagents, offers robust signal‐to‐noise with minimal sample requirements, uses a conventional fluorescence microplate reader, and can be adapted for high‐throughput workflows. We determined the nucleosome‐binding affinities of several chromatin proteins and complexes, which are consistent with measurements obtained through orthogonal biophysical methods. We also developed a TR‐FRET competition assay for high‐resolution footprinting of chromatin protein–nucleosome interactions. Finally, we set up a TR‐FRET competition assay using the LANA peptide to quantitate nucleosome acidic patch binding. We applied this assay to establish a proof‐of‐principle for regulation of nucleosome acidic patch binding by methylation of chromatin protein arginine anchors. Overall, our TR‐FRET assays allow facile, high‐throughput quantification of chromatin interactions and are poised to complement mechanistic chromatin biochemistry, structural biology, and drug discovery programs. [ABSTRACT FROM AUTHOR]

Mesenchymal stem/stromal cells (MSCs) hold great promise and clinical efficacy in bone/cartilage regeneration. With a deeper understanding of stem cell biology over the past decade, epigenetics stands out as one of the most promising ways to control MSCs differentiation. Trithorax group (TrxG) proteins, including the COMPASS family, ASH1L, CBP/p300 as histone modifying factors, and the SWI/SNF complexes as chromatin remodelers, play an important role in gene expression regulation during the process of stem cell differentiation. This review summarises the components and functions of TrxG complexes. We provide an overview of the regulation mechanisms of TrxG in MSCs osteogenic and chondrogenic differentiation, and discuss the prospects of epigenetic regulation mediated by TrxG in bone and cartilage regeneration. [ABSTRACT FROM AUTHOR]

In the last 15 years, increasing evidence linking epigenetics to various aspects of cancer biology has prompted the investigation of histone post‐translational modifications (PTMs) and histone variants in the context of clinical samples. The studies performed so far demonstrated the potential of this type of investigations for the discovery of both potential epigenetic biomarkers for patient stratification and novel epigenetic mechanisms potentially targetable for cancer therapy. Although traditionally the analysis of histones in clinical samples was performed through antibody‐based methods, mass spectrometry (MS) has emerged as a more powerful tool for the unbiased, comprehensive, and quantitative investigation of histone PTMs and variants. MS has been extensively used for the analysis of epigenetic marks in cell lines and animal tissue and, thanks to recent technological advances, is now ready to be applied also to clinical samples. In this review, we will provide an overview on the quantitative MS‐based analysis of histones, their PTMs and their variants in cancer clinical samples, highlighting current achievements and future perspectives for this novel field of research. Among the different MS‐based approaches currently available for histone PTM profiling, we will focus on the 'bottom‐up' strategy, namely the analysis of short proteolytic peptides, as it has been already successfully employed for the analysis of clinical samples. [ABSTRACT FROM AUTHOR]

Since the discovery of recurrent mutations in histone H3 variants in paediatric brain tumours, so‐called 'oncohistones' have been identified in various cancers. While their mechanism of action remains under active investigation, several studies have shed light on how they promote genome‐wide epigenetic perturbations. These findings converge on altered post‐translational modifications on two key lysine (K) residues of the H3 tail, K27 and K36, which regulate several cellular processes, including those linked to cell differentiation during development. We will review how these oncohistones affect the methylation of cognate residues, but also disrupt the distribution of opposing chromatin marks, creating genome‐wide epigenetic changes which participate in the oncogenic process. Ultimately, tumorigenesis is promoted through the maintenance of a progenitor state at the expense of differentiation in defined cellular and developmental contexts. As these epigenetic disruptions are reversible, improved understanding of oncohistone pathogenicity can result in needed alternative therapies. [ABSTRACT FROM AUTHOR]

Mouse embryonic stem cells (mESCs) can adopt naïve, ground, and paused pluripotent states that give rise to unique transcriptomes. Here, we use transient transcriptome sequencing (TT‐seq) to define both coding and non‐coding transcription units (TUs) in these three pluripotent states and combine TT‐seq with RNA polymerase II occupancy profiling to unravel the kinetics of RNA metabolism genome‐wide. Compared to the naïve state (serum), RNA synthesis and turnover rates are globally reduced in the ground state (2i) and the paused state (mTORi). The global reduction in RNA synthesis goes along with a genome‐wide decrease of polymerase elongation velocity, which is related to epigenomic features and alterations in the Pol II termination window. Our data suggest that transcription activity is the main determinant of steady state mRNA levels in the naïve state and that genome‐wide changes in transcription kinetics invoke ground and paused pluripotent states. SYNOPSIS: Genome‐wide analyses of RNA metabolism kinetics in naïve, ground and paused pluripotent mouse embryonic stem cells (mESCs) indicate that the three states show markedly different global transcriptional kinetics. Transient transcriptome sequencing (TT‐seq) is used to annotate transcription units in mESC pluripotent states.Transcription reduction in the early state transitions is liked to lower total RNA abundance and turnover rates.Genome‐wide changes in transcription kinetics invoke ground and paused pluripotent states.The estimated elongation velocity connects with RNA Pol II pause‐release and termination distance. [ABSTRACT FROM AUTHOR]

Histone chaperones modulate the stability of histones beginning from histone synthesis, through incorporation into DNA, and during recycling during transcription and replication. Following histone removal from DNA, chaperones regulate histone storage and degradation. Here, we demonstrate that UBR7 is a histone H3.1 chaperone that modulates the supply of pre‐existing post‐nucleosomal histone complexes. We demonstrate that UBR7 binds to post‐nucleosomal H3K4me3 and H3K9me3 histones via its UBR box and PHD. UBR7 binds to the non‐nucleosomal histone chaperone NASP. In the absence of UBR7, the pool of NASP‐bound post‐nucleosomal histones accumulate and chromatin is depleted of H3K4me3‐modified histones. We propose that the interaction of UBR7 with NASP and histones opposes the histone storage functions of NASP and that UBR7 promotes reincorporation of post‐nucleosomal H3 complexes. SYNOPSIS: Histone chaperones facilitate deposition and recycling of histones. Here, UBR7 is found as a new chaperone recognizing post‐nucleosomal H3.1 and promoting its reincorporation, with implications for maintenance of epigenetic information. UBR7 binds non‐nucleosomal histone H3.1.The UBR7 PHD binds soluble histones methylated on Lys4 or Lys9, representing pre‐existing histone H3.UBR7 associates with the histone chaperone NASP.UBR7 opposes soluble H3/H4 buffering by NASP and promotes reincorporation of post‐nucleosomal histone H3. [ABSTRACT FROM AUTHOR]

Pediatric high‐grade gliomas (pHGG) comprise a deadly, heterogenous category of pediatric gliomas with a clear need for more effective treatment options. Advances in high‐throughput molecular techniques have enhanced molecular understanding of these tumors, but outcomes are still poor, and treatments beyond resection and radiation have not yet been clearly established as standard of care. In this review, we first discuss the history of treatment approaches to pHGG to this point. We then review four distinct categories of pHGG, including histone 3‐mutant, IDH‐mutant, histone 3/IDH‐wildtype, and radiation‐induced pHGG. We discuss the molecular understanding of each subgroup and targeted treatment options in development. Finally, we look at the development and current status of two novel approaches to pHGG as a whole: localized convection‐enhanced chemotherapy delivery and immunotherapy, including checkpoint inhibitors, vaccine therapy, and CAR‐T cells. Through this review, we demonstrate the potential for rational, molecularly driven, subtype‐specific therapy to be used with other novel approaches in combinations that could meaningfully improve the prognosis in pHGG. [ABSTRACT FROM AUTHOR]

Testis‐specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis‐specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis. SYNOPSIS: The eutherian testis‐specific histone variant H2A.B is aberrantly expressed in Hodgkin Lymphoma and enhances ribosomal biogenesis by stimulating both RNA Pol I transcription and the transcription of ribosomal protein genes. H2A.B is required for the high cell proliferation rate of Hodgkin Lymphoma cell lines.H2A.B is enriched in rDNA chromatin and interacts with RNA Pol I.H2A.B is required for transcription and alternative splicing of genes that are linked to cancer such as ribosomal proteins.The chromatin accessibility of active genes is enhanced by H2A.B. [ABSTRACT FROM AUTHOR]

Methylation of cytosine in CpG dinucleotides and histone lysine and arginine residues is a chromatin modification that critically contributes to the regulation of genome integrity, replication, and accessibility. A strong correlation exists between the genome‐wide distribution of DNA and histone methylation, suggesting an intimate relationship between these epigenetic marks. Indeed, accumulating literature reveals complex mechanisms underlying the molecular crosstalk between DNA and histone methylation. These in vitro and in vivo discoveries are further supported by the finding that genes encoding DNA‐ and histone‐modifying enzymes are often mutated in overlapping human diseases. Here, we summarize recent advances in understanding how DNA and histone methylation cooperate to maintain the cellular epigenomic landscape. We will also discuss the potential implication of these insights for understanding the etiology of, and developing biomarkers and therapies for, human congenital disorders and cancers that are driven by chromatin abnormalities. [ABSTRACT FROM AUTHOR]

Chemical modifications of histone tails influence genome accessibility and the transcriptional state of eukaryotic cells. Lysine acetylation is one of the most common modifications and acetyllysine‐binding bromodomains (BDs) provide a means for acetyllysine marks to be translated into meaningful cellular responses. Here, we have investigated the mechanism underlying the reported association between the Bromodomain and Extra Terminal (BET) family of BD proteins and the essential histone variant H2A.Z. We use NMR spectroscopy to demonstrate a physical interaction between the N‐terminal tail of H2A.Z and the BDs of BRD2, BRD3, and BRD4, and show that the interaction is dependent on lysine acetylation in H2A.Z. The BDs preferentially engage a diacetylated H2A.Z‐K4acK7ac motif that is reminiscent of sequences found in other biologically important BET BD target proteins, including histones and transcription factors. A H2A.Z‐K7acK11ac motif can also bind BET BDs—with a preference for the second BD of each protein. Chemical shift perturbation mapping of the interactions, together with an X‐ray crystal structure of BRD2‐BD1 bound to H2A.Z‐K4acK7ac, shows that H2A.Z binds the canonical AcK binding pocket of the BDs. This mechanism mirrors the conserved binding mode that is unique to the BET BDs, in which two acetylation marks are read simultaneously by a single BD. Our findings provide structural corroboration of biochemical and cell biological data that link H2A.Z and BET‐family proteins, suggesting that the function of H2A.Z is enacted through interactions with these chromatin readers. PDB Code(s): 7JX7; [ABSTRACT FROM AUTHOR]

Chromatin integrity is essential for cellular homeostasis. Polycomb group proteins modulate chromatin states and transcriptionally repress developmental genes to maintain cell identity. They also repress repetitive sequences such as major satellites and constitute an alternative state of pericentromeric constitutive heterochromatin at paternal chromosomes (pat‐PCH) in mouse pre‐implantation embryos. Remarkably, pat‐PCH contains the histone H3.3 variant, which is absent from canonical PCH at maternal chromosomes, which is marked by histone H3 lysine 9 trimethylation (H3K9me3), HP1, and ATRX proteins. Here, we show that SUMO2‐modified CBX2‐containing Polycomb Repressive Complex 1 (PRC1) recruits the H3.3‐specific chaperone DAXX to pat‐PCH, enabling H3.3 incorporation at these loci. Deficiency of Daxx or PRC1 components Ring1 and Rnf2 abrogates H3.3 incorporation, induces chromatin decompaction and breakage at PCH of exclusively paternal chromosomes, and causes their mis‐segregation. Complementation assays show that DAXX‐mediated H3.3 deposition is required for chromosome stability in early embryos. DAXX also regulates repression of PRC1 target genes during oogenesis and early embryogenesis. The study identifies a novel critical role for Polycomb in ensuring heterochromatin integrity and chromosome stability in mouse early development. Synopsis: Formation of pericentromeric heterochromatin (PCH) in early mouse embryos involves selective incorporation of the histone variant H3.3. Polycomb repressive complex 1 (PRC1) facilitates this by recruitment of H3.3 chaperone/remodeler DAXX‐ATRX in a SUMOylation‐dependent manner. Histone chaperone DAXX controls H3.3 deposition into pericentromeric heterochromatin exclusively at paternal chromosomes of early mouse embryos.Canonical PRC1, but not PRC2, is required for recruitment of DAXX and H3.3 deposition at paternal PCH.SUMOylation of the PRC1 component CBX2 mediates interaction between CBX2 and DAXX.DAXX contributes to PRC1‐dependent gene repression in oocytes. [ABSTRACT FROM AUTHOR]

α‐Tthalassemia mental retardation X‐linked (ATRX) is a chromatin remodeler frequently mutated in many cancers. Despite the binding pattern of ATRX in heterochromatin, ATRX‐mediated epigenomic changes in cancer cells have not been profiled, especially for the heterochromatin regions. Here, we profiled genome‐wide maps of chromatin accessibility in ATRX‐intact and ATRX‐null human cancer cells. We found extensive changes in chromatin accessibility in both repetitive DNA regions and non‐repetitive regulatory regions following ATRX loss. These changes are highly correlated with changes in transcription, which lead to alterations in cancer‐related signalling pathways, such as upregulation of the TGF‐β pathway and downregulation of the cadherin family of proteins. These findings indicate that ATRX deficiency induces epigenomic changes and promotes tumorigenesis through both genome instability and shifts in transcription. [ABSTRACT FROM AUTHOR]

Secretory proteins are of particular importance to apicomplexan parasites and comprise over 15% of the genomes of the human pathogens that cause diseases like malaria, toxoplasmosis and babesiosis as well as other diseases of agricultural significance. Here, we developed an approach that allows us to control the trafficking destination of secretory proteins in the human malaria parasite Plasmodium falciparum. Based on the unique structural requirements of apicoplast transit peptides, we designed three conditional localization domains (CLD1, 2 and 3) that can be used to control protein trafficking via the addition of a cell permeant ligand. Studies comparing the trafficking dynamics of each CLD show that CLD2 has the most optimal trafficking efficiency. To validate this system, we tested whether CLD2 could conditionally localize a biotin ligase called holocarboxylase synthetase 1 (HCS1) without interfering with the function of the enzyme. In a parasite line expressing CLD2‐HCS1, we were able to control protein biotinylation in the apicoplast in a ligand‐dependent manner, demonstrating the full functionality of the CLD tool. We have developed and validated a novel molecular tool that may be used in future studies to help elucidate the function of secretory proteins in malaria parasites. [ABSTRACT FROM AUTHOR]

Proteins in the cell have to be eliminated once their function is no longer desired or they become damaged. Most regulated protein degradation is achieved by a large enzymatic complex called the proteasome. Many proteasome substrates are targeted for degradation by the covalent attachment of ubiquitin molecules. Ubiquitinated proteins can be bound by the proteasome, but for proteolysis to occur the proteasome needs to find a disordered tail somewhere in the target at which it initiates degradation. The initiation step contributes to the specificity of proteasomal degradation. Here, we review how the proteasome selects initiation sites within its substrates and discuss how the initiation step affects physiological processes. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs, produced from distinct regions of the chromosomes, are emerging as new key players in several important biological processes. The long noncoding RNAs add a new layer of complexity to cellular regulatory pathways, from transcription to cellular trafficking or chromatin remodeling. More than 25 years ago, the discovery of a transcriptional activity at telomeres of protozoa ended the long‐lasting belief that telomeres were transcriptionally silent. Since then, progressively accumulating evidences established that production of TElomeric Repeat‐containing RNA (TERRA) was a general feature of eukaryotic cells. Whether TERRA molecules always originate from the telomeres or whether they can be transcribed from internal telomeric repeats as well is however still a matter of debate. Whether TERRA transcripts always localize to telomeres and play similar roles in all eukaryotic cells is also unclear. We review the studies on TERRA localization in the cell, its composition and some aspects of its transcriptional regulation to summarize the current knowledge and controversies about the genomic origin of TERRA, with a focus on human and mouse TERRA. [ABSTRACT FROM AUTHOR]

Ganglioglioma (GG) is a grade I tumor characterized by alterations in the MAPK pathway, including BRAF V600E mutation. Recently, diffuse midline glioma with an H3 K27M mutation was added to the WHO 2016 classification as a new grade IV entity. As co-occurrence of H3 K27M and BRAF V600E mutations has been reported in midline tumors and anaplastic GG, we searched for BRAF V600E and H3 K27M mutations in a series of 54 paediatric midline grade I GG (midline GG) to determine the frequency of double mutations and its relevance for prognosis. Twenty-seven patients (50%) possessed the BRAF V600E mutation. The frequency of the co-occurrence of H3F3A/BRAF mutations at diagnosis was 9.3%. No H3 K27M mutation was detected in the absence of the BRAF V600E mutation. Double-immunostaining revealed that BRAF V600E and H3 K27M mutant proteins were present in both the glial and neuronal components. Immunopositivity for the BRAF V600E mutant protein correlated with BRAF mutation status as detected by massARRAY or digital droplet PCR. The median follow-up of patients with double mutation was 4 years. One patient died of progressive disease 8 years after diagnosis, whereas the four other patients were all alive with stable disease at the last clinical follow-up (at 9 months, 1 year and 7 years) without adjuvant therapy. We demonstrate in this first series of midline GGs that the H3 K27M mutation can occur in association with the BRAF V600E mutation in grade I glioneuronal tumors. Despite the presence of H3 K27M mutations, these cases should not be graded and treated as grade IV tumors because they have a better spontaneous outcome than classic diffuse midline H3 K27M-mutant glioma. These data suggest that H3 K27M cannot be considered a specific hallmark of grade IV diffuse gliomas and highlight the importance of integrated histomolecular diagnosis in paediatric brain tumors. [ABSTRACT FROM AUTHOR]

Cellular differentiation, developmental processes, and environmental factors challenge the integrity of the proteome in every eukaryotic cell. The maintenance of protein homeostasis, or proteostasis, involves folding and degradation of damaged proteins, and is essential for cellular function, organismal growth, and viability . Misfolded proteins that cannot be refolded by chaperone machineries are degraded by specialized proteolytic systems. A major degradation pathway regulating cellular proteostasis is the ubiquitin (Ub)/proteasome system ( UPS), which regulates turnover of damaged proteins that accumulate upon stress and during aging. Despite a large number of structurally unrelated substrates, Ub conjugation is remarkably selective. Substrate selectivity is mainly provided by the group of E3 enzymes. Several observations indicate that numerous E3 Ub ligases intimately collaborate with molecular chaperones to maintain the cellular proteome. In this review, we provide an overview of specialized quality control E3 ligases playing a critical role in the degradation of damaged proteins. The process of substrate recognition and turnover, the type of chaperones they team up with, and the potential pathogeneses associated with their malfunction will be further discussed. [ABSTRACT FROM AUTHOR]

Interferon-induced transmembrane protein 1 ( IFITM1), a member of the IFITM protein family, is a component of a multimeric complex involved in the transduction of antiproliferation and cell adhesion signals. IFITM1 is thought to play a role in antiproliferation and immune surveillance, and has been shown to restrict infection by numerous viruses. It is highly expressed in human embryonic stem cells ( hESCs) but its role in hESCs remains to be elucidated. In this study, knockout of IFITM1 mediated by CRISPR/Cas9 in hESCs did not affect self-renewal, pluripotency, telomerase activity or telomeres. However expression of human endogenous retroviruses ( HERVs) was higher than in wild-type hESCs, and there was also a reduced level of trimethylation of histone H3 on lysine 9 at HERV loci. These data show that IFITM1 suppresses HERVs in hESCs by regulating epigenetic modifications. [ABSTRACT FROM AUTHOR]

ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres ( TTAGGG)n. ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere ( ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA- DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX. Here, we show loss of ATRX is also associated with increased R-loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology-directed repair previously observed upon loss of ATRX function. [ABSTRACT FROM AUTHOR]

This study aims (1) to evaluate ATRX expression in different grades and subtypes of gliomas and correlate with other hallmark genetic alterations, (2) to identify and characterize mosaic/heterogeneous staining in gliomas in terms of mutation status. One hundred seventy six cases of glioma were assessed for ATRX immunohistochemistry and subdivided into positive, negative and mosaic/heterogeneous staining patterns. Five cases with heterogeneous staining were further subjected to next generation sequencing. Higher frequency of ATRX immune-negativity was detected in grade II/III astrocytic, oligoastrocytic tumors and secondary glioblastomas (GBMs), while infrequent in primary GBMs and rare in oligodendrogliomas. Loss of expression was significantly associated with IDH1 and/or TP53 mutation, while mutually exclusive with 1p/19q codeletion. Mosaic/heterogeneous staining was detected exclusively in GBMs (21.2%). Two different types of mosaic staining were identified (1) Admixture of positive and negative nuclei or intermixed mosaic and (2) Separate fragments with positive and negative/intermixed mosaic staining. ATRX mutation was identified in 2/5 (40%) cases with mosaic staining while one case showed DAXX mutation. All these cases were characterized by distinctly separate immune-negative and positive/intermixed foci. Hence, it is suggested that cases with heterogeneous staining (especially those with distinctly negative fragments) should be subjected to mutation analysis. [ABSTRACT FROM AUTHOR]

Cancer genome sequencing projects have identified hundreds of genetic alterations, often at low frequencies, raising questions as to their functional relevance. One exemplar gene is HUWE1, which has been found to be mutated in numerous studies. However, due to the large size of this gene and a lack of functional analysis of identified mutations, their significance to carcinogenesis is unclear. To determine the importance of HUWE1, we chose to examine its function in colorectal cancer, where it is mutated in up to 15 per cent of tumours. Modelling of identified mutations showed that they inactivate the E3 ubiquitin ligase activity of HUWE1. Genetic deletion of Huwe1 rapidly accelerated tumourigenic in mice carrying loss of the intestinal tumour suppressor gene Apc, with a dramatic increase in tumour initiation. Mechanistically, this phenotype was driven by increased MYC and rapid DNA damage accumulation leading to loss of the second copy of Apc. The increased levels of DNA damage sensitised Huwe1-deficient tumours to DNA-damaging agents and to deletion of the anti-apoptotic protein MCL1. Taken together, these data identify HUWE1 as a bona fide tumour suppressor gene in the intestinal epithelium and suggest a potential vulnerability of HUWE1-mutated tumours to DNA-damaging agents and inhibitors of anti-apoptotic proteins. [ABSTRACT FROM AUTHOR]

Somatic mutations of the H3F3A and HIST1H3B genes encoding the histone H3 variants, H3.3 and H3.1, were recently identified in high-grade gliomas arising in the thalamus, pons and spinal cord of children and young adults. However, the complete range of patients and locations in which these tumors arise, as well as the morphologic spectrum and associated genetic alterations remain undefined. Here, we describe a series of 47 diffuse midline gliomas with histone H3-K27M mutation. The 25 male and 22 female patients ranged in age from 2 to 65 years (median = 14). Tumors were centered not only in the pons, thalamus, and spinal cord, but also in the third ventricle, hypothalamus, pineal region and cerebellum. Patients with pontine tumors were younger (median = 7 years) than those with thalamic (median = 24 years) or spinal (median = 25 years) tumors. A wide morphologic spectrum was encountered including gliomas with giant cells, epithelioid and rhabdoid cells, primitive neuroectodermal tumor (PNET)-like foci, neuropil-like islands, pilomyxoid features, ependymal-like areas, sarcomatous transformation, ganglionic differentiation and pleomorphic xanthoastrocytoma (PXA)-like areas. In this series, histone H3-K27M mutation was mutually exclusive with IDH1 mutation and EGFR amplification, rarely co-occurred with BRAF-V600E mutation, and was commonly associated with p53 overexpression, ATRX loss (except in pontine gliomas), and monosomy 10. [ABSTRACT FROM AUTHOR]

Immunoglobulin diversification is driven by activation-induced deaminase ( AID), which converts cytidine to uracil within the Ig variable (IgV) regions. Central to the recruitment of AID to the IgV genes are factors that regulate the generation of single-stranded DNA (ss DNA), the enzymatic substrate of AID. Here, we report that chicken DT40 cells lacking variant histone H3.3 exhibit reduced IgV sequence diversification. We show that this results from impairment of the ability of AID to access the IgV genes due to reduced formation of ss DNA during IgV transcription. Loss of H3.3 also diminishes IgV R-loop formation. However, reducing IgV R-loops by RNase HI overexpression in wild-type cells does not affect IgV diversification, showing that these structures are not necessary intermediates for AID access. Importantly, the reduction in the formation of AID-accessible ss DNA in cells lacking H3.3 is independent of any effect on the level of transcription or the kinetics of RNAPII elongation, suggesting the presence of H3.3 in the nucleosomes of the IgV genes increases the chances of the IgV DNA becoming single-stranded, thereby creating an effective AID substrate. [ABSTRACT FROM AUTHOR]

Bidirectional transport of membrane organelles along microtubules ( MTs) is driven by plus-end directed kinesins and minus-end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT-based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug-of-war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug-of-war between opposing MT motors alone, by attaching a large number of kinesin-1 motors to organelles transported by dynein to minus-ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus-end-directed dynein-dependent MT runs, leading to a reversal of the overall direction of dynein-driven organelles in vivo. Therefore, in the absence of external regulators tug-of-war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo. [ABSTRACT FROM AUTHOR]

Pluripotent embryonic stem cells (ESCs) are characterized by distinct epigenetic features including a relative enrichment of histone modifications related to active chromatin. Among these is tri-methylation of lysine 4 on histone H3 (H3K4me3). Several thousands of the H3K4me3-enriched promoters in pluripotent cells also contain a repressive histone mark, namely H3K27me3, a situation referred to as "bivalency". While bivalent promoters are not unique to pluripotent cells, they are relatively enriched in these cell types, largely marking developmental and lineage-specific genes which are silent but poised for immediate action. The H3K4me3 and H3K27me3 modifications are catalyzed by lysine methyltransferases which are usually found within, although not entirely limited to, the Trithorax group (TrxG) and Polycomb group (PcG) protein complexes, respectively, but these do not provide selective bivalent specificity. Recent studies highlight the family of ATPdependent chromatin remodeling proteins as regulators of bivalent domains. Here, we discuss bivalency in general, describe the machineries that catalyze bivalent chromatin domains, and portray the emerging connection between bivalency and the action of different families of chromatin remodelers, namely INO80, esBAF, and NuRD, in pluripotent cells. We posit that chromatin remodeling proteins may enable "bivalent specificity", often selectively acting on, or selectively depleted from, bivalent domains. [ABSTRACT FROM AUTHOR]

Key points RGK (Rad, Rem, Rem2, Gem/Kir) proteins are small monomeric G-proteins implicated in various cellular functions and disease states. All RGK proteins potently inhibit voltage-gated calcium (CaV) channels., It is unclear whether RGK proteins are regulated by guanine nucleotide binding in a manner that conforms to a canonical G-protein regulation paradigm., We utilized strategic Rad and Rem mutants together with a range of functional readouts (CaV1.2 currents, Ca2+ transients, CaVβ binding) in heterologous cells and cardiomyocytes to determine whether the function of these RGK proteins is regulated in a canonical manner., Our results demonstrate that Rad and Rem are non-canonical G-proteins with respect to the regulatory role of their guanine nucleotide binding domain in CaV1.2 channel regulation., Our findings offer deepened insights into cellular mechanisms governing RGK regulation and function, and contribute towards our understanding of their pathophysiological roles., Abstract Rad and Rem are Ras-like G-proteins linked to diverse cardiovascular functions and pathophysiology. Understanding how Rad and Rem are regulated is important for deepened insights into their pathophysiological roles. As in other Ras-like G-proteins, Rad and Rem contain a conserved guanine-nucleotide binding domain (G-domain). Canonically, G-domains are key control modules, functioning as nucleotide-regulated switches of G-protein activity. Whether Rad and Rem G-domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (CaV1.2 currents, Ca2+ transients, CaVβ binding) as biosensors to probe the role of the G-domain in regulation of Rad and Rem function. We utilized RadS105N and RemT94N, which are the cognate mutants to RasS17N, a dominant-negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over-expression of either RadS105N or RemT94N strongly inhibited reconstituted CaV1.2 currents to the same extent as their wild-type (wt) counterparts, contrasting with reports that RadS105N is functionally inert in HEK293 cells. Adenovirus-mediated expression of either wt Rad or RadS105N in cardiomyocytes dramatically blocked L-type calcium current ( ICa,L) and inhibited Ca2+-induced Ca2+ release, contradicting reports that RadS105N acts as a dominant negative in heart. By contrast, RemT94N was significantly less effective than wt Rem at inhibiting ICa,L and Ca2+ transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both RadS105N and RemT94N had moderately reduced binding affinity for CaVβs relative to their wt counterparts. The results indicate Rad and Rem are non-canonical G-proteins with respect to the regulatory role of their G-domain in CaV1.2 regulation. [ABSTRACT FROM AUTHOR]

Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics. [ABSTRACT FROM AUTHOR]

Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K-M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K-M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation ( ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice. [ABSTRACT FROM AUTHOR]

More than 50% of mammalian genomes consist of retrotransposon sequences. Silencing of retrotransposons by heterochromatin is essential to ensure genomic stability and transcriptional integrity. Here, we identified a short sequence element in intracisternal A particle ( IAP) retrotransposons that is sufficient to trigger heterochromatin formation. We used this sequence in a genome-wide sh RNA screen and identified the chromatin remodeler Atrx as a novel regulator of IAP silencing. Atrx binds to IAP elements and is necessary for efficient heterochromatin formation. In addition, Atrx facilitates a robust and largely inaccessible heterochromatin structure as Atrx knockout cells display increased chromatin accessibility at retrotransposons and non-repetitive heterochromatic loci. In summary, we demonstrate a direct role of Atrx in the establishment and robust maintenance of heterochromatin. [ABSTRACT FROM AUTHOR]

The H3.3 histone variant has been a subject of increasing interest in the field of chromatin studies due to its two distinguishing features. First, its incorporation into chromatin is replication independent unlike the replication-coupled deposition of its canonical counterparts H3.1/2. Second, H3.3 has been consistently associated with an active state of chromatin. In accordance, this histone variant should be expected to be causally involved in the regulation of gene expression, or more generally, its incorporation should have downstream consequences for the structure and function of chromatin. This, however, leads to an apparent paradox: In cells that slowly replicate in the organism, H3.3 will accumulate with time, opening the way to aberrant effects on heterochromatin. Here, we review the indications that H3.3 is expected both to be incorporated in the heterochromatin of slowly replicating cells and to retain its functional downstream effects. Implications for organismal aging are discussed. [ABSTRACT FROM AUTHOR]

Cell identity is a reflection of a cell type-specific gene expression profile, and consequently, cell type-specific transcription factor networks are considered to be at the heart of a given cellular phenotype. Although generally stable, cell identity can be reprogrammed in vitro by forced changes to the transcriptional network, the most dramatic example of which was shown by the induction of pluripotency in somatic cells by the ectopic expression of defined transcription factors alone. Although changes to cell fate can be achieved in this way, the efficiency of such conversion remains very low, in large part due to specific chromatin signatures constituting an epigenetic barrier to the transcription factor-mediated reprogramming processes. Here we discuss the two-way relationship between transcription factor binding and chromatin structure during cell fate reprogramming. We additionally explore the potential roles and mechanisms by which histone variants, chromatin remodelling enzymes, and histone and DNA modifications contribute to the stability of cell identity and/or provide a permissive environment for cell fate change during cellular reprogramming. [ABSTRACT FROM AUTHOR]

Polycomb group proteins (PcGs) are a large protein family that includes diverse biochemical features assembled together in two large multiprotein complexes. These complexes maintain gene transcriptional repression in a cell type specific manner by modifying the surrounding chromatin to control development, differentiation and cell proliferation. PcGs are also involved in several diseases. PcGs are often directly or indirectly implicated in cancer development for which they have been proposed as potential targets for cancer therapeutic strategies. However, in the last few years a series of discoveries about the basic properties of PcGs and the identification of specific genetic alterations affecting specific Polycomb proteins in different tumours have converged to challenge old dogmas about PcG biological and molecular functions. In this review, we analyse these new data in the context of the old knowledge, highlighting the controversies and providing new models of interpretation and ideas that will perhaps bring some order among apparently contradicting observations. [ABSTRACT FROM AUTHOR]

Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein ( YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3-dependent intellectual disability. [ABSTRACT FROM AUTHOR]

The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death. [ABSTRACT FROM AUTHOR]

Clathrin-mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein-protein interactions between up to 60 different proteins. However, little is known about the spatio-temporal regulation of these interactions. Using fluorescence (cross)-correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self-interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein-protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo. [ABSTRACT FROM AUTHOR]

Histone deposition onto DNA assisted by specific chaperones forms the chromatin basic unit and serves to package the genome within the cell nucleus. The resulting chromatin organization, often referred to as the epigenome, contributes to a unique transcriptional program that defines cell identity. Importantly, during cellular life, substantial alterations in chromatin structure may arise due to cell stress, including DNA damage, which not only challenges the integrity of the genome but also threatens the epigenome. Considerable efforts have been made to decipher chromatin dynamics in response to genotoxic stress, and to assess how it affects both genome and epigenome stability. Here, we review recent advances in understanding the mechanisms of DNA damage-induced chromatin plasticity in mammalian cells. We focus specifically on the dynamics of histone H3 variants in response to UV irradiation, and highlight the role of their dedicated chaperones in restoring both chromatin structure and function. Finally, we discuss how, in addition to restoring chromatin integrity, the cellular networks that signal and repair DNA damage may also provide a window of opportunity for modulating the information conveyed by chromatin. [ABSTRACT FROM AUTHOR]

Actively transcribed genes are enriched with the histone variant H3.3. Although H3.3 deposition has been linked to transcription, mechanisms controlling this process remain elusive. We investigated the role of the histone methyltransferase Wolf-Hirschhorn syndrome candidate 1 (WHSC1) (NSD2/MMSET) in H3.3 deposition into interferon (IFN) response genes. IFN treatment triggered robust H3.3 incorporation into activated genes, which continued even after cessation of transcription. Likewise, UV radiation caused H3.3 deposition in UV-activated genes. However, in Whsc1−/− cells IFN- or UV-triggered H3.3 deposition was absent, along with a marked reduction in IFN- or UV-induced transcription. We found that WHSC1 interacted with the bromodomain protein 4 (BRD4) and the positive transcription elongation factor b (P-TEFb) and facilitated transcriptional elongation. WHSC1 also associated with HIRA, the H3.3-specific histone chaperone, independent of BRD4 and P-TEFb. WHSC1 and HIRA co-occupied IFN-stimulated genes and supported prolonged H3.3 incorporation, leaving a lasting transcriptional mark. Our results reveal a previously unrecognized role of WHSC1, which links transcriptional elongation and H3.3 deposition into activated genes through two molecularly distinct pathways. [ABSTRACT FROM AUTHOR]

Faithful transmission of chromosomes during eukaryotic cell division requires sister chromatids to be paired from their generation in S phase until their separation in M phase. Cohesion is mediated by the cohesin complex, whose Smc1, Smc3 and Scc1 subunits form a tripartite ring that entraps both DNA double strands. Whereas centromeric cohesin is removed in late metaphase by Scc1 cleavage, metazoan cohesin at chromosome arms is displaced already in prophase by proteolysis-independent signalling. Which of the three gates is triggered by the prophase pathway to open has remained enigmatic. Here, we show that displacement of human cohesin from early mitotic chromosomes requires dissociation of Smc3 from Scc1 but no opening of the other two gates. In contrast, loading of human cohesin onto chromatin in telophase occurs through the Smc1-Smc3 hinge. We propose that the use of differently regulated gates for loading and release facilitates unidirectionality of DNA's entry into and exit from the cohesin ring. [ABSTRACT FROM AUTHOR]