Pervasive structural heterogeneity rewires glioblastoma chromosomes to sustain patient-specific transcriptional programs.

Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compile a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generate and analyze 5 kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map thousands of standalone and complex structural variants (SVs) and the multitude of neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can relate to patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.

Enhancer-promoter contact formation requires RNAPII and antagonizes loop extrusion.

Homotypic chromatin interactions and loop extrusion are thought to be the two main drivers of mammalian chromosome folding. Here we tested the role of RNA polymerase II (RNAPII) across different scales of interphase chromatin organization in a cellular system allowing for its rapid, auxin-mediated degradation. We combined Micro-C and computational modeling to characterize subsets of loops differentially gained or lost upon RNAPII depletion. Gained loops, extrusion of which was antagonized by RNAPII, almost invariably formed by engaging new or rewired CTCF anchors. Lost loops selectively affected contacts between enhancers and promoters anchored by RNAPII, explaining the repression of most genes. Surprisingly, promoter-promoter interactions remained essentially unaffected by polymerase depletion, and cohesin occupancy was sustained. Together, our findings reconcile the role of RNAPII in transcription with its direct involvement in setting-up regulatory three-dimensional chromatin contacts genome wide, while also revealing an impact on cohesin loop extrusion.

RNA polymerase II is required for spatial chromatin reorganization following exit from mitosis.

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces marked chromosome condensation, but following reentry into the G1 phase of the cell cycle, chromosomes reestablish their interphase organization. Here, we tested the role of RNA polymerase II (RNAPII) in this transition using a cell line that allows its auxin-mediated degradation. In situ Hi-C showed that RNAPII is required for both compartment and loop establishment following mitosis. RNAPs often counteract loop extrusion, and in their absence, longer and more prominent loops arose. Evidence from chromatin binding, super-resolution imaging, and in silico modeling allude to these effects being a result of RNAPII-mediated cohesin loading upon G1 reentry. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.

HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence.

Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome-wide in two primary cell lines. We integrated ChIP-seq and Hi-C with graph theory to uncover clustering of HMGB1-marked topological domains that harbor genes involved in paracrine senescence. Using simplified Cross-Linking and Immuno-Precipitation and functional tests, we show that HMGB1 is also a bona fide RNA-binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate availability of SASP-relevant transcripts. Our findings reveal a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell-autonomous and paracrine senescence.

Technologies to study spatial genome organization: beyond 3C.

The way that chromatin is organized in three-dimensional nuclear space is now acknowledged as a factor critical for the major cell processes, like transcription, replication and cell division. Researchers have been armed with new molecular and imaging technologies to study this structure-to-function link of genomes, spearheaded by the introduction of the 'chromosome conformation capture' technology more than a decade ago. However, this technology is not without shortcomings, and novel variants and orthogonal approaches are being developed to overcome these. As a result, the field of nuclear organization is constantly fueled by methods of increasing resolution and/or throughput that strive to eliminate systematic biases and increase precision. In this review, we attempt to highlight the most recent advances in technology that promise to provide novel insights on how chromosomes fold and function.

The histone methyltransferase DOT1L is required for proper DNA damage response, DNA repair, and modulates chemotherapy responsiveness.

BACKGROUND: Disruptor of telomeric silencing 1-like (DOT1L) is a non-SET domain containing methyltransferase known to catalyze mono-, di-, and tri-methylation of histone 3 on lysine 79 (H3K79me). DOT1L-mediated H3K79me has been implicated in chromatin-associated functions including gene transcription, heterochromatin formation, and DNA repair. Recent studies have uncovered a role for DOT1L in the initiation and progression of leukemia and other solid tumors. The development and availability of small molecule inhibitors of DOT1L may provide new and unique therapeutic options for certain types or subgroups of cancer. METHODS: In this study, we examined the role of DOT1L in DNA double-strand break (DSB) response and repair by depleting DOT1L using siRNA or inhibiting its methyltransferase activity using small molecule inhibitors in colorectal cancer cells. Cells were treated with different agents to induce DNA damage in DOT1L-depleted or -inhibited cells and analyzed for DNA repair efficiency and survival. Further, rectal cancer patient samples were analyzed for H3K79me3 levels in order to determine whether it may serve as a potential marker for personalized therapy. RESULTS: Our results indicate that DOT1L is required for a proper DNA damage response following DNA double-strand breaks by regulating the phosphorylation of the variant histone H2AX (γH2AX) and repair via homologous recombination (HR). Importantly, we show that small molecule inhibitors of DOT1L combined with chemotherapeutic agents that are used to treat colorectal cancers show additive effects. Furthermore, examination of H3K79me3 levels in rectal cancer patients demonstrates that lower levels correlate with a poorer prognosis. CONCLUSIONS: In this study, we conclude that DOT1L plays an important role in an early DNA damage response and repair of DNA double-strand breaks via the HR pathway. Moreover, DOT1L inhibition leads to increased sensitivity to chemotherapeutic agents and PARP inhibition, which further highlights its potential clinical utility. Our results further suggest that H3K79me3 can be useful as a predictive and or prognostic marker for rectal cancer patients.