A New Facet of Vitamin B12: Gene Regulation by Cobalamin-Based Photoreceptors.

Living organisms sense and respond to light, a crucial environmental factor, using photoreceptors, which rely on bound chromophores such as retinal, flavins, or linear tetrapyrroles for light sensing. The discovery of photoreceptors that sense light using 5'-deoxyadenosylcobalamin, a form of vitamin B12 that is best known as an enzyme cofactor, has expanded the number of known photoreceptor families and unveiled a new biological role of this vitamin. The prototype of these B12-dependent photoreceptors, the transcriptional repressor CarH, is widespread in bacteria and mediates light-dependent gene regulation in a photoprotective cellular response. CarH activity as a transcription factor relies on the modulation of its oligomeric state by 5'-deoxyadenosylcobalamin and light. This review surveys current knowledge about these B12-dependent photoreceptors, their distribution and mode of action, and the structural and photochemical basis of how they orchestrate signal transduction and control gene expression.

DNA-PKcs-mediated transcriptional regulation of TOP2B drives chemoresistance in acute myeloid leukemia.

Anthracyclines, topoisomerase II enzyme poisons that cause DNA damage, are the mainstay of acute myeloid leukemia (AML) treatment. However, acquired resistance to anthracyclines leads to relapse, which currently lacks effective treatment and is the cause of poor survival in individuals with AML. Therefore, the identification of the mechanisms underlying anthracycline resistance remains an unmet clinical need. Here, using patient-derived primary cultures and clinically relevant cellular models that recapitulate acquired anthracycline resistance in AML, we have found that GCN5 (also known as KAT2A) mediates transcriptional upregulation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in AML relapse, independently of the DNA-damage response. We demonstrate that anthracyclines fail to induce DNA damage in resistant cells, owing to the loss of expression of their target enzyme, TOP2B; this was caused by DNA-PKcs directly binding to its promoter upstream region as a transcriptional repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone, and abrogated their tumorigenic potential in a xenograft mouse model. Taken together, our findings identify a GCN5-DNA-PKcs-TOP2B transcriptional regulatory axis as the mechanism underlying anthracycline resistance, and demonstrate the therapeutic potential of DNA-PKcs inhibition to re-sensitize resistant AML relapse cells to anthracycline.

The MAPK-Alfin-like 7 module negatively regulates ROS scavenging genes to promote NLR-mediated immunity.

Nucleotide-binding leucine-rich repeat (NLR) receptor-mediated immunity includes rapid production of reactive oxygen species (ROS) and transcriptional reprogramming, which is controlled by transcription factors (TFs). Although some TFs have been reported to participate in NLR-mediated immune response, most TFs are transcriptional activators, and whether and how transcriptional repressors regulate NLR-mediated plant defenses remains largely unknown. Here, we show that the Alfin-like 7 (AL7) interacts with N NLR and functions as a transcriptional repressor. Knockdown and knockout of AL7 compromise N NLR-mediated resistance against tobacco mosaic virus, whereas AL7 overexpression enhances defense, indicating a positive regulatory role for AL7 in immunity. AL7 binds to the promoters of ROS scavenging genes to inhibit their transcription during immune responses. Mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK) directly interact with and phosphorylate AL7, which impairs the AL7-N interaction and enhances its DNA binding activity, which promotes ROS accumulation and enables immune activation. In addition to N, AL7 is also required for the function of other Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeats (TNLs) including Roq1 and RRS1-R/RPS4. Our findings reveal a hitherto unknown MAPK-AL7 module that negatively regulates ROS scavenging genes to promote NLR-mediated immunity.

Canalizing cell fate by transcriptional repression.

Precision in the establishment and maintenance of cellular identities is crucial for the development of multicellular organisms and requires tight regulation of gene expression. While extensive research has focused on understanding cell type-specific gene activation, the complex mechanisms underlying the transcriptional repression of alternative fates are not fully understood. Here, we provide an overview of the repressive mechanisms involved in cell fate regulation. We discuss the molecular machinery responsible for suppressing alternative fates and highlight the crucial role of sequence-specific transcription factors (TFs) in this process. Depletion of these TFs can result in unwanted gene expression and increased cellular plasticity. We suggest that these TFs recruit cell type-specific repressive complexes to their cis-regulatory elements, enabling them to modulate chromatin accessibility in a context-dependent manner. This modulation effectively suppresses master regulators of alternative fate programs and their downstream targets. The modularity and dynamic behavior of these repressive complexes enables a limited number of repressors to canalize and maintain major and minor cell fate decisions at different stages of development.

Hypoxia causes pancreatic ß-cell dysfunction and impairs insulin secretion by activating the transcriptional repressor BHLHE40.

Hypoxia can occur in pancreatic ß-cells in type 2 diabetes. Although hypoxia exerts deleterious effects on ß-cell function, the associated mechanisms are largely unknown. Here, we show that the transcriptional repressor basic helix-loop-helix family member e40 (BHLHE40) is highly induced in hypoxic mouse and human ß-cells and suppresses insulin secretion. Conversely, BHLHE40 deficiency in hypoxic MIN6 cells or ß-cells of ob/ob mice reverses defects in insulin secretion. Mechanistically, BHLHE40 represses the expression of Mafa, encoding the transcription factor musculoaponeurotic fibrosarcoma oncogene family A (MAFA), by attenuating the binding of pancreas/duodenum homeobox protein 1 (PDX1) to its enhancer region. Impaired insulin secretion in hypoxic ß-cells was recovered by MAFA re-expression. Collectively, our work identifies BHLHE40 as a key hypoxia-induced transcriptional repressor in ß-cells that inhibit insulin secretion by suppressing MAFA expression.

CsMYBL2 homologs modulate the light and temperature stress-regulated anthocyanin and catechins biosynthesis in tea plants (Camellia sinensis).

Anthocyanin and catechin production in tea (Camellia sinensis) leaves can positively affect tea quality; however, their regulatory mechanisms are not fully understood. Here we report that, while the CsMYB75- or CsMYB86-directed MYB-bHLH-WD40 (MBW) complexes differentially activate anthocyanin or catechin biosynthesis in tea leaves, respectively, CsMYBL2a and CsMYBL2b homologs negatively modified the light- and temperature-induced anthocyanin and catechin production in both Arabidopsis and tea plants. The MBW complexes activated both anthocyanin synthesis genes and the downstream repressor genes CsMYBL2a and CsMYBL2b. Overexpression of CsMYBL2b, but not CsMYBL2a, repressed Arabidopsis leaf anthocyanin accumulation and seed coat proanthocyanin production. CsMYBL2b strongly and CsMYBL2a weakly repressed the activating effects of CsMYB75/CsMYB86 on CsDFR and CsANS, due to their different EAR and TLLLFR domains and interactions with CsTT8/CsGL3, interfering with the functions of activating MBW complexes. CsMYBL2b and CsMYBL2a in tea leaves play different roles in fine-tuning CsMYB75/CsMYB86-MBW activation of biosynthesis of anthocyanins and catechins, respectively. The CsbZIP1-CsmiR858a-CsMYBL2 module mediated the UV-B- or cold-activated CsMYB75/CsMYB86 regulation of anthocyanin/catechin biosynthesis by repressing CsMYBL2a and CsMYBL2b. Similarly, the CsCOP1-CsbZIP1-CsPIF3 module, and BR signaling as well, mediated the high temperature repression of anthocyanin and catechin biosynthesis through differentially upregulating CsMYBL2b and CsMYBL2a, respectively. The present study provides new insights into the complex regulatory networks in environmental stress-modified flavonoid production in tea plant leaves.

Tartrolon sensing and detoxification by the Listeria monocytogenes timABR resistance operon.

Listeria monocytogenes is a foodborne bacterium that naturally occurs in the soil. Originating from there, it contaminates crops and infects farm animals and their consumption by humans may lead to listeriosis, a systemic life-threatening infectious disease. The adaptation of L. monocytogenes to such contrastive habitats is reflected by the presence of virulence genes for host infection and other genes for survival under environmental conditions. Among the latter are ABC transporters for excretion of antibiotics produced by environmental competitors; however, most of these transporters have not been characterized. Here, we generated a collection of promoter-lacZ fusions for genes encoding ABC-type drug transporters of L. monocytogenes and screened this reporter strain collection for induction using a library of natural compounds produced by various environmental microorganisms. We found that the timABR locus (lmo1964-lmo1962) was induced by the macrodiolide antibiotic tartrolon B, which is synthesized by the soil myxobacterium Sorangium cellulosum. Tartrolon B resistance of L. monocytogenes was dependent on timAB, encoding the ATPase and the permease component of a novel ABC transporter. Moreover, transplantation of timAB was sufficient to confer tartrolon B resistance to Bacillus subtilis. Expression of the timABR locus was found to be auto-repressed by the TimR repressor, whose repressing activity was lost in the presence of tartrolon B. We also demonstrate that tartrolon sensitivity was suppressed by high external potassium concentrations, suggesting that tartrolon acts as potassium ionophore. Our results help to map the ecological interactions of an important human pathogen with its co-residing species within their joint natural reservoir.

Structural insights into BirA from Haemophilus influenzae, a bifunctional protein as a biotin protein ligase and a transcriptional repressor.

Biotin is an essential coenzyme involved in various metabolic processes across all known organisms, with biotinylation being crucial for the activity of carboxylases. BirA from Haemophilus influenzae is a bifunctional protein that acts as a biotin protein ligase and a transcriptional repressor. This study reveals the crystal structures of Hin BirA in both its apo- and holo-(biotinyl-5'-AMP bound) forms. As a class II BirA, it consists of three domains: N-terminal DNA binding domain, central catalytic domain, and C-terminal SH3-like domain. The structural analysis shows that the biotin-binding loop forms an ordered structure upon biotinyl-5'-AMP binding. This facilitates its interaction with the ligand and promotes protein dimerization. Comparative studies with other BirA homologs from different organisms indicate that the residues responsible for binding biotinyl-5'-AMP are highly conserved. This study also utilized AlphaFold2 to model the potential heterodimeric interaction between Hin BirA and biotin carboxyl carrier protein, thereby providing insights into the structural basis for biotinylation. These findings enhance our understanding of the structural and functional characteristics of Hin BirA, highlighting its potential as a target for novel antibiotics that disrupt the bacterial biotin synthesis pathways.

Domain analysis of Drosophila Blimp-1 reveals the importance of its repression function and instability in determining pupation timing.

The PRDM family transcription repressor Blimp-1 is present in almost all multicellular organisms and plays important roles in various developmental processes. This factor has several conserved motifs among different species, but the function of each motif is unclear. Drosophila Blimp-1 plays an important role in determining pupation timing by acting as an unstable transcriptional repressor of the ßftz-f1 gene. Thus, Drosophila provides a good system for analyzing the molecular and biological functions of each region in Blimp-1. Various Blimp-1 mutants carrying deletions at the conserved motifs were induced under the control of the heat shock promoter in prepupae, and the expression patterns of ßFTZ-F1 and Blimp-1 and pupation timing were observed. The results showed that the regions with strong and weak repressor functions exist within the proline-rich middle section of the factor and near the N-terminal conserved motif, respectively. Rapid degradation was supported by multiple regions that were mainly located in a large proline-rich region. Results revealed that pupation timing was affected by the repression ability and stability of Blimp-1. This suggests that both the repression function and instability of Blimp-1 are indispensable for the precise determination of pupation timing.

Architecting a transcriptional repressor-based genetic inverter for tryptophan derived pathway regulation in Escherichia coli.

Efficient microbial cell factories require intricate and precise metabolic regulations for optimized production, which can be significantly aided by implementing regulatory genetic circuits with versatile functions. However, constructing functionally diverse genetic circuits in host strains is challenging. Especially, functional diversification based on transcriptional repressors has been rarely explored due to the difficulty in inverting their repression properties. To address this, we proposed a design logic to create transcriptional repressor-based genetic inverters for functional enrichment. As proof of concept, a tryptophan-inducible genetic inverter was constructed by integrating two sets of transcriptional repressors, PtrpO1-TrpR1 and PtetO1-TetR. In this genetic inverter, the repression of TetR towards PtetO1 could be alleviated by the tryptophan-TrpR1 complex in the presence of tryptophan, leading to the activated output. Subsequently, we optimized the dynamic performance of the inverter and constructed tryptophan-triggered dynamic activation systems. Further coupling of the original repression function of PtrpO1-TrpR1 with inverter variants realized the tryptophan-triggered bifunctional regulation system. Finally, the dynamic regulation systems enabled tryptophan production monitoring. These systems also remarkably increased the titers of the tryptophan derivatives tryptamine and violacein by 2.0-fold and 7.4-fold, respectively. The successful design and application of the genetic inverter enhanced the applicability of transcriptional repressors.

A peptide encoded by pri-miRNA-31 represses autoimmunity by promoting Treg differentiation.

Recent evidence has revealed that small polypeptides (containing fewer than 100 amino acids) can be translated from noncoding RNAs (ncRNAs), which are usually defined as RNA molecules that do not encode proteins. However, studies on functional products translated from primary transcripts of microRNA (pri-miRNA) are quite limited. Here, we describe a peptide termed miPEP31 that is encoded by pri-miRNA-31. miPEP31 is highly expressed in Foxp3+ regulatory T cells (Tregs ) and significantly promotes the differentiation of Tregs without affecting their inhibitory ability. Our results show that miPEP31 is a cell-penetrating peptide both in vitro and in vivo. miPEP31 downregulates miR-31 expression, enhances peripheral Treg induction, and dramatically suppresses experimental autoimmune encephalomyelitis. Mechanistically, we show that miPEP31 acts as a transcriptional repressor inhibiting the expression of miRNA-31, a negative regulator of Tregs . Our results reveal an indispensable role of miPEP31 in maintaining immune homeostasis by promoting Treg differentiation and also present a potential therapeutic peptide for modulating miRNA expression and treating autoimmune diseases.

Characterization of the ZCTs, a subgroup of Cys2-His2 zinc finger transcription factors regulating alkaloid biosynthesis in Catharanthus roseus.

KEY MESSAGE: The C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the terpenoid indole alkaloid pathway when highly expressed. Catharanthus roseus is the sole known producer of the anti-cancer terpenoid indole alkaloids (TIAs), vinblastine and vincristine. While the enzymatic steps of the pathway have been elucidated, an understanding of its regulation is still emerging. The present study characterizes an important subgroup of Cys2-His2 zinc finger transcription factors known as Zinc finger Catharanthus Transcription factors (ZCTs). We identified three new ZCT members (named ZCT4, ZCT5, and ZCT6) that clustered with the putative repressors of the TIA pathway, ZCT1, ZCT2, and ZCT3. We characterized the role of these six ZCTs as potential redundant regulators of the TIA pathway, and their tissue-specific and jasmonate-responsive expression. These ZCTs share high sequence conservation in their two Cys2-His2 zinc finger domains but differ in the spacer length and sequence between these zinc fingers. The transient overexpression of ZCTs in seedlings significantly repressed the promoters of the terpenoid (pLAMT) and condensation branch (pSTR1) of the TIA pathway, consistent with that previously reported for ZCT1, ZCT2, and ZCT3. In addition, ZCTs significantly repressed and indirectly activated several promoters of the vindoline pathway (not previously studied). The ZCTs differed in their tissue-specific expression but similarly increased with jasmonate in a dosage-dependent manner (except for ZCT5). We showed significant activation of the pZCT1 and pZCT3 promoters by the de-repressed CrMYC2a, suggesting that the jasmonate-responsive expression of the ZCTs can be mediated by CrMYC2a. In summary, the C. roseus ZCTs are jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the TIA pathway when highly expressed.

Bach1 derepression is neuroprotective in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by the loss of nigrostriatal dopaminergic neurons. Mounting evidence suggests that Nrf2 is a promising target for neuroprotective interventions in PD. However, electrophilic chemical properties of the canonical Nrf2-based drugs cause irreversible alkylation of cysteine residues on cellular proteins resulting in side effects. Bach1 is a known transcriptional repressor of the Nrf2 pathway. We report that Bach1 levels are up-regulated in PD postmortem brains and preclinical models. Bach1 knockout (KO) mice were protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity and associated oxidative damage and neuroinflammation. Functional genomic analysis demonstrated that the neuroprotective effects in Bach1 KO mice was due to up-regulation of Bach1-targeted pathways that are associated with both Nrf2-dependent antioxidant response element (ARE) and Nrf2-independent non-ARE genes. Using a proprietary translational technology platform, a drug library screen identified a substituted benzimidazole as a Bach1 inhibitor that was validated as a nonelectrophile. Oral administration of the Bach1 inhibitor attenuated MPTP neurotoxicity in pre- and posttreatment paradigms. Bach1 inhibitor-induced neuroprotection was associated with the up-regulation of Bach1-targeted pathways in concurrence with the results from Bach1 KO mice. Our results suggest that genetic deletion as well as pharmacologic inhibition of Bach1 by a nonelectrophilic inhibitor is a promising therapeutic approach for PD.

A class of independently evolved transcriptional repressors in plant RNA viruses facilitates viral infection and vector feeding.

Plant viruses employ diverse virulence strategies to achieve successful infection, but there are few known general strategies of viral pathogenicity and transmission used by widely different plant viruses. Here, we report a class of independently evolved virulence factors in different plant RNA viruses which possess active transcriptional repressor activity. Rice viruses in the genera Fijivirus, Tenuivirus, and Cytorhabdovirus all have transcriptional repressors that interact in plants with the key components of jasmonic acid (JA) signaling, namely mediator subunit OsMED25, OsJAZ proteins, and OsMYC transcription factors. These transcriptional repressors can directly disassociate the OsMED25-OsMYC complex, inhibit the transcriptional activation of OsMYC, and then combine with OsJAZ proteins to cooperatively attenuate the JA pathway in a way that benefits viral infection. At the same time, these transcriptional repressors efficiently enhanced feeding by the virus insect vectors by repressing JA signaling. Our findings reveal a common strategy in unrelated plant viruses in which viral transcriptional repressors hijack and repress the JA pathway in favor of both viral pathogenicity and vector transmission.

Roles of ß-Cell Hypoxia in the Progression of Type 2 Diabetes.

Type 2 diabetes is a chronic disease marked by hyperglycemia; impaired insulin secretion by pancreatic ß-cells is a hallmark of this disease. Recent studies have shown that hypoxia occurs in the ß-cells of patients with type 2 diabetes and hypoxia, in turn, contributes to the insulin secretion defect and ß-cell loss through various mechanisms, including the activation of hypoxia-inducible factors, induction of transcriptional repressors, and activation of AMP-activated protein kinase. This review focuses on advances in our understanding of the contribution of ß-cell hypoxia to the development of ß-cell dysfunction in type 2 diabetes. A better understanding of ß-cell hypoxia might be useful in the development of new strategies for treating type 2 diabetes.

SifR is an Rrf2-family quinone sensor associated with catechol iron uptake in Streptococcus pneumoniae D39.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive commensal and human respiratory pathogen. How this bacterium satisfies its nutritional iron (Fe) requirement in the context of endogenously produced hydrogen peroxide is not well understood. Here, we characterize a novel virulence-associated Rrf2-family transcriptional repressor that we term SifR (streptococcal IscR-like family transcriptional repressor) encoded by spd_1448 and conserved in Streptococci. Global transcriptomic analysis of a ΔsifR strain defines the SifR regulon as genes encoding a candidate catechol dioxygenase CatE, an uncharacterized oxidoreductase YwnB, a candidate flavin-dependent ferric reductase YhdA, a candidate heme-based ferric reductase domain-containing protein and the Piu (pneumococcus iron uptake) Fe transporter (piuBCDA). Previous work established that membrane-anchored PiuA binds FeIII-bis-catechol or monocatechol complexes with high affinity, including the human catecholamine stress hormone, norepinephrine. We demonstrate that SifR senses quinone via a single conserved cysteine that represses its regulon when in the reduced form. Upon reaction with catechol-derived quinones, we show that SifR dissociates from the DNA leading to regulon derepression, allowing the pneumococcus to access a catechol-derived source of Fe while minimizing reactive electrophile stress induced by quinones. Consistent with this model, we show that CatE is an FeII-dependent 2,3-catechol dioxygenase with broad substrate specificity, YwnB is an NAD(P)H-dependent quinone reductase capable of reducing the oxidized and cyclized norepinephrine, adrenochrome, and YhdA is capable of reducing a number of FeIII complexes, including PiuA-binding transport substrates. These findings are consistent with a model where FeIII-catechol complexes serve as significant nutritional Fe sources in the host.

Unmasking the biological function and regulatory mechanism of NOC2L: a novel inhibitor of histone acetyltransferase.

NOC2 like nucleolar associated transcriptional repressor (NOC2L) was recently identified as a novel inhibitor of histone acetyltransferase (INHAT). NOC2L is found to have two INHAT function domains and regulates histone acetylation in a histone deacetylases (HDAC) independent manner, which is distinct from other INHATs. In this review, we summarize the biological function of NOC2L in histone acetylation regulation, P53-mediated transcription, ribosome RNA processing, certain development events and carcinogenesis. We propose that NOC2L may be explored as a potential biomarker and a therapeutic target in clinical practice.

Tree peony PsMYB44 negatively regulates petal blotch distribution by inhibiting dihydroflavonol-4-reductase gene expression.

BACKGROUND AND AIMS: The tree peony (Paeonia suffruticosa Andr.) has been widely cultivated as a field plant, and petal blotch is one of its important traits, which not only promotes proliferation but also confers high ornamental value. However, the regulatory network controlling blotch formation remains elusive owing to the functional differences and limited conservation of transcriptional regulators in dicots. METHODS: We performed phylogenetic analysis to identify MYB44-like transcription factors in P. suffruticosa blotched cultivar 'High noon' petals. A candidate MYB44-like transcription factor, PsMYB44, was analysed via expression pattern analysis, subcellular localization, target gene identification, gene silencing in P. suffruticosa petals and heterologous overexpression in tobacco. KEY RESULTS: A blotch formation-related MYB44-like transcription factor, PsMYB44, was cloned. The C-terminal of the PsMYB44 amino acid sequence had a complete C2 motif that affects anthocyanin biosynthesis, and PsMYB44 was clustered in the MYB44-like transcriptional repressor branch. PsMYB44 was located in the nucleus, and its spatial and temporal expression patterns were negatively correlated with blotch formation. Furthermore, a yeast one-hybrid assay showed that PsMYB44 could target the promoter of the late anthocyanin biosynthesis-related dihydroflavonol-4-reductase (DFR) gene, and a dual-luciferase assay demonstrated that PsMYB44 could repress PsDFR promoter activity. On the one hand, overexpression of PsMYB44 significantly faded the red colour of tobacco flowers and decreased the anthocyanin content by 42.3 % by downregulating the expression level of the tobacco NtDFR gene. On the other hand, PsMYB44-silenced P. suffruticosa petals had a redder blotch colour, which was attributed to the fact that silencing PsMYB44 redirected metabolic flux to the anthocyanin biosynthesis branch, thereby promoting more anthocyanin accumulation in the petal base. CONCLUSION: These results demonstrated that PsMYB44 negatively regulated the biosynthesis of anthocyanin by directly binding to the PsDFR promoter and subsequently inhibiting blotch formation, which helped to elucidate the molecular regulatory network of anthocyanin-mediated blotch formation in plants.

Integrative multi-omic analysis reveals neurodevelopmental gene dysregulation in CIC-knockout and IDH1-mutant cells.

Capicua (CIC)'s transcriptional repressor function is implicated in neurodevelopment and in oligodendroglioma (ODG) aetiology. However, CIC's role in these contexts remains obscure, primarily from our currently limited knowledge regarding its biological functions. Moreover, CIC mutations in ODG invariably co-occur with a neomorphic IDH1/2 mutation, yet the functional relationship between these two genetic events is unknown. Here, we analysed models derived from an E6/E7/hTERT-immortalized (i.e. p53- and RB-deficient) normal human astrocyte cell line. To examine the consequences of CIC loss, we compared transcriptomic and epigenomic profiles between CIC wild-type and knockout cell lines, with and without mutant IDH1 expression. Our analyses revealed dysregulation of neurodevelopmental genes in association with CIC loss. CIC ChIP-seq was also performed to expand upon the currently limited ensemble of known CIC target genes. Among the newly identified direct CIC target genes were EPHA2 and ID1, whose functions are linked to neurodevelopment and the tumourigenicity of in vivo glioma tumour models. NFIA, a known mediator of gliogenesis, was discovered to be uniquely overexpressed in CIC-knockout cells expressing mutant IDH1-R132H protein. These results identify neurodevelopment and specific genes within this context as candidate targets through which CIC alterations may contribute to the progression of IDH-mutant gliomas. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

dCas9 techniques for transcriptional repression in mammalian cells: Progress, applications and challenges.

Innovative loss-of-function techniques developed in recent years have made it much easier to target specific genomic loci at transcriptional levels. CRISPR interference (CRISPRi) has been proven to be the most effective and specific tool to knock down any gene of interest in mammalian cells. The catalytically deactivated Cas9 (dCas9) can be fused with transcription repressors to downregulate gene expression specified by sgRNA complementary to target genomic sequence. Although CRISPRi has huge potential for gene knockdown, there is still a lack of systematic guidelines for efficient and widespread use. Here we describe the working mechanism and development of CRISPRi, designing principles of sgRNA, delivery methods and applications in mammalian cells in detail. Finally, we propose possible solutions and future directions with regard to current challenges.