M1BP cooperates with CP190 to activate transcription at TAD borders and promote chromatin insulator activity

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.

ECOA-7. Conserved cell-lineage controlled chromatin accessibility in human and mouse glioblastoma stem cells predicts functionally distinct subgroups

There is ample support for developmental control of glioblastoma stem cells (GSCs), and a deeper knowledge of their epigenetic regulation could be central to more efficient glioblastoma (GBM) therapies. For this purpose, we analyzed the chromatin-accessibility landscape of nine mouse GSC cultures of defined cell of origin and 60 patient-derived GSC cultures by assay for transposase-accessible chromatin using sequencing (ATAC-seq). This uncovered an epigenetic variability of both mouse and human GSC cultures that differed from transcriptome clusters. Both mouse and human chromatin accessibility-guided clusters were predominantly determined by distal regulatory elements, displayed unique sets of transcription factor motif enrichment, and exhibited different functional and drug-response properties. Cross-species analysis of distal regulatory element regions in accessible chromatin of mouse and human cultures revealed conserved epigenetic regulation of GSCs.

Rational gRNA Design Based on Transcription Factor Binding Data

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has become a standard tool in many genome engineering endeavors. The endonuclease-deficient version of Cas9 (dCas9) is also a powerful programmable tool for gene regulation. In this study, we made use of Saccharomyces cerevisiae transcription factor binding data to obtain a better understanding of the interplay between transcription factor binding and binding of dCas9 fused to an activator domain, VPR. More specifically, we targeted dCas9-VPR towards binding sites of Gcr1-Gcr2 and Tye7 present in several promoters of genes encoding enzymes engaged in the central carbon metabolism. From our data, we observed an upregulation of gene expression when dCas9-VPR was targeted next to a transcription factor binding motif, whereas downregulation or no change was observed when dCas9 was bound on a transcription factor motif. This suggests a steric competition between dCas9 and the specific transcription factor. Integrating transcription factor binding data, therefore, proved to be useful for designing gRNAs for CRISPRi/a applications.

spatzie: An R package for identifying significant transcription factor motif co-enrichment from enhancer-promoter interactions

Genomic interactions provide important context to our understanding of the state of the genome. One question is whether specific transcription factor interactions give rise to genome organization. We introduce spatzie, an R package and a website that implements statistical tests for significant transcription factor motif cooperativity between enhancer-promoter interactions. We conducted controlled experiments under realistic simulated data from ChIP-seq to confirm spatzie is capable of discovering co-enriched motif interactions even in noisy conditions. We then use spatzie to investigate cell type specific transcription factor cooperativity within recent human ChIA-PET enhancer-promoter interaction data. The method is available online at https://spatzie.mit.edu.

Integrating transcription factor abundance with chromatin accessibility in human erythroid lineage commitment

We present InTAC-seq, a method for simultaneous quantification of genome-wide open chromatin and intracellular protein abundance in fixed cells. Using InTAC we directly observe variation in chromatin accessibility and transcription factor motif occupancy driven by differences in transcription factor protein abundance. By purifying bone marrow progenitor cells based on GATA1 protein expression, we establish its role in both functional and epigenetic restriction of erythroid cell identity in human hematopoiesis.

C11orf95-RELA fusion drives aberrant gene expression through the unique epigenetic regulation for ependymoma formation

Recurrent C11orf95-RELA fusions (RELAFUS) are the hallmark of supratentorial ependymomas. The presence of RELA as the fusion partner indicates a close association of aberrant NF-κB activity with tumorigenesis. However, the oncogenic role of the C11orf95 has not been determined. Here, we performed ChIP-seq analyses to explore genomic regions bound by RELAFUS and H3K27ac proteins in human 293T and mouse ependymoma cells. We then utilized published RNA-Seq data from human and mouse RELAFUS tumors and identified target genes that were directly regulated by RELAFUS in these tumors. Subsequent transcription factor motif analyses of RELAFUS target genes detected a unique GC-rich motif recognized by the C11orf95 moiety, that is present in approximately half of RELAFUS target genes. Luciferase assays confirmed that a promoter carrying this motif is sufficient to drive RELAFUS-dependent gene expression. Further, the RELAFUS target genes were found to be overlapped with Rela target genes primarily via non-canonical NF-κB binding sites. Using a series of truncation and substitution mutants of RELAFUS, we also show that the activation domain in the RELAFUS moiety is necessary for the regulation of gene expression of these RELAFUS target genes. Lastly, we performed an anti-cancer drug screening with mouse ependymoma cells and identified potential anti-ependymoma drugs that are related to the oncogenic mechanism of RELAFUS. These findings suggested that RELAFUS might induce ependymoma formation through oncogenic pathways orchestrated by both C11orf95 and RELA target genes. Thus, our study unveils a complex gene function of RELAFUS as an oncogenic transcription factor in RELAFUS positive ependymomas.

“High resolution discovery of regulatory DNA with synthetic wild-type and ablated genome constructs”

ABSTRACTGenomic enhancer elements play a role in modulating gene expression by interacting with effector proteins known as transcription factors. We present a new computational method, “STARR-scan”, which identifies transcription factor motif appearances in synthetic DNA constructs that are correlated with enhancer activity. Using a tiled STARR-seq assay, we demonstrate that STARR-scan can suggest the biological function of transcription factors previously suspected to activate the APOBEC-3B gene. Using the same methodology, STARR-scan can suggest transcription factors with previously unknown biological activity which may regulate the APOBEC-3B gene. These novel factors may have biological significance in our understanding of cancer biology.

M1BP cooperates with CP190 to activate transcription at TAD borders and promote chromatin insulator activity

ABSTRACTGenome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP is required for CP190 chromatin binding at many shared sites, and CP190 also affects M1BP chromatin association. Both factors are required for Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP alters local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.