Transcription factors remain essential yet intractable targets for drug discovery owing to their flat, dynamic interfaces. We present an intracellular cyclization strategy that enables in-cell generation of conformationally constrained peptide libraries. Bis-alkylating reagents traverse bacterial membranes and selectively bridge cysteine pairs, permitting post-translational peptide stapling during in vivo screening. Integrated with the transcription block survival (TBS) assay, this intracellular-cyclization TBS (icTBS) platform simultaneously selects both peptide sequence and optimal constraint site, eliminating iterative synthesis. Libraries directed against the oncogenic transcription factor CREB1 yielded three nanomolar-affinity antagonists, with cyclized variants selected by icTBS displaying enhanced functional activity in cellular assays. The lead peptide penetrated melanoma and colorectal cancer cells, suppressed CREB1-dependent transcription, reduced oncogenic protein expression, and triggered apoptosis. icTBS thus provides a general, genetically encoded route to discover constrained peptide therapeutics that disrupt protein-DNA interfaces previously considered "undruggable."

The development of an intracellular peptide library screening platform is described to identify covalent transcription factor (TF) antagonists. The Transcription Block Survival (TBS) assay and subsequent hit refinement previously produced potent but reversible antagonists of the oncogenic TF cJun. TBS moves beyond a target binding readout to ensure loss of TF function by blocking TF-DNA binding. Here, the TBS methodology is significantly expanded to identify covalent and highly selective inhibitors. A 131,072-member library is probed containing a Cys option at nine positions within a non-reducing cell line. This identified a single Cys residue with the appropriate geometry for disulphide bond formation with cJun C269 in its DNA binding domain. The selection of a unique Cys in the antagonist indicates both target shutdown and concomitant disulphide formation in a single step, resulting in increased potency. Substituting Cys with an electrophile generates an irreversible yet highly selective covalent cJun inhibitor capable of penetrating human melanoma cells in culture and depleting oncogenic cJun levels to inhibit cell viability, with enhanced efficacy compared to a previous cJun-targeting peptide. This enhanced covalent-TBS screening pipeline provides a robust approach to profile target protein surfaces for ligandable cysteines, producing covalent and selective antagonists with appropriately positioned warheads.

Neural stem cells (NSCs) in the subventricular zone (SVZ) produce neurons throughout life. However, the epigenetic mechanisms that maintain NSCs and control neurogenesis remain unclear. We previously showed the long non-coding RNA (lncRNA) Paupar and KAP1 transcription co-factor control neuroblastoma cell growth. In the last stages of olfactory bulb (OB) neurogenesis, these two molecules affect rates of neurogenesis. Here we investigated upstream functions with inducible knockout and in vivo knockdown electroporation and molecular analysis. KAP1 and Paupar are expressed in the SVZ, physically interact in SVZ cells, and regulate NSC behavior. KAP1 directly maintains the NSC pool and regulates the expression of genes involved in the cell cycle. Paupar, in turn, maintains stem cell quiescence by restricting lineage progression from stem cells to transit amplifying progenitors (TAPs). These results suggest important roles for KAP1 and Paupar in the control of SVZ NSC maintenance and SVZ-OB neurogenesis.

Long non-coding RNAs (lncRNAs) play crucial roles in regulating gene expression. Some are essential for organismal development and physiology, and they can contribute to diseases including cancer. Whilst most lncRNAs exhibit little sequence similarity, conservation of lncRNA transcription relative to neighbouring protein-coding genes suggests potential functional significance. Most positionally equivalent lncRNAs are uncharacterized and it remains unclear whether they exert similar roles in distant species. Here, we identified melanoma-associated lncRNAs predicted to be components of the MITF gene regulatory network in human melanoma that have positionally equivalent transcripts in zebrafish. We prioritized the cancer-associated lncRNA Differentiation Antagonizing Non-Protein Coding RNA (DANCR) as an exemplar for functional investigation. DANCR is a multi-exonic, cytoplasmically-enriched lncRNA and small RNA host gene transcribed from syntenic regions in the human and zebrafish genomes. MITF and c-MYC, key melanoma transcription factors, regulate human DANCR expression and melanoma patients with high DANCR display significantly decreased survival. DANCR is a melanoma oncogene that controls cancer-associated gene expression networks to promote human melanoma cell proliferation and migration. Zebrafish dancr is essential for embryonic development. It is dynamically expressed across multiple different cell types in the developing embryo, transcriptionally activated by mitfa during early zebrafish development and it regulates genes involved in cell death. Our work suggests that cancer-critical lncRNAs such as DANCR, expressed from similar regions in vertebrate genomes, may control related genes and processes involved in both embryonic development and tumorigenesis across species.

Long non-coding RNAs (lncRNAs) can regulate gene expression. Some are essential for organismal development and physiology and can contribute to diseases including cancer. Whilst most lncRNAs exhibit little sequence similarity, conservation of lncRNA transcription relative to neighbouring protein-coding genes suggests potential functional significance. Most positionally equivalent lncRNAs are uncharacterized and it remains unclear whether they exert similar roles in distant species. Here, we identified syntenic melanoma-associated lncRNAs predicted to be components of the MITF gene regulatory network in human melanoma, with positionally equivalent transcripts in zebrafish. We prioritized Differentiation Antagonizing Non-Protein Coding RNA (DANCR), a cancer-associated lncRNA critical for maintaining somatic progenitor cells in human models, for functional investigation. DANCR is a multi-exonic, cytoplasmically-enriched lncRNA transcribed from syntenic regions in the human and zebrafish genomes. MITF and c-MYC, key melanoma transcription factors, regulate human DANCR expression and melanoma patients with high DANCR display significantly decreased survival. DANCR is a melanoma oncogene that controls cancer-associated gene expression networks and promotes human melanoma cell proliferation and migration. Zebrafish dancr is dynamically expressed across multiple different cell types in the developing embryo, regulates genes involved in cell death, and is essential for embryonic development. Our work suggests that cancer-critical lncRNAs such as DANCR, expressed from similar regions in vertebrate genomes, may regulate related genes and processes involved in both embryonic development and tumorigenesis across species. ### Competing Interest Statement The authors have declared no competing interest.

Sequence of primers used in the study

Differential gene expression DESeq2 analysis for siEZH2 vs siNC.

Long non-coding RNA (lncRNA) function is mediated by the process of transcription or through transcript-dependent associations with proteins or nucleic acids to control gene regulatory networks. Many lncRNAs are transcribed in the ventricular-subventricular zone (V-SVZ), a postnatal neural stem cell niche. lncRNAs in the V-SVZ are implicated in neurodevelopmental disorders, cancer, and brain disease, but their functions are poorly understood. V-SVZ neurogenesis capacity declines with age due to stem cell depletion and resistance to neural stem cell activation. Here we analyzed V-SVZ transcriptomics by pooling current single-cell RNA-seq data. They showed consistent lncRNA expression during stem cell activation, lineage progression, and aging. In conjunction with epigenetic and genetic data, we predicted V-SVZ lncRNAs that regulate stem cell activation and differentiation. Some of the lncRNAs validate known epigenetic mechanisms, but most remain uninvestigated. Our analysis points to several lncRNAs that likely participate in key aspects of V-SVZ stem cell activation and neurogenesis in health and disease.

Differential gene expression DESeq2 analysis for siSCIRT vs siNC using two independent siRNAs against SCIRT (Sheet #1 contains siSCIRT #1 data; Sheet #2 contains siSCIRT #2 data).

Table S2. Pathway enrichment analyses for genes up- and down-regulated after growing 16h or 5d in spheres

Significant marker genes of cluster 3 after scRNA-seq analysis of Breast Cancer primary samples

Supplementary material and methods

Top 100 gene significantly up-regulated upon siSCIRT treatment (Overlap between siSCIRT #1 and siSCIRT #2). Genes are ranked from the most up-regulated to the least up-regulated.

Significant CHART-seq SCIRT peaks identified using HOMER

Differential gene expression DESeq2 analysis for 16h Spheres vs Adh (Sheet #1) and 5d Spheres vs Adh

Central nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map the cis-regulatory interaction landscape of the key neuro-developmental Paupar-Pax6 lncRNA-mRNA locus. The results define chromatin architecture changes associated with high Paupar-Pax6 expression in neurons and identify both promoter selective as well as shared cis-regulatory-promoter interactions involved in regulating Paupar-Pax6 co-expression. We discovered that the TCF7L2 transcription factor, a regulator of chromatin architecture and major effector of the Wnt signalling pathway, binds to a subset of these candidate cis-regulatory elements to coordinate Paupar and Pax6 co-expression. We describe distinct roles for Paupar in Pax6 expression control and show that the Paupar DNA locus contains a TCF7L2 bound transcriptional silencer whilst the Paupar transcript can act as an activator of Pax6. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain.

Abstract In many tumors, cells transition reversibly between slow-proliferating tumor-initiating cells (TIC) and their differentiated, faster-growing progeny. Yet, how transcriptional regulation of cell-cycle and self-renewal genes is orchestrated during these conversions remains unclear. In this study, we show that as breast TIC form, a decrease in cell-cycle gene expression and increase in self-renewal gene expression are coregulated by SOX2 and EZH2, which colocalize at CpG islands. This pattern was negatively controlled by a novel long noncoding RNA (lncRNA) that we named Stem Cell Inhibitory RNA Transcript (SCIRT), which was markedly upregulated in tumorspheres but colocalized with and counteracted EZH2 and SOX2 during cell-cycle and self-renewal regulation to restrain tumorigenesis. SCIRT specifically interacted with EZH2 to increase EZH2 affinity to FOXM1 without binding the latter. In this manner, SCIRT induced transcription at cell-cycle gene promoters by recruiting FOXM1 through EZH2 to antagonize EZH2-mediated effects at target genes. Conversely, on stemness genes, FOXM1 was absent and SCIRT antagonized EZH2 and SOX2 activity, balancing toward repression. These data suggest that the interaction of an lncRNA with EZH2 can alter the affinity of EZH2 for its protein-binding partners to regulate cancer cell state transitions. Significance: These findings show that a novel lncRNA SCIRT counteracts breast tumorigenesis by opposing transcriptional networks associated with cell cycle and self-renewal. See related commentary by Pardini and Dragomir, p. 535

AbstractCentral nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map thecis-regulatory interaction landscape of the key neuro-developmentalPaupar-Pax6lncRNA-mRNA locus. The results defined chromatin architecture changes associated with highPaupar-Pax6expression in neurons and identified both promoter selective as well as sharedcis-regulatory interactions with thePauparandPax6promoters involved in regulatingPaupar-Pax6co-expression in neuronal cells. The TCF7L2 transcription factor, a major regulator of chromatin architecture and effector of the Wnt signalling pathway, binds to a subset of these candidatecis-regulatory elements to coordinatePauparandPax6co-expression. We identify a functional TCF7L2 boundcis-regulatory element within thePaupargene, suggesting that thePauparDNA locus itself regulatesPax6expression in addition to its previously described transcriptdependent modes of action. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain.

The MITF and SOX10 transcription factors regulate the expression of genes important for melanoma proliferation, invasion and metastasis. Despite growing evidence of the contribution of long noncoding RNAs (lncRNAs) in cancer, including melanoma, their functions within MITF-SOX10 transcriptional programmes remain poorly investigated. Here we identify 245 candidate melanoma associated lncRNAs whose loci are co-occupied by MITF-SOX10 and that are enriched at active enhancer-like regions. Our work suggests that one of these, Disrupted In Renal Carcinoma 3 (DIRC3), may be a clinically important MITF-SOX10 regulated tumour suppressor. DIRC3 depletion in human melanoma cells leads to increased anchorage-independent growth, a hallmark of malignant transformation, whilst melanoma patients classified by low DIRC3 expression have decreased survival. DIRC3 is a nuclear lncRNA that activates expression of its neighbouring IGFBP5 tumour suppressor through modulating chromatin structure and suppressing SOX10 binding to putative regulatory elements within the DIRC3 locus. In turn, DIRC3 dependent regulation of IGFBP5 impacts the expression of genes involved in cancer associated processes and is needed for DIRC3 control of anchorage-independent growth. Our work indicates that lncRNA components of MITF-SOX10 networks are an important new class of melanoma regulators and candidate therapeutic targets that can act not only as downstream mediators of MITF-SOX10 function but as feedback regulators of MITF-SOX10 activity.

Many long non‐coding RNAs (lncRNAs) are expressed during central nervous system (CNS) development, yet their in vivo roles and mechanisms of action remain poorly understood. Paupar, a CNS‐expressed lncRNA, controls neuroblastoma cell growth by binding and modulating the activity of transcriptional regulatory elements in a genome‐wide manner. We show here that the Paupar lncRNA directly binds KAP1, an essential epigenetic regulatory protein, and thereby regulates the expression of shared target genes important for proliferation and neuronal differentiation. Paupar promotes KAP1 chromatin occupancy and H3K9me3 deposition at a subset of distal targets, through the formation of a ribonucleoprotein complex containing Paupar, KAP1 and the PAX6 transcription factor. Paupar‐KAP1 genome‐wide co‐occupancy reveals a fourfold enrichment of overlap between Paupar and KAP1 bound sequences, the majority of which also appear to associate with PAX6. Furthermore, both Paupar and Kap1 loss‐of‐function in vivo disrupt olfactory bulb neurogenesis. These observations provide important conceptual insights into the trans‐acting modes of lncRNA‐mediated epigenetic regulation and the mechanisms of KAP1 genomic recruitment, and identify Paupar and Kap1 as regulators of neurogenesis in vivo.