Dataset Information

Topologically Associated Domains Delineate Susceptibility to Somatic Hypermutation

ABSTRACT: Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes of activated B cells to support the process of antibody affinity maturation but also causes "off-target" mutations in other parts of the genome. We have used sensitive lentiviral SHM reporter vectors and a mutationally active human B cell line to identify dozens of regions of the genome that are intrinsically susceptible to SHM ("hot" regions) and many hundreds of regions that are resistant to SHM ("cold" regions). Hot and cold regions are frequently contained within topologically associated domains (TADs). Comparison of hot and cold TADs reveals that while overall levels of transcription are equal, hot TADs are enriched for NIPBL (a component of the cohesin loader), super enhancers, markers of paused/stalled RNA polymerase 2, and multiple transcription factors implicated in B cell development and targeting of SHM. We demonstrate that at least some hot TADs contain enhancer elements that possess SHM targeting activity and that insertion of a strong Ig SHM-targeting element into a cold TAD renders it hot. Our findings lead to a model for SHM susceptibility involving the cooperative action of cis-acting SHM targeting elements and the dynamic and architectural properties of TADs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE139810 | GEO | 2019/12/04

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. Sequencing of the IgL V region and mutationally active GFP transgene in WT, Bcl6-/- Pax5R, and Bcl6-/- Pax5R Bcl6R chicken DT40 cells for evidence of AID dependent mutations. ChIP-seq proiles of RNA Pol II, Spt5, and pSer5 Pol II at a GFP transgene in WT, Bcl6-/- Pax5R, and Bcl6-/- Pax5R Bcl6R chicken DT40 cells.

Project description:Intratumoral heterogeneity (ITH)—defined as genetic and cellular diversity within a tumor—is linked to a failure of immunotherapy in multiple cancer types and to an inferior anti-tumor immune response even in the absence of therapy. To determine how tumor heterogeneity shapes the immune microenvironment and impacts responses to therapy, we modeled heterogeneous tumors comprised of a pro-inflammatory (“hot”) and an immunosuppressive (“cold”) tumor population. This was done by mixing two squamous skin carcinoma cell lines that alone give rise to tumors with a reproducible immunologically “hot” or “cold” immune phenotype respectively, and labeling these with YFP or RFP fluorescent tags to enable precise spatial tracking. The resulting mixed-population tumors were made up of a patchwork of distinct regions that could be classified as being comprised of YFP+(“hot”) cells, RFP+(“cold”) cells, or a mixture of YFP+and RFP+cells. We find the spatial organization of tumor cells creates a blueprint for the spatial organization and functional activity of infiltrating immune cells, defining the architecture of both tumor-infiltrating T cell and myeloid cell compartments. Using a combination of microdissection techniques and single-cell spatial transcriptomics, we show that YFP regions harbor a higher frequency of Th1 cells and IFNψ+CD8 T cells compared to RFP regions, whereas immunosuppressive macrophages preferentially accumulate in RFP regions. Total CD4 T cells were also enriched in YFP regions, but by contrast, total CD8 T cells—despite their superior function in YFP regions—were of low abundance throughout the heterogeneous tumors. We identify the chemokine Cx3cl1, produced at higher levels by our “cold” tumors, as a mediator of intratumoral macrophage abundance, and show that overexpression of Cx3cl1 in pro-inflammatory (“hot”) tumor cells leads to an increase in immunosuppressive CD206Himacrophages. We further interrogated the impact of this spatial patterning of immune cells on the responses of heterogeneous tumors to checkpoint blockade. We find that combination of PD-1 blockade and CD40 agonist is able to increase the Th1 response in “cold” RFP regions, however the T cell response in “cold” regions remains inferior to “hot” regions and treatment achieves only a modest reduction in tumor growth. Collectively, our results demonstrate that the spatial organization of heterogeneous tumor cells has a profound impact on directing the spatial organization and function of tumor-infiltrating immune cells as well as on responses to immunotherapy.

Dataset Information

Topologically Associated Domains Delineate Susceptibility to Somatic Hypermutation

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets