Overview of chromatin regulatory processes during surface ectodermal development and homeostasis.

The ectoderm is the outermost of the three germ layers of the early embryo that arise during gastrulation. Once the germ layers are established, the complex interplay of cellular proliferation, differentiation, and migration results in organogenesis. The ectoderm is the progenitor of both the surface ectoderm and the neural ectoderm. Notably, the surface ectoderm develops into the epidermis and its associated appendages, nails, external exocrine glands, olfactory epithelium, and the anterior pituitary. Specification, development, and homeostasis of these organs demand a tightly orchestrated gene expression program that is often dictated by epigenetic regulation. In this review, we discuss the recent discoveries that have highlighted the importance of chromatin regulatory mechanisms mediated by transcription factors, histone and DNA modifications that aid in the development of surface ectodermal organs and maintain their homeostasis post-development.

Fluorescent Reporter Systems to Investigate Chromatin Effector Proteins in Living Cells.

Epigenetic research faces the challenge of the high complexity and tight regulation in chromatin modification networks. Although many isolated mechanisms of chromatin-mediated gene regulation have been described, solid approaches for the comprehensive analysis of specific processes as parts of the bigger epigenome network are missing. In order to expand the toolbox of methods by a system that will help to capture and describe the complexity of transcriptional regulation, we describe here a robust protocol for the generation of stable reporter systems for transcriptional activity and summarize their applications. The system allows for the induced recruitment of a chromatin regulator to a fluorescent reporter gene, followed by the detection of transcriptional changes using flow cytometry. The reporter gene is integrated into an endogenous chromatin environment, thus enabling the detection of regulatory dependencies of the investigated chromatin regulator on endogenous cofactors. The system allows for an easy and dynamic readout at the single-cell level and the ability to compensate for cell-to-cell variances of transcription. The modular design of the system enables the simple adjustment of the method for the investigation of different chromatin regulators in a broad panel of cell lines. We also summarize applications of this technology to characterize the silencing velocity of different chromatin effectors, removal of activating histone modifications, analysis of stability and reversibility of epigenome modifications, the investigation of the effects of small molecule on chromatin effectors and of functional effector-coregulator relationships. The presented method allows to investigate the complexity of transcriptional regulation by epigenetic effector proteins in living cells.

HIRA complex deposition of histone H3.3 is driven by histone tetramerization and histone-DNA binding.

The HIRA histone chaperone complex is comprised of four protein subunits: HIRA, UBN1, CABIN1, and transiently associated ASF1a. All four subunits have been demonstrated to play a role in deposition of the histone variant H3.3 onto areas of actively transcribed euchromatin in cells. The mechanism by which these subunits function together to drive histone deposition has remained poorly understood. Here we present biochemical and biophysical data supporting a model whereby ASF1a delivers histone H3.3/H4 dimers to the HIRA complex, H3.3/H4 tetramerization drives the association of two HIRA/UBN1 complexes, and the affinity of the histones for DNA drives release of ASF1a and subsequent histone deposition. These findings have implications for understanding how other histone chaperone complexes may mediate histone deposition.

Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer.

Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.

Decoding Chromatin Ubiquitylation: A Chemical Biology Perspective.

Since Strahl and Allis proposed the "language of covalent histone modifications", a host of experimental studies have shed light on the different facets of chromatin regulation by epigenetic mechanisms. Initially proposed as a concept for controlling gene transcription, the regulation of deposition and removal of histone post-translational modifications (PTMs), such as acetylation, methylation, and phosphorylation, have been implicated in many chromatin regulation pathways. However, large PTMs such as ubiquitylation challenge research on many levels due to their chemical complexity. In recent years, chemical tools have been developed to generate chromatin in defined ubiquitylation states in vitro. Chemical biology approaches are now used to link specific histone ubiquitylation marks with downstream chromatin regulation events on the molecular level. Here, we want to highlight how chemical biology approaches have empowered the mechanistic study of chromatin ubiquitylation in the context of gene regulation and DNA repair with attention to future challenges.

The chromatin remodeler Ino80 regulates yeast stress tolerance and cell metabolism through modulating nitrogen catabolite repression.

Chromatin remodelers are important in maintaining the dynamic chromatin state in eukaryotic cells, which is essential for epigenetic regulation. Among the remodelers, the multi-subunits complex INO80 plays crucial roles in transcriptional regulation. However, current knowledge of chromatin regulation of the core subunit Ino80 on stress adaptation remains mysterious. Here we revealed that overexpressing the chromatin remodeler Ino80 elevated tolerance to multiple stresses in budding yeast Saccharomyces cerevisiae. Analyses of differential chromatin accessibility and global transcription levels revealed an enrichment of genes involved in NCR (nitrogen catabolite repression) under acetic acid stress. We demonstrated that Ino80 overexpression reduced the histone H3 occupancy in the promoter region of the glutamate dehydrogenase gene GDH2 and the allantoinase gene DAL1. Consistently, the decreased occupancy of nucleosome was revealed in the Ino80-inactivation mutant. Further analyses showed that Ino80 was recruited to the specific DNA locus in the promoter region of GDH2. Consistently, Ino80 overexpression facilitated the utilization of non-preferred nitrogen source to enhance ethanol yield under prolonged acetic acid stress. These results demonstrate that Ino80 plays a crucial role in coordinating carbon and nitrogen metabolism during stress adaptation.

The complex nature of lncRNA-mediated chromatin dynamics in multiple myeloma.

Extensive genome-wide sequencing efforts have unveiled the intricate regulatory potential of long non-protein coding RNAs (lncRNAs) within the domain of haematological malignancies. Notably, lncRNAs have been found to directly modulate chromatin architecture, thereby impacting gene expression and disease progression by interacting with DNA, RNA, and proteins in a tissue- or condition-specific manner. Furthermore, recent studies have highlighted the intricate epigenetic control of lncRNAs in cancer. Consequently, this provides a rationale to explore the possibility of therapeutically targeting lncRNAs themselves or the epigenetic mechanisms that govern their activity. Within the scope of this review, we will assess the current state of knowledge regarding the epigenetic regulation of lncRNAs and how, in turn, lncRNAs contribute to chromatin remodelling in the context of multiple myeloma.

Conserved functions of chromatin regulators in basal Archaeplastida.

Chromatin is a dynamic network that regulates genome organization and gene expression. Different types of chromatin regulators are highly conserved among Archaeplastida, including unicellular algae, while some chromatin genes are only present in land plant genomes. Here, we review recent advances in understanding the function of conserved chromatin factors in basal land plants and algae. We focus on the role of Polycomb-group genes which mediate H3K27me3-based silencing and play a role in balancing gene dosage and regulating haploid-to-diploid transitions by tissue-specific repression of the transcription factors KNOX and BELL in many representatives of the green lineage. Moreover, H3K27me3 predominantly occupies repetitive elements which can lead to their silencing in a unicellular alga and basal land plants, while it covers mostly protein-coding genes in higher land plants. In addition, we discuss the role of nuclear matrix constituent proteins as putative functional lamin analogs that are highly conserved among land plants and might have an ancestral function in stress response regulation. In summary, our review highlights the importance of studying chromatin regulation in a wide range of organisms in the Archaeplastida.

Programmed DNA elimination in Mesorhabditis nematodes.

Some species undergo programmed DNA elimination (PDE), whereby portions of the genome are systematically destroyed in somatic cells. PDE has emerged independently in several phyla, but its function is unknown. Although the mechanisms are partially solved in ciliates, PDE remains mysterious in metazoans because the study species were not yet amenable to functional approaches. We fortuitously discovered massive PDE in the free-living nematode genus Mesorhabditis, from the same family as C. elegans. As such, these species offer many experimental advantages to start elucidating the PDE mechanisms in an animal. Here, we used cytology to describe the dynamics of chromosome fragmentation and destruction in early embryos. Elimination occurs once in development, at the third embryonic cell division in the somatic blastomeres. Chromosomes are first fragmented during S phase. Next, some of the fragments fail to align on the mitotic spindle and remain outside the re-assembled nuclei after mitosis. These fragments are gradually lost after a few cell cycles. The retained fragments form new mini chromosomes, which are properly segregated in the subsequent cell divisions. With genomic approaches, we found that Mesorhabditis mainly eliminate repeated regions and also about a hundred genes. Importantly, none of the eliminated protein-coding genes are shared between closely related Mesorhabditis species. Our results strongly suggest PDE has not been selected for regulating genes with important biological functions in Mesorhabditis but rather mainly to irreversibly remove repeated sequences in the soma. We propose that PDE may target genes, provided their elimination in the soma is invisible to selection.

CTCF and Its Multi-Partner Network for Chromatin Regulation.

Architectural proteins are essential epigenetic regulators that play a critical role in organizing chromatin and controlling gene expression. CTCF (CCCTC-binding factor) is a key architectural protein responsible for maintaining the intricate 3D structure of chromatin. Because of its multivalent properties and plasticity to bind various sequences, CTCF is similar to a Swiss knife for genome organization. Despite the importance of this protein, its mechanisms of action are not fully elucidated. It has been hypothesized that its versatility is achieved through interaction with multiple partners, forming a complex network that regulates chromatin folding within the nucleus. In this review, we delve into CTCF's interactions with other molecules involved in epigenetic processes, particularly histone and DNA demethylases, as well as several long non-coding RNAs (lncRNAs) that are able to recruit CTCF. Our review highlights the importance of CTCF partners to shed light on chromatin regulation and pave the way for future exploration of the mechanisms that enable the finely-tuned role of CTCF as a master regulator of chromatin.

The bromo-adjacent homology domains of PBRM1 associate with histone tails and contribute to PBAF-mediated gene regulation.

A critical component of gene regulation is recognition of histones and their post-translational modifications by transcription-associated proteins or complexes. Although many histone-binding reader modules have been characterized, the bromo-adjacent homology (BAH) domain family of readers is still poorly characterized. A pre-eminent member of this family is PBRM1 (BAF180), a component of the PBAF chromatin-remodeling complex. PBRM1 contains two adjacent BAH domains of unknown histone-binding potential. We evaluated the tandem BAH domains for their capacity to associate with histones and to contribute to PBAF-mediated gene regulation. The BAH1 and BAH2 domains of human PBRM1 broadly interacted with histone tails, but they showed a preference for unmodified N-termini of histones H3 and H4. Molecular modeling and comparison of the BAH1 and BAH2 domains with other BAH readers pointed to a conserved binding mode via an extended open pocket and, in general, an aromatic cage for histone lysine binding. Point mutants that were predicted to disrupt the interaction between the BAH domains and histones reduced histone binding in vitro and resulted in dysregulation of genes targeted by PBAF in cellulo. Although the BAH domains in PBRM1 were important for PBAF-mediated gene regulation, we found that overall chromatin targeting of PBRM1 was not dependent on BAH-histone interaction. Our findings identify a function of the PBRM1 BAH domains in PBAF activity that is likely mediated by histone tail interaction.

The prognostic and biological importance of chromatin regulation-related genes for lung cancer is examined using bioinformatics and experimentally confirmed.

BACKGROUND: The pathogenesis and clinical diagnosis of lung adenocarcinoma (LUAD), a malignant illness with substantial morbidity and mortality, are still being investigated. Genes involved in chromatin regulation are crucial in the biological function of LUAD. METHODS: The prognostic prediction model for LUAD was developed using multivariables and least absolute shrinkage and selection operator (LASSO) regression. It consisted of 10 chromatin regulators. The LUAD has been divided into two groups, high- and low-risk, using a predictive model. The model was shown to be accurate in predicting survival by the nomogram, receiver operating characteristic (ROC) curves, and principal component analysis (PCA). An analysis of differences in immune-cell infiltration, immunologicalfunction, and clinical traits between low- and high-risk populations was conducted. Protein-protein interaction (PPI) networks and Gene Ontology (GO) pathways of differentially expressed genes (DEGs) in the high versus low risk group were also examined to investigate the association between genes and biological pathways. The biological roles of chromatin regulators (CRs) in LUAD were finally estimated using colony formation and cell movement. The important genes' mRNA expression has been measured using real-time polymerase chain reaction (RT-PCR). RESULTS AND CONCLUSION: Risk score and stage based on the model could be seen as separate prognostic indicators for patients with LUAD. The main signaling pathway difference across various risk groups was in cell cycle. The immunoinfiltration profile of the tumor microenvironment (TME) and individuals with different risk levels were correlated, suggesting that the interaction of immune cells with the tumor led to the creation of a favorable immunosuppressive microenvironment. These discoveries aid in the creation of individualized therapies for LUAD patients.

Differential Occupancy and Regulatory Interactions of KDM6A in Bladder Cell Lines.

Epigenetic deregulation is a critical theme which needs further investigation in bladder cancer research. One of the most highly mutated genes in bladder cancer is KDM6A, which functions as an H3K27 demethylase and is one of the MLL3/4 complexes. To decipher the role of KDM6A in normal versus tumor settings, we identified the genomic landscape of KDM6A in normal, immortalized, and cancerous bladder cells. Our results showed differential KDM6A occupancy in the genes involved in cell differentiation, chromatin organization, and Notch signaling depending on the cell type and the mutation status of KDM6A. Transcription factor motif analysis revealed HES1 to be enriched at KDM6A peaks identified in the T24 bladder cancer cell line; moreover, it has a truncating mutation in KDM6A and lacks a demethylase domain. Our co-immunoprecipitation experiments revealed TLE co-repressors and HES1 as potential truncated and wild-type KDM6A interactors. With the aid of structural modeling, we explored how truncated KDM6A could interact with TLE and HES1, as well as RUNX and HHEX transcription factors. These structures provide a solid means of studying the functions of KDM6A independently of its demethylase activity. Collectively, our work provides important contributions to the understanding of KDM6A malfunction in bladder cancer.

Clusters of co-abundant proteins in the brain cortex associated with fronto-temporal lobar degeneration.

BACKGROUND: Frontotemporal lobar degeneration (FTLD) is characterized pathologically by neuronal and glial inclusions of hyperphosphorylated tau or by neuronal cytoplasmic inclusions of TDP43. This study aimed at deciphering the molecular mechanisms leading to these distinct pathological subtypes. METHODS: To this end, we performed an unbiased mass spectrometry-based proteomic and systems-level analysis of the middle frontal gyrus cortices of FTLD-tau (n = 6), FTLD-TDP (n = 15), and control patients (n = 5). We validated these results in an independent patient cohort (total n = 24). RESULTS: The middle frontal gyrus cortex proteome was most significantly altered in FTLD-tau compared to controls (294 differentially expressed proteins at FDR = 0.05). The proteomic modifications in FTLD-TDP were more heterogeneous (49 differentially expressed proteins at FDR = 0.1). Weighted co-expression network analysis revealed 17 modules of co-regulated proteins, 13 of which were dysregulated in FTLD-tau. These modules included proteins associated with oxidative phosphorylation, scavenger mechanisms, chromatin regulation, and clathrin-mediated transport in both the frontal and temporal cortex of FTLD-tau. The most strongly dysregulated subnetworks identified cyclin-dependent kinase 5 (CDK5) and polypyrimidine tract-binding protein 1 (PTBP1) as key players in the disease process. Dysregulation of 9 of these modules was confirmed in independent validation data sets of FLTD-tau and control temporal and frontal cortex (total n = 24). Dysregulated modules were primarily associated with changes in astrocyte and endothelial cell protein abundance levels, indicating pathological changes in FTD are not limited to neurons. CONCLUSIONS: Using this innovative workflow and zooming in on the most strongly dysregulated proteins of the identified modules, we were able to identify disease-associated mechanisms in FTLD-tau with high potential as biomarkers and/or therapeutic targets.

The molecular consequences of androgen activity in the human breast.

Estrogen and progesterone have been extensively studied in the mammary gland, but the molecular effects of androgen remain largely unexplored. Transgender men are recorded as female at birth but identify as male and may undergo gender-affirming androgen therapy to align their physical characteristics and gender identity. Here we perform single-cell-resolution transcriptome, chromatin, and spatial profiling of breast tissues from transgender men following androgen therapy. We find canonical androgen receptor gene targets are upregulated in cells expressing the androgen receptor and that paracrine signaling likely drives sex-relevant androgenic effects in other cell types. We also observe involution of the epithelium and a spatial reconfiguration of immune, fibroblast, and vascular cells, and identify a gene regulatory network associated with androgen-induced fat loss. This work elucidates the molecular consequences of androgen activity in the human breast at single-cell resolution.

Pioneer factor ASCL1 cooperates with the mSWI/SNF complex at distal regulatory elements to regulate human neural differentiation.

Pioneer transcription factors are thought to play pivotal roles in developmental processes by binding nucleosomal DNA to activate gene expression, though mechanisms through which pioneer transcription factors remodel chromatin remain unclear. Here, using single-cell transcriptomics, we show that endogenous expression of neurogenic transcription factor ASCL1, considered a classical pioneer factor, defines a transient population of progenitors in human neural differentiation. Testing ASCL1's pioneer function using a knockout model to define the unbound state, we found that endogenous expression of ASCL1 drives progenitor differentiation by cis-regulation both as a classical pioneer factor and as a nonpioneer remodeler, where ASCL1 binds permissive chromatin to induce chromatin conformation changes. ASCL1 interacts with BAF SWI/SNF chromatin remodeling complexes, primarily at targets where it acts as a nonpioneer factor, and we provide evidence for codependent DNA binding and remodeling at a subset of ASCL1 and SWI/SNF cotargets. Our findings provide new insights into ASCL1 function regulating activation of long-range regulatory elements in human neurogenesis and uncover a novel mechanism of its chromatin remodeling function codependent on partner ATPase activity.

Long non-coding RNAs reveal new regulatory mechanisms controlling gene expression.

A plethora of non-coding RNAs have been found in eukaryotes, notably with the advent of modern sequencing technologies to analyze the transcriptome. Apart from the well-known housekeeping RNA genes (such as the ribosomal RNA or the transfer RNA), many thousands of transcripts detected are not evidently linked to a protein-coding gene. These, so called non-coding RNAs, may code for crucial regulators of gene expression, the small si/miRNAs, for small peptides (translated under specific conditions) or may act as long RNA molecules (antisense, intronic or intergenic long non-coding RNAs or lncRNAs). The lncRNAs interact with members of multiple machineries involved in gene regulation. In this review, we discussed about how plant lncRNAs permitted to discover new regulatory mechanisms acting in epigenetic control, chromatin 3D structure and alternative splicing. These novel regulations diversified the expression patterns and protein variants of target protein-coding genes and are an important element of the response of plants to environmental stresses and their adaptation to changing conditions.

Une pléthore d'ARN non codants a été découverte chez les eucaryotes, notamment avec l'avènement des technologies modernes de séquençage pour analyser le transcriptome. En dehors des gènes ARN domestiques bien connus (tels que l'ARN ribosomal ou l'ARN de transfert), plusieurs milliers de transcrits détectés ne sont pas manifestement liés à un gène codant pour une protéine. Ces ARN, appelés ARN non codants, peuvent coder pour des régulateurs cruciaux de l'expression des gènes, les petits si/miARN, pour de petits peptides (traduits dans des conditions spécifiques) ou peuvent agir comme de longues molécules d'ARN (ARN non codants antisens, introniques ou intergéniques ou lncRNA). Les lncRNAs interagissent avec les membres des multiples machineries impliquées dans la régulation des gènes. Dans cette revue, nous avons discuté de la façon dont les lncRNAs des plantes ont permis de découvrir de nouveaux mécanismes de régulation agissant dans le contrôle épigénétique, la structure 3D de la chromatine et l'épissage alternatif. Ces nouvelles régulations ont diversifié les profils d'expression des gènes codants pour les protéines cibles et constituent un élément important de la réponse des plantes aux stress environnementaux et de leur adaptation aux conditions changeantes.

Repeat Element Activation-Driven Inflammation: Role of NFκB and Implications in Normal Development and Cancer?

The human genome is composed of unique DNA sequences that encode proteins and unique sequence noncoding RNAs that are essential for normal development and cellular differentiation. The human genome also contains over 50% of genome sequences that are repeat in nature (tandem and interspersed repeats) that are now known to contribute dynamically to genetic diversity in populations, to be transcriptionally active under certain physiological conditions, and to be aberrantly active in disease states including cancer, where consequences are pleiotropic with impact on cancer cell phenotypes and on the tumor immune microenvironment. Repeat element-derived RNAs play unique roles in exogenous and endogenous cell signaling under normal and disease conditions. A key component of repeat element-derived transcript-dependent signaling occurs via triggering of innate immune receptor signaling that then feeds forward to inflammatory responses through interferon and NFκB signaling. It has recently been shown that cancer cells display abnormal transcriptional activity of repeat elements and that this is linked to either aggressive disease and treatment failure or to improved prognosis/treatment response, depending on cell context and the amplitude of the so-called 'viral mimicry' response that is engaged. 'Viral mimicry' refers to a cellular state of active antiviral response triggered by endogenous nucleic acids often derived from aberrantly transcribed endogenous retrotransposons and other repeat elements. In this paper, the literature regarding transcriptional activation of repeat elements and engagement of inflammatory signaling in normal (focusing on hematopoiesis) and cancer is reviewed with an emphasis on the role of innate immune receptor signaling, in particular by dsRNA receptors of the RIG-1 like receptor family and interferons/NFκB. How repeat element-derived RNA reprograms cell identity through RNA-guided chromatin state modulation is also discussed.

MyoD-Induced Trans-Differentiation: A Paradigm for Dissecting the Molecular Mechanisms of Cell Commitment, Differentiation and Reprogramming.

The discovery of the skeletal muscle-specific transcription factor MyoD represents a milestone in the field of transcriptional regulation during differentiation and cell-fate reprogramming. MyoD was the first tissue-specific factor found capable of converting non-muscle somatic cells into skeletal muscle cells. A unique feature of MyoD, with respect to other lineage-specific factors able to drive trans-differentiation processes, is its ability to dramatically change the cell fate even when expressed alone. The present review will outline the molecular strategies by which MyoD reprograms the transcriptional regulation of the cell of origin during the myogenic conversion, focusing on the activation and coordination of a complex network of co-factors and epigenetic mechanisms. Some molecular roadblocks, found to restrain MyoD-dependent trans-differentiation, and the possible ways for overcoming these barriers, will also be discussed. Indeed, they are of critical importance not only to expand our knowledge of basic muscle biology but also to improve the generation skeletal muscle cells for translational research.