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.

A Phosphorylated Histone H2A Variant Displays Properties of Chromatin Insulator Proteins in Drosophila

Chromatin insulators are responsible for mediating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of topologically associating domains (TADs). Here, we demonstrate an interaction between proteins that associate with the gypsy insulator and the phosphorylated histone variant H2Av (γH2Av), a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome. Mutation of insulator components prevents stable H2Av phosphorylation in polytene chromatin. Phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av is a component of insulator bodies, and that phosphatase activity is required for insulator body dissolution after recovery from osmotic stress. We further demonstrate a tight association between γH2Av and TAD boundaries. Together, our results indicate a novel mechanism linking insulator function with a histone H2A variant and with genome stability.

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.

The cHS4 Chromatin Insulator Reduces the Rate of Retroviral Vector-Mediated Gene Dysregulation Associated with Aberrant Vector Transcription

Integrating gammaretroviral vectors can dysregulate the expression of cellular genes through a variety of mechanisms, leading to genotoxicity and malignant transformation. Although most attention has focused on the activation of cellular genes by vector enhancers, aberrant fusion transcripts involving cellular gene sequences and vector promoters, vector splice elements, and vector transcription termination sequences have also been mechanistically associated with dysregulated expression of cellular genes. Chromatin insulators have emerged as an effective tool for reducing the frequency of vector-mediated genotoxicity and malignant transformation and have been shown to block the activation of cellular genes by vector enhancers. We report here evidence that flanking a gammaretroviral reporter vector with the cHS4 chromatin insulator also reduces the frequency of vector-mediated cellular gene dysregulation associated with aberrant vector transcripts, including vector transcription run-through and aberrant splicing. We demonstrate that the cHS4 element does not function to terminate transcription directly, implicating other mechanisms for this activity.

The Sea Urchin sns5 Chromatin Insulator Improves the Likelihood of Lentiviral Vectors in Erythroid Milieu By Organizing an Independent Chromatin Domain at the Integration Site

Retroviral vectors are currently the most suitable vehicles for therapeutic gene transfer in hematopoietic stem cells. However, these vectors are known to integrate rather randomly throughout the genome, suffering the so called chromosomal position effects (PE). Such a critical occurrence most probably depends upon the ability of heterochromatin to spread in the inserted vector sequences. Moreover, the use of transgenes imply genotoxicity effects, since the cis-regulatory sequences harbored by the vector can disturb the proper transcription of the resident genes neighboring the integration site, potentially leading to malignant transformation. Due to their enhancer blocker activity, the incorporation of chromatin insulators in flanking position to the transferred unit can reduce the mentioned dangerous effects. Moreover, by acting as barriers to the spread of heterochromatin, chromatin insulators can also mitigate vector silencing. We have previously shown that the sea urchin sns5 chromatin insulator activity is conserved in mouse and human erythroid milieu: it blocks the βglobin-LCR-HS2 enhancer/globin promoter interaction when placed between them. In addition, when placed in flanking location of a γ-retrovirus vector, sns5 impedes PE variegation and improves vector-specific expression following integration in the erythroid genome. Importantly, by binding both erythroid-specific and ubiquitous factors, sns5 favors the accumulation inside the provirus locus of epigenetic marks commonly associated to an euchromatic state (Acuto S. et al., BCMD 2005; D'Apolito D. et al., 2009; Di Caro D. et al., J Mol Biol 2004; Cavalieri V. et al., NAR 2009). In this study we extend these findings, demonstrating that sns5 works as chromatin insulator also when placed in flanking position of a GFP transgene contained in a lentivirus vector (LV-GFP). A large panel of mouse erythroleukemic clones (MELC) was generated after transduction with uninsulated and sns5 -insulated LV-GFP. Individual clones were screened for single vector integrants (by Q-PCR), and for GFP-expression (by cytofluorimetry). Our results shown that the inclusion of the sns5 element in a forward orientation increased the fraction of vector expressing cells (89% for the insulated vector vs 42% for the uninsulated ones). The clonal variegation of expression, assessed as frequency of clones that showed a percentage of GFP-negative cells in the progeny, decreased in clones transduced with the insulated vectors (7.4% vs 13,9%). It has been suggested that chromatin insulators could shape the architecture of topologically independent chromosome domains. High resolution mapping of chromosomal domains in drosophila and higher eukaryotes highlighted that chromatin insulators play a critical role in shaping the architectural genome organization both in a local chromosome environment and in long range chromosomal interaction. Intriguingly, by using the Chromosome Conformation Capture (3C) technology, we demonstrated that the sns5 -flanked LV-GFP integrated at a single copy in the erythroid cell genome is organized into an independent chromatin loop at the integration site. Worth to mention, no looping was detected in the absence of sns5, indicating that the two flanking copies of sns5 are specifically involved in the reorganization of the chromatin structure at the provirus locus. In conclusion our results not only confirm the conserved and striking boundary function of sns5, but also provide a new clue concerning the molecular mechanism that allows this function to occur. On these basis, our findings reassure the use of sns5 to improve both efficacy and safety of lentiviral vectors for gene therapy. This work was funded by the Assessorato Regionale della Salute, Regione Siciliana (PO FESR 4.1.1.1 RIMEDRI) Disclosures No relevant conflicts of interest to declare.