CCDC6-RET fusion protein regulates Ras/MAPK signaling through the fusion- GRB2-SHC1 signal niche.

Rearranged during transfection (RET) rearrangement oncoprotein-mediated Ras/MAPK signaling cascade is constitutively activated in cancers. Here, we demonstrate a unique signal niche. The niche is a ternary complex based on the chimeric RET liquid-liquid phase separation. The complex comprises the rearranged kinase (RET fusion); the adaptor (GRB2), and the effector (SHC1). Together, they orchestrate the Ras/MAPK signal cascade, which is dependent on tyrosine kinase. CCDC6-RET fusion undergoes LLPS requiring its kinase domain and its fusion partner. The CCDC6-RET fusion LLPS promotes the autophosphorylation of RET fusion, with enhanced kinase activity, which is necessary for the formation of the signaling niche. Within the signal niche, the interactions among the constituent components are reinforced, and the signal transduction efficiency is amplified. The specific RET fusion-related signal niche elucidates the mechanism of the constitutive activation of the Ras/MAPK signaling pathway. Beyond just focusing on RET fusion itself, exploration of the ternary complex potentially unveils a promising avenue for devising therapeutic strategies aimed at treating RET fusion-driven diseases.

RNA m6A modification, signals for degradation or stabilisation?

The RNA modification N6-methyladenosine (m6A) is conserved across eukaryotes, and profoundly influences RNA metabolism, including regulating RNA stability. METTL3 and METTL14, together with several accessory components, form a 'writer' complex catalysing m6A modification. Conversely, FTO and ALKBH5 function as demethylases, rendering m6A dynamic. Key to understanding the functional significance of m6A is its 'reader' proteins, exemplified by YTH-domain-containing proteins (YTHDFs) canonical reader and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) non-canonical reader. These proteins play a crucial role in determining RNA stability: YTHDFs mainly promote mRNA degradation through different cytoplasmic pathways, whereas IGF2BPs function to maintain mRNA stability. Additionally, YTHDC1 functions within the nucleus to degrade or protect certain m6A-containing RNAs, and other non-canonical readers also contribute to RNA stability regulation. Notably, m6A regulates retrotransposon LINE1 RNA stability and/or transcription via multiple mechanisms. However, conflicting observations underscore the complexities underlying m6A's regulation of RNA stability depending upon the RNA sequence/structure context, developmental stage, and/or cellular environment. Understanding the interplay between m6A and other RNA regulatory elements is pivotal in deciphering the multifaceted roles m6A plays in RNA stability regulation and broader cellular biology.

Small Molecule Inhibitors as Therapeutic Agents Targeting Oncogenic Fusion Proteins: Current Status and Clinical.

Oncogenic fusion proteins, arising from chromosomal rearrangements, have emerged as prominent drivers of tumorigenesis and crucial therapeutic targets in cancer research. In recent years, the potential of small molecular inhibitors in selectively targeting fusion proteins has exhibited significant prospects, offering a novel approach to combat malignancies harboring these aberrant molecular entities. This review provides a comprehensive overview of the current state of small molecular inhibitors as therapeutic agents for oncogenic fusion proteins. We discuss the rationale for targeting fusion proteins, elucidate the mechanism of action of inhibitors, assess the challenges associated with their utilization, and provide a summary of the clinical progress achieved thus far. The objective is to provide the medicinal community with current and pertinent information and to expedite the drug discovery programs in this area.

Xist-mediated silencing requires additive functions of SPEN and Polycomb together with differentiation-dependent recruitment of SmcHD1.

X chromosome inactivation (XCI) is mediated by the non-coding RNA Xist, which directs chromatin modification and gene silencing in cis. The RNA binding protein SPEN and associated corepressors have a central role in Xist-mediated gene silencing. Other silencing factors, notably the Polycomb system, have been reported to function downstream of SPEN. In recent work, we found that SPEN has an additional role in correct localization of Xist RNA in cis, indicating that its contribution to chromatin-mediated gene silencing needs to be reappraised. Making use of a SPEN separation-of-function mutation, we show that SPEN and Polycomb pathways, in fact, function in parallel to establish gene silencing. We also find that differentiation-dependent recruitment of the chromosomal protein SmcHD1 is required for silencing many X-linked genes. Our results provide important insights into the mechanism of X inactivation and the coordination of chromatin-based gene regulation with cellular differentiation and development.

Role of Histone Post-Translational Modifications in Inflammatory Diseases.

Inflammation is a defensive reaction for external stimuli to the human body and generally accompanied by immune responses, which is associated with multiple diseases such as atherosclerosis, type 2 diabetes, Alzheimer's disease, psoriasis, asthma, chronic lung diseases, inflammatory bowel disease, and multiple virus-associated diseases. Epigenetic mechanisms have been demonstrated to play a key role in the regulation of inflammation. Common epigenetic regulations are DNA methylation, histone modifications, and non-coding RNA expression; among these, histone modifications embrace various post-modifications including acetylation, methylation, phosphorylation, ubiquitination, and ADP ribosylation. This review focuses on the significant role of histone modifications in the progression of inflammatory diseases, providing the potential target for clinical therapy of inflammation-associated diseases.

The zinc-finger protein OEF-1 stabilizes histone modification patterns and promotes efficient splicing in the Caenorhabditis elegans germline.

To ensure stable transmission of genetic information to the next generation, germ cells frequently silence sex chromosomes, as well as autosomal loci that promote inappropriate differentiation programs. In Caenorhabditis elegans, silenced and active genomic domains are established in germ cells by the histone modification complexes MES-2/3/6 and MES-4, which promote silent and active chromatin states, respectively. These states are generally mutually exclusive and modulation of one state influences the pattern of the other. Here, we identify the zinc-finger protein OEF-1 as a novel modifier of this epigenetic balance in the C. elegans germline. Loss of oef-1 genetically enhances mes mutant phenotypes. Moreover, OEF-1 binding correlates with the active modification H3K36me3 and sustains H3K36me3 levels in the absence of MES-4 activity. OEF-1 also promotes efficient mRNA splicing activity, a process that is influenced by H3K36me3 levels. Finally, OEF-1 limits deposition of the silencing modification H3K27me3 on the X chromosome and at repressed autosomal loci. We propose that OEF-1 might act as an intermediary to mediate the downstream effects of H3K36me3 that promote transcript integrity, and indirectly affect gene silencing as a consequence.

Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading.

X-inactive specific transcript (Xist) RNA directs the process of X chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual-color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, which enabled us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we find that the protein SPEN, a key factor for Xist-mediated gene silencing, has a distinct function in Xist RNA localization, stability, and coupling behaviors. Our results provide insights toward understanding the distinct dynamic properties of Xist RNA.

Acute depletion of METTL3 implicates N 6-methyladenosine in alternative intron/exon inclusion in the nascent transcriptome.

RNA N 6-methyladenosine (m6A) modification plays important roles in multiple aspects of RNA regulation. m6A is installed cotranscriptionally by the METTL3/14 complex, but its direct roles in RNA processing remain unclear. Here, we investigate the presence of m6A in nascent RNA of mouse embryonic stem cells. We find that around 10% of m6A peaks are located in alternative introns/exons, often close to 5' splice sites. m6A peaks significantly overlap with RBM15 RNA binding sites and the histone modification H3K36me3. Acute depletion of METTL3 disrupts inclusion of alternative introns/exons in the nascent transcriptome, particularly at 5' splice sites that are proximal to m6A peaks. For terminal or variable-length exons, m6A peaks are generally located on or immediately downstream from a 5' splice site that is suppressed in the presence of m6A and upstream of a 5' splice site that is promoted in the presence of m6A. Genes with the most immediate effects on splicing include several components of the m6A pathway, suggesting an autoregulatory function. Collectively, our findings demonstrate crosstalk between the m6A machinery and the regulation of RNA splicing.

The PWWP2A Histone Deacetylase Complex Represses Intragenic Spurious Transcription Initiation in mESCs.

Transcriptional fidelity depends on accurate promoter selection and initiation from the correct sites. In yeast, H3K36me3-mediated recruitment of the Rpd3S HDAC complex to gene bodies suppresses spurious transcription initiation. Here we describe an equivalent pathway in metazoans. PWWP2A/B is an H3K36me3 reader that forms a stable complex with HDAC1/2. We used CAGE-seq to profile all transcription initiation sites in wild-type mESCs and cells lacking PWWP2A/B. Loss of PWWP2A/B enhances spurious initiation from intragenic sites present in wild-type mESCs, and this effect is associated with increased levels of initiating Pol-II and histone acetylation. Spurious initiation events in Pwwp2a/b DKO mESCs do not overlap in genomic location or chromatin features with spurious sites that arise in Dnmt3b KO mESCs, previously reported to function in the suppression of intragenic transcriptional initiation, suggesting these pathways function cooperatively in maintaining the fidelity of transcription initiation in metazoans.

Progress toward understanding chromosome silencing by Xist RNA.

The X inactive-specific transcript (Xist) gene is the master regulator of X chromosome inactivation in mammals. Xist produces a long noncoding (lnc)RNA that accumulates over the entire length of the chromosome from which it is transcribed, recruiting factors to modify underlying chromatin and silence X-linked genes in cis Recent years have seen significant progress in identifying important functional elements in Xist RNA, their associated RNA-binding proteins (RBPs), and the downstream pathways for chromatin modification and gene silencing. In this review, we summarize progress in understanding both how these pathways function in Xist-mediated silencing and the complex interplay between them.

LRIK interacts with the Ku70-Ku80 heterodimer enhancing the efficiency of NHEJ repair.

Despite recent advances in our understanding of the function of long noncoding RNAs (lncRNAs), their roles and functions in DNA repair pathways remain poorly understood. By screening a panel of uncharacterized lncRNAs to identify those whose transcription is induced by double-strand breaks (DSBs), we identified a novel lncRNA referred to as LRIK that interacts with Ku, which enhances the ability of the Ku heterodimer to detect the presence of DSBs. Here, we show that depletion of LRIK generates significantly enhanced sensitivity to DSB-inducing agents and reduced DSB repair efficiency. In response to DSBs, LRIK enhances the recruitment of repair factors at DSB sites and facilitates γH2AX signaling. Our results demonstrate that LRIK is necessary for efficient repairing DSBs via nonhomologous end-joining pathway.

The role of the Xist 5' m6A region and RBM15 in X chromosome inactivation.

Background: X chromosome inactivation in mammals is regulated by the non-coding (nc) RNA, Xist, which represses the chromosome from which it is transcribed.  High levels of the N6-methyladenosine (m6A) RNA modification occur within Xist exon I, close to the 5' end of the transcript, and also further 3', in Xist exon VII. The m6A modification is catalysed by the METTL3/14 complex that is directed to specific targets, including Xist, by the RNA binding protein RBM15/15B. m6A modification of Xist RNA has been reported to be important for Xist-mediated gene silencing.  Methods: We use CRISPR/Cas9 mediated mutagenesis to delete sequences around the 5' m6A region in interspecific XX mouse embryonic stem cells (mESCs).  Following induction of Xist RNA expression, we assay chromosome silencing using allelic RNA-seq and Xist m6A distribution using m6A-seq. Additionally, we use Xist RNA FISH to analyse the effect of deleting the 5' m6A region on the function of the endogenous Xist promoter. We purify epitope tagged RBM15 from mESCs, and then apply MS/MS analysis to define the RBM15 interactome. Results: We show that a deletion encompassing the entire Xist 5' m6A region results in a modest reduction in Xist-mediated silencing, and that the 5' m6A region overlaps essential DNA elements required for activation of the endogenous Xist promoter. Deletion of the Xist A-repeat, to which RBM15 binds, entirely abolishes deposition of m6A in the Xist 5' m6A region without affecting the modification in exon VII. We show that in mESCs, RBM15 interacts with the m6A complex, the SETD1B histone modifying complex, and several proteins linked to RNA metabolism. Conclusions: Our findings support that RBM15 binding to the Xist A-repeat recruits the m6A complex to the 5' Xist m6A region and that this region plays a role in Xist-mediated chromosome silencing.

Systematic allelic analysis defines the interplay of key pathways in X chromosome inactivation.

Xist RNA, the master regulator of X chromosome inactivation, acts in cis to induce chromosome-wide silencing. Whilst recent studies have defined candidate silencing factors, their relative contribution to repressing different genes, and their relationship with one another is poorly understood. Here we describe a systematic analysis of Xist-mediated allelic silencing in mouse embryonic stem cell-based models. Using a machine learning approach we identify distance to the Xist locus and prior gene expression levels as key determinants of silencing efficiency. We go on to show that Spen, recruited through the Xist A-repeat, plays a central role, being critical for silencing of all except a subset of weakly expressed genes. Polycomb, recruited through the Xist B/C-repeat, also plays a key role, favouring silencing of genes with pre-existing H3K27me3 chromatin. LBR and the Rbm15/m6A-methyltransferase complex make only minor contributions to gene silencing. Together our results provide a comprehensive model for Xist-mediated chromosome silencing.

Isolated C. elegans germ nuclei exhibit distinct genomic profiles of histone modification and gene expression.

BACKGROUND: The wide variety of specialized permissive and repressive mechanisms by which germ cells regulate developmental gene expression are not well understood genome-wide. Isolation of germ cells with high integrity and purity from living animals is necessary to address these open questions, but no straightforward methods are currently available. RESULTS: Here we present an experimental paradigm that permits the isolation of nuclei from C. elegans germ cells at quantities sufficient for genomic analyses. We demonstrate that these nuclei represent a very pure population and are suitable for both transcriptome analysis (RNA-seq) and chromatin immunoprecipitation (ChIP-seq) of histone modifications. From these data, we find unexpected germline- and soma-specific patterns of gene regulation. CONCLUSIONS: This new capacity removes a major barrier in the field to dissect gene expression mechanisms in the germ line of C. elegans. Consequent discoveries using this technology will be relevant to conserved regulatory mechanisms across species.

TRIM67 Activates p53 to Suppress Colorectal Cancer Initiation and Progression.

Tripartite motif (TRIM) family proteins participate in a variety of important cellular processes, including apoptosis, cell-cycle arrest, DNA repair, and senescence. In this study, we demonstrated that a novel TRIM family member, TRIM67, was commonly silenced in colorectal cancer and its downregulation was associated with poor survival. Trim67 knockout in ApcMin/+ mice increased the incidence, multiplicity, and burden of colorectal tumors. Similarly, colon-specific knockout of Trim67 significantly accelerated azoxymethane-induced colorectal cancer in mice. RNA sequencing revealed that the antitumor effect of TRIM67 was mediated by activation of the p53 signaling pathway. TRIM67 interacted directly with the C-terminus of p53, inhibiting p53 degradation by its ubiquitin ligase MDM2. TRIM67 was also a transcriptional target of p53; upon cellular stress, p53 bound to the TRIM67 promoter and induced significant upregulation of TRIM67, thereby forming a TRIM67/p53 self-amplifying loop that boosts p53-induced cell growth inhibition and apoptosis. Consequently, loss of this p53-positive regulatory program profoundly compromised p53-mediated responses to chemotherapy-induced DNA damage. Dampened p53 response was also observed in tumors of Trim67 knockout mice and Trim67 knockout embryonic fibroblasts. TRIM67 reactivation restored p53 activation and sensitized colorectal cancer cells to chemotherapy in vitro and in vivo. TRIM67 thus functions as a pivotal tumor suppressor in colorectal cancer and is a potential target for improving chemotherapy responsiveness. SIGNIFICANCE: The TRIM67/p53 axis represents a novel therapeutic target that could be harnessed to improve chemotherapy efficacy in colorectal cancer expressing wild-type p53 but with repressed p53 signaling.

m6A modification of non-coding RNA and the control of mammalian gene expression.

The biology of non-coding RNA (ncRNA) and the regulation of mammalian gene expression is a rapidly expanding field. In this review, we consider how recent advances in technology, enabling the precise mapping of modifications to RNA transcripts, has provided new opportunities to dissect post-transcriptional gene regulation. With this has come the realisation that in the absence of translation, the modification of ncRNAs may play a fundamental role in their regulation, protein interactome and subsequent downstream effector functions. We focus upon modification of RNA by N6-methyladenosine (m6A); its readers, writers and erasers, before considering the differing role of m6A modified lncRNAs MALAT1 and Xist. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes.

Transcriptional regulation by chromatin is a highly dynamic process directed through the recruitment and coordinated action of epigenetic modifiers and readers of these modifications. Using an unbiased proteomic approach to find interactors of H3K36me3, a modification enriched on active chromatin, here we identify PWWP2A and HDAC2 among the top interactors. PWWP2A and its paralog PWWP2B form a stable complex with NuRD subunits MTA1/2/3:HDAC1/2:RBBP4/7, but not with MBD2/3, p66α/ß, and CHD3/4. PWWP2A competes with MBD3 for binding to MTA1, thus defining a new variant NuRD complex that is mutually exclusive with the MBD2/3 containing NuRD. In mESCs, PWWP2A/B is most enriched at highly transcribed genes. Loss of PWWP2A/B leads to increases in histone acetylation predominantly at highly expressed genes, accompanied by decreases in Pol II elongation. Collectively, these findings suggest a role for PWWP2A/B in regulating transcription through the fine-tuning of histone acetylation dynamics at actively transcribed genes.

hnRNPK Recruits PCGF3/5-PRC1 to the Xist RNA B-Repeat to Establish Polycomb-Mediated Chromosomal Silencing.

The Polycomb-repressive complexes PRC1 and PRC2 play a key role in chromosome silencing induced by the non-coding RNA Xist. Polycomb recruitment is initiated by the PCGF3/5-PRC1 complex, which catalyzes chromosome-wide H2A lysine 119 ubiquitylation, signaling recruitment of other PRC1 complexes, and PRC2. However, the molecular mechanism for PCGF3/5-PRC1 recruitment by Xist RNA is not understood. Here we define the Xist RNA Polycomb Interaction Domain (XR-PID), a 600 nt sequence encompassing the Xist B-repeat element. Deletion of XR-PID abolishes Xist-dependent Polycomb recruitment, in turn abrogating Xist-mediated gene silencing and reversing Xist-induced chromatin inaccessibility. We identify the RNA-binding protein hnRNPK as the principal XR-PID binding factor required to recruit PCGF3/5-PRC1. Accordingly, synthetically tethering hnRNPK to Xist RNA lacking XR-PID is sufficient for Xist-dependent Polycomb recruitment. Our findings define a key pathway for Polycomb recruitment by Xist RNA, providing important insights into mechanisms of chromatin modification by non-coding RNA.

A Systematic RNAi Screen Reveals a Novel Role of a Spindle Assembly Checkpoint Protein BuGZ in Synaptic Transmission in C. elegans.

Synaptic vesicles (SV) store various neurotransmitters that are released at the synapse. The molecular mechanisms of biogenesis, exocytosis, and endocytosis for SV, however, remain largely elusive. In this study, using Complex Object Parametric Analysis and Sorter (COPAS) to monitor the fluorescence of synapto-pHluorin (SpH), we performed a whole-genome RNAi screen in C. elegans to identify novel genetic modulators in SV cycling. One hundred seventy six genes that up-regulating SpH fluorescence and 96 genes that down-regulating SpH fluorescence were identified after multi-round screen. Among these genes, B0035.1 (bugz-1) encodes ortholog of mammalian C2H2 zinc-finger protein BuGZ/ZNF207, which is a spindle assembly checkpoint protein essential for mitosis in human cells. Combining electrophysiology, imaging and behavioral assays, we reveal that depletion of BuGZ-1 results in defects in locomotion. We further demonstrate that BuGZ-1 promotes SV recycling by regulating the expression levels of endocytosis-related genes such as rab11.1. Therefore, we have identified a bunch of potential genetic modulators in SV cycling, and revealed an unexpected role of BuGZ-1 in regulating synaptic transmission.